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Sample records for acid-inducible gene-i rig-i

  1. Antiviral activity of human oligoadenylate synthetases-like (OASL) is mediated by enhancing retinoic acid-inducible gene I (RIG-I) signaling

    PubMed Central

    Zhu, Jianzhong; Zhang, Yugen; Ghosh, Arundhati; Cuevas, Rolando A.; Forero, Adriana; Dhar, Jayeeta; Ibsen, Mikkel Søes; Schmid-Burgk, Jonathan Leo; Schmidt, Tobias; Ganapathiraju, Madhavi K.; Fujita, Takashi; Hartmann, Rune; Barik, Sailen; Hornung, Veit; Coyne, Carolyn B.; Sarkar, Saumendra N.

    2014-01-01

    SUMMARY Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN), and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein had antiviral activity and mediated RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone marrow derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL; Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation. PMID:24931123

  2. Muscovy duck retinoic acid-induced gene I (MdRIG-I) functions in innate immunity against H9N2 avian influenza viruses (AIV) infections.

    PubMed

    Cheng, Yuqiang; Huang, Qingqing; Ji, Wenhui; Du, Bin; Fu, Qiang; An, Huiting; Li, Jing; Wang, Hengan; Yan, Yaxian; Ding, Chan; Sun, Jianhe

    2015-02-15

    Retinoic acid inducible gene I (RIG-I) is a cytosolic pattern recognition receptor that senses pathogen-associated molecular patterns (PAMPs). Muscovy duck (Cairina moschata) is a large duck different from other species of ducks, and is more susceptible to some microbial pathogens. In this study, the Muscovy duck RIG-I gene (MdRIG-I) was identified. Quantitative RT-PCR showed that MdRIG-I mRNA was widely expressed in different tissues, especially in those with mucosa. RIG-I null DF-1 cells transfected with DNA constructs encoding MdRIG-I or CARDs domain can activate IRF-3 and NF-κB to up-regulated activity of IFN-β promoter. The components of the signaling pathway downstream of RIG-I in mammalian cells including IRF-3, NF-κB, IFN-β and the IFN-stimulated genes Mx-1, PKR and MDA5 were significantly up-regulated in CARDs-overexpressing-DF-1 cells. Implicating RIG-I in the antiviral response to an infection in vivo, we found that RIG-I expression in brain, spleen, lung and bursa were up-regulated in ducks challenged with H9N2 avian influenza virus (AIV), whose six internal genes were closely related to the H7N9 and H10N8 AIV. In vitro, DF-1 cells transfected with MdRIG-I plasmid can respond significantly to H9N2 AIV, evident through enhancement of IFN-β promoter activity and decreased virus titer. Altogether, these results indicated that MdRIG-I is a novel member of RLR gene family, engaging in the early stage of antiviral innate immunity.

  3. Retinoic acid-induced gene-I (RIG-I) associates with nucleotide-binding oligomerization domain-2 (NOD2) to negatively regulate inflammatory signaling.

    PubMed

    Morosky, Stefanie A; Zhu, Jianzhong; Mukherjee, Amitava; Sarkar, Saumendra N; Coyne, Carolyn B

    2011-08-12

    Cytoplasmic caspase recruiting domain (CARD)-containing molecules often function in the induction of potent antimicrobial responses in order to protect mammalian cells from invading pathogens. Retinoic acid-induced gene-I (RIG-I) and nucleotide binding oligomerization domain 2 (NOD2) serve as key factors in the detection of viral and bacterial pathogens, and in the subsequent initiation of innate immune signals to combat infection. RIG-I and NOD2 share striking similarities in their cellular localization, both localize to membrane ruffles in non-polarized epithelial cells and both exhibit a close association with the junctional complex of polarized epithelia. Here we show that RIG-I and NOD2 not only colocalize to cellular ruffles and cell-cell junctions, but that they also form a direct interaction that is mediated by the CARDs of RIG-I and multiple regions of NOD2. Moreover, we show that RIG-I negatively regulates ligand-induced nuclear factor-κB (NF-κB) signaling mediated by NOD2, and that NOD2 negatively regulates type I interferon induction by RIG-I. We also show that the three main Crohn disease-associated mutants of NOD2 (1007fs, R702W, G908R) form an interaction with RIG-I and negatively regulate its signaling to a greater extent than wild-type NOD2. Our results show that in addition to their role in innate immune recognition, RIG-I and NOD2 form a direct interaction at actin-enriched sites within cells and suggest that this interaction may impact RIG-I- and NOD2-dependent innate immune signaling.

  4. Tumor suppressor activity of RIG-I

    PubMed Central

    Li, Xian-Yang; Guo, He-Zhou; Zhu, Jiang

    2014-01-01

    Retinoic acid inducible gene-I (RIG-I), named for the observation that its mRNA expression is highly upregulated in the progression of all-trans retinoic acid (ATRA)-induced maturation of acute promyelocytic leukemia (APL) cells, has been well documented as a pivotal virus-associated molecular pattern recognition receptor (PRR) responsible for triggering innate immunity. Upon recognizing viral RNA ligands, RIG-I experiences a series of programmed conformational changes and modifications that unleash its activity through the formation of complexes with various binding partners. Such partners include the mitochondria membrane-anchored protein IPS-1 (also named MAVS/VISA/Cardif) that activates both the IRF3/7 and NF-κB pathways. These partnerships and resulting pathway activations underlie the synthesis of type I interferon and other inflammatory factors. Recent studies have demonstrated that RIG-I is also involved in the regulation of basic cellular processes outside of innate immunity against viral infections, such as hematopoietic proliferation and differentiation, maintenance of leukemic stemness, and tumorigenesis of hepatocellular carcinoma. In this review, we will highlight recent studies leading up to the recognition that RIG-I performs an essential function as a tumor suppressor and try to reconcile this activity of RIG-I with its well-known role in protecting cells against viral infection. PMID:27308362

  5. Endothelial RIG-I activation impairs endothelial function

    SciTech Connect

    Asdonk, Tobias; Nickenig, Georg; Zimmer, Sebastian

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer RIG-I activation impairs endothelial function in vivo. Black-Right-Pointing-Pointer RIG-I activation alters HCAEC biology in vitro. Black-Right-Pointing-Pointer EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 {mu}g of the RIG-ligand 3pRNA (RNA with triphosphate at the 5 Prime end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

  6. Dependence of RIG-I Nucleic Acid-Binding and ATP Hydrolysis on Activation of Type I Interferon Response

    PubMed Central

    Baek, Yu Mi; Yoon, Soojin; Hwang, Yeo Eun

    2016-01-01

    Exogenous nucleic acids induce an innate immune response in mammalian host cells through activation of the retinoic acid-inducible gene I (RIG-I). We evaluated RIG-I protein for RNA binding and ATPase stimulation with RNA ligands to investigate the correlation with the extent of immune response through RIG-I activation in cells. RIG-I protein favored blunt-ended, double-stranded RNA (dsRNA) ligands over sticky-ended dsRNA. Moreover, the presence of the 5'-triphosphate (5'-ppp) moiety in dsRNA further enhanced binding affinity to RIG-I. Two structural motifs in RNA, blunt ends in dsRNA and 5'-ppp, stimulated the ATP hydrolysis activity of RIG-I. These structural motifs also strongly induced IFN expression as an innate immune response in cells. Therefore, we suggest that IFN induction through RIG-I activation is mainly determined by structural motifs in dsRNA that increase its affinity for RIG-I protein and stimulate ATPase activity in RIG-I. PMID:27574504

  7. Negative Role of RIG-I Serine 8 Phosphorylation in the Regulatin of Interferon-beta Production

    SciTech Connect

    E Nistal-Villan; M Gack; G Martinez-Delgado; N Maharaj; K Inn; H Yang; R Wang; A Aggarwal; J Jung; A Garcia-Sastre

    2011-12-31

    RIG-I (retinoic acid-inducible gene I) and TRIM25 (tripartite motif protein 25) have emerged as key regulatory factors to induce interferon (IFN)-mediated innate immune responses to limit viral replication. Upon recognition of viral RNA, TRIM25 E3 ligase binds the first caspase recruitment domain (CARD) of RIG-I and subsequently induces lysine 172 ubiquitination of the second CARD of RIG-I, which is essential for the interaction with downstream MAVS/IPS-1/CARDIF/VISA and, thereby, IFN-beta mRNA production. Although ubiquitination has emerged as a major factor involved in RIG-I activation, the potential contribution of other post-translational modifications, such as phosphorylation, to the regulation of RIG-I activity has not been addressed. Here, we report the identification of serine 8 phosphorylation at the first CARD of RIG-I as a negative regulatory mechanism of RIG-I-mediated IFN-beta production. Immunoblot analysis with a phosphospecific antibody showed that RIG-I serine 8 phosphorylation steady-state levels were decreased upon stimulation of cells with IFN-beta or virus infection. Substitution of serine 8 in the CARD RIG-I functional domain with phosphomimetic aspartate or glutamate results in decreased TRIM25 binding, RIG-I ubiquitination, MAVS binding, and downstream signaling. Finally, sequence comparison reveals that only primate species carry serine 8, whereas other animal species carry an asparagine, indicating that serine 8 phosphorylation may represent a primate-specific regulation of RIG-I activation. Collectively, these data suggest that the phosphorylation of RIG-I serine 8 operates as a negative switch of RIG-I activation by suppressing TRIM25 interaction, further underscoring the importance of RIG-I and TRIM25 connection in type I IFN signal transduction.

  8. RIG-I Signaling Is Critical for Efficient Polyfunctional T Cell Responses during Influenza Virus Infection

    PubMed Central

    Kandasamy, Matheswaran; Suryawanshi, Amol; Tundup, Smanla; Perez, Jasmine T.; Schmolke, Mirco; Manicassamy, Santhakumar; Manicassamy, Balaji

    2016-01-01

    Retinoic acid inducible gene-I (RIG-I) is an innate RNA sensor that recognizes the influenza A virus (IAV) RNA genome and activates antiviral host responses. Here, we demonstrate that RIG-I signaling plays a crucial role in restricting IAV tropism and regulating host immune responses. Mice deficient in the RIG-I-MAVS pathway show defects in migratory dendritic cell (DC) activation, viral antigen presentation, and priming of CD8+ and CD4+ T cell responses during IAV infection. These defects result in decreased frequency of polyfunctional effector T cells and lowered protection against heterologous IAV challenge. In addition, our data show that RIG-I activation is essential for protecting epithelial cells and hematopoietic cells from IAV infection. These diverse effects of RIG-I signaling are likely imparted by the actions of type I interferon (IFN), as addition of exogenous type I IFN is sufficient to overcome the defects in antigen presentation by RIG-I deficient BMDC. Moreover, the in vivo T cell defects in RIG-I deficient mice can be overcome by the activation of MDA5 –MAVS via poly I:C treatment. Taken together, these findings demonstrate that RIG-I signaling through MAVS is critical for determining the quality of polyfunctional T cell responses against IAV and for providing protection against subsequent infection from heterologous or novel pandemic IAV strains. PMID:27438481

  9. Host and Viral Modulation of RIG-I-Mediated Antiviral Immunity

    PubMed Central

    Liu, Yiliu; Olagnier, David; Lin, Rongtuan

    2017-01-01

    Innate immunity is the first line of defense against invading pathogens. Rapid and efficient detection of pathogen-associated molecular patterns via pattern-recognition receptors is essential for the host to mount defensive and protective responses. Retinoic acid-inducible gene-I (RIG-I) is critical in triggering antiviral and inflammatory responses for the control of viral replication in response to cytoplasmic virus-specific RNA structures. Upon viral RNA recognition, RIG-I recruits the mitochondrial adaptor protein mitochondrial antiviral signaling protein, which leads to a signaling cascade that coordinates the induction of type I interferons (IFNs), as well as a large variety of antiviral interferon-stimulated genes. The RIG-I activation is tightly regulated via various posttranslational modifications for the prevention of aberrant innate immune signaling. By contrast, viruses have evolved mechanisms of evasion, such as sequestrating viral structures from RIG-I detections and targeting receptor or signaling molecules for degradation. These virus–host interactions have broadened our understanding of viral pathogenesis and provided insights into the function of the RIG-I pathway. In this review, we summarize the recent advances regarding RIG-I pathogen recognition and signaling transduction, cell-intrinsic control of RIG-I activation, and the viral antagonism of RIG-I signaling. PMID:28096803

  10. RIG-I Signaling Is Critical for Efficient Polyfunctional T Cell Responses during Influenza Virus Infection.

    PubMed

    Kandasamy, Matheswaran; Suryawanshi, Amol; Tundup, Smanla; Perez, Jasmine T; Schmolke, Mirco; Manicassamy, Santhakumar; Manicassamy, Balaji

    2016-07-01

    Retinoic acid inducible gene-I (RIG-I) is an innate RNA sensor that recognizes the influenza A virus (IAV) RNA genome and activates antiviral host responses. Here, we demonstrate that RIG-I signaling plays a crucial role in restricting IAV tropism and regulating host immune responses. Mice deficient in the RIG-I-MAVS pathway show defects in migratory dendritic cell (DC) activation, viral antigen presentation, and priming of CD8+ and CD4+ T cell responses during IAV infection. These defects result in decreased frequency of polyfunctional effector T cells and lowered protection against heterologous IAV challenge. In addition, our data show that RIG-I activation is essential for protecting epithelial cells and hematopoietic cells from IAV infection. These diverse effects of RIG-I signaling are likely imparted by the actions of type I interferon (IFN), as addition of exogenous type I IFN is sufficient to overcome the defects in antigen presentation by RIG-I deficient BMDC. Moreover, the in vivo T cell defects in RIG-I deficient mice can be overcome by the activation of MDA5 -MAVS via poly I:C treatment. Taken together, these findings demonstrate that RIG-I signaling through MAVS is critical for determining the quality of polyfunctional T cell responses against IAV and for providing protection against subsequent infection from heterologous or novel pandemic IAV strains.

  11. What Really Rigs Up RIG-I?

    PubMed

    Barik, Sailen

    2016-01-01

    RIG-I (retinoic acid-inducible gene 1) is an archetypal member of the cytoplasmic DEAD-box dsRNA helicase family (RIG-I-like receptors or RLRs), the members of which play essential roles in the innate immune response of the metazoan cell. RIG-I functions as a pattern recognition receptor that detects nonself RNA as a pathogen-associated molecular pattern (PAMP). However, the exact molecular nature of the viral RNAs that act as a RIG-I ligand has remained a mystery and a matter of debate. In this article, we offer a critical review of the actual viral RNAs that act as PAMPs to activate RIG-I, as seen from the perspective of a virologist, including a recent report that the viral Leader-read-through transcript is a novel and effective RIG-I ligand.

  12. Syndecan-4 negatively regulates antiviral signalling by mediating RIG-I deubiquitination via CYLD

    PubMed Central

    Lin, Wei; Zhang, Jing; Lin, Haiyan; Li, Zexing; Sun, Xiaofeng; Xin, Di; Yang, Meng; Sun, Liwei; Li, Lin; Wang, Hongmei; Chen, Dahua; Sun, Qinmiao

    2016-01-01

    Retinoic acid-inducible gene I (RIG-I) plays important roles in pathogen recognition and antiviral signalling transduction. Here we show that syndecan-4 (SDC4) is a RIG-I-interacting partner identified in a yeast two-hybrid screen. We find that SDC4 negatively regulates the RIG-I-mediated antiviral signalling in a feedback-loop control manner. The genetic evidence obtained by using knockout mice further emphasizes this biological role of SDC4 in antiviral signalling. Mechanistically, we show that SDC4 interacts with both RIG-I and deubiquitinase CYLD via its carboxyl-terminal intracellular region. SDC4 likely promotes redistribution of RIG-I and CYLD in a perinuclear pattern post viral infection, and thus enhances the RIG-I–CYLD interaction and potentiates the K63-linked deubiquitination of RIG-I. Collectively, our findings uncover a mechanism by which SDC4 antagonizes the activation of RIG-I in a CYLD-mediated deubiquitination-dependent process, thereby balancing antiviral signalling to avoid deleterious effects on host cells. PMID:27279133

  13. Hepatitis C Virus Frameshift/Alternate Reading Frame Protein Suppresses Interferon Responses Mediated by Pattern Recognition Receptor Retinoic-Acid-Inducible Gene-I

    PubMed Central

    Park, Seung Bum; Seronello, Scott; Mayer, Wasima; Ojcius, David M.

    2016-01-01

    Hepatitis C virus (HCV) actively evades host interferon (IFN) responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I)-mediated IFN induction. Expression of frameshift/alternate reading frame protein (F/ARFP) from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP) and poly(IC). The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in the full-length HCV sequence (JFH1 genotype 2a strain) were made to introduce premature termination codons in the -2/+1 reading frame coding for F/ARFP while preserving the original reading frame, which enhanced IFNα and IFNβ induction by HCV. The potentiation of IFN response by the F/ARFP mutations was diminished in Huh7.5 cells, which already have a defective RIG-I, and by decreasing RIG-I expression in Huh7 cells. Furthermore, adding F/ARFP back via trans-complementation suppressed IFN induction in the F/ARFP mutant. The F/ARFP mutants, on the other hand, were not resistant to exogenous IFNα. Finally, HCV-infected human liver samples showed significant F/ARFP antibody reactivity, compared to HCV-uninfected control livers. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress type I and III IFN induction occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of pattern recognition receptor modulation by HCV and suggests a biological function of the HCV alternate reading frame in the modulation of host innate immunity. PMID:27404108

  14. Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5′-diphosphates

    PubMed Central

    Deddouche, Safia; Pruijssers, Andrea J.; Zillinger, Thomas; Goldeck, Marion; Schuberth, Christine; Van der Veen, Annemarthe G.; Fujimura, Tsutomu; Rehwinkel, Jan; Iskarpatyoti, Jason A.; Barchet, Winfried; Ludwig, Janos; Dermody, Terence S.; Hartmann, Gunther; Reis e Sousa, Caetano

    2014-01-01

    SUMMARY Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection1,2. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α/β; hereafter IFN), key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5′-end (reviewed in 1-3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5′-diphosphates (5′pp). Genomes from mammalian reoviruses with 5′pp termini, 5′pp-RNA isolated from yeast L-A virus, and base-paired 5′pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5′pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5′pp. Such RNAs are found in some viruses but not uninfected cells, indicating that recognition of 5′pp-RNA, like that of 5′ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system. PMID:25119032

  15. RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling

    PubMed Central

    Durbin, Ann Fiegen; Wang, Chen; Marcotrigiano, Joseph

    2016-01-01

    ABSTRACT Invading pathogen nucleic acids are recognized and bound by cytoplasmic (retinoic acid-inducible gene I [RIG-I]-like) and membrane-bound (Toll-like) pattern recognition receptors to activate innate immune signaling. Modified nucleotides, when present in RNA molecules, diminish the magnitude of these signaling responses. However, mechanisms explaining the blunted signaling have not been elucidated. In this study, we used several independent biological assays, including inhibition of virus replication, RIG-I:RNA binding assays, and limited trypsin digestion of RIG-I:RNA complexes, to begin to understand how RNAs containing modified nucleotides avoid or suppress innate immune signaling. The experiments were based on a model innate immune activating RNA molecule, the polyU/UC RNA domain of hepatitis C virus, which was transcribed in vitro with canonical nucleotides or with one of eight modified nucleotides. The approach revealed signature assay responses associated with individual modified nucleotides or classes of modified nucleotides. For example, while both N-6-methyladenosine (m6A) and pseudouridine nucleotides correlate with diminished signaling, RNA containing m6A modifications bound RIG-I poorly, while RNA containing pseudouridine bound RIG-I with high affinity but failed to trigger the canonical RIG-I conformational changes associated with robust signaling. These data advance understanding of RNA-mediated innate immune signaling, with additional relevance for applying nucleotide modifications to RNA therapeutics. PMID:27651356

  16. Pigeon RIG-I Function in Innate Immunity against H9N2 IAV and IBDV.

    PubMed

    Xu, Wenping; Shao, Qiang; Zang, Yunlong; Guo, Qiang; Zhang, Yongchao; Li, Zandong

    2015-07-22

    Retinoic acid-inducible gene I (RIG-I), a cytosolic pattern recognition receptor (PRR), can sense various RNA viruses, including the avian influenza virus (AIV) and infectious bursal disease virus (IBDV), and trigger the innate immune response. Previous studies have shown that mammalian RIG-I (human and mice) and waterfowl RIG-I (ducks and geese) are essential for type I interferon (IFN) synthesis during AIV infection. Like ducks, pigeons are also susceptible to infection but are ineffective propagators and disseminators of AIVs, i.e., "dead end" hosts for AIVs and even highly pathogenic avian influenza (HPAI). Consequently, we sought to identify pigeon RIG-I and investigate its roles in the detection of A/Chicken/Shandong/ZB/2007 (H9N2) (ZB07), Gansu/Tianshui (IBDV TS) and Beijing/CJ/1980 (IBDV CJ-801) strains in chicken DF-1 fibroblasts or human 293T cells. Pigeon mRNA encoding the putative pigeon RIG-I analogs was identified. The exogenous expression of enhanced green fluorescence protein (EGFP)-tagged pigeon RIG-I and caspase activation and recruitment domains (CARDs), strongly induced antiviral gene (IFN-β, Mx, and PKR) mRNA synthesis, decreased viral gene (M gene and VP2) mRNA expression, and reduced the viral titers of ZB07 and IBDV TS/CJ-801 virus strains in chicken DF-1 cells, but not in 293T cells. We also compared the antiviral abilities of RIG-I proteins from waterfowl (duck and goose) and pigeon. Our data indicated that waterfowl RIG-I are more effective in the induction of antiviral genes and the repression of ZB07 and IBDV TS/CJ-801 strain replication than pigeon RIG-I. Furthermore, chicken melanoma differentiation associated gene 5(MDA5)/ mitochondrial antiviral signaling (MAVS) silencing combined with RIG-I transfection suggested that pigeon RIG-I can restore the antiviral response in MDA5-silenced DF-1 cells but not in MAVS-silenced DF-1 cells. In conclusion, these results demonstrated that pigeon RIG-I and CARDs have a strong antiviral ability

  17. Systems-based analysis of RIG-I-dependent signalling identifies KHSRP as an inhibitor of RIG-I receptor activation.

    PubMed

    Soonthornvacharin, Stephen; Rodriguez-Frandsen, Ariel; Zhou, Yingyao; Galvez, Felipe; Huffmaster, Nicholas J; Tripathi, Shashank; Balasubramaniam, Vinod R M T; Inoue, Atsushi; de Castro, Elisa; Moulton, Hong; Stein, David A; Sánchez-Aparicio, María Teresa; De Jesus, Paul D; Nguyen, Quy; König, Renate; Krogan, Nevan J; García-Sastre, Adolfo; Yoh, Sunnie M; Chanda, Sumit K

    2017-03-01

    Retinoic acid-inducible gene I (RIG-I) receptor recognizes 5'-triphosphorylated RNA and triggers a signalling cascade that results in the induction of type-I interferon (IFN)-dependent responses. Its precise regulation represents a pivotal balance between antiviral defences and autoimmunity. To elucidate the cellular cofactors that regulate RIG-I signalling, we performed two global RNA interference analyses to identify both positive and negative regulatory nodes operating on the signalling pathway during virus infection. These factors were integrated with experimentally and computationally derived interactome data to build a RIG-I protein interaction network. Our analysis revealed diverse cellular processes, including the unfolded protein response, Wnt signalling and RNA metabolism, as critical cellular components governing innate responses to non-self RNA species. Importantly, we identified K-Homology Splicing Regulatory Protein (KHSRP) as a negative regulator of this pathway. We find that KHSRP associates with the regulatory domain of RIG-I to maintain the receptor in an inactive state and attenuate its sensing of viral RNA (vRNA). Consistent with increased RIG-I antiviral signalling in the absence of KHSRP, viral replication is reduced when KHSRP expression is knocked down both in vitro and in vivo. Taken together, these data indicate that KHSRP functions as a checkpoint regulator of the innate immune response to pathogen challenge.

  18. Z proteins of New World arenaviruses bind RIG-I and interfere with type I interferon induction.

    PubMed

    Fan, Lina; Briese, Thomas; Lipkin, W Ian

    2010-02-01

    The retinoic acid-inducible gene I product (RIG-I) is a cellular sensor of RNA virus infection that regulates the cellular beta interferon (IFN-beta) response. The nucleoproteins (NP) of arenaviruses are reported to antagonize the IFN response by inhibiting interferon regulatory factor 3 (IRF-3). Here, we demonstrate that the Z proteins of four New World (NW) arenaviruses, Guanarito virus (GTOV), Junin virus (JUNV), Machupo virus (MAVC), and Sabia virus (SABV), bind to RIG-I, resulting in downregulation of the IFN-beta response. We show that expression of the four NW arenavirus Z proteins inhibits IFN-beta mRNA induction in A549 cells in response to RNA bearing 5' phosphates (5'pppRNA). NW arenavirus Z proteins interact with RIG-I in coimmunoprecipitation studies and colocalize with RIG-I. Furthermore, expression of Z proteins interferes with the interaction between RIG-I and MAVS. Z expression also impedes the nuclear factor kappa light chain enhancer of activated B cells (NF-kappaB) and IRF-3 activation. Our results indicate that NW arenavirus Z proteins, but not Z protein of the Old World (OW) arenavirus lymphocytic choriomeningitis virus (LCMV) or Lassa virus, bind to RIG-I and inhibit downstream activation of the RIG-I signaling pathway, preventing the transcriptional induction of IFN-beta.

  19. Activation of Duck RIG-I by TRIM25 Is Independent of Anchored Ubiquitin

    PubMed Central

    Miranzo-Navarro, Domingo; Magor, Katharine E.

    2014-01-01

    Retinoic acid inducible gene I (RIG-I) is a viral RNA sensor crucial in defense against several viruses including measles, influenza A and hepatitis C. RIG-I activates type-I interferon signalling through the adaptor for mitochondrial antiviral signaling (MAVS). The E3 ubiquitin ligase, tripartite motif containing protein 25 (TRIM25), activates human RIG-I through generation of anchored K63-linked polyubiquitin chains attached to lysine 172, or alternatively, through the generation of unanchored K63-linked polyubiquitin chains that interact non-covalently with RIG-I CARD domains. Previously, we identified RIG-I of ducks, of interest because ducks are the host and natural reservoir of influenza viruses, and showed it initiates innate immune signaling leading to production of interferon-beta (IFN-β). We noted that K172 is not conserved in RIG-I of ducks and other avian species, or mouse. Because K172 is important for both mechanisms of activation of human RIG-I, we investigated whether duck RIG-I was activated by TRIM25, and if other residues were the sites for attachment of ubiquitin. Here we show duck RIG-I CARD domains are ubiquitinated for activation, and ubiquitination depends on interaction with TRIM25, as a splice variant that cannot interact with TRIM25 is not ubiquitinated, and cannot be activated. We expressed GST-fusion proteins of duck CARD domains and characterized TRIM25 modifications of CARD domains by mass spectrometry. We identified two sites that are ubiquitinated in duck CARD domains, K167 and K193, and detected K63 linked polyubiquitin chains. Site directed mutagenesis of each site alone, does not alter the ubiquitination profile of the duck CARD domains. However, mutation of both sites resulted in loss of all attached ubiquitin and polyubiquitin chains. Remarkably, the double mutant duck RIG-I CARD still interacts with TRIM25, and can still be activated. Our results demonstrate that anchored ubiquitin chains are not necessary for TRIM25

  20. RIG-I detects HIV-1 infection and mediates type I interferon response in human macrophages from patients with HIV-1-associated neurocognitive disorders

    PubMed Central

    Wang, M.Q.; Huang, Y.L.; Huang, J.; Zheng, J.L.; Qian, G.X.

    2016-01-01

    The aim of this study was to explore the precise role of retinoic acid-inducible gene-I (RIG-I) signaling in human immunodeficiency virus type 1 (HIV-1)-infected macrophages from patients with HIV-1-associated neurocognitive disorders (HAND). Postmortem brain tissues were collected from patients with HIV-1-associated dementia and were compared to samples collected from HIV serum-positive patients without dementia and HIV serum-negative patients. A human monocyte-derived macrophage (MDM) primary culture system was established to evaluate the expression of RIG-I in these samples. Knockdown of RIG-I pathways genes was employed and STAT1 expression and phosphorylation levels were examined to explore the molecular mechanisms of HAND. The expression of RIG-I in postmortem brain tissue from HAND patients was significantly higher than in patients who were HIV serum-positive without dementia or HIV serum-negative. Moreover, we demonstrated that HIV-1 infection could result in a significant increase in the level of RIG-I in human MDMs. Moreover, a correlation was found between the increase in RIG-I expression and STAT1 expression and phosphorylation. Accordingly, knockdown of RIG-I decreased the phosphorylation of STAT1 and downregulated interferon-related genes. These observations highlight the importance of RIG-I signaling in anti-HIV innate immunity in macrophages, which may be beneficial for the treatment of HIV and aid in the understanding of the neuropathogenesis of HAND. PMID:26535695

  1. Structural features of influenza A virus panhandle RNA enabling the activation of RIG-I independently of 5′-triphosphate

    PubMed Central

    Lee, Mi-Kyung; Kim, Hee-Eun; Park, Eun-Byeol; Lee, Janghyun; Kim, Ki-Hun; Lim, Kyungeun; Yum, Seoyun; Lee, Young-Hoon; Kang, Suk-Jo; Lee, Joon-Hwa; Choi, Byong-Seok

    2016-01-01

    Retinoic acid-inducible gene I (RIG-I) recognizes specific molecular patterns of viral RNAs for inducing type I interferon. The C-terminal domain (CTD) of RIG-I binds to double-stranded RNA (dsRNA) with the 5′-triphosphate (5′-PPP), which induces a conformational change in RIG-I to an active form. It has been suggested that RIG-I detects infection of influenza A virus by recognizing the 5′-triphosphorylated panhandle structure of the viral RNA genome. Influenza panhandle RNA has a unique structure with a sharp helical bending. In spite of extensive studies of how viral RNAs activate RIG-I, whether the structural elements of the influenza panhandle RNA confer the ability to activate RIG-I signaling has been poorly explored. Here, we investigated the dynamics of the influenza panhandle RNA in complex with RIG-I CTD using NMR spectroscopy and showed that the bending structure of the panhandle RNA negates the requirement of a 5′-PPP moiety for RIG-I activation. PMID:27288441

  2. Establishing the role of ATP for the function of the RIG-I innate immune sensor

    PubMed Central

    Rawling, David C; Fitzgerald, Megan E; Pyle, Anna Marie

    2015-01-01

    Retinoic acid-inducible gene I (RIG-I) initiates a rapid innate immune response upon detection and binding to viral ribonucleic acid (RNA). This signal activation occurs only when pathogenic RNA is identified, despite the ability of RIG-I to bind endogenous RNA while surveying the cytoplasm. Here we show that ATP binding and hydrolysis by RIG-I play a key role in the identification of viral targets and the activation of signaling. Using biochemical and cell-based assays together with mutagenesis, we show that ATP binding, and not hydrolysis, is required for RIG-I signaling on viral RNA. However, we show that ATP hydrolysis does provide an important function by recycling RIG-I and promoting its dissociation from non-pathogenic RNA. This activity provides a valuable proof-reading mechanism that enhances specificity and prevents an antiviral response upon encounter with host RNA molecules. DOI: http://dx.doi.org/10.7554/eLife.09391.001 PMID:26371557

  3. RIG-I activation induces the release of extracellular vesicles with antitumor activity

    PubMed Central

    Daßler-Plenker, Juliane; Reiners, Katrin S.; van den Boorn, Jasper G.; Hansen, Hinrich P.; Putschli, Bastian; Barnert, Sabine; Schuberth-Wagner, Christine; Schubert, Rolf; Tüting, Thomas; Hallek, Michael; Schlee, Martin; Hartmann, Gunther; Pogge von Strandmann, Elke; Coch, Christoph

    2016-01-01

    ABSTRACT Activation of the innate immune receptor retinoic acid-inducible gene I (RIG-I) by its specific ligand 5′-triphosphate-RNA (3pRNA) triggers antitumor immunity predominantly via NK cell activation and direct apoptosis induction in tumor cells. However, how NK cells are mobilized to attack the tumor cells remains elusive. Here, we show that RIG-I activation induced the secretion of extracellular vesicles (EVs) from melanoma cells, which by themselves revealed antitumor activity in vitro and in vivo. RIG-I-induced EVs from melanoma cells exhibited an increased expression of the NKp30-ligand (BAG6, BAT3) on their surface triggering NK cell-mediated lysis of melanoma cells via activation of the cytotoxicity NK cell-receptor NKp30. Moreover, systemic administration of RIG-I-induced melanoma-EVs showed a potent antitumor activity in a melanoma mouse model in vivo. In conclusion, our data establish a new RIG-I-dependent pathway leading to NK cell-mediated tumor cell killing. PMID:27853642

  4. RIG-I Mediates an Antiviral Response to Crimean-Congo Hemorrhagic Fever Virus

    PubMed Central

    Spengler, Jessica R.; Patel, Jenish R.; Chakrabarti, Ayan K.; Zivcec, Marko; García-Sastre, Adolfo; Spiropoulou, Christina F.

    2015-01-01

    ABSTRACT In the cytoplasm, the retinoic acid-inducible gene I (RIG-I) senses the RNA genomes of several RNA viruses. RIG-I binds to viral RNA, eliciting an antiviral response via the cellular adaptor MAVS. Crimean-Congo hemorrhagic fever virus (CCHFV), a negative-sense RNA virus with a 5′-monophosphorylated genome, is a highly pathogenic zoonotic agent with significant public health implications. We found that, during CCHFV infection, RIG-I mediated a type I interferon (IFN) response via MAVS. Interfering with RIG-I signaling reduced IFN production and IFN-stimulated gene expression and increased viral replication. Immunostimulatory RNA was isolated from CCHFV-infected cells and from virion preparations, and RIG-I coimmunoprecipitation of infected cell lysates isolated immunostimulatory CCHFV RNA. This report serves as the first description of a pattern recognition receptor for CCHFV and highlights a critical signaling pathway in the antiviral response to CCHFV. IMPORTANCE CCHFV is a tick-borne virus with a significant public health impact. In order for cells to respond to virus infection, they must recognize the virus as foreign and initiate antiviral signaling. To date, the receptors involved in immune recognition of CCHFV are not known. Here, we investigate and identify RIG-I as a receptor involved in initiating an antiviral response to CCHFV. This receptor initially was not expected to play a role in CCHFV recognition because of characteristics of the viral genome. These findings are important in understanding the antiviral response to CCHFV and support continued investigation into the spectrum of potential viruses recognized by RIG-I. PMID:26223644

  5. Foot-and-Mouth Disease Virus Viroporin 2B Antagonizes RIG-I-Mediated Antiviral Effects by Inhibition of Its Protein Expression

    PubMed Central

    Zhu, Zixiang; Wang, Guoqing; Yang, Fan; Cao, Weijun; Mao, Ruoqing; Du, Xiaoli; Zhang, Xiangle; Li, Chuntian; Li, Dan; Zhang, Keshan; Shu, Hongbing; Liu, Xiangtao

    2016-01-01

    ABSTRACT The role of retinoic acid-inducible gene I (RIG-I) in foot-and-mouth disease virus (FMDV)-infected cells remains unknown. Here, we showed that RIG-I inhibits FMDV replication in host cells. FMDV infection increased the transcription of RIG-I, while it decreased RIG-I protein expression. A detailed analysis revealed that FMDV leader proteinase (Lpro), as well as 3C proteinase (3Cpro) and 2B protein, decreased RIG-I protein expression. Lpro and 3Cpro are viral proteinases that can cleave various host proteins and are responsible for several of the viral polyprotein cleavages. However, for the first time, we observed 2B-induced reduction of host protein. Further studies showed that 2B-mediated reduction of RIG-I is specific to FMDV, but not other picornaviruses, including encephalomyocarditis virus, enterovirus 71, and coxsackievirus A16. Moreover, we found the decreased protein level of RIG-I is independent of the cleavage of eukaryotic translation initiation factor 4 gamma, the induction of cellular apoptosis, or the association of proteasome, lysosome, and caspase pathways. A direct interaction was observed between RIG-I and 2B. The carboxyl-terminal amino acids 105 to 114 and amino acids 135 to 144 of 2B were essential for the reduction of RIG-I, while residues 105 to 114 were required for the interaction. These data suggest the antiviral role of RIG-I against FMDV and a novel antagonistic mechanism of FMDV that is mediated by 2B protein. IMPORTANCE This study demonstrated that RIG-I could suppress FMDV replication during virus infection. FMDV infection increased the transcriptional expression of RIG-I, while it decreased RIG-I protein expression. FMDV 2B protein interacted with RIG-I and induced reduction of RIG-I. 2B-induced reduction of RIG-I was independent of the induction of the cleavage of eukaryotic translation initiation factor 4 gamma or cellular apoptosis. In addition, proteasome, lysosome, and caspase pathways were not involved in this process

  6. Structural basis of RNA recognition and activation by innate immune receptor RIG-I

    SciTech Connect

    Jiang, Fuguo; Ramanathan, Anand; Miller, Matthew T.; Tang, Guo-Qing; Gale, Jr., Michael; Patel, Smita S.; Marcotrigiano, Joseph

    2012-05-29

    Retinoic-acid-inducible gene-I (RIG-I; also known as DDX58) is a cytoplasmic pathogen recognition receptor that recognizes pathogen-associated molecular pattern (PAMP) motifs to differentiate between viral and cellular RNAs. RIG-I is activated by blunt-ended double-stranded (ds)RNA with or without a 5'-triphosphate (ppp), by single-stranded RNA marked by a 5'-ppp and by polyuridine sequences. Upon binding to such PAMP motifs, RIG-I initiates a signalling cascade that induces innate immune defences and inflammatory cytokines to establish an antiviral state. The RIG-I pathway is highly regulated and aberrant signalling leads to apoptosis, altered cell differentiation, inflammation, autoimmune diseases and cancer. The helicase and repressor domains (RD) of RIG-I recognize dsRNA and 5'-ppp RNA to activate the two amino-terminal caspase recruitment domains (CARDs) for signalling. Here, to understand the synergy between the helicase and the RD for RNA binding, and the contribution of ATP hydrolysis to RIG-I activation, we determined the structure of human RIG-I helicase-RD in complex with dsRNA and an ATP analogue. The helicase-RD organizes into a ring around dsRNA, capping one end, while contacting both strands using previously uncharacterized motifs to recognize dsRNA. Small-angle X-ray scattering, limited proteolysis and differential scanning fluorimetry indicate that RIG-I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full-length RIG-I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG-I helicase-RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including RNA

  7. Grass Carp Laboratory of Genetics and Physiology 2 Serves As a Negative Regulator in Retinoic Acid-Inducible Gene I- and Melanoma Differentiation-Associated Gene 5-Mediated Antiviral Signaling in Resting State and Early Stage of Grass Carp Reovirus Infection

    PubMed Central

    Rao, Youliang; Wan, Quanyuan; Yang, Chunrong; Su, Jianguo

    2017-01-01

    Laboratory of genetics and physiology 2 (LGP2) is a key component of RIG-I-like receptors (RLRs). However, the lack of the caspase recruitment domains (CARDs) results in its controversial functional performance as a negative or positive regulator in antiviral responses. Especially, no sufficient evidence uncovers the functional mechanisms of LGP2 in RLR signaling pathways in teleost. Here, negative regulation mechanism of LGP2 in certain situations in retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5)-mediated antiviral responses was identified in Ctenopharyngodon idella kidney cells. LGP2 overexpression inhibits synthesis and phosphorylation of interferon regulatory factor 3/7 (IRF3/7), and mRNA levels and promoter activities of IFNs and NF-κBs in resting state and early phase of grass carp reovirus (GCRV) infection. Knockdown of LGP2 obtains opposite effects. Luciferase report assay indicates that LGP2 works at the upstream of RIG-I and MDA5. LGP2 binds to RIG-I and MDA5 with diverse domain preference and which is independent of GCRV infection. Furthermore, LGP2 restrains K63-linked ubiquitination of RIG-I and MDA5 in various degrees. These differences result in disparate repressive mechanisms of LGP2 to RIG-I- and MDA5-mediated signal activations of IFN-β promoter stimulator 1 and mediator of IRF3 activation. Interestingly, LGP2 also inhibits K48-linked RIG-I and MDA5 ubiquitination to suppress proteins degradation, which guarantees the basal protein levels for subsequently rapid signal activation. All these results reveal a mechanism that LGP2 functions as a suppressor in RLR signaling pathways to maintain cellular homeostasis in resting state and early phase during GCRV infection.

  8. Grass Carp Laboratory of Genetics and Physiology 2 Serves As a Negative Regulator in Retinoic Acid-Inducible Gene I- and Melanoma Differentiation-Associated Gene 5-Mediated Antiviral Signaling in Resting State and Early Stage of Grass Carp Reovirus Infection.

    PubMed

    Rao, Youliang; Wan, Quanyuan; Yang, Chunrong; Su, Jianguo

    2017-01-01

    Laboratory of genetics and physiology 2 (LGP2) is a key component of RIG-I-like receptors (RLRs). However, the lack of the caspase recruitment domains (CARDs) results in its controversial functional performance as a negative or positive regulator in antiviral responses. Especially, no sufficient evidence uncovers the functional mechanisms of LGP2 in RLR signaling pathways in teleost. Here, negative regulation mechanism of LGP2 in certain situations in retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5)-mediated antiviral responses was identified in Ctenopharyngodon idella kidney cells. LGP2 overexpression inhibits synthesis and phosphorylation of interferon regulatory factor 3/7 (IRF3/7), and mRNA levels and promoter activities of IFNs and NF-κBs in resting state and early phase of grass carp reovirus (GCRV) infection. Knockdown of LGP2 obtains opposite effects. Luciferase report assay indicates that LGP2 works at the upstream of RIG-I and MDA5. LGP2 binds to RIG-I and MDA5 with diverse domain preference and which is independent of GCRV infection. Furthermore, LGP2 restrains K63-linked ubiquitination of RIG-I and MDA5 in various degrees. These differences result in disparate repressive mechanisms of LGP2 to RIG-I- and MDA5-mediated signal activations of IFN-β promoter stimulator 1 and mediator of IRF3 activation. Interestingly, LGP2 also inhibits K48-linked RIG-I and MDA5 ubiquitination to suppress proteins degradation, which guarantees the basal protein levels for subsequently rapid signal activation. All these results reveal a mechanism that LGP2 functions as a suppressor in RLR signaling pathways to maintain cellular homeostasis in resting state and early phase during GCRV infection.

  9. Exonuclease domain of the Lassa virus nucleoprotein is critical to avoid RIG-I signaling and to inhibit the innate immune response.

    PubMed

    Reynard, Stéphanie; Russier, Marion; Fizet, Alexandra; Carnec, Xavier; Baize, Sylvain

    2014-12-01

    Lassa virus (LASV), which causes a viral hemorrhagic fever, inhibits the innate immune response. The exonuclease (ExoN) domain of its nucleoprotein (NP) is implicated in the suppression of retinoic acid-inducible gene I (RIG-I) signaling. We show here that a LASV in which ExoN function has been abolished strongly activates innate immunity and that this effect is dependent on RIG-I signaling. These results highlight the key role of NP ExoN function in the immune evasion that occurs during LASV infection.

  10. Role of Toll-like receptors and retinoic acid inducible gene I in endogenous production of type I interferon in dermatomyositis.

    PubMed

    Li, Ling; Dai, Tingjun; Lv, Jingwei; Ji, Kunqian; Liu, Junling; Zhang, Bin; Yan, Chuanzhu

    2015-08-15

    To explore the possible mechanisms implicated in the endogenous production of type I interferons within the muscle tissue of dermatomyositis (DM) patients. We detected the co-localization of plasmacytoid dendritic cells (pDCs) with Toll-like receptors (TLRs) and retinoic acid inducible gene (RIG)-I by immunohistochemistry and immunofluorescence. Western blotting confirmed the expression of TLRs and RIG-I. TLR-3 and RIG-I was preferentially expressed in the perifascicular atrophy fibers of DM. TLR-7 was only in inflammatory infiltrates of a few DM patients. TLR-4 and TLR-9 was expressed mainly in inflammatory infiltrates. Immunofluorescence showed extensive co-localization of BDCA-2 with TLR-9 and little co-localization with TLR-7. Western blotting showed upregulation of expression of TLRs and RIG-I in DM compared with the controls. Our findings indicate that endogenous production of type I IFN in DM is generated by pDCs, mainly through the TLR-9 pathway and in part by TLR-7. TLR-3 and RIG-I are implicated in the formation of perifascicular atrophy in DM.

  11. RIG-I is required for VSV-induced cytokine production by murine glia and acts in combination with DAI to initiate responses to HSV-1.

    PubMed

    Crill, Emma K; Furr-Rogers, Samantha R; Marriott, Ian

    2015-12-01

    A defining feature of viral central nervous system (CNS) infection is the rapid onset of severe neuroinflammation. However, the mechanisms underlying glial responses to replicative neurotropic viruses are only now becoming apparent with the discovery of a number of cytosolic sensors for viral nucleic acids. We have described the expression by murine and human glial cells of two disparate pattern recognition receptors, retinoic acid inducible gene-I (RIG-I) and DNA-dependent activator of interferon regulatory factors (DAI), receptors for viral RNA and DNA moieties, respectively. In the present study, we demonstrate the functional significance of RIG-I expression in primary murine microglia and astrocytes. Our data indicate that murine glial immune responses to a model neurotropic RNA virus, vesicular stomatitis virus, are RIG-I dependent and independent of levels of DAI expression or RNA polymerase III activity. In contrast, maximal glial inflammatory and antiviral responses to the DNA virus herpes simplex virus-1 (HSV-1) are dependent on the expression of both RIG-I and DAI, and require RNA polymerase III activity. These findings indicate that the RNA sensor, RIG-I, acts in parallel with DAI in an RNA polymerase III-dependent manner to initiate glial responses to HSV-1. We therefore suggest that RIG-I plays a significant role in the detection of both RNA and DNA pathogens by microglia and astrocytes.

  12. Importance of interferon inducible trans-membrane proteins and retinoic acid inducible gene I for influenza virus replication: A review.

    PubMed

    Suo, Siqingaowa; Ren, Xiaofeng

    2016-01-01

    Understanding the interplay between Influenza viruses and host cells is key to elucidating the pathogenesis of these viruses. Several host factors have been identified that exert antiviral functions; however, influenza viruses continue to replicate utilizing host cell machinery. Herein, we review the mechanisms of action of two host-derived proteins on conferring cellular resistance to the influenza virus; (1) the interferon inducible trans-membrane proteins, 1, 2 and 3, a recently identified family of early restriction factors; and (2) retinoic acid inducible gene I, a key mediator of antiviral immunity. These data may contribute to the design of novel and efficient anti-influenza treatments.

  13. Sequence-Specific Modifications Enhance the Broad-Spectrum Antiviral Response Activated by RIG-I Agonists

    PubMed Central

    Chiang, Cindy; Beljanski, Vladimir; Yin, Kevin; Olagnier, David; Ben Yebdri, Fethia; Steel, Courtney; Goulet, Marie-Line; DeFilippis, Victor R.; Streblow, Daniel N.; Haddad, Elias K.; Trautmann, Lydie; Ross, Ted; Lin, Rongtuan

    2015-01-01

    ABSTRACT The cytosolic RIG-I (retinoic acid-inducible gene I) receptor plays a pivotal role in the initiation of the immune response against RNA virus infection by recognizing short 5′-triphosphate (5′ppp)-containing viral RNA and activating the host antiviral innate response. In the present study, we generated novel 5′ppp RIG-I agonists of varieous lengths, structures, and sequences and evaluated the generation of the antiviral and inflammatory responses in human epithelial A549 cells, human innate immune primary cells, and murine models of influenza and chikungunya viral pathogenesis. A 99-nucleotide, uridine-rich hairpin 5′pppRNA termed M8 stimulated an extensive and robust interferon response compared to other modified 5′pppRNA structures, RIG-I aptamers, or poly(I·C). Interestingly, manipulation of the primary RNA sequence alone was sufficient to modulate antiviral activity and inflammatory response, in a manner dependent exclusively on RIG-I and independent of MDA5 and TLR3. Both prophylactic and therapeutic administration of M8 effectively inhibited influenza virus and dengue virus replication in vitro. Furthermore, multiple strains of influenza virus that were resistant to oseltamivir, an FDA-approved therapeutic treatment for influenza, were highly sensitive to inhibition by M8. Finally, prophylactic M8 treatment in vivo prolonged survival and reduced lung viral titers of mice challenged with influenza virus, as well as reducing chikungunya virus-associated foot swelling and viral load. Altogether, these results demonstrate that 5′pppRNA can be rationally designed to achieve a maximal RIG-I-mediated protective antiviral response against human-pathogenic RNA viruses. IMPORTANCE The development of novel therapeutics to treat human-pathogenic RNA viral infections is an important goal to reduce spread of infection and to improve human health and safety. This study investigated the design of an RNA agonist with enhanced antiviral and inflammatory

  14. Downregulation of MicroRNA miR-526a by Enterovirus Inhibits RIG-I-Dependent Innate Immune Response

    PubMed Central

    Xu, Changzhi; He, Xiang; Zheng, Zirui; Zhang, Zhe; Wei, Congwen; Guan, Kai; Hou, Lihua; Zhang, Buchang; Zhu, Lin; Cao, Yuan; Zhang, Yanhong; Cao, Ye; Ma, Shengli; Wang, Penghao; Zhang, Pingping; Xu, Quanbin; Ling, Youguo

    2014-01-01

    ABSTRACT Retinoic acid-inducible gene I (RIG-I) is an intracellular RNA virus sensor that induces type I interferon-mediated host-protective innate immunity against viral infection. Although cylindromatosis (CYLD) has been shown to negatively regulate innate antiviral response by removing K-63-linked polyubiquitin from RIG-I, the regulation of its expression and the underlying regulatory mechanisms are still incompletely understood. Here we show that RIG-I activity is regulated by inhibition of CYLD expression mediated by the microRNA miR-526a. We found that viral infection specifically upregulates miR-526a expression in macrophages via interferon regulatory factor (IRF)-dependent mechanisms. In turn, miR-526a positively regulates virus-triggered type I interferon (IFN-I) production, thus suppressing viral replication, the underlying mechanism of which is the enhancement of RIG-I K63-linked ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein, while ectopic miR-526a expression inhibits the replication of EV71 virus. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and suggest a novel mechanism for the evasion of the innate immune response controlled by EV71. IMPORTANCE RNA virus infection upregulates the expression of miR-526a in macrophages through IRF-dependent pathways. In turn, miR-526a positively regulates virus-triggered type I IFN production and inhibits viral replication, the underlying mechanism of which is the enhancement of RIG-I K-63 ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein; cells with overexpressed miR-526a were highly resistant to EV71 infection. The collective results of this study

  15. Cigarette smoke attenuates the RIG-I-initiated innate antiviral response to influenza infection in two murine models.

    PubMed

    Wu, Wenxin; Zhang, Wei; More, Sunil; Booth, J Leland; Duggan, Elizabeth S; Liu, Lin; Zhao, Yan D; Metcalf, Jordan P

    2014-12-01

    Cigarette smoke (CS) exposure increases the frequency and severity of respiratory tract infections. Despite this association, the mechanisms underlying the increased susceptibility to respiratory virus infection are poorly understood. Retinoic acid-inducible gene I (RIG-I) is an important regulator of influenza virus-induced expression of antiviral cytokines, mainly interferons (IFNs), which are necessary to clear viral infections. In this study, we compared the innate cytokine responses of two mouse CS exposure models following a challenge with influenza A virus (IAV): 1) exposure of the mice to cigarette smoke extract (CSE) intratracheally and 2) exposure of the mice to CS in a whole body exposure chamber. Both intratracheal CSE treatment and whole body CS exposure caused antiviral immunosuppression in these mice, and both CS exposure methods inhibited RIG-I induction. CS attenuated influenza-induced antiviral IFNs and IP-10 expression in vivo. However, we did not find that CS inhibited induction of the proinflammatory cytokines IL-6 and TNF-α, whose expression was induced by IAV. Interestingly, IAV infection also increased Toll-like receptor 3 (TLR3) expression in mouse lung, but CS exposure did not impact TLR3 induction in these mice. Together, the results support our previous finding in a human lung organ culture model that the suppression of RIG-I induction and antiviral cytokine responses by CS are likely important in the enhanced susceptibility of smokers to influenza infection in the lung.

  16. Pathogen-Associated Molecular Pattern Recognition of Hepatitis C Virus Transmitted/Founder Variants by RIG-I Is Dependent on U-Core Length

    PubMed Central

    Kell, Alison; Stoddard, Mark; Li, Hui; Marcotrigiano, Joe; Shaw, George M.

    2015-01-01

    ABSTRACT Despite the introduction of direct-acting antiviral (DAA) drugs against hepatitis C virus (HCV), infection remains a major public health concern because DAA therapeutics do not prevent reinfection and patients can still progress to chronic liver disease. Chronic HCV infection is supported by a variety of viral immune evasion strategies, but, remarkably, 20% to 30% of acute infections spontaneously clear prior to development of adaptive immune responses, thus implicating innate immunity in resolving acute HCV infection. However, the virus-host interactions regulating acute infection are unknown. Transmission of HCV involves one or a few transmitted/founder (T/F) variants. In infected hepatocytes, the retinoic acid-inducible gene I (RIG-I) protein recognizes 5′ triphosphate (5′ppp) of the HCV RNA and a pathogen-associated molecular pattern (PAMP) motif located within the 3′ untranslated region consisting of poly-U/UC. PAMP binding activates RIG-I to induce innate immune signaling and type 1 interferon antiviral defenses. HCV poly-U/UC sequences can differ in length and complexity, suggesting that PAMP diversity in T/F genomes could regulate innate immune control of acute HCV infection. Using 14 unique poly-U/UC sequences from HCV T/F genomes recovered from acute-infection patients, we tested whether RIG-I recognition and innate immune activation correlate with PAMP sequence characteristics. We show that T/F variants are recognized by RIG-I in a manner dependent on length of the U-core motif of the poly-U/UC PAMP and are recognized by RIG-I to induce innate immune responses that restrict acute infection. PAMP recognition of T/F HCV variants by RIG-I may therefore impart innate immune signaling and HCV restriction to impact acute-phase-to-chronic-phase transition. IMPORTANCE Recognition of nonself molecular patterns such as those seen with viral nucleic acids is an essential step in triggering the immune response to virus infection. Innate immunity is

  17. NV Proteins of Fish Novirhabdovirus Recruit Cellular PPM1Bb Protein Phosphatase and Antagonize RIG-I-Mediated IFN Induction.

    PubMed

    Biacchesi, Stéphane; Mérour, Emilie; Chevret, Didier; Lamoureux, Annie; Bernard, Julie; Brémont, Michel

    2017-03-09

    Non virion (NV) protein expression is critical for fish Novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV), in vivo pathogenesis. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomic and identified several NV-associated cellular partners underlying cellular pathways as potential viral targets. Among these cell partners, we showed that NV proteins specifically interact with a protein phosphatase, Mg(2+)/Mn(2+)-dependent, 1Bb (PPM1Bb) and recruit it in the close vicinity of mitochondria, a subcellular compartment important for retinoic acid-inducible gene-I- (RIG-I)-mediated interferon induction pathway. PPM1B proteins belong to the PP2C family of serine/threonine (Ser/Thr) protein phosphatase and have recently been shown to negatively regulate the host antiviral response via dephosphorylating Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1). We demonstrated that NV proteins and PPM1Bb counteract RIG-I- and TBK1-dependent interferon (IFN) and IFN-stimulated gene promoter induction in fish cells and, hence, the establishment of an antiviral state. Furthermore, the expression of VHSV NV strongly reduced TBK1 phosphorylation and thus its activation. Our findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPM1Bb protein phosphatase to subvert innate immune recognition.

  18. NV Proteins of Fish Novirhabdovirus Recruit Cellular PPM1Bb Protein Phosphatase and Antagonize RIG-I-Mediated IFN Induction

    PubMed Central

    Biacchesi, Stéphane; Mérour, Emilie; Chevret, Didier; Lamoureux, Annie; Bernard, Julie; Brémont, Michel

    2017-01-01

    Non virion (NV) protein expression is critical for fish Novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV), in vivo pathogenesis. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomic and identified several NV-associated cellular partners underlying cellular pathways as potential viral targets. Among these cell partners, we showed that NV proteins specifically interact with a protein phosphatase, Mg2+/Mn2+-dependent, 1Bb (PPM1Bb) and recruit it in the close vicinity of mitochondria, a subcellular compartment important for retinoic acid-inducible gene-I- (RIG-I)-mediated interferon induction pathway. PPM1B proteins belong to the PP2C family of serine/threonine (Ser/Thr) protein phosphatase and have recently been shown to negatively regulate the host antiviral response via dephosphorylating Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1). We demonstrated that NV proteins and PPM1Bb counteract RIG-I- and TBK1-dependent interferon (IFN) and IFN-stimulated gene promoter induction in fish cells and, hence, the establishment of an antiviral state. Furthermore, the expression of VHSV NV strongly reduced TBK1 phosphorylation and thus its activation. Our findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPM1Bb protein phosphatase to subvert innate immune recognition. PMID:28276468

  19. SARS Coronavirus Nucleocapsid Inhibits Type I Interferon Production by Interfering with TRIM25-Mediated RIG-I Ubiquitination.

    PubMed

    Hu, Yong; Li, Wei; Gao, Ting; Cui, Yan; Jin, Yanwen; Li, Ping; Ma, Qingjun; Liu, Xuan; Cao, Cheng

    2017-02-01

    Severe acute respiratory syndrome (SARS) is a respiratory disease caused by a coronavirus (SARS-CoV) that is characterized by atypical pneumonia. The nucleocapsid protein (N protein) of SARS-CoV plays an important role in inhibition of type I interferon (IFN) production via an unknown mechanism. In this study, the SARS-CoV N protein was found to bind to the SPRY domain of the tripartite motif protein 25 (TRIM25) E3 ubiquitin ligase, thereby interfering with the association between TRIM25 and retinoic acid-inducible gene I (RIG-I) and inhibiting TRIM25-mediated RIG-I ubiquitination and activation. Type I IFN production induced by poly I:C or Sendai virus (SeV) was suppressed by the SARS-CoV N protein. SARS-CoV replication was increased by over-expression of the full-length N protein but not N (1-361), which could not interact with TRIM25. These findings provide an insightful interpretation of the SARS-CoV-mediated host innate immune suppression caused by the N protein.

  20. Evolution of viral sensing RIG-I-like receptor genes in Leporidae genera Oryctolagus, Sylvilagus, and Lepus.

    PubMed

    Lemos de Matos, Ana; McFadden, Grant; Esteves, Pedro J

    2014-01-01

    One of the most severe European rabbit (Oryctolagus cuniculus) pathogens is myxoma virus (MYXV), a rabbit-specific leporipoxvirus that causes the highly lethal disease myxomatosis. Other leporid genera, Sylvilagus and Lepus, encompass species with variable susceptibilities to MYXV, but these do not develop the lethal form of the disease. The protective role of the retinoic acid-inducible gene-I (RIG-I/DDX58) in sensing MYXV in nonpermissive human myeloid cells prompted the study of the RIG-I-like receptor (RLR) family evolution in the three leporid genera. This viral-sensor family also includes the melanoma differentiation-associated factor 5 (MDA5/IFIH1), and the laboratory of genetics and physiology 2 (LGP2/DHX58). Considering specifically the MYXV susceptible host (European rabbit) and one of the virus natural long-term hosts (Sylvilagus bachmani, brush rabbit), the amino acid differences of positively selected sites in RIG-I between the two species were located in the protein region responsible for viral RNA recognition and binding, the repressor domain. Such differences might play a determinant role in how MYXV is sensed. When looking for episodic selection on MDA5 and LGP2 of the eastern cottontail (Sylvilagus floridanus), we also uncovered evidence of selective pressures that might be exerted by a species-specific leporipoxvirus, the Shope fibroma virus. Finally, a putative alternative splicing case was identified in Oryctolagus and Lepus MDA5 isoforms, corresponding to the deletion of one specific exon. This study provided the first insights into the evolution of the leporid RLR gene family that helps illuminate the origins of the species-specific innate responses to pathogens and more specifically to MYXV.

  1. Influenza A(H1N1)pdm09 Virus Suppresses RIG-I Initiated Innate Antiviral Responses in the Human Lung

    PubMed Central

    Booth, J. Leland; Metcalf, Jordan P.

    2012-01-01

    Influenza infection is a major cause of morbidity and mortality. Retinoic acid-inducible gene I (RIG-I) is believed to play an important role in the recognition of, and response to, influenza virus and other RNA viruses. Our study focuses on the hypothesis that pandemic H1N1/09 influenza virus alters the influenza-induced proinflammatory response and suppresses host antiviral activity. We first compared the innate response to a clinical isolate of influenza A(H1N1)pdm09 virus, OK/09, a clinical isolate of seasonal H3N2 virus, OK/06, and to a laboratory adapted seasonal H1N1 virus, PR8, using a unique human lung organ culture model. Exposure of human lung tissue to either pandemic or seasonal influenza virus resulted in infection and replication in alveolar epithelial cells. Pandemic virus induces a diminished RIG-I mRNA and antiviral cytokine response than seasonal virus in human lung. The suppression of antiviral response and RIG-I mRNA expression was confirmed at the protein level by ELISA and western blot. We performed a time course of RIG-I and interferon-β (IFN-β) mRNA induction by the two viruses. RIG-I and IFN-β induction by OK/09 was of lower amplitude and shorter duration than that caused by PR8. In contrast, the pandemic virus OK/09 caused similar induction of proinflammatory cytokines, IL-8 and IL-6, at both the transcriptional and translational level as PR8 in human lung. Differential antiviral responses did not appear to be due to a difference in cellular infectivity as immunohistochemistry showed that both viruses infected alveolar macrophages and epithelial cells. These findings show that influenza A(H1N1)pdm09 virus suppresses anti-viral immune responses in infected human lung through inhibition of viral-mediated induction of the pattern recognition receptor, RIG-I, though proinflammatory cytokine induction was unaltered. This immunosuppression of the host antiviral response by pandemic virus may have contributed to the more serious lung

  2. MDA5 Induces a Stronger Interferon Response than RIG-I to GCRV Infection through a Mechanism Involving the Phosphorylation and Dimerization of IRF3 and IRF7 in CIK Cells

    PubMed Central

    Wan, Quanyuan; Yang, Chunrong; Rao, Youliang; Liao, Zhiwei; Su, Jianguo

    2017-01-01

    Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are critical cytosolic sensors that trigger the production of interferons (IFNs). Though their recognition functions are well identified, their unique roles in the downstream signal transduction remain to be elucidated. Herein, we report the differential effect between grass carp (Ctenopharyngodon idella) MDA5 (CiMDA5) and CiRIG-I on the production of various IFNs upon grass carp reovirus (GCRV) infection in C. idella kidney (CIK) cell line. In CIK cells, grass carp IFN1 (CiIFN1) and CiIFN3 are relatively highly expressed while CiIFN2 and CiIFN4 are relatively slightly expressed. Following GCRV infection, CiMDA5 induces a more extensive type I IFN response than CiRIG-I. Further investigation reveals that both CiMDA5 and CiRIG-I facilitate the expression and total phosphorylation levels of grass carp IFN regulatory factor (IRF) 3 (CiIRF3) and CiIRF7 upon GCRV infection or poly(I:C) stimulation. However, the difference is that CiRIG-I decreases the threonine phosphorylation level of CiIRF7. As a consequence, CiMDA5 enhances the heterodimerization of CiIRF3 and CiIRF7 and homodimerization of CiIRF7, whereas CiRIG-I facilitates the heterodimerization but attenuates homodimerization of CiIRF7. Moreover, the present study suggests that CiIRF3 and CiIRF7 heterodimers and CiIRF7 homodimers are able to induce more extensive IFN-I responses than CiIRF3 homodimers under GCRV infection. Additionally, CiMDA5 induces a stronger type II IFN (IFN-II) response against GCRV infection than CiRIG-I. Collectively, these results demonstrate that CiMDA5 plays a more potent role than CiRIG-I in IFN response to GCRV infection through differentially regulating the phosphorylation and dimerization of CiIRF3 and CiIRF7. PMID:28286505

  3. MDA5 Induces a Stronger Interferon Response than RIG-I to GCRV Infection through a Mechanism Involving the Phosphorylation and Dimerization of IRF3 and IRF7 in CIK Cells.

    PubMed

    Wan, Quanyuan; Yang, Chunrong; Rao, Youliang; Liao, Zhiwei; Su, Jianguo

    2017-01-01

    Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are critical cytosolic sensors that trigger the production of interferons (IFNs). Though their recognition functions are well identified, their unique roles in the downstream signal transduction remain to be elucidated. Herein, we report the differential effect between grass carp (Ctenopharyngodon idella) MDA5 (CiMDA5) and CiRIG-I on the production of various IFNs upon grass carp reovirus (GCRV) infection in C. idella kidney (CIK) cell line. In CIK cells, grass carp IFN1 (CiIFN1) and CiIFN3 are relatively highly expressed while CiIFN2 and CiIFN4 are relatively slightly expressed. Following GCRV infection, CiMDA5 induces a more extensive type I IFN response than CiRIG-I. Further investigation reveals that both CiMDA5 and CiRIG-I facilitate the expression and total phosphorylation levels of grass carp IFN regulatory factor (IRF) 3 (CiIRF3) and CiIRF7 upon GCRV infection or poly(I:C) stimulation. However, the difference is that CiRIG-I decreases the threonine phosphorylation level of CiIRF7. As a consequence, CiMDA5 enhances the heterodimerization of CiIRF3 and CiIRF7 and homodimerization of CiIRF7, whereas CiRIG-I facilitates the heterodimerization but attenuates homodimerization of CiIRF7. Moreover, the present study suggests that CiIRF3 and CiIRF7 heterodimers and CiIRF7 homodimers are able to induce more extensive IFN-I responses than CiIRF3 homodimers under GCRV infection. Additionally, CiMDA5 induces a stronger type II IFN (IFN-II) response against GCRV infection than CiRIG-I. Collectively, these results demonstrate that CiMDA5 plays a more potent role than CiRIG-I in IFN response to GCRV infection through differentially regulating the phosphorylation and dimerization of CiIRF3 and CiIRF7.

  4. RIG-I inhibits pancreatic β cell proliferation through competitive binding of activated Src

    PubMed Central

    Pan, Yi; Li, GuangMing; Zhong, HengGao; Chen, MeiJuan; Chen, TingTing; Gao, LiLi; Wu, HuiWen; Guo, Jun

    2016-01-01

    Nutrition is a necessary condition for cell proliferation, including pancreatic β cells; however, over-nutrition, and the resulting obesity and glucolipotoxicity, is a risk factor for the development of Type 2 diabetes mellitus (DM), and causes inhibition of pancreatic β-cells proliferation and their loss of compensation for insulin resistance. Here, we showed that Retinoic acid (RA)-inducible gene I (RIG-I) responds to nutrient signals and induces loss of β cell mass through G1 cell cycle arrest. Risk factors for type 2 diabetes (e.g., glucolipotoxicity, TNF-α and LPS) activate Src in pancreatic β cells. Elevated RIG-I modulated the interaction of activated Src and STAT3 by competitive binding to STAT3. Elevated RIG-I downregulated the transcription of SKP2, and increased the stability and abundance of P27 protein in a STAT3-dependent manner, which was associated with inhibition of β cell growth elicited by Src. These results supported a role for RIG-I in β cell mass loss under conditions of metabolic surplus and suggested that RIG-I-induced blocking of Src/STAT3 signalling might be involved in G1 phase cycle arrest through the Skp2/P27 pathway in pancreatic β cells. PMID:27349479

  5. Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Nsp4 Cleaves VISA to Impair Antiviral Responses Mediated by RIG-I-like Receptors

    PubMed Central

    Huang, Chen; Du, Yinping; Yu, Zhibin; Zhang, Qiong; Liu, Yihao; Tang, Jun; Shi, Jishu; Feng, Wen-hai

    2016-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most significant etiological agents in the swine industry worldwide. It has been reported that PRRSV infection can modulate host immune responses, and innate immune evasion is thought to play a vital role in PRRSV pathogenesis. In this study, we demonstrated that highly pathogenic PRRSV (HP-PRRSV) infection specifically down-regulated virus-induced signaling adaptor (VISA), a unique adaptor molecule that is essential for retinoic acid induced gene-I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) signal transduction. Moreover, we verified that nsp4 inhibited IRF3 activation induced by signaling molecules, including RIG-I, MDA5, VISA, and TBK1, but not IRF3. Subsequently, we demonstrated that HP-PRRSV nsp4 down-regulated VISA and suppressed type I IFN induction. Importantly, VISA was cleaved by nsp4 and released from mitochondrial membrane, which interrupted the downstream signaling of VISA. However, catalytically inactive mutant of nsp4 abolished its ability to cleave VISA. Interestingly, nsp4 of typical PRRSV strain CH-1a had no effect on VISA. Taken together, these findings reveal a strategy evolved by HP-PRRSV to counteract anti-viral innate immune signaling, which complements the known PRRSV-mediated immune-evasion mechanisms. PMID:27329948

  6. Emerging Role of Ubiquitination in Antiviral RIG-I Signaling

    PubMed Central

    Maelfait, Jonathan

    2012-01-01

    Summary: Detection of viruses by the innate immune system involves the action of specialized pattern recognition receptors. Intracellular RIG-I receptors sense the presence of viral nucleic acids in infected cells and trigger signaling pathways that lead to the production of proinflammatory and antiviral proteins. Over the past few years, posttranslational modification of RIG-I and downstream signaling proteins by different types of ubiquitination has been found to be a key event in the regulation of RIG-I-induced NF-κB and interferon regulatory factor 3 (IRF3) activation. Multiple ubiquitin ligases, deubiquitinases, and ubiquitin binding scaffold proteins contribute to both positive and negative regulation of the RIG-I-induced antiviral immune response. A better understanding of the function and activity of these proteins might eventually lead to the development of novel therapeutic approaches for management of viral diseases. PMID:22390971

  7. Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs

    PubMed Central

    Coffman, Stephanie R.; Lu, Jinfeng; Guo, Xunyang; Zhong, Jing; Broitman-Maduro, Gina; Li, Wan-Xiang; Lu, Rui; Maduro, Morris

    2017-01-01

    ABSTRACT Dicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans. Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans. Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1, three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors. PMID:28325765

  8. Cigarette smoke extract suppresses the RIG-I-initiated innate immune response to influenza virus in the human lung

    PubMed Central

    Wu, Wenxin; Patel, Krupa B.; Booth, J. Leland; Zhang, Wei

    2011-01-01

    Cigarette smoking is the major cause of chronic obstructive pulmonary disease (COPD) and predisposes subjects to severe respiratory tract infections. Epidemiological studies have shown that cigarette smokers are seven times more likely to contract influenza infection than nonsmokers. The mechanisms underlying this increased susceptibility are poorly characterized. Retinoic acid-inducible gene (RIG)-I is believed to play an important role in the recognition of, and response to, influenza virus and other RNA viruses. Our study focused on how cigarette smoke extract (CSE) alters the influenza-induced proinflammatory response and suppresses host antiviral activity in the human lung using a unique lung organ culture model. We first determined that treatment with 2–20% CSE did not induce cytotoxicity as assessed by LDH release. However, CSE treatment inhibited influenza-induced IFN-inducible protein 10 protein and mRNA expression. Induction of the major antiviral cytokine IFN-β mRNA was also decreased by CSE. CSE also blunted viral-mediated RIG-I mRNA and protein expression. Inhibition of viral-mediated RIG-I induction by CSE was prevented by the antioxidants N-acetyl-cysteine and glutathione. These findings show that CSE suppresses antiviral and innate immune responses in influenza virus-infected human lungs through oxidative inhibition of viral-mediated induction of the pattern recognition receptor RIG-I. This immunosuppressive effect of CSE may play a role in the enhanced susceptibility of smokers to serious influenza infection in the lung. PMID:21335520

  9. Structural insights into RNA recognition by RIG-I

    PubMed Central

    Luo, Dahai; Ding, Steve C.; Vela, Adriana; Kohlway, Andrew; Lindenbach, Brett D.; Pyle, Anna Marie

    2011-01-01

    Summary Intracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP-2) recognize viral RNAs as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral immune response. To understand the molecular basis of this process, we determined the crystal structure of RIG-I in complex with double-stranded RNA. The dsRNA is sheathed within a network of protein domains that include a conserved “helicase” domain (regions HEL1 and HEL2), a specialized insertion domain (HEL2i), and a C-terminal regulatory domain (CTD). A V-shaped pincer connects HEL2 and the CTD by gripping an α-helical shaft that extends from HEL1. In this way, the pincer coordinates functions of all the domains and couples RNA binding with ATP hydrolysis. RIG-I falls within the Dicer-RIG-I clade of super family 2 of helicases and this structure reveals complex interplay between motor domains, accessory mechanical domains and RNA that has implications for understanding the nanomechanical function this protein family and other ATPases more broadly. PMID:22000018

  10. Intracellular pathogen detection by RIG-I-like receptors

    PubMed Central

    Dixit, Evelyn; Kagan, Jonathan C.

    2014-01-01

    The RIG-I-like receptors (RLR) RIG-I, MDA5 and LGP2 trigger innate immune responses against viral infections that serve to limit virus replication and to stimulate adaptive immunity. RLRs are cytosolic sensors for virus-derived RNA and thus responsible for intracellular immune surveillance against infection. RLR signaling requires the adapter protein MAVS to induce type I interferon, interferon-stimulated genes and proinflammatory cytokines. This review focusses on the molecular and cell biological requirements for RLR signal transduction. PMID:23611287

  11. RIG-I and TLR3 are both required for maximum interferon induction by influenza virus in human lung alveolar epithelial cells.

    PubMed

    Wu, Wenxin; Zhang, Wei; Duggan, Elizabeth S; Booth, J Leland; Zou, Ming-Hui; Metcalf, Jordan P

    2015-08-01

    Pattern recognition receptors, such as retinoic acid-inducible protein I (RIG-I), Toll-like receptors 3 and 7 (TLR3 and 7), and nucleotide-binding oligomerization domain containing protein 2 (NOD2), play important roles in the recognition of influenza A virus (IAV), but their role in interferon (IFN) induction is still unclear, particularly in human lung. We investigated IFN induction by IAV in the A549 cell line as well as in primary human alveolar epithelial cells (AEC). TLR3/7, NOD2, RIG-I, and IFN expression levels were measured by qRT-PCR and ELISA in cells infected with IAV PR8. We found that TLR7 and NOD2 were not involved in IFN induction by IAV in these cells. Neither RIG-I nor TLR3 siRNA alone completely blocked IFN induction. However, double knockdown of RIG-I and TLR3 completely inhibited IFN induction by influenza. Thus, signaling through both RIG-I and TLR3 is important for IFN induction by IAV in human lung AEC.

  12. A RING finger protein 114 (RNF114) homolog from Chinese sturgeon (Acipenser sinensis) possesses immune-regulation properties via modulating RIG-I signaling pathway-mediated interferon expression.

    PubMed

    Liao, Zhiyong; Chen, Xiaojun; Nie, Dongsong; Wang, Jiajia; Wu, Mingjiang

    2014-12-01

    Ubiquitin ligases play important roles in immune regulation. The human RNF114 (RING finger protein 114), an ubiquitin ligase, was recently reported to be involved in immune response to double-stranded RNA in disease pathogenesis. Here, we identified a RNF114 homolog in Chinese sturgeon (Acipenser sinensis) and investigated its potential role in immune response. The full-length cDNA of Chinese sturgeon RNF114 (csRNF114) contains an open reading frame (ORF) of 681 nucleotides coding a protein of 227 amino acids. csRNF114 shares the highest identity of 76% at amino acid level to other RNF114 homologs, clustering with bony fish RNF114s based on phylogenetic analysis. The main structural features of csRNF114, including a C3HC4 (Cys3-His-Cys4) RING domain, a C2HC (Cys2-His-Cys)-type zinc finger motif, a C2H2 (Cys2-His2)-type zinc finger motif, and a UIM (ubiquitin-interacting motif), take csRNF114 as an ubiquitin ligase. csRNF114 mRNA was widely expressed in various tissues and significantly up-regulated in poly(I:C)-treated Chinese sturgeon. Over-expression of csRNF114 in HEK293T cells significantly promoted both basal and poly(I:C)-induced activation of interferon regulatory transcription factor 3 (IRF3) and nuclear factor-κB (NF-κB) downstream retinoic acid inducible gene I (RIG-I) signaling pathway and expression of target genes type I interferon (IFN), which was nearly abolished by knockdown of RIG-I with specific human siRNA and by mutation of the C3HC4 RING domain (C28A/C31A) in csRNF114 as well. Furthermore, csRNF114 associated with ubiquitinated proteins in HEK293T cells, for which the C3HC4 RING domain was essential. These data suggested that an ubiquitin ligase RNF114 homolog with a potential role in antiviral response possibly through modulating RIG-I signaling pathway was cloned from Chinese sturgeon, which might contribute to our understanding of the immune biology of Chinese sturgeon.

  13. RIG-I self-oligomerization is either dispensable or very transient for signal transduction.

    PubMed

    Louber, Jade; Kowalinski, Eva; Bloyet, Louis-Marie; Brunel, Joanna; Cusack, Stephen; Gerlier, Denis

    2014-01-01

    Effective host defence against viruses depends on the rapid triggering of innate immunity through the induction of a type I interferon (IFN) response. To this end, microbe-associated molecular patterns are detected by dedicated receptors. Among them, the RIG-I-like receptors RIG-I and MDA5 activate IFN gene expression upon sensing viral RNA in the cytoplasm. While MDA5 forms long filaments in vitro upon activation, RIG-I is believed to oligomerize after RNA binding in order to transduce a signal. Here, we show that in vitro binding of synthetic RNA mimicking that of Mononegavirales (Ebola, rabies and measles viruses) leader sequences to purified RIG-I does not induce RIG-I oligomerization. Furthermore, in cells devoid of endogenous functional RIG-I-like receptors, after activation of exogenous Flag-RIG-I by a 62-mer-5'ppp-dsRNA or by polyinosinic:polycytidylic acid, a dsRNA analogue, or by measles virus infection, anti-Flag immunoprecipitation and specific elution with Flag peptide indicated a monomeric form of RIG-I. Accordingly, when using the Gaussia Luciferase-Based Protein Complementation Assay (PCA), a more sensitive in cellula assay, no RIG-I oligomerization could be detected upon RNA stimulation. Altogether our data indicate that the need for self-oligomerization of RIG-I for signal transduction is either dispensable or very transient.

  14. RIG-I Self-Oligomerization Is Either Dispensable or Very Transient for Signal Transduction

    PubMed Central

    Louber, Jade; Kowalinski, Eva; Bloyet, Louis-Marie; Brunel, Joanna; Cusack, Stephen; Gerlier, Denis

    2014-01-01

    Effective host defence against viruses depends on the rapid triggering of innate immunity through the induction of a type I interferon (IFN) response. To this end, microbe-associated molecular patterns are detected by dedicated receptors. Among them, the RIG-I-like receptors RIG-I and MDA5 activate IFN gene expression upon sensing viral RNA in the cytoplasm. While MDA5 forms long filaments in vitro upon activation, RIG-I is believed to oligomerize after RNA binding in order to transduce a signal. Here, we show that in vitro binding of synthetic RNA mimicking that of Mononegavirales (Ebola, rabies and measles viruses) leader sequences to purified RIG-I does not induce RIG-I oligomerization. Furthermore, in cells devoid of endogenous functional RIG-I-like receptors, after activation of exogenous Flag-RIG-I by a 62-mer-5′ppp-dsRNA or by polyinosinic:polycytidylic acid, a dsRNA analogue, or by measles virus infection, anti-Flag immunoprecipitation and specific elution with Flag peptide indicated a monomeric form of RIG-I. Accordingly, when using the Gaussia Luciferase-Based Protein Complementation Assay (PCA), a more sensitive in cellula assay, no RIG-I oligomerization could be detected upon RNA stimulation. Altogether our data indicate that the need for self-oligomerization of RIG-I for signal transduction is either dispensable or very transient. PMID:25259935

  15. Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs.

    PubMed

    Coffman, Stephanie R; Lu, Jinfeng; Guo, Xunyang; Zhong, Jing; Jiang, Hongshan; Broitman-Maduro, Gina; Li, Wan-Xiang; Lu, Rui; Maduro, Morris; Ding, Shou-Wei

    2017-03-21

    Dicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1, three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors.IMPORTANCE The helicase and C-terminal domains of mammalian RLRs sense intracellular viral RNAs to initiate the interferon-regulated innate immunity against RNA virus infection. Both of the domains from

  16. The powerstroke and camshaft of the RIG-I antiviral RNA detection machine.

    PubMed

    O'Neill, Luke A J; Bowie, Andrew G

    2011-10-14

    The innate immune sensor RIG-I responds to infection by binding to viral double-stranded RNA (dsRNA). In this issue of Cell, Kowalinski et al. (2011) and Luo et al. (2011) reveal the structure of RIG-I, and in combination with functional analyses, they show how RIG-I recognizes viral RNA to initiate signaling and a type I interferon response.

  17. Pyruvate Carboxylase Activates the RIG-I-like Receptor-Mediated Antiviral Immune Response by Targeting the MAVS signalosome

    PubMed Central

    Cao, Zhongying; Zhou, Yaqin; Zhu, Shengli; Feng, Jian; Chen, Xueyuan; Liu, Shi; Peng, Nanfang; Yang, Xiaodan; Xu, Gang; Zhu, Ying

    2016-01-01

    When retinoic acid-inducible gene 1 protein (RIG-I)-like receptors sense viral dsRNA in the cytosol, RIG-I and melanoma differentiation-associated gene 5 (MDA5) are recruited to the mitochondria to interact with mitochondrial antiviral signaling protein (MAVS) and initiate antiviral immune responses. In this study, we demonstrate that the biotin-containing enzyme pyruvate carboxylase (PC) plays an essential role in the virus-triggered activation of nuclear factor kappa B (NF-κB) signaling mediated by MAVS. PC contributes to the enhanced production of type I interferons (IFNs) and pro-inflammatory cytokines, and PC knockdown inhibits the virus-triggered innate immune response. In addition, PC shows extensive antiviral activity against RNA viruses, including influenza A virus (IAV), human enterovirus 71 (EV71), and vesicular stomatitis virus (VSV). Furthermore, PC mediates antiviral action by targeting the MAVS signalosome and induces IFNs and pro-inflammatory cytokines by promoting phosphorylation of NF-κB inhibitor-α (IκBα) and the IκB kinase (IKK) complex, as well as NF-κB nuclear translocation, which leads to activation of interferon-stimulated genes (ISGs), including double-stranded RNA-dependent protein kinase (PKR) and myxovirus resistance protein 1 (Mx1). Our findings suggest that PC is an important player in host antiviral signaling. PMID:26906558

  18. USP3 inhibits type I interferon signaling by deubiquitinating RIG-I-like receptors

    PubMed Central

    Cui, Jun; Song, Yanxia; Li, Yinyin; Zhu, Qingyuan; Tan, Peng; Qin, Yunfei; Wang, Helen Y; Wang, Rong-Fu

    2014-01-01

    Lysine 63 (K63)-linked ubiquitination of RIG-I plays a critical role in the activation of type I interferon pathway, yet the molecular mechanism responsible for its deubiquitination is still poorly understood. Here we report that the deubiquitination enzyme ubiquitin-specific protease 3 (USP3) negatively regulates the activation of type I interferon signaling by targeting RIG-I. Knockdown of USP3 specifically enhanced K63-linked ubiquitination of RIG-I, upregulated the phosphorylation of IRF3 and augmented the production of type I interferon cytokines and antiviral immunity. We further show that there is no interaction between USP3 and RIG-I-like receptors (RLRs) in unstimulated or uninfected cells, but upon viral infection or ligand stimulation, USP3 binds to the caspase activation recruitment domain of RLRs and then cleaves polyubiquitin chains through cooperation of its zinc-finger Ub-binding domain and USP catalytic domains. Mutation analysis reveals that binding of USP3 to polyubiquitin chains on RIG-I is a prerequisite step for its cleavage of polyubiquitin chains. Our findings identify a previously unrecognized role of USP3 in RIG-I activation and provide insights into the mechanisms by which USP3 inhibits RIG-I signaling and antiviral immunity. PMID:24366338

  19. Cancer therapies activate RIG-I-like receptor pathway through endogenous non-coding RNAs

    PubMed Central

    Ranoa, Diana Rose E.; Parekh, Akash D.; Pitroda, Sean P.; Huang, Xiaona; Darga, Thomas; Wong, Anthony C.; Huang, Lei; Andrade, Jorge; Staley, Jonathan P.; Satoh, Takashi; Akira, Shizuo

    2016-01-01

    Emerging evidence indicates that ionizing radiation (IR) and chemotherapy activate Type I interferon (IFN) signaling in tumor and host cells. However, the mechanism of induction is poorly understood. We identified a novel radioprotective role for the DEXH box RNA helicase LGP2 (DHX58) through its suppression of IR-induced cytotoxic IFN-beta [1]. LGP2 inhibits activation of the RIG-I-like receptor (RLR) pathway upon binding of viral RNA to the cytoplasmic sensors RIG-I (DDX58) and MDA5 (IFIH1) and subsequent IFN signaling via the mitochondrial adaptor protein MAVS (IPS1). Here we show that MAVS is necessary for IFN-beta induction and interferon-stimulated gene expression in the response to IR. Suppression of MAVS conferred radioresistance in normal and cancer cells. Germline deletion of RIG-I, but not MDA5, protected mice from death following total body irradiation, while deletion of LGP2 accelerated the death of irradiated animals. In human tumors depletion of RIG-I conferred resistance to IR and different classes of chemotherapy drugs. Mechanistically, IR stimulated the binding of cytoplasmic RIG-I with small endogenous non-coding RNAs (sncRNAs), which triggered IFN-beta activity. We demonstrate that the small nuclear RNAs U1 and U2 translocate to the cytoplasm after IR treatment, thus stimulating the formation of RIG-I: RNA complexes and initiating downstream signaling events. Taken together, these findings suggest that the physiologic responses to radio-/chemo-therapy converge on an antiviral program in recruitment of the RLR pathway by a sncRNA-dependent activation of RIG-I which commences cytotoxic IFN signaling. Importantly, activation of interferon genes by radiation or chemotherapy is associated with a favorable outcome in patients undergoing treatment for cancer. To our knowledge, this is the first demonstration of a cell-intrinsic response to clinically relevant genotoxic treatments mediated by an RNA-dependent mechanism. PMID:27034163

  20. Paramyxovirus V proteins interact with the RNA Helicase LGP2 to inhibit RIG-I-dependent interferon induction.

    PubMed

    Childs, Kay; Randall, Richard; Goodbourn, Stephen

    2012-04-01

    RIG-I and mda-5 are activated by viral RNA and stimulate type I interferon production. Laboratory of genetics and physiology 2 (LGP2) shares homology with RIG-I and mda-5 but lacks the CARD domains required for signaling. The V proteins of paramyxoviruses limit interferon induction by binding mda-5 and preventing its activation; however, they do not bind RIG-I and have not been considered inhibitors of RIG-I signaling. Here we uncover a novel mechanism of RIG-I inhibition in which the V protein of parainfluenzavirus type 5 (PIV5; formerly known as simian virus type 5 [SV5]) interacts with LGP2 and cooperatively inhibits induction by RIG-I ligands. A complex between RIG-I and LGP2 is observed in the presence of PIV5-V, and we propose that this complex is refractory to activation by RIG-I ligands. The V proteins from other paramyxoviruses also bind LGP2 and demonstrate LGP2-dependent inhibition of RIG-I signaling. This is significant, because it demonstrates a general mechanism for the targeting of the RIG-I pathway by paramyxoviruses.

  1. Influenza virus adaptation PB2-627K modulates nucleocapsid inhibition by the pathogen sensor RIG-I

    PubMed Central

    Weber, Michaela; Sediri, Hanna; Felgenhauer, Ulrike; Binzen, Ina; Bänfer, Sebastian; Jacob, Ralf; Brunotte, Linda; García-Sastre, Adolfo; Schmid-Burgk, Jonathan L.; Schmidt, Tobias; Hornung, Veit; Kochs, Georg; Schwemmle, Martin; Klenk, Hans-Dieter; Weber, Friedemann

    2015-01-01

    Summary The cytoplasmic RNA helicase RIG-I mediates innate sensing of RNA viruses. The genomes of influenza A virus (FLUAV) are encapsidated by the nucleoprotein and associated with RNA polymerase, posing potential barriers to RIG-I sensing. We show that RIG-I recognizes the 5’-triphosphorylated dsRNA on FLUAV nucleocapsids but that polymorphisms at position 627 of the viral polymerase subunit PB2 modulate RIG-I sensing. Compared to mammalian-adapted PB2-627K, avian FLUAV nucleocapsids possessing PB2-627E are prone to increased RIG-I recognition, and RIG-I-deficiency partially restores PB2-627E virus infection of mammalian cells. Heightened RIG-I sensing of PB2-627E nucleocapsids correlates with previously established lower affinity of 627E-containing PB2 for nucleoprotein and is increased by further nucleocapsid instability. The effect of RIG-I on PB2-627E nucleocapsids is independent of antiviral signaling, suggesting that RIG-I-nucleocapsid binding alone can inhibit infection. These results indicate that RIG-I is a direct avian FLUAV restriction factor and highlight nucleocapsid disruption as an antiviral strategy. PMID:25704008

  2. A Hierarchical Mechanism of RIG-I Ubiquitination Provides Sensitivity, Robustness and Synergy in Antiviral Immune Responses

    NASA Astrophysics Data System (ADS)

    Sun, Xiaoqiang; Xian, Huifang; Tian, Shuo; Sun, Tingzhe; Qin, Yunfei; Zhang, Shoutao; Cui, Jun

    2016-07-01

    RIG-I is an essential receptor in the initiation of the type I interferon (IFN) signaling pathway upon viral infection. Although K63-linked ubiquitination plays an important role in RIG-I activation, the optimal modulation of conjugated and unanchored ubiquitination of RIG-I as well as its functional implications remains unclear. In this study, we determined that, in contrast to the RIG-I CARD domain, full-length RIG-I must undergo K63-linked ubiquitination at multiple sites to reach full activity. A systems biology approach was designed based on experiments using full-length RIG-I. Model selection for 7 candidate mechanisms of RIG-I ubiquitination inferred a hierarchical architecture of the RIG-I ubiquitination mode, which was then experimentally validated. Compared with other mechanisms, the selected hierarchical mechanism exhibited superior sensitivity and robustness in RIG-I-induced type I IFN activation. Furthermore, our model analysis and experimental data revealed that TRIM4 and TRIM25 exhibited dose-dependent synergism. These results demonstrated that the hierarchical mechanism of multi-site/type ubiquitination of RIG-I provides an efficient, robust and optimal synergistic regulatory module in antiviral immune responses.

  3. A Hierarchical Mechanism of RIG-I Ubiquitination Provides Sensitivity, Robustness and Synergy in Antiviral Immune Responses

    PubMed Central

    Sun, Xiaoqiang; Xian, Huifang; Tian, Shuo; Sun, Tingzhe; Qin, Yunfei; Zhang, Shoutao; Cui, Jun

    2016-01-01

    RIG-I is an essential receptor in the initiation of the type I interferon (IFN) signaling pathway upon viral infection. Although K63-linked ubiquitination plays an important role in RIG-I activation, the optimal modulation of conjugated and unanchored ubiquitination of RIG-I as well as its functional implications remains unclear. In this study, we determined that, in contrast to the RIG-I CARD domain, full-length RIG-I must undergo K63-linked ubiquitination at multiple sites to reach full activity. A systems biology approach was designed based on experiments using full-length RIG-I. Model selection for 7 candidate mechanisms of RIG-I ubiquitination inferred a hierarchical architecture of the RIG-I ubiquitination mode, which was then experimentally validated. Compared with other mechanisms, the selected hierarchical mechanism exhibited superior sensitivity and robustness in RIG-I-induced type I IFN activation. Furthermore, our model analysis and experimental data revealed that TRIM4 and TRIM25 exhibited dose-dependent synergism. These results demonstrated that the hierarchical mechanism of multi-site/type ubiquitination of RIG-I provides an efficient, robust and optimal synergistic regulatory module in antiviral immune responses. PMID:27387525

  4. Both RIG-I and MDA5 detect alphavirus replication in concentration-dependent mode

    SciTech Connect

    Akhrymuk, Ivan; Frolov, Ilya; Frolova, Elena I.

    2016-01-15

    Alphaviruses are a family of positive-strand RNA viruses that circulate on all continents between mosquito vectors and vertebrate hosts. Despite a significant public health threat, their biology is not sufficiently investigated, and the mechanisms of alphavirus replication and virus–host interaction are insufficiently understood. In this study, we have applied a variety of experimental systems to further understand the mechanism by which infected cells detect replicating alphaviruses. Our new data strongly suggest that activation of the antiviral response by alphavirus-infected cells is determined by the integrity of viral genes encoding proteins with nuclear functions, and by the presence of two cellular pattern recognition receptors (PRRs), RIG-I and MDA5. No type I IFN response is induced in their absence. The presence of either of these PRRs is sufficient for detecting virus replication. However, type I IFN activation in response to pathogenic alphaviruses depends on the basal levels of RIG-I or MDA5. - Highlights: • Both RIG-I and MDA5 detect alphavirus replication. • Alphavirus-induced transcriptional shutoff affects type I IFN induction. • Sensing of alphavirus replication by RIG-I and MDA5 depends on their concentrations. • High basal level of RIG-I and MDA5 allows IFN induction by pathogenic alphaviruses. • This dependence determines the discrepancy between the in vivo and in vitro data.

  5. Leader-Containing Uncapped Viral Transcript Activates RIG-I in Antiviral Stress Granules

    PubMed Central

    Oh, Seong-Wook; Onomoto, Koji; Wakimoto, Mai; Onoguchi, Kazuhide; Ishidate, Fumiyoshi; Fujiwara, Takahiro; Yoneyama, Mitsutoshi; Kato, Hiroki; Fujita, Takashi

    2016-01-01

    RIG-I triggers antiviral responses by recognizing viral RNA (vRNA) in the cytoplasm. However, the spatio-temporal dynamics of vRNA sensing and signal transduction remain elusive. We investigated the time course of events in cells infected with Newcastle disease virus (NDV), a non-segmented negative-strand RNA virus. RIG-I was recruited to viral replication complexes (vRC) and triggered minimal primary type I interferon (IFN) production. RIG-I subsequently localized to antiviral stress granules (avSG) induced after vRC formation. The inhibition of avSG attenuated secondary IFN production, suggesting avSG as a platform for efficient vRNA detection. avSG selectively captured positive-strand vRNA, and poly(A)+ RNA induced IFN production. Further investigations suggested that uncapped vRNA derived from read-through transcription was sensed by RIG-I in avSG. These results highlight how viral infections stimulate host stress responses, thereby selectively recruiting uncapped vRNA to avSG, in which RIG-I and other components cooperate in an efficient antiviral program. PMID:26862753

  6. Comparative analysis of viral RNA signatures on different RIG-I-like receptors

    PubMed Central

    Sanchez David, Raul Y; Combredet, Chantal; Sismeiro, Odile; Dillies, Marie-Agnès; Jagla, Bernd; Coppée, Jean-Yves; Mura, Marie; Guerbois Galla, Mathilde; Despres, Philippe; Tangy, Frédéric; Komarova, Anastassia V

    2016-01-01

    The RIG-I-like receptors (RLRs) play a major role in sensing RNA virus infection to initiate and modulate antiviral immunity. They interact with particular viral RNAs, most of them being still unknown. To decipher the viral RNA signature on RLRs during viral infection, we tagged RLRs (RIG-I, MDA5, LGP2) and applied tagged protein affinity purification followed by next-generation sequencing (NGS) of associated RNA molecules. Two viruses with negative- and positive-sense RNA genome were used: measles (MV) and chikungunya (CHIKV). NGS analysis revealed that distinct regions of MV genome were specifically recognized by distinct RLRs: RIG-I recognized defective interfering genomes, whereas MDA5 and LGP2 specifically bound MV nucleoprotein-coding region. During CHIKV infection, RIG-I associated specifically to the 3’ untranslated region of viral genome. This study provides the first comparative view of the viral RNA ligands for RIG-I, MDA5 and LGP2 in the presence of infection. DOI: http://dx.doi.org/10.7554/eLife.11275.001 PMID:27011352

  7. RIG-I detects infection with live Listeria by sensing secreted bacterial nucleic acids

    PubMed Central

    Abdullah, Zeinab; Schlee, Martin; Roth, Susanne; Mraheil, Mobarak Abu; Barchet, Winfried; Böttcher, Jan; Hain, Torsten; Geiger, Sergej; Hayakawa, Yoshihiro; Fritz, Jörg H; Civril, Filiz; Hopfner, Karl-Peter; Kurts, Christian; Ruland, Jürgen; Hartmann, Gunther; Chakraborty, Trinad; Knolle, Percy A

    2012-01-01

    Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill-defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG-I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG-I-dependent IL-1β-production and inflammasome activation. The signalling molecule CARD9 contributed to IL-1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG-I provides a mechanistic explanation for efficient induction of immunity by live bacteria. PMID:23064150

  8. Rhodiola inhibits dengue virus multiplication by inducing innate immune response genes RIG-I, MDA5 and ISG in human monocytes.

    PubMed

    Diwaker, Drishya; Mishra, K P; Ganju, Lilly; Singh, S B

    2014-08-01

    Recognition of virus infection by retinoic acid-inducible gene (RIG) I and melanoma differentiation-associated protein (MDA) 5, which are RNA helicases, and interferon-stimulated gene (ISG) 15 activates cascades of signal transduction pathways leading to production of type I interferons and proinflammatory cytokines that orchestrate the elimination of the viruses. However, it has been demonstrated that RNA-helicase-mediated innate immunity plays an essential role in defending the host from infection. In our efforts to identify plant-derived antivirals that selectively enhance ISG- and RNA-helicase-mediated antiviral immune responses, we identified a plant, rhodiola, that significantly promoted ISG, RIG-I and MDA 5 gene expression and an antiviral immune response against dengue virus (DENV) infection. Rhodiola induced interferon (IFN) β and other cytokines, including IL-1β, TNF-α, IL-6 and IL-8, in infected cells. It was also found that rhodiola upregulated phosphorylated eIF-2α, PKR and NF-kB in infected cells. In addition, the number of NK cells was also increased by rhodiola treatment in dengue-virus-infected human PBMCs. Treatment with a crude extract of rhodiola (RAE) resulted in effects in the 20 % range, which is similar to the magnitude of the same effects observed in DENV infections. Taken together, our results imply that rhodiola induces pharmacological modulation of RIG-I, MDA 5 and ISG signal transduction pathways in favor of the induction of a beneficial antiviral immune response against dengue virus, which can be a novel therapeutic strategy for management of infection.

  9. In Vivo Ligands of MDA5 and RIG-I in Measles Virus-Infected Cells

    PubMed Central

    Hembach, Katharina; Baum, Alina; García-Sastre, Adolfo; Söding, Johannes; Conzelmann, Karl-Klaus

    2014-01-01

    RIG-I-like receptors (RLRs: RIG-I, MDA5 and LGP2) play a major role in the innate immune response against viral infections and detect patterns on viral RNA molecules that are typically absent from host RNA. Upon RNA binding, RLRs trigger a complex downstream signaling cascade resulting in the expression of type I interferons and proinflammatory cytokines. In the past decade extensive efforts were made to elucidate the nature of putative RLR ligands. In vitro and transfection studies identified 5′-triphosphate containing blunt-ended double-strand RNAs as potent RIG-I inducers and these findings were confirmed by next-generation sequencing of RIG-I associated RNAs from virus-infected cells. The nature of RNA ligands of MDA5 is less clear. Several studies suggest that double-stranded RNAs are the preferred agonists for the protein. However, the exact nature of physiological MDA5 ligands from virus-infected cells needs to be elucidated. In this work, we combine a crosslinking technique with next-generation sequencing in order to shed light on MDA5-associated RNAs from human cells infected with measles virus. Our findings suggest that RIG-I and MDA5 associate with AU-rich RNA species originating from the mRNA of the measles virus L gene. Corresponding sequences are poorer activators of ATP-hydrolysis by MDA5 in vitro, suggesting that they result in more stable MDA5 filaments. These data provide a possible model of how AU-rich sequences could activate type I interferon signaling. PMID:24743923

  10. Loss of RIG-I leads to a functional replacement with MDA5 in the Chinese tree shrew

    PubMed Central

    Xu, Ling; Yu, Dandan; Fan, Yu; Peng, Li; Wu, Yong; Yao, Yong-Gang

    2016-01-01

    The function of the RIG-I-like receptors (RLRs; including RIG-I, MDA5, and LGP2) as key cytoplasmic sensors of viral pathogen-associated molecular patterns (PAMPs) has been subjected to numerous pathogenic challenges and has undergone a dynamic evolution. We found evolutionary evidence that RIG-I was lost in the Chinese tree shrew lineage. Along with the loss of RIG-I, both MDA5 (tMDA5) and LGP2 (tLGP2) have undergone strong positive selection in the tree shrew. tMDA5 or tMDA5/tLGP2 could sense Sendai virus (an RNA virus posed as a RIG-I agonist) for inducing type I IFN, although conventional RIG-I and MDA5 were thought to recognize distinct RNA structures and viruses. tMDA5 interacted with adaptor tMITA (STINGTMEM173/ERIS), which was reported to bind only with RIG-I. The positively selected sites in tMDA5 endowed the substitute function for the lost RIG-I. These findings provided insights into the adaptation and functional diversity of innate antiviral activity in vertebrates. PMID:27621475

  11. Innate Immunity at Mucosal Surfaces: the IRE1-RIDD-RIG-I Pathway

    PubMed Central

    Lencer, Wayne I.; DeLuca, Heidi; Grey, Michael J.; Cho, Jin Ah

    2015-01-01

    Recent studies have linked the ER stress sensor IRE1α with the RIG-I pathway, which triggers an inflammatory response upon detection of viral RNAs. In response to ER dysfunction, IRE1α cleaves mRNA into single-strand fragments that lack markers of self, which activate RIG-I. Certain microbial products from mucosal pathogens activate this pathway by binding IRE1α directly, and the discovery that IRE1 is amplified at mucosal surfaces by gene duplication suggests an important role for IRE1 in mucosal immunity. Here, we review evidence in support of this hypothesis, and propose a model wherein IRE1 surveys the integrity of the ER, acting as a guard receptor and a pattern recognition receptor, capable both of sensing cellular stress caused by microbial infection and of responding to pathogens directly. PMID:26093676

  12. Both RIG-I and MDA5 detect alphavirus replication in concentration-dependent mode.

    PubMed

    Akhrymuk, Ivan; Frolov, Ilya; Frolova, Elena I

    2016-01-01

    Alphaviruses are a family of positive-strand RNA viruses that circulate on all continents between mosquito vectors and vertebrate hosts. Despite a significant public health threat, their biology is not sufficiently investigated, and the mechanisms of alphavirus replication and virus-host interaction are insufficiently understood. In this study, we have applied a variety of experimental systems to further understand the mechanism by which infected cells detect replicating alphaviruses. Our new data strongly suggest that activation of the antiviral response by alphavirus-infected cells is determined by the integrity of viral genes encoding proteins with nuclear functions, and by the presence of two cellular pattern recognition receptors (PRRs), RIG-I and MDA5. No type I IFN response is induced in their absence. The presence of either of these PRRs is sufficient for detecting virus replication. However, type I IFN activation in response to pathogenic alphaviruses depends on the basal levels of RIG-I or MDA5.

  13. Both RIG-I and MDA5 detect alphavirus replication in concentration-dependent mode

    PubMed Central

    Akhrymuk, Ivan; Frolov, Ilya; Frolova, Elena I.

    2015-01-01

    Alphaviruses are a family of positive-strand RNA viruses that circulate on all continents between mosquito vectors and vertebrate hosts. Despite a significant public health threat, their biology is not sufficiently investigated, and the mechanisms of alphavirus replication and virus-host interaction are insufficiently understood. In this study, we have applied a variety of experimental systems to further understand the mechanism by which infected cells detect replicating alphaviruses. Our new data strongly suggest that activation of the antiviral response by alphavirus-infected cells is determined by the integrity of viral genes encoding proteins with nuclear functions, and by the presence of two cellular pattern recognition receptors (PRRs), RIG-I and MDA5. No type I IFN response is induced in their absence. The presence of either of these PRRs is sufficient for detecting virus replication. However, type I IFN activation in response to pathogenic alphaviruses depends on the basal levels of RIG-I or MDA5. PMID:26550947

  14. Innate immunity to RNA virus is regulated by temporal and reversible sumoylation of RIG-I and MDA5.

    PubMed

    Hu, Ming-Ming; Liao, Chen-Yang; Yang, Qing; Xie, Xue-Qin; Shu, Hong-Bing

    2017-04-03

    Sensing of viral RNA by the cytosolic receptors RIG-I and melanoma differentiation-associated gene 5 (MDA5) leads to innate antiviral response. How RIG-I and MDA5 are dynamically regulated in innate antiviral response is not well understood. Here, we show that TRIM38 positively regulates MDA5- and RIG-I-mediated induction of downstream genes and acts as a SUMO E3 ligase for their dynamic sumoylation at K43/K865 and K96/K888, respectively, before and after viral infection. The sumoylation of MDA5 and RIG-I suppresses their K48-linked polyubiquitination and degradation in uninfected or early-infected cells. Sumoylation of the caspase recruitment domains of MDA5 and RIG-I is also required for their dephosphorylation by PP1 and activation upon viral infection. At the late phase of viral infection, both MDA5 and RIG-I are desumoylated by SENP2, resulting in their K48-linked polyubiquitination and degradation. These findings suggest that dynamic sumoylation and desumoylation of MDA5 and RIG-I modulate efficient innate immunity to RNA virus and its timely termination.

  15. DHX36 enhances RIG-I signaling by facilitating PKR-mediated antiviral stress granule formation.

    PubMed

    Yoo, Ji-Seung; Takahasi, Kiyohiro; Ng, Chen Seng; Ouda, Ryota; Onomoto, Koji; Yoneyama, Mitsutoshi; Lai, Janice Ching; Lattmann, Simon; Nagamine, Yoshikuni; Matsui, Tadashi; Iwabuchi, Kuniyoshi; Kato, Hiroki; Fujita, Takashi

    2014-03-01

    RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN) responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) activation, which has been shown to be essential for the formation of antiviral stress granule (avSG). We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR).

  16. Ubiquitin-Induced Oligomerization of the RNA Sensors RIG-I and MDA5 Activates Antiviral Innate Immune Response

    PubMed Central

    Jiang, Xiaomo; Kinch, Lisa; Brautigam, Chad A.; Chen, Xiang; Du, Fenghe; Grishin, Nick; Chen, Zhijian J.

    2012-01-01

    SUMMARY RIG-I and MDA5 detect viral RNA in the cytoplasm and activate signaling cascades leading to the production of type-I interferons. RIG-I is activated through sequential binding of viral RNA and unanchored lysine-63 (K63) polyubiquitin chains, but how polyubiquitin activates RIG-I and whether MDA5 is activated through a similar mechanism remain unresolved. Here we showed that the CARD domains of MDA5 bound to K63 polyubiquitin and that this binding was essential for MDA5 to activate the transcription factor IRF3. Mutations of conserved residues in MDA5 and RIG-I that disrupt their ubiquitin binding also abrogated their ability to activate IRF3. Polyubiquitin binding induced the formation of a large complex consisting of four RIG-I and four ubiquitin chains. This hetero-tetrameric complex was highly potent in activating the antiviral signaling cascades. These results suggest a unified mechanism of RIG-I and MDA5 activation and reveal a unique mechanism by which ubiquitin regulates cell signaling and immune response. PMID:22705106

  17. ATP hydrolysis by the viral RNA sensor RIG-I prevents unintentional recognition of self-RNA

    PubMed Central

    Lässig, Charlotte; Matheisl, Sarah; Sparrer, Konstantin MJ; de Oliveira Mann, Carina C; Moldt, Manuela; Patel, Jenish R; Goldeck, Marion; Hartmann, Gunther; García-Sastre, Adolfo; Hornung, Veit; Conzelmann, Karl-Klaus; Beckmann, Roland; Hopfner, Karl-Peter

    2015-01-01

    The cytosolic antiviral innate immune sensor RIG-I distinguishes 5′ tri- or diphosphate containing viral double-stranded (ds) RNA from self-RNA by an incompletely understood mechanism that involves ATP hydrolysis by RIG-I's RNA translocase domain. Recently discovered mutations in ATPase motifs can lead to the multi-system disorder Singleton-Merten Syndrome (SMS) and increased interferon levels, suggesting misregulated signaling by RIG-I. Here we report that SMS mutations phenocopy a mutation that allows ATP binding but prevents hydrolysis. ATPase deficient RIG-I constitutively signals through endogenous RNA and co-purifies with self-RNA even from virus infected cells. Biochemical studies and cryo-electron microscopy identify a 60S ribosomal expansion segment as a dominant self-RNA that is stably bound by ATPase deficient RIG-I. ATP hydrolysis displaces wild-type RIG-I from this self-RNA but not from 5' triphosphate dsRNA. Our results indicate that ATP-hydrolysis prevents recognition of self-RNA and suggest that SMS mutations lead to unintentional signaling through prolonged RNA binding. DOI: http://dx.doi.org/10.7554/eLife.10859.001 PMID:26609812

  18. Mutual antagonism between the Ebola virus VP35 protein and the RIG-I activator PACT determines infection outcome.

    PubMed

    Luthra, Priya; Ramanan, Parameshwaran; Mire, Chad E; Weisend, Carla; Tsuda, Yoshimi; Yen, Benjamin; Liu, Gai; Leung, Daisy W; Geisbert, Thomas W; Ebihara, Hideki; Amarasinghe, Gaya K; Basler, Christopher F

    2013-07-17

    The cytoplasmic pattern recognition receptor RIG-I is activated by viral RNA and induces type I IFN responses to control viral replication. The cellular dsRNA binding protein PACT can also activate RIG-I. To counteract innate antiviral responses, some viruses, including Ebola virus (EBOV), encode proteins that antagonize RIG-I signaling. Here, we show that EBOV VP35 inhibits PACT-induced RIG-I ATPase activity in a dose-dependent manner. The interaction of PACT with RIG-I is disrupted by wild-type VP35, but not by VP35 mutants that are unable to bind PACT. In addition, PACT-VP35 interaction impairs the association between VP35 and the viral polymerase, thereby diminishing viral RNA synthesis and modulating EBOV replication. PACT-deficient cells are defective in IFN induction and are insensitive to VP35 function. These data support a model in which the VP35-PACT interaction is mutually antagonistic and plays a fundamental role in determining the outcome of EBOV infection.

  19. Mutual Antagonism between the Ebola Virus VP35 Protein and the RIG-I Activator PACT Determines Infection Outcome

    PubMed Central

    Luthra, Priya; Ramanan, Parameshwaran; Mire, Chad E.; Weisend, Carla; Tsuda, Yoshimi; Yen, Benjamin; Liu, Gai; Leung, Daisy W.; Geisbert, Thomas W.; Ebihara, Hideki; Amarasinghe, Gaya K.; Basler, Christopher F.

    2013-01-01

    SUMMARY The cytoplasmic pattern recognition receptor RIG-I is activated by viral RNA and induces type I IFN responses to control viral replication. The cellular dsRNA binding protein PACT can also activate RIG-I. To counteract innate antiviral responses, some viruses, including Ebola virus (EBOV), encode proteins that antagonize RIG-I signaling. Here, we show that EBOV VP35 inhibits PACT-induced RIG-I ATPase activity in a dose-dependent manner. The interaction of PACT with RIG-I is disrupted by wild-type VP35, but not by VP35 mutants that are unable to bind PACT. In addition, PACT-VP35 interaction impairs the association between VP35 and the viral polymerase, thereby diminishing viral RNA synthesis and modulating EBOV replication. PACT-deficient cells are defective in IFN induction and are insensitive to VP35 function. These data support a model in which the VP35-PACT interaction is mutually antagonistic and plays a fundamental role in determining the outcome of EBOV infection. PMID:23870315

  20. The SKIV2L RNA exosome limits activation of the RIG-I-like receptors

    PubMed Central

    Eckard, Sterling C.; Rice, Gillian I.; Fabre, Alexandre; Badens, Catherine; Gray, Elizabeth E.; Hartley, Jane L.; Crow, Yanick J.; Stetson, Daniel B.

    2014-01-01

    Innate immune sensors of intracellular nucleic acids must be regulated to prevent inappropriate activation by endogenous DNA and RNA. The exonuclease Trex1 regulates the DNA sensing pathway by metabolizing potential DNA ligands that trigger it. However, an analogous mechanism for regulating the RIG-I-like receptors (RLRs) that detect RNA remains unknown. We show that the SKIV2L RNA exosome potently limits the activation of RLRs. We find that the unfolded protein response (UPR), which generates endogenous RLR ligands through IRE-1 endonuclease cleavage of cellular RNAs, triggers type I interferon (IFN) production in SKIV2L-depleted cells. Humans with SKIV2L deficiency have a type I IFN signature in their peripheral blood. Our findings reveal a mechanism for intracellular metabolism of immunostimulatory RNA, with implications for specific autoimmune disorders. PMID:25064072

  1. The Long Noncoding RNA NEAT1 Exerts Antihantaviral Effects by Acting as Positive Feedback for RIG-I Signaling.

    PubMed

    Ma, Hongwei; Han, Peijun; Ye, Wei; Chen, Hesong; Zheng, Xuyang; Cheng, Linfeng; Zhang, Liang; Yu, Lan; Wu, Xing'an; Xu, Zhikai; Lei, Yingfeng; Zhang, Fanglin

    2017-05-01

    Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs (lncRNAs) play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus (HTNV) infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon (IFN-β) production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I-IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein (SFPQ) by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs (lncRNAs) as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon (IFN) responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I-IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by

  2. The Unfolded Protein Response Element IRE1α Senses Bacterial Proteins Invading the ER to Activate RIG-I and Innate Immune Signaling

    PubMed Central

    Cho, Jin A.; Lee, Ann-Hwee; Platzer, Barbara; Cross, Benedict C.S.; Gardner, Brooke M.; De Luca, Heidi; Luong, Phi; Harding, Heather P.; Glimcher, Laurie H.; Walter, Peter; Fiebiger, Edda; Ron, David; Kagan, Jonathan C.; Lencer, Wayne I.

    2013-01-01

    SUMMARY The plasma membrane and all membrane-bound organelles except for the Golgi and endoplasmic reticulum (ER) are equipped with pattern-recognition molecules to sense microbes or their products and induce innate immunity for host defense. Here, we report that inositol-requiring-1α (IRE1α), an ER protein that signals in the unfolded protein response (UPR), is activated to induce inflammation by binding a portion of cholera toxin as it co-opts the ER to cause disease. Other known UPR transducers, including the IRE1α-dependent transcription factor XBP1, are dispensable for this signaling. The inflammatory response depends instead on the RNase activity of IRE1α to degrade endogenous mRNA, a process termed regulated IRE1α-dependent decay (RIDD) of mRNA. The mRNA fragments produced engage retinoic-acid inducible gene 1 (RIG-I), a cyto-solic sensor of RNA viruses, to activate NF-κB and interferon pathways. We propose IRE1α provides for a generalized mechanism of innate immune surveillance originating within the ER lumen. PMID:23684307

  3. Structural and functional analysis reveals that human OASL binds dsRNA to enhance RIG-I signaling.

    PubMed

    Ibsen, Mikkel Søes; Gad, Hans Henrik; Andersen, Line Lykke; Hornung, Veit; Julkunen, Ilkka; Sarkar, Saumendra N; Hartmann, Rune

    2015-05-26

    The oligoadenylate synthetase (OAS) enzymes are cytoplasmic dsRNA sensors belonging to the antiviral innate immune system. Upon binding to viral dsRNA, the OAS enzymes synthesize 2'-5' linked oligoadenylates (2-5As) that initiate an RNA decay pathway to impair viral replication. The human OAS-like (OASL) protein, however, does not harbor the catalytic activity required for synthesizing 2-5As and differs from the other human OAS family members by having two C-terminal ubiquitin-like domains. In spite of its lack of enzymatic activity, human OASL possesses antiviral activity. It was recently demonstrated that the ubiquitin-like domains of OASL could substitute for K63-linked poly-ubiquitin and interact with the CARDs of RIG-I and thereby enhance RIG-I signaling. However, the role of the OAS-like domain of OASL remains unclear. Here we present the crystal structure of the OAS-like domain, which shows a striking similarity with activated OAS1. Furthermore, the structure of the OAS-like domain shows that OASL has a dsRNA binding groove. We demonstrate that the OAS-like domain can bind dsRNA and that mutating key residues in the dsRNA binding site is detrimental to the RIG-I signaling enhancement. Hence, binding to dsRNA is an important feature of OASL that is required for enhancing RIG-I signaling.

  4. A deletion polymorphism in the Caenorhabditis elegans RIG-I homolog disables viral RNA dicing and antiviral immunity

    PubMed Central

    Ashe, Alyson; Bélicard, Tony; Le Pen, Jérémie; Sarkies, Peter; Frézal, Lise; Lehrbach, Nicolas J; Félix, Marie-Anne; Miska, Eric A

    2013-01-01

    RNA interference defends against viral infection in plant and animal cells. The nematode Caenorhabditis elegans and its natural pathogen, the positive-strand RNA virus Orsay, have recently emerged as a new animal model of host-virus interaction. Using a genome-wide association study in C. elegans wild populations and quantitative trait locus mapping, we identify a 159 base-pair deletion in the conserved drh-1 gene (encoding a RIG-I-like helicase) as a major determinant of viral sensitivity. We show that DRH-1 is required for the initiation of an antiviral RNAi pathway and the generation of virus-derived siRNAs (viRNAs). In mammals, RIG-I-domain containing proteins trigger an interferon-based innate immunity pathway in response to RNA virus infection. Our work in C. elegans demonstrates that the RIG-I domain has an ancient role in viral recognition. We propose that RIG-I acts as modular viral recognition factor that couples viral recognition to different effector pathways including RNAi and interferon responses. DOI: http://dx.doi.org/10.7554/eLife.00994.001 PMID:24137537

  5. Induction of interferon-λ contributes to TLR3 and RIG-I activation-mediated inhibition of herpes simplex virus type 2 replication in human cervical epithelial cells

    PubMed Central

    Zhou, Li; Li, Jie-Liang; Zhou, Yu; Liu, Jin-Biao; Zhuang, Ke; Gao, Jian-Feng; Liu, Shi; Sang, Ming; Wu, Jian-Guo; Ho, Wen-Zhe

    2015-01-01

    STUDY HYPOTHESIS Is it possible to immunologically activate human cervical epithelial cells to produce antiviral factors that inhibit herpes simplex virus type 2 (HSV-2) replication? STUDY FINDING Our results indicate that human cervical epithelial cells possess a functional TLR3/RIG-I signaling system, the activation of which can mount an Interferon-λ (IFN-λ)-mediated anti-HSV-2 response. WHAT IS KNOWN ALREADY There is limited information about the role of cervical epithelial cells in genital innate immunity against HSV-2 infection. STUDY DESIGN, SAMPLES/MATERIALS, METHODS We examined the expression of toll-like receptors (TLRs) and retinoic acid-inducible I (RIG-I) in End1/E6E7 cells by real-time PCR. The IFN-λ induced by TLR3 and RIG-I activation of End1/E6E7 cells was also examined by real-time PCR and ELISA. HSV-2 infection of End1/E6E7 cells was evaluated by the real-time PCR detection of HSV-2 gD expression. The antibody to IL-10Rβ was used to determine whether IFN-λ contributes to TLR3/RIG-I mediated HSV-2 inhibition. Expression of interferon regulatory factor 3 (IRF3), IRF7, IFN-stimulated gene 56 (ISG56), 2′-5′-oligoadenylate synthetase I (OAS-1) and myxovirus resistance A (MxA) were determined by the real-time PCR and western blot. End1/E6E7 cells were transfected with shRNA to knockdown the IRF3, IRF7 or RIG-I expression. Student's t-test and post Newman–Keuls test were used to analyze stabilized differences in the immunological parameters above between TLR3/RIG-I-activated cells and control cells. MAIN RESULTS AND THE ROLE OF CHANCE Human cervical epithelial cells expressed functional TLR3 and RIG-I, which could be activated by poly I:C and 5′ppp double-strand RNAs (5′ppp dsRNA), resulting in the induction of endogenous interferon lambda (IFN-λ). The induced IFN-λ contributed to TLR3/RIG-I-mediated inhibition of HSV-2 replication in human cervical epithelial cells, as an antibody to IL-10Rβ, an IFN-λ receptor subunit, could

  6. Overexpression of the Transcription Factor Sp1 Activates the OAS-RNAse L-RIG-I Pathway

    PubMed Central

    Dupuis-Maurin, Valéryane; Brinza, Lilia; Baguet, Joël; Plantamura, Emilie; Schicklin, Stéphane; Chambion, Solène; Macari, Claire; Tomkowiak, Martine; Deniaud, Emmanuelle; Leverrier, Yann

    2015-01-01

    Deregulated expression of oncogenes or transcription factors such as specificity protein 1 (Sp1) is observed in many human cancers and plays a role in tumor maintenance. Paradoxically in untransformed cells, Sp1 overexpression induces late apoptosis but the early intrinsic response is poorly characterized. In the present work, we studied increased Sp1 level consequences in untransformed cells and showed that it turns on an early innate immune transcriptome. Sp1 overexpression does not activate known cellular stress pathways such as DNA damage response or endoplasmic reticulum stress, but induces the activation of the OAS-RNase L pathway and the generation of small self-RNAs, leading to the upregulation of genes of the antiviral RIG-I pathway at the transcriptional and translational levels. Finally, Sp1-induced intrinsic innate immune response leads to the production of the chemokine CXCL4 and to the recruitment of inflammatory cells in vitro and in vivo. Altogether our results showed that increased Sp1 level in untransformed cells constitutes a novel danger signal sensed by the OAS-RNase L axis leading to the activation of the RIG-I pathway. These results suggested that the OAS-RNase L-RIG-I pathway may be activated in sterile condition in absence of pathogen. PMID:25738304

  7. Overexpression of the transcription factor Sp1 activates the OAS-RNAse L-RIG-I pathway.

    PubMed

    Dupuis-Maurin, Valéryane; Brinza, Lilia; Baguet, Joël; Plantamura, Emilie; Schicklin, Stéphane; Chambion, Solène; Macari, Claire; Tomkowiak, Martine; Deniaud, Emmanuelle; Leverrier, Yann; Marvel, Jacqueline; Michallet, Marie-Cécile

    2015-01-01

    Deregulated expression of oncogenes or transcription factors such as specificity protein 1 (Sp1) is observed in many human cancers and plays a role in tumor maintenance. Paradoxically in untransformed cells, Sp1 overexpression induces late apoptosis but the early intrinsic response is poorly characterized. In the present work, we studied increased Sp1 level consequences in untransformed cells and showed that it turns on an early innate immune transcriptome. Sp1 overexpression does not activate known cellular stress pathways such as DNA damage response or endoplasmic reticulum stress, but induces the activation of the OAS-RNase L pathway and the generation of small self-RNAs, leading to the upregulation of genes of the antiviral RIG-I pathway at the transcriptional and translational levels. Finally, Sp1-induced intrinsic innate immune response leads to the production of the chemokine CXCL4 and to the recruitment of inflammatory cells in vitro and in vivo. Altogether our results showed that increased Sp1 level in untransformed cells constitutes a novel danger signal sensed by the OAS-RNase L axis leading to the activation of the RIG-I pathway. These results suggested that the OAS-RNase L-RIG-I pathway may be activated in sterile condition in absence of pathogen.

  8. 55 Amino acid linker between helicase and carboxyl terminal domains of RIG-I functions as a critical repression domain and determines inter-domain conformation.

    PubMed

    Kageyama, Maiko; Takahasi, Kiyohiro; Narita, Ryo; Hirai, Reiko; Yoneyama, Mitsutoshi; Kato, Hiroki; Fujita, Takashi

    2011-11-11

    In virus-infected cells, viral RNA with non-self structural pattern is recognized by DExD/Hbox RNA helicase, RIG-I. Once RIG-I senses viral RNA, it triggers a signaling cascade, resulting in the activation of genes including type I interferon, which activates antiviral responses. Overexpression of N-terminal caspase activation and recruitment domain (CARD) is sufficient to activate signaling; however basal activity of full-length RIG-I is undetectable. The repressor domain (RD), initially identified as a.a. 735-925, is responsible for diminished basal activity; therefore, it is suggested that RIG-I is under auto-repression in uninfected cells and the repression is reversed upon its encounter with viral RNA. In this report, we further delimited RD to a.a. 747-801, which corresponds to a linker connecting the helicase and the C-terminal domain (CTD). Alanine substitutions of the conserved residues in the linker conferred constitutive activity to full-length RIG-I. We found that the constitutive active mutants do not exhibit ATPase activity, suggesting that ATPase is required for de-repression but not signaling itself. Furthermore, trypsin digestion of recombinant RIG-I revealed that the wild-type, but not linker mutant conforms to the trypsin-resistant structure, containing CARD and helicase domain. The result strongly suggests that the linker is responsible for maintaining RIG-I in a "closed" structure to minimize unwanted production of interferon in uninfected cells. These findings shed light on the structural regulation of RIG-I function.

  9. Toll-like Receptor 3, RIG-I-like Receptors and the NLRP3 Inflammasome: Key Modulators of Innate Immune Responses to Double-stranded RNA Viruses

    PubMed Central

    Yu, Man; Levine, Stewart J.

    2011-01-01

    Double-stranded RNA (dsRNA), the genetic material for many RNA viruses, induces robust host immune responses via pattern recognition receptors, which include Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I-like receptors (RLRs) and the multi-protein NLRP3 inflammasome complex. The engagement of dsRNA receptors or inflammasome activation by viral dsRNA initiates complex intracellular signaling cascades that play essential roles in inflammation and innate immune responses, as well as the resultant development of adaptive immunity. This review focuses on signaling pathways mediated by TLR3, RLRs and the NLRP3 inflammasome, as well as the potential use of agonists and antagonists that target these pathways to treat disease. PMID:21466970

  10. Dephosphorylation of the RNA sensors RIG-I and MDA5 by the phosphatase PP1 is essential for innate immune signaling

    PubMed Central

    Wies, Effi; Wang, May K.; Maharaj, Natalya P.; Chen, Kan; Zhou, Shenghua; Finberg, Robert W.; Gack, Michaela U.

    2013-01-01

    SUMMARY RIG-I and MDA5 have emerged as key cytosolic sensors for the detection of RNA viruses, leading to antiviral interferon (IFN) production. Recent studies highlighted the importance of posttranslational modification for controlling RIG-I antiviral activity. However, the regulation of MDA5 signal-transducing ability remains unclear. Here we show that MDA5 signaling activity is regulated by a dynamic balance between phosphorylation and dephosphorylation of its CARD domains. Employing a phosphatome RNAi screen, we identified PP1α and PP1γ as primary phosphatases responsible for MDA5 and RIG-I dephosphorylation, leading to their activation. Silencing of PP1α and PP1γ enhanced RIG-I and MDA5 CARD phosphorylation and reduced antiviral IFN-β production. PP1α and PP1γ depleted cells were impaired in their ability to induce interferon-stimulated gene expression, which resulted in enhanced RNA virus replication. This work identifies PP1α and PP1γ as regulators of antiviral innate immune responses to various RNA viruses, including influenza virus, paramyxovirus, dengue virus and picornavirus. PMID:23499489

  11. Invertebrate Iridescent Virus 6, a DNA Virus, Stimulates a Mammalian Innate Immune Response through RIG-I-Like Receptors

    PubMed Central

    Ahlers, Laura R. H.; Bastos, Reginaldo G.; Hiroyasu, Aoi

    2016-01-01

    Insects are not only major vectors of mammalian viruses, but are also host to insect-restricted viruses that can potentially be transmitted to mammals. While mammalian innate immune responses to arboviruses are well studied, less is known about how mammalian cells respond to viruses that are restricted to infect only invertebrates. Here we demonstrate that IIV-6, a DNA virus of the family Iridoviridae, is able to induce a type I interferon-dependent antiviral immune response in mammalian cells. Although IIV-6 is a DNA virus, we demonstrate that the immune response activated during IIV-6 infection is mediated by the RIG-I-like receptor (RLR) pathway, and not the canonical DNA sensing pathway via cGAS/STING. We further show that RNA polymerase III is required for maximal IFN-β secretion, suggesting that viral DNA is transcribed by this enzyme into an RNA species capable of activating the RLR pathway. Finally, we demonstrate that the RLR-driven mammalian innate immune response to IIV-6 is functionally capable of protecting cells from subsequent infection with the arboviruses Vesicular Stomatitis virus and Kunjin virus. These results represent a novel example of an invertebrate DNA virus activating a canonically RNA sensing pathway in the mammalian innate immune response, which reduces viral load of ensuing arboviral infection. PMID:27824940

  12. PACT- and RIG-I-Dependent Activation of Type I Interferon Production by a Defective Interfering RNA Derived from Measles Virus Vaccine

    PubMed Central

    Ho, Ting-Hin; Kew, Chun; Lui, Pak-Yin; Chan, Chi-Ping; Satoh, Takashi; Akira, Shizuo

    2015-01-01

    ABSTRACT The live attenuated measles virus vaccine is highly immunostimulatory. Identification and characterization of its components that activate the innate immune response might provide new strategies and agents for the rational design and development of chemically defined adjuvants. In this study, we report on the activation of type I interferon (IFN) production by a defective interfering (DI) RNA isolated from the Hu-191 vaccine strain of measles virus. We found that the Hu-191 virus induced IFN-β much more potently than the Edmonston strain. In the search for IFN-inducing species in Hu-191, we identified a DI RNA specifically expressed by this strain. This DI RNA, which was of the copy-back type, was predicted to fold into a hairpin structure with a long double-stranded stem region of 206 bp, and it potently induced the expression of IFN-β. Its IFN-β-inducing activity was further enhanced when both cytoplasmic RNA sensor RIG-I and its partner, PACT, were overexpressed. On the contrary, this activity was abrogated in cells deficient in PACT or RIG-I. The DI RNA was found to be associated with PACT in infected cells. In addition, both the 5′-di/triphosphate end and the double-stranded stem region on the DI RNA were essential for its activation of PACT and RIG-I. Taken together, our findings support a model in which a viral DI RNA is sensed by PACT and RIG-I to initiate an innate antiviral response. Our work might also provide a foundation for identifying physiological PACT ligands and developing novel adjuvants or antivirals. IMPORTANCE The live attenuated measles virus vaccine is one of the most successful human vaccines and has largely contained the devastating impact of a highly contagious virus. Identifying the components in this vaccine that stimulate the host immune response and understanding their mechanism of action might help to design and develop better adjuvants, vaccines, antivirals, and immunotherapeutic agents. We identified and characterized

  13. Junín Virus Infection Activates the Type I Interferon Pathway in a RIG-I-Dependent Manner

    PubMed Central

    Yun, Nadezdha E.; Seregin, Alexey V.; Poussard, Allison L.; Walker, Aida G.; Brasier, Allan R.; Zhao, Yingxin; Tian, Bing; de la Torre, Juan Carlos; Paessler, Slobodan

    2012-01-01

    Junín virus (JUNV), an arenavirus, is the causative agent of Argentine hemorrhagic fever, an infectious human disease with 15–30% case fatality. The pathogenesis of AHF is still not well understood. Elevated levels of interferon and cytokines are reported in AHF patients, which might be correlated to the severity of the disease. However the innate immune response to JUNV infection has not been well evaluated. Previous studies have suggested that the virulent strain of JUNV does not induce IFN in human macrophages and monocytes, whereas the attenuated strain of JUNV was found to induce IFN response in murine macrophages via the TLR-2 signaling pathway. In this study, we investigated the interaction between JUNV and IFN pathway in human epithelial cells highly permissive to JUNV infection. We have determined the expression pattern of interferon-stimulated genes (ISGs) and IFN-β at both mRNA and protein levels during JUNV infection. Our results clearly indicate that JUNV infection activates the type I IFN response. STAT1 phosphorylation, a downstream marker of activation of IFN signaling pathway, was readily detected in JUNV infected IFN-competent cells. Our studies also demonstrated for the first time that RIG-I was required for IFN production during JUNV infection. IFN activation was detected during infection by either the virulent or attenuated vaccine strain of JUNV. Curiously, both virus strains were relatively insensitive to human IFN treatment. Our studies collectively indicated that JUNV infection could induce host type I IFN response and provided new insights into the interaction between JUNV and host innate immune system, which might be important in future studies on vaccine development and antiviral treatment. PMID:22629479

  14. Functional Effects of Toll-Like Receptor (TLR)3, 7, 9, RIG-I and MDA-5 Stimulation in Nasal Epithelial Cells

    PubMed Central

    Tengroth, Lotta; Millrud, Camilla Rydberg; Kvarnhammar, Anne Månsson; Kumlien Georén, Susanna; Latif, Leith; Cardell, Lars-Olaf

    2014-01-01

    Background The human nasal epithelium is an important physical barrier, and a part of the innate immune defense that protect against pathogens. The epithelial cells recognize microbial components by pattern-recognition receptors (PRRs), and thereby trigger an immune response. Even though TLR3, TLR7, TLR9, RIG-I and MDA-5 are all known to respond to viral stimulation, their potential role in chronic airway inflammation triggered by local cytokine release remains to be established. Methods mRNA and corresponding protein expression of TLR3, TLR7, TLR9, RIG-I and MDA-5 were analyzed in nasal biopsies and various upper airway epithelial cell lines using real-time reverse transcription PCR, immunohistochemistry and flow cytometry. Ligand induced, cytokine release, was evaluated with ELISA. Results Nasal biopsies were found to express TLR3, TLR7, TLR9, RIG-I and MDA-5, with the most abundant expression in the surface epithelium. These receptors were verified in primary human nasal epithelial cell (HNEC) as well as in the airway epithelial cell lines Detroit-562 and FaDu. Poly(I:C) (TLR3) and R-837 (TLR7) stimulation increased secretion of IL-6 and GM-CSF from the nasal mucosa and the epithelial cell lines. CpG (TLR9) stimulation caused release of IL-8 in the nasal mucosa and in FaDu. Poly(I:C)/LyoVec (RIG-I/MDA-5) stimulation activated the secretion of IFN-β in the nasal mucosa. A corresponding release was also detected from HNEC and Detroit-562. Conclusion The nasal epithelium has the ability to recognize viral intrusion through TLR and RLR receptors, and the subsequent response might have a role in exacerbation of inflammatory diseases like allergic rhinitis and chronic rhinosinusitis. PMID:24886842

  15. De Novo Transcriptome Analysis Provides Insights into Immune Related Genes and the RIG-I-Like Receptor Signaling Pathway in the Freshwater Planarian (Dugesia japonica)

    PubMed Central

    Deng, Hongkuan; Zhang, Yichao; Sun, Xiaowen; Zhu, Guangzhong; Liu, Baohua; Zhao, Bosheng

    2016-01-01

    Background The freshwater planarian Dugesia japonica (D. japonica) possesses extraordinary ability to regenerate lost organs or body parts. Interestingly, in the process of regeneration, there is little wound infection, suggesting that D. japonica has a formidable innate immune system. The importance of immune system prompted us to search for immune-related genes and RIG-I-like receptor signaling pathways. Results Transcriptome sequencing of D. japonica was performed on an IlluminaHiSeq2000 platform. A total of 27,180 transcripts were obtained by Trinity assembler. CEGMA analysis and mapping of all trimmed reads back to the assembly result showed that our transcriptome assembly covered most of the whole transcriptome. 23,888 out of 27,180 transcripts contained ORF (open reading fragment), and were highly similar to those in Schistosoma mansoni using BLASTX analysis. 8,079 transcripts (29.7%) and 8,668 (31.9%) were annotated by Blast2GO and KEGG respectively. A DYNLRB-like gene was cloned to verify its roles in the immune response. Finally, the expression patterns of 4 genes (RIG-I, TRAF3, TRAF6, P38) in the RIG-I-like receptor signaling pathway were detected, and the results showed they are very likely to be involved in planarian immune response. Conclusion RNA-Seq analysis based on the next-generation sequencing technology was an efficient approach to discover critical genes and to understand their corresponding biological functions. Through GO and KEGG analysis, several critical and conserved signaling pathways and genes related to RIG-I-like receptor signaling pathway were identified. Four candidate genes were selected to identify their expression dynamics in the process of pathogen stimulation. These annotated transcripts of D. japonica provide a useful resource for subsequent investigation of other important pathways. PMID:26986572

  16. Herpes simplex virus 1 tegument protein US11 downmodulates the RLR signaling pathway via direct interaction with RIG-I and MDA-5.

    PubMed

    Xing, Junji; Wang, Shuai; Lin, Rongtuan; Mossman, Karen L; Zheng, Chunfu

    2012-04-01

    The interferon (IFN)-mediated antiviral response is a major defense of the host immune system. In order to complete their life cycle, viruses must modulate host IFN-mediated immune responses. Herpes simplex virus 1 (HSV-1) is a large DNA virus containing more than 80 genes, many of which encode proteins that are involved in virus-host interactions and show immune modulatory capabilities. In this study, we demonstrate that the US11 protein, an RNA binding tegument protein of HSV-1, is a novel antagonist of the beta IFN (IFN-β) pathway. US11 significantly inhibited Sendai virus (SeV)-induced IFN-β production, and its double-stranded RNA (dsRNA) binding domain was indispensable for this inhibition activity. Additionally, wild-type HSV-1 coinfection showed stronger inhibition than US11 mutant HSV-1 in SeV-induced IFN-β production. Coimmunoprecipitation analysis demonstrated that the US11 protein in HSV-1-infected cells interacts with endogenous RIG-I and MDA-5 through its C-terminal RNA-binding domain, which was RNA independent. Expression of US11 in both transfected and HSV-1-infected cells interferes with the interaction between MAVS and RIG-I or MDA-5. Finally, US11 dampens SeV-mediated IRF3 activation. Taken together, the combined data indicate that HSV-1 US11 binds to RIG-I and MDA-5 and inhibits their downstream signaling pathway, preventing the production of IFN-β, which may contribute to the pathogenesis of HSV-1 infection.

  17. Seasonal and pandemic influenza H1N1 viruses induce differential expression of SOCS-1 and RIG-I genes and cytokine/chemokine production in macrophages

    PubMed Central

    Ramírez-Martínez, Gustavo; Cruz-Lagunas, Alfredo; Jiménez-Alvarez, Luis; Espinosa, Enrique; Ortíz-Quintero, Blanca; Santos-Mendoza, Teresa; Herrera, María Teresa; Canché-Pool, Elsy; Mendoza, Criselda; Bañales, José L.; García-Moreno, Sara A.; Morán, Juan; Cabello, Carlos; Orozco, Lorena; Aguilar-Delfín, Irma; Hidalgo-Miranda, Alfredo; Romero, Sandra; Suratt, Benjamin T.; Selman, Moisés; Zúñiga, Joaquín

    2014-01-01

    Background Infection with pandemic (pdm) A/H1N1 virus induces high levels of pro-inflammatory mediators in blood and lungs of experimental animals and humans. Methods To compare the involvement of seasonal A/PR/8/34 and pdm A/H1N1 virus strains in the regulation of inflammatory responses, we analyzed the changes in the whole-genome expression induced by these strains in macrophages and A549 epithelial cells. We also focused on the functional implications (cytokine production) of the differential induction of suppressors of cytokine signaling (SOCS)-1, SOCS-3, retinoid-inducible gene (RIG)-I and interferon receptor 1 (IFNAR1) genes by these viral strains in early stages of the infection. Results We identified 130 genes differentially expressed by pdm A/H1N1 and A/PR/8/34 infections in macrophages. mRNA levels of SOCS-1 and RIG-I were up-regulated in macrophages infected with the A/PR/8/34 but not with pdm A/H1N1 virus. mRNA levels of SOCS-3 and IFNAR1 induced by A/PR/8/34 and pdm A/H1N1 strains in macrophages, as well as in A549 cells were similar. We found higher levels of IL-6, TNF-α, IL-10, CCL3, CCL5, CCL4 and CXCL8 (p<0.05) in supernatants from cultures of macrophages infected with the pdm A/H1N1 virus compared to those infected with the A/PR/8/34 strain, coincident with the lack of SOCS-1 and RIG-I expression. In contrast, levels of INF-α were higher in cultures of macrophages 48 h after infection with the A/PR/8/34 strain than with the pdm A/H1N1 virus. Conclusions These findings suggest that factors inherent to the pdm A/H1N1 viral strain may increase the production of inflammatory mediators by inhibiting SOCS-1 and modifying the expression of antiviral immunity-related genes, including RIG-I, in human macrophages. PMID:23434273

  18. Rhinovirus and dsRNA Induce RIG-I-Like Receptors and Expression of Interferon β and λ1 in Human Bronchial Smooth Muscle Cells

    PubMed Central

    Calvén, Jenny; Yudina, Yuliana; Uller, Lena

    2013-01-01

    Rhinovirus (RV) infections cause exacerbations and development of severe asthma highlighting the importance of antiviral interferon (IFN) defence by airway cells. Little is known about bronchial smooth muscle cell (BSMC) production of IFNs and whether BSMCs have dsRNA-sensing receptors besides TLR3. dsRNA is a rhinoviral replication intermediate and necrotic cell effect mimic that mediates innate immune responses in bronchial epithelial cells. We have explored dsRNA-evoked IFN-β and IFN-λ1 production in human BSMCs and potential involvement of TLR3 and RIG-I-like receptors (RLRs). Primary BSMCs were stimulated with 0.1–10 µg/ml dsRNA, 0.1–1 µg/ml dsRNA in complex with the transfection agent LyoVec (dsRNA/LyoVec; selectively activating cytosolic RLRs) or infected with 0.05–0.5 MOI RV1B. Both dsRNA stimuli evoked early (3 h), concentration-dependent IFN-β and IFN-λ1 mRNA expression, which with dsRNA/LyoVec was much greater, and with dsRNA was much less, after 24 h. The effects were inhibited by dexamethasone. Further, dsRNA and dsRNA/LyoVec concentration-dependently upregulated RIG-I and MDA5 mRNA and protein. dsRNA and particularly dsRNA/LyoVec caused IFN-β and IFN-λ1 protein production (24 h). dsRNA- but not dsRNA/LyoVec-induced IFN expression was partly inhibited by chloroquine that suppresses endosomal TLR3 activation. RV1B dose-dependently increased BSMC expression of RIG-I, MDA5, IFN-β, and IFN-λ1 mRNA. We suggest that BSMCs express functional RLRs and that both RLRs and TLR3 are involved in viral stimulus-induced BSMC expression of IFN-β and IFN-λ1. PMID:23658644

  19. TRIM21 Promotes cGAS and RIG-I Sensing of Viral Genomes during Infection by Antibody-Opsonized Virus

    PubMed Central

    Watkinson, Ruth E.; McEwan, William A.; Tam, Jerry C. H.; Vaysburd, Marina; James, Leo C.

    2015-01-01

    Encapsidation is a strategy almost universally employed by viruses to protect their genomes from degradation and from innate immune sensors. We show that TRIM21, which targets antibody-opsonized virions for proteasomal destruction, circumvents this protection, enabling the rapid detection and degradation of viral genomes before their replication. TRIM21 triggers an initial wave of cytokine transcription that is antibody, rather than pathogen, driven. This early response is augmented by a second transcriptional program, determined by the nature of the infecting virus. In this second response, TRIM21-induced exposure of the viral genome promotes sensing of DNA and RNA viruses by cGAS and RIG-I. This mechanism allows early detection of an infection event and drives an inflammatory response in mice within hours of viral challenge. PMID:26506431

  20. Nucleic acid-induced antiviral immunity in invertebrates: an evolutionary perspective.

    PubMed

    Wang, Pei-Hui; Weng, Shao-Ping; He, Jian-Guo

    2015-02-01

    Nucleic acids derived from viral pathogens are typical pathogen associated molecular patterns (PAMPs). In mammals, the recognition of viral nucleic acids by pattern recognition receptors (PRRs), which include Toll-like receptors (TLRs) and retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), induces the release of inflammatory cytokines and type I interferons (IFNs) through the activation of nuclear factor κB (NF-κB) and interferon regulatory factor (IRF) 3/7 pathways, triggering the host antiviral state. However, whether nucleic acids can induce similar antiviral immunity in invertebrates remains ambiguous. Several studies have reported that nucleic acid mimics, especially dsRNA mimic poly(I:C), can strongly induce non-specific antiviral immune responses in insects, shrimp, and oyster. This behavior shows multiple similarities to the hallmarks of mammalian IFN responses. In this review, we highlight the current understanding of nucleic acid-induced antiviral immunity in invertebrates. We also discuss the potential recognition and regulatory mechanisms that confer non-specific antiviral immunity on invertebrate hosts.

  1. Apoptosis, Toll-like, RIG-I-like and NOD-like Receptors Are Pathways Jointly Induced by Diverse Respiratory Bacterial and Viral Pathogens.

    PubMed

    Martínez, Isidoro; Oliveros, Juan C; Cuesta, Isabel; de la Barrera, Jorge; Ausina, Vicente; Casals, Cristina; de Lorenzo, Alba; García, Ernesto; García-Fojeda, Belén; Garmendia, Junkal; González-Nicolau, Mar; Lacoma, Alicia; Menéndez, Margarita; Moranta, David; Nieto, Amelia; Ortín, Juan; Pérez-González, Alicia; Prat, Cristina; Ramos-Sevillano, Elisa; Regueiro, Verónica; Rodriguez-Frandsen, Ariel; Solís, Dolores; Yuste, José; Bengoechea, José A; Melero, José A

    2017-01-01

    Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here.

  2. Apoptosis, Toll-like, RIG-I-like and NOD-like Receptors Are Pathways Jointly Induced by Diverse Respiratory Bacterial and Viral Pathogens

    PubMed Central

    Martínez, Isidoro; Oliveros, Juan C.; Cuesta, Isabel; de la Barrera, Jorge; Ausina, Vicente; Casals, Cristina; de Lorenzo, Alba; García, Ernesto; García-Fojeda, Belén; Garmendia, Junkal; González-Nicolau, Mar; Lacoma, Alicia; Menéndez, Margarita; Moranta, David; Nieto, Amelia; Ortín, Juan; Pérez-González, Alicia; Prat, Cristina; Ramos-Sevillano, Elisa; Regueiro, Verónica; Rodriguez-Frandsen, Ariel; Solís, Dolores; Yuste, José; Bengoechea, José A.; Melero, José A.

    2017-01-01

    Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here. PMID:28298903

  3. Hantaan Virus Infection Induces CXCL10 Expression through TLR3, RIG-I, and MDA-5 Pathways Correlated with the Disease Severity

    PubMed Central

    Liu, Bei; Ma, Ying; Yi, Jing; Zhang, Chunmei; Zhang, Yun; Xu, Zhuwei; Wang, Jiuping; Yang, Kun; Yang, Angang; Jin, Boquan

    2014-01-01

    Hantaan virus (HTNV) is a major agent causing hemorrhagic fever with renal syndrome (HFRS). Although the pathogenesis of HFRS is unclear, some reports have suggested that the abundant production of proinflammatory cytokines and uncontrolled inflammatory responses may contribute to the development of HFRS. CXCL10 is one of these cytokines and is found to be involved in the pathogenesis of many virus infectious diseases. However, the role of CXCL10 in the pathogenesis of HFRS and the molecular regulation mechanism of CXCL10 in HTNV infection remain unknown. In this study, we report that CXCL10 expresses highly in the HFRS patients' sera and the elevated CXCL10 is positively correlated with the severity of HFRS. We find that HTNV, a single-strand RNA virus, can act as a double-strand RNA to activate the TLR3, RIG-I, and MDA-5 signaling pathways. Through the downstream transcription factors of these pathways, NF-κB and IRF7, which bind directly to the CXCL10's promoter, the expression of CXCL10 is increased. Our results may help to better understand the role of CXCL10 in the development of HFRS and may provide some novel insights into the immune response of HTNV infection. PMID:24701034

  4. RIG-I-like helicases induce immunogenic cell death of pancreatic cancer cells and sensitize tumors toward killing by CD8+ T cells

    PubMed Central

    Duewell, P; Steger, A; Lohr, H; Bourhis, H; Hoelz, H; Kirchleitner, S V; Stieg, M R; Grassmann, S; Kobold, S; Siveke, J T; Endres, S; Schnurr, M

    2014-01-01

    Pancreatic cancer is characterized by a microenvironment suppressing immune responses. RIG-I-like helicases (RLH) are immunoreceptors for viral RNA that induce an antiviral response program via the production of type I interferons (IFN) and apoptosis in susceptible cells. We recently identified RLH as therapeutic targets of pancreatic cancer for counteracting immunosuppressive mechanisms and apoptosis induction. Here, we investigated immunogenic consequences of RLH-induced tumor cell death. Treatment of murine pancreatic cancer cell lines with RLH ligands induced production of type I IFN and proinflammatory cytokines. In addition, tumor cells died via intrinsic apoptosis and displayed features of immunogenic cell death, such as release of HMGB1 and translocation of calreticulin to the outer cell membrane. RLH-activated tumor cells led to activation of dendritic cells (DCs), which was mediated by tumor-derived type I IFN, whereas TLR, RAGE or inflammasome signaling was dispensable. Importantly, CD8α+ DCs effectively engulfed apoptotic tumor material and cross-presented tumor-associated antigen to naive CD8+ T cells. In comparison, tumor cell death mediated by oxaliplatin, staurosporine or mechanical disruption failed to induce DC activation and antigen presentation. Tumor cells treated with sublethal doses of RLH ligands upregulated Fas and MHC-I expression and were effectively sensitized towards Fas-mediated apoptosis and cytotoxic T lymphocyte (CTL)-mediated lysis. Vaccination of mice with RLH-activated tumor cells induced protective antitumor immunity in vivo. In addition, MDA5-based immunotherapy led to effective tumor control of established pancreatic tumors. In summary, RLH ligands induce a highly immunogenic form of tumor cell death linking innate and adaptive immunity. PMID:25012502

  5. Signaling through RIG-I and type I interferon receptor: Immune activation by Newcastle disease virus in man versus immune evasion by Ebola virus (Review).

    PubMed

    Schirrmacher, Volker

    2015-07-01

    In this review, two types of RNA viruses are compared with regard to the type I interferon (IFN) response in order to obtain a better understanding of the molecular mechanisms of immune activation or evasion. Upon human infection, both viruses exert either beneficial or detrimental effects. The Newcastle disease virus (NDV), is a type strain for avian paramyxoviruses, while the Ebola virus (EBOV), is a virus affecting primates. During evolution, both viruses specifically adapted to their respective hosts, acquiring sophisticated viral escape mechanisms. Two types of receptors play an important role in the life cycle of these two viruses: cytoplasmic retinoic acid‑inducible gene I (RIG‑I) and membrane expressed type I IFN receptor (IFNAR). In mouse and human cells, NDV is a strong inducer of the type I IFN response. The early phase of this is initiated by signaling through RIG‑I and the late response by signaling through IFNAR. EBOV does not induce type I IFN responses in humans as it has viral proteins that specifically and strongly interfere with RIG‑I and IFNAR signaling, as well as immune activation. In this review, we discuss whether the beneficial effects of one virus can be exploited in the fight against the detrimental effects of the other.

  6. Nonstructural Protein 11 of Porcine Reproductive and Respiratory Syndrome Virus Suppresses Both MAVS and RIG-I Expression as One of the Mechanisms to Antagonize Type I Interferon Production

    PubMed Central

    Sun, Yan; Ke, Hanzhong; Han, Mingyuan; Chen, Ning; Fang, Weihuan; Yoo, Dongwan

    2016-01-01

    Type I interferons (IFN-α/β) play a key role in antiviral defense, and porcine reproductive and respiratory syndrome virus (PRRSV) is known to down-regulate the IFN response in virus-infected cells and pigs. In this study, we showed that the overexpression of nsp11 of PRRSV induced a strong suppression of IFN production. Nsp11 suppressed both IRF3 and NF-κB activities when stimulated with a dsRNA analogue and TNF-α, respectively. This suppression was RLR dependent, since the transcripts and proteins of MAVS and RIG-I, two critical factors in RLR-mediated pathway, were both found to be reduced in the presence of overexpressed nsp11. Since nsp11 is an endoribonuclease (EndoU), the structure function relationship was examined using a series of nsp11 EndoU mutant plasmids. The mutants that impaired the EndoU activity failed to suppress IFN and led to the normal expression of MAVS. Seven single amino acid substitutions (4 in subdomain A and 3 in subdomain B) plus one insertion (frame-shift in nsp11) were then introduced into PRRSV infectious cDNA clones to generate nsp11 mutant viruses. Unfortunately, all EndoU knock-out nsp11 mutant viruses appeared replication-defective and no progenies were produced. Three mutations in EndoU subdomain A expressed the N and nsp2/3 proteins but their infectivity diminished after 2 passages. Taken together, our data show that PRRSV nsp11 endoribonuclease activity is critical for both viral replication and IFN antagonism. More importantly, the endoribonuclease activity of nsp11 demonstrates the substrate specificity towards MAVS and RIG-I (transcripts and proteins) over p65 and IRF3 in the context of gene transfection and overexpression. This is likely a mechanism of nsp11 suppression of type I IFN production. PMID:27997564

  7. 5'-triphosphate-siRNA activates RIG-I-dependent type I interferon production and enhances inhibition of hepatitis B virus replication in HepG2.2.15 cells.

    PubMed

    Chen, Xiaojuan; Qian, Yuanyu; Yan, Fei; Tu, Jian; Yang, Xingxing; Xing, Yaling; Chen, Zhongbin

    2013-12-05

    Hepatitis B virus (HBV) infection often results in acute or chronic viral hepatitis and other liver diseases including cirrhosis and hepatocellular carcinoma. Current therapies for HBV usually have severe side effects and can cause development of drug-resistant mutants. An alternative and safe immunotherapeutic approach for HBV infection is urgently needed for effective anti-HBV therapy. In this study, we propose a new strategy for anti-HBV therapy that activates type-I interferon (IFN) antiviral innate immunity through stimulating pattern-recognition receptors with RNA interference (RNAi) using a 5'-end triphosphate-modified small interfering RNA (3p-siRNA). We designed and generated a 3p-siRNA targeting overlapping region of S gene and P gene of the HBV genome at the 5'-end of pregenomic HBV RNA. Our results demonstrated that 3p-siRNA induced a RIG-I-dependent antiviral type-I IFN response when transfected into HepG2.2.15 cells that support HBV replication. The 3p-siRNA significantly inhibited HBsAg and HBeAg secretion from HepG2.2.15 cells in a RIG-I-dependent manner, and the antiviral effect of 3p-siRNA was superior to that of siRNA. Furthermore, 3p-siRNA had more pronounced inhibition effects on the replication of HBV DNA and the transcription of mRNA than that of siRNA. Finally, 3p-siRNA displayed antiviral activity with long-term suppression of HBV replication. In conclusion, our findings suggest that 3p-siRNA could act as a powerful bifunctional antiviral molecule with potential for developing a promising therapeutic against chronic HBV infection.

  8. Hypochlorous and peracetic acid induced oxidation of dairy proteins.

    PubMed

    Kerkaert, Barbara; Mestdagh, Frédéric; Cucu, Tatiana; Aedo, Philip Roger; Ling, Shen Yan; De Meulenaer, Bruno

    2011-02-09

    Hypochlorous and peracetic acids, both known disinfectants in the food industry, were compared for their oxidative capacity toward dairy proteins. Whey proteins and caseins were oxidized under well controlled conditions at pH 8 as a function of the sanitizing concentration. Different markers for protein oxidation were monitored. The results established that the protein carbonyl content was a rather unspecific marker for protein oxidation, which did not allow one to differentiate the oxidant used especially at the lower concentrations. Cysteine, tryptophan, and methionine were proven to be the most vulnerable amino acids for degradation upon hypochlorous and peracetic acid treatment, while tyrosine was only prone to degradation in the presence of hypochlorous acid. Hypochlorous acid induced oxidation gave rise to protein aggregation, while during peracetic acid induced oxidation, no high molecular weight aggregates were observed. Protein aggregation upon hypochlorous acid oxidation could primarily be linked to tryptophan and tyrosine degradation.

  9. Protective Mechanisms of Nitrone Antioxidants in Kanic Acid Induced Neurodegeneration

    DTIC Science & Technology

    2004-01-01

    L., Hong, J.S. (1996) Expression of) FosB in the rat hippocampus and striatum after systemic administration of kainic acid. Neurosci. Abstr. 22...gene expression in the hippocampus . Immunohistochemical methods and electromobility gel shift assays (EMSAs) demonstrate the concerted activation of...acid-induced neurodegenerative diseases. The major focus will be on the pathophysiological changes in the hippocampus . Special attention will be given

  10. Fine-Tuning of the RIG-I-Like Receptor/Interferon Regulatory Factor 3-Dependent Antiviral Innate Immune Response by the Glycogen Synthase Kinase 3/β-Catenin Pathway.

    PubMed

    Khan, Kashif Aziz; Dô, Florence; Marineau, Alexandre; Doyon, Priscilla; Clément, Jean-François; Woodgett, James R; Doble, Bradley W; Servant, Marc J

    2015-09-01

    Induction of an antiviral innate immune response relies on pattern recognition receptors, including retinoic acid-inducible gene 1-like receptors (RLR), to detect invading pathogens, resulting in the activation of multiple latent transcription factors, including interferon regulatory factor 3 (IRF3). Upon sensing of viral RNA and DNA, IRF3 is phosphorylated and recruits coactivators to induce type I interferons (IFNs) and selected sets of IRF3-regulated IFN-stimulated genes (ISGs) such as those for ISG54 (Ifit2), ISG56 (Ifit1), and viperin (Rsad2). Here, we used wild-type, glycogen synthase kinase 3α knockout (GSK-3α(-/-)), GSK-3β(-/-), and GSK-3α/β double-knockout (DKO) embryonic stem (ES) cells, as well as GSK-3β(-/-) mouse embryonic fibroblast cells in which GSK-3α was knocked down to demonstrate that both isoforms of GSK-3, GSK-3α and GSK-3β, are required for this antiviral immune response. Moreover, the use of two selective small-molecule GSK-3 inhibitors (CHIR99021 and BIO-acetoxime) or ES cells reconstituted with the catalytically inactive versions of GSK-3 isoforms showed that GSK-3 activity is required for optimal induction of antiviral innate immunity. Mechanistically, GSK-3 isoform activation following Sendai virus infection results in phosphorylation of β-catenin at S33/S37/T41, promoting IRF3 DNA binding and activation of IRF3-regulated ISGs. This study identifies the role of a GSK-3/β-catenin axis in antiviral innate immunity.

  11. Valproic acid-induced hyperammonaemic coma and unrecognised portosystemic shunt.

    PubMed

    Nzwalo, Hipólito; Carrapatoso, Leonor; Ferreira, Fátima; Basilio, Carlos

    2013-06-01

    Hyperammonaemic encephalopathy is a rare and potentially fatal complication of valproic acid treatment. The clinical presentation of hyperammonaemic encephalopathy is wide and includes seizures and coma. We present a case of hyperammonaemic coma precipitated by sodium valproate use for symptomatic epilepsy in a patient with unrecognised portosystemic shunt, secondary to earlier alcoholism. The absence of any stigmata of chronic liver disease and laboratory markers of liver dysfunction delayed the recognition of this alcohol-related complication. The portal vein bypass led to a refractory, valproic acid-induced hyperammonaemic coma. The patient fully recovered after dialysis treatment.

  12. Increased isoprostane levels in oleic acid-induced lung injury

    SciTech Connect

    Ono, Koichi; Koizumi, Tomonobu; Tsushima, Kenji; Yoshikawa, Sumiko; Yokoyama, Toshiki; Nakagawa, Rikimaru; Obata, Toru

    2009-10-16

    The present study was performed to examine a role of oxidative stress in oleic acid-induced lung injury model. Fifteen anesthetized sheep were ventilated and instrumented with a lung lymph fistula and vascular catheters for blood gas analysis and measurement of isoprostanes (8-epi prostaglandin F2{alpha}). Following stable baseline measurements, oleic acid (0.08 ml/kg) was administered and observed 4 h. Isoprostane was measured by gas chromatography mass spectrometry with the isotope dilution method. Isoprostane levels in plasma and lung lymph were significantly increased 2 h after oleic acid administration and then decreased at 4 h. The percent increases in isoprostane levels in plasma and lung lymph at 2 h were significantly correlated with deteriorated oxygenation at the same time point, respectively. These findings suggest that oxidative stress is involved in the pathogenesis of the pulmonary fat embolism-induced acute lung injury model in sheep and that the increase relates with the deteriorated oxygenation.

  13. [Sunitinib and zoledronic acid induced osteonecrosis of the jaw].

    PubMed

    Soós, Balázs; Vajta, László; Szalma, József

    2015-11-15

    The tendency for bisphosphonate and non-bisphosphonate (eg.: antiresorptive or anti-angiogenesis drugs) induced osteonecrosis is increasing. Treatment of these patients is a challenge both for dentists and for oral and maxillofacial surgeons. Cooperation with the drug prescribing general medicine colleagues to prevent osteonecrosis is extremely important. Furthermore, prevention should include dental focus elimination, oral hygienic instructions and education, dental follow-up and, in case of manifest necrosis, referral to maxillofacial departments. Authors outline the difficulties of conservative and surgical treatment of a patient with sunitinib and zoledronic acid induced osteonecrosis. The patient became symptomless and the operated area healed entirely six and twelve months postoperatively. A long term success further follow-up is necessary to verify long-term success.

  14. Computerized image analysis for acetic acid induced intraepithelial lesions

    NASA Astrophysics Data System (ADS)

    Li, Wenjing; Ferris, Daron G.; Lieberman, Rich W.

    2008-03-01

    Cervical Intraepithelial Neoplasia (CIN) exhibits certain morphologic features that can be identified during a visual inspection exam. Immature and dysphasic cervical squamous epithelium turns white after application of acetic acid during the exam. The whitening process occurs visually over several minutes and subjectively discriminates between dysphasic and normal tissue. Digital imaging technologies allow us to assist the physician analyzing the acetic acid induced lesions (acetowhite region) in a fully automatic way. This paper reports a study designed to measure multiple parameters of the acetowhitening process from two images captured with a digital colposcope. One image is captured before the acetic acid application, and the other is captured after the acetic acid application. The spatial change of the acetowhitening is extracted using color and texture information in the post acetic acid image; the temporal change is extracted from the intensity and color changes between the post acetic acid and pre acetic acid images with an automatic alignment. The imaging and data analysis system has been evaluated with a total of 99 human subjects and demonstrate its potential to screening underserved women where access to skilled colposcopists is limited.

  15. Oleic acid-induced mucosal injury in developing piglet intestine.

    PubMed

    Velasquez, O R; Henninger, K; Fowler, M; Tso, P; Crissinger, K D

    1993-03-01

    A role for luminal nutrients, in particular products of lipid digestion, in the pathogenesis of mucosal injury to developing intestine has been postulated. We evaluated changes in mucosal permeability and light and electron microscopic histology induced by luminal perfusion with the long-chain fatty acid oleate in developing piglet intestine as a function of age and concentration of the fatty acid. 51Cr-labeled EDTA plasma-to-lumen clearance was measured in jejunum and ileum of 1-day-, 3-day-, 2-wk-, and 1-mo-old piglets during sequential perfusion with saline control (20 min); 0, 1, 5, and 10 mM oleic acid/10 mM taurocholate in saline (20 min); and normal saline (60 min). The jejunum of piglets < or = 2 wk showed significantly greater increases in mucosal permeability compared with 1-mo-old animals after perfusion with oleic acid. This effect was dependent on the luminal concentration of the fatty acid and was associated with mucosal injury evident under light and electron microscopy. In contrast, the overall response in ileum was more attenuated compared with jejunum. Thus oleic acid, a common dietary fatty acid, induces dose- and age-dependent injury in developing piglet intestine. Investigation of the mechanisms of this injury may provide the basis for dietary modifications directed at decreasing the risk of mucosal injury during enteral feeding in neonatal intestine.

  16. Unsaturated fatty acids induce non-canonical autophagy

    PubMed Central

    Niso-Santano, Mireia; Malik, Shoaib Ahmad; Pietrocola, Federico; Bravo-San Pedro, José Manuel; Mariño, Guillermo; Cianfanelli, Valentina; Ben-Younès, Amena; Troncoso, Rodrigo; Markaki, Maria; Sica, Valentina; Izzo, Valentina; Chaba, Kariman; Bauvy, Chantal; Dupont, Nicolas; Kepp, Oliver; Rockenfeller, Patrick; Wolinski, Heimo; Madeo, Frank; Lavandero, Sergio; Codogno, Patrice; Harper, Francis; Pierron, Gérard; Tavernarakis, Nektarios; Cecconi, Francesco; Maiuri, Maria Chiara; Galluzzi, Lorenzo; Kroemer, Guido

    2015-01-01

    To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1+/− mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus. PMID:25586377

  17. Sphingoid bases inhibit acid-induced demineralization of hydroxyapatite.

    PubMed

    Valentijn-Benz, Marianne; van 't Hof, Wim; Bikker, Floris J; Nazmi, Kamran; Brand, Henk S; Sotres, Javier; Lindh, Liselott; Arnebrant, Thomas; Veerman, Enno C I

    2015-01-01

    Calcium hydroxyapatite (HAp), the main constituent of dental enamel, is inherently susceptible to the etching and dissolving action of acids, resulting in tooth decay such as dental caries and dental erosion. Since the prevalence of erosive wear is gradually increasing, there is urgent need for agents that protect the enamel against erosive attacks. In the present study we studied in vitro the anti-erosive effects of a number of sphingolipids and sphingoid bases, which form the backbone of sphingolipids. Pretreatment of HAp discs with sphingosine, phytosphingosine (PHS), PHS phosphate and sphinganine significantly protected these against acid-induced demineralization by 80 ± 17%, 78 ± 17%, 78 ± 7% and 81 ± 8%, respectively (p < 0.001). On the other hand, sphingomyelin, acetyl PHS, octanoyl PHS and stearoyl PHS had no anti-erosive effects. Atomic force measurement revealed that HAp discs treated with PHS were almost completely and homogeneously covered by patches of PHS. This suggests that PHS and other sphingoid bases form layers on the surface of HAp, which act as diffusion barriers against H(+) ions. In principle, these anti-erosive properties make PHS and related sphingosines promising and attractive candidates as ingredients in oral care products.

  18. Acid-induced unfolding mechanism of recombinant human endostatin.

    PubMed

    Li, Bing; Wu, Xiaoyu; Zhou, Hao; Chen, Qianjie; Luo, Yongzhang

    2004-03-09

    Endostatin is a potent angiogenesis inhibitor. The structure of endostatin is unique in that its secondary structure is mainly irregular loops and beta-sheets and contains only a small fraction of alpha-helices with two pairs of disulfide bonds in a nested pattern. We choose human endostatin as a model system to study the folding mechanism of this kind. Nuclear magnetic resonance (NMR), tryptophan emission fluorescence, and circular dichroism (CD) were used to monitor the unfolding process of endostatin upon acid titration. Urea-induced unfolding was used to measure the stability of endostatin under different conditions. Our results show that endostatin is very acid-resistant; some native structure still remains even at pH 2 as evidenced by (1)H NMR. Trifluoroethanol (TFE) destabilizes native endostatin, while it makes endostatin even more acid-resistant in the low pH region. Stability measurement of endostatin suggests that endostatin is still in native structure at pH 3.5 despite the decreased stability. Acid-induced unfolding of endostatin is reversible, although it requires a long time to reach equilibrium below pH 3. Surprisingly, the alpha-helical content of endostatin is increased when it is unfolded at pH 1.6, and the alpha-helical content of the polypeptide chain of unfolded endostatin increases linearly with TFE concentration in the range of 0-30%. This observation indicates that the polypeptide chain of unfolded endostatin has an intrinsic alpha-helical propensity. Our discoveries may provide clues for refolding endostatin more efficiently. The acid-resistance property of endostatin may have biological significance in that it cannot be easily digested by proteases in an acidic environment such as in a lysosome in the cell.

  19. High dose of ascorbic acid induces cell death in mesothelioma cells.

    PubMed

    Takemura, Yukitoshi; Satoh, Motohiko; Satoh, Kiyotoshi; Hamada, Hironobu; Sekido, Yoshitaka; Kubota, Shunichiro

    2010-04-02

    Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. Recently, high dose of ascorbate in cancer treatment has been reexamined. We studied whether high dose of ascorbic acid induced cell death of four human mesothelioma cell lines. High dose of ascorbic acid induced cell death of all mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism that ascorbic acid induced reactive oxygen species and impaired mitochondrial membrane potential. In vivo experiment, intravenous administration of ascorbic acid significantly decreased the growth rate of mesothelioma tumor inoculated in mice. These data suggest that ascorbic acid may have benefits for patients with mesothelioma.

  20. The nucleolar protein GLTSCR2 is required for efficient viral replication

    PubMed Central

    Wang, Peng; Meng, Wen; Han, Shi-Chong; Li, Cui-Cui; Wang, Xiao-Jun; Wang, Xiao-Jia

    2016-01-01

    Glioma tumor suppressor candidate region gene 2 protein (GLTSCR2) is a nucleolar protein. In the investigation of the role of GLTSCR2 that played in the cellular innate immune response to viral infection, we found GLTSCR2 supported viral replication of rhabdovirus, paramyxovirus, and coronavirus in cells. Viral infection induced translocation of GLTSCR2 from nucleus to cytoplasm that enabled GLTSCR2 to attenuate type I interferon IFN-β and support viral replication. Cytoplasmic GLTSCR2 was able to interact with retinoic acid-inducible gene I (RIG-I) and the ubiquitin-specific protease 15 (USP15), and the triple interaction induced USP15 activity to remove K63-linked ubiquitination of RIG-I, leading to attenuation of RIG-I and IFN-β. Blocking cytoplasmic translocation of GLTSCR2, by deletion of its nuclear export sequence (NES), abrogated its ability to attenuate IFN-β and support viral replication. GLTSCR2-mediated attenuation of RIG-I and IFN-β led to alleviation of host cell innate immune response to viral infection. Our findings suggested that GLTSCR2 contributed to efficient viral replication, and GLTSCR2 should be considered as a potential target for therapeutic control of viral infection. PMID:27824081

  1. Tranexamic acid induces kaolin intake stimulating a pathway involving tachykinin neurokinin 1 receptors in rats.

    PubMed

    Kakiuchi, Hitoshi; Kawarai-Shimamura, Asako; Kuwagata, Makiko; Orito, Kensuke

    2014-01-15

    Tranexamic acid suppresses post-partum haemorrhage and idiopathic menorrhagia through its anti-fibrinolytic action. Although it is clinically useful, it is associated with high risks of side effects such as emesis. Understanding the mechanisms underlying tranexamic acid-induced emesis is very important to explore appropriate anti-emetic drugs for the prevention and/or suppression of emesis. In this study, we examined the receptors involved in tranexamic acid-induced kaolin intake in rats, which reflects the drug's clinical emetogenic potential in humans. Further, we examined the brain regions activated by administration of tranexamic acid and elucidated pivotal pathways of tranexamic acid-induced kaolin intake. We examined the effects of ondansetron, a 5-hydroxytryptamine 3 receptor antagonist, domperidone, a dopamine 2 receptor antagonist, and aprepitant, a tachykinin neurokinin 1 (NK1) receptor antagonist, on tranexamic acid-induced kaolin intake in rats. Then, we determined the brain regions that showed increased numbers of c-Fos immunoreactive cells. Finally, we examined the effects of an antagonist(s) that reduced tranexamic acid-induced kaolin intake on the increase in c-Fos immunoreactive cells. Aprepitant significantly decreased tranexamic acid-induced kaolin intake. However, neither ondansetron nor domperidone decreased kaolin intake. Tranexamic acid significantly increased c-Fos immunoreactive cells by approximately 5.5-fold and 22-fold in the area postrema and nucleus of solitary tract, respectively. Aprepitant decreased the number of c-Fos immunoreactive cells in both areas. Tranexamic acid induced kaolin intake possibly via stimulation of tachykinin NK1 receptors in rats. The tachykinin NK1 receptor could be targeted to prevent and/or suppress emesis in patients receiving tranexamic acid.

  2. MICROARRAY ANALYSIS OF DICHLOROACETIC ACID-INDUCED CHANGES IN GENE EXPRESSION

    EPA Science Inventory


    MICROARRAY ANALYSIS OF DICHLOROACETIC ACID-INDUCED CHANGES IN GENE EXPRESSION

    Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated the hepatocarcinogenicity of DCA in rodents when administered in dri...

  3. Homologous RIG-I–like helicase proteins direct RNAi-mediated antiviral immunity in C. elegans by distinct mechanisms

    PubMed Central

    Guo, Xunyang; Zhang, Rui; Wang, Jeffrey; Ding, Shou-Wei; Lu, Rui

    2013-01-01

    RNAi-mediated antiviral immunity in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), which encodes the helicase and C-terminal domains homologous to the mammalian retinoic acid inducible gene I (RIG-I)-like helicase (RLH) family of cytosolic immune receptors. Here we show that the antiviral function of DRH-1 requires the RIG-I homologous domains as well as its worm-specific N-terminal domain. We also demonstrate that the helicase and C-terminal domains encoded by either worm DRH-2 or human RIG-I can functionally replace the corresponding domains of DRH-1 to mediate antiviral RNAi in C. elegans. Notably, substitutions in a three-residue motif of the C-terminal regulatory domain of RIG-I that physically interacts with viral double-stranded RNA abolish the antiviral activity of C-terminal regulatory domains of both RIG-I and DRH-1 in C. elegans. Genetic analysis revealed an essential role for both DRH-1 and DRH-3 in C. elegans antiviral RNAi targeting a natural viral pathogen. However, Northern blot and small RNA deep sequencing analyses indicate that DRH-1 acts to enhance production of viral primary siRNAs, whereas DRH-3 regulates antiviral RNAi by participating in the biogenesis of secondary siRNAs after Dicer-dependent production of primary siRNAs. We propose that DRH-1 facilitates the acquisition of viral double-stranded RNA by the worm dicing complex for the subsequent processing into primary siRNAs. The strong parallel for the antiviral function of RLHs in worms and mammals suggests that detection of viral double-stranded RNA may activate completely unrelated effector mechanisms or, alternatively, that the mammalian RLHs have a conserved activity to stimulate production of viral siRNAs for antiviral immunity by an RNAi effector mechanism. PMID:24043766

  4. Gene I, a potential cell-to-cell movement locus of cauliflower mosaic virus, encodes an RNA-binding protein

    SciTech Connect

    Citovsky, V.; Knorr, D.; Zambryski, P. )

    1991-03-15

    Cauliflower mosaic virus (CaMV) is a double-stranded DNA (dsDNA) pararetrovirus capable of cell-to-cell movement presumably through intercellular connections, the plasmodesmata, of the infected plant. This movement is likely mediated by a specific viral protein encoded by the gene I locus. Here we report that the purified gene I protein binds RNA and single-stranded DNA (ssDNA) but not dsDNA regardless of nucleotide sequence specificity. The binding is highly cooperative, and the affinity of the gene I protein for RNA is 10-fold higher than for ssDNA. CaMV replicates by reverse transcription of a 35S RNA that is homologous to the entire genome. The authors propose that the 35S RNA may be involved in cell-to-cell movement of CaMV as an intermediate that is transported through plasmodesmata as an RNA-gene I protein complex.

  5. Association of ACE Gene I/D polymorphism with migraine in Kashmiri population

    PubMed Central

    Wani, Irfan Yousuf; Sheikh, Saleem; Shah, Zafar Amin; Pandith, Arshid A.; Wani, Mushtaq; Asimi, Ravouf; Wani, Maqbool; Sheikh, Shahnawaz; Mehraj, Iqra

    2016-01-01

    Introduction: Migraine is a complex, recurrent headache disorder that is one of the most common complaints in neurology practice. The role of various genes in its pathogenesis is being studied. We did this study to see whether an association exists between ACE gene I/D polymorphism and migraine in our region. Materials and Methods: The study included 100 patients diagnosed with migraine and 121 healthy controls. The study subject were age and gender matched. The analysis was based on Polymerase Chain Reaction (PCR) and included following steps: DNA extraction from blood, PCR and Restriction Fragment Length Polymorphism (RFLP). Results: Out of 100 cases, 69 were females and 31 were males. Fifty-seven were having migraine without aura and 43 had migraine with aura. 45 of the cases had II polymorphism, 40 had ID polymorphism and 15 had DD polymorphism in ACE gene. Conclusion: We were not able to find a statistically significant association between ACE gene I/D polymorphism with migraine. The reason for difference in results between our study and other studies could be because of different ethnicity in study populations. So a continuous research is needed in this regard in order to find the genes and different polymorphism that increase the susceptibility of Kashmiri population to migraine. PMID:27011636

  6. Protective Effect of Ocimum basilicum Essential Oil Against Acetic Acid-Induced Colitis in Rats.

    PubMed

    Rashidian, Amir; Roohi, Parnia; Mehrzadi, Saeed; Ghannadi, Ali Reza; Minaiyan, Mohsen

    2016-10-01

    Ocimum basilicum L has been traditionally used for the treatment of inflammatory bowel disease in Iran. This study investigates the ameliorative effect of Ocimum basilicum essential oil on an acetic acid-induced colitis model in rats. Ocimum basilicum essential oil with 2 doses (200 and 400 μL/kg) significantly ameliorated wet weight/length ratio of colonic tissue compared to the control group. Higher doses of essential oil (200 and 400 μL/kg) significantly reduced ulcer severity, ulcer area, and ulcer index. On the other hand, histological examination revealed the diminution of total colitis index as a marker for inflammatory cell infiltration in the colonic segments of rats treated with Ocimum basilicum essential oil (200 and 400 μL/kg). The increased level of myeloperoxidase was significantly decreased after the treatment with the essential oil (200 and 400 μL/kg). These results suggest that Ocimum basilicum exhibits protective effect against acetic acid-induced colitis.

  7. SCH 58261 differentially influences quinolinic acid-induced effects in striatal and in hippocampal slices.

    PubMed

    Tebano, Maria Teresa; Domenici, Maria Rosaria; Popoli, Patrizia

    2002-08-30

    The influence of the adenosine A(2A) receptor antagonist SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-trizolo[1,5-c] pyrimidine) (50, 200 nM, 1 microM) on quinolinic acid effects has been studied in rat striatal and hippocampal slices. Quinolinic acid induced disappearance of field potentials at concentrations of 500 microM and 2 mM in hippocampal and corticostriatal slices, respectively. We found that 1 microM SCH 58261 prevented quinolinic acid-induced field potential disappearance in corticostriatal but not in hippocampal slices. This finding demonstrates that the peculiar binding profile of SCH 58261 and the predominance in the hippocampus of "atypical" adenosine A(2A) receptor population (not recognized by SCH 58261) could have a functional relevance in the occurrence of region-specific neuroprotective effects.

  8. Effects of sodium bicarbonate on butyric acid-induced epithelial cell damage in vitro.

    PubMed

    Takigawa, Satoko; Sugano, Naoyuki; Ochiai, Kuniyasu; Arai, Noriyuki; Ota, Noriko; Ito, Koichi

    2008-12-01

    Butyric acid is detected in periodontal pockets and is thought to be involved in the initiation and progression of periodontal disease. We examined the effects of sodium bicarbonate on the butyric acid-induced epithelial cell damage. The human gingival carcinoma cell line Ca9-22 was cultured in medium that contained butyric acid with or without sodium bicarbonate. The viability of cells treated with sodium bicarbonate was significantly higher than that of cells treated with butyric acid alone. The effects of butyric acid on ICAM-1 expression were significantly improved by sodium bicarbonate. Within the limitations of this in vitro study, sodium bicarbonate was indicated to be a useful therapeutic agent to reduce the butyric acid-induced periodontal tissue damage.

  9. Acetic acid induces a programmed cell death process in the food spoilage yeast Zygosaccharomyces bailii.

    PubMed

    Ludovico, Paula; Sansonetty, Filipe; Silva, Manuel T; Côrte-Real, Manuela

    2003-03-01

    Here we show that 320-800 mM acetic acid induces in Zygosaccharomyces bailii a programmed cell death (PCD) process that is inhibited by cycloheximide, is accompanied by structural and biochemical alterations typical of apoptosis, and occurs in cells with preserved mitochondrial and plasma membrane integrity (as revealed by rhodamine 123 (Rh123) and propidium iodide (PI) staining, respectively). Mitochondrial ultrastructural changes, namely decrease of the cristae number, formation of myelinic bodies and swelling were also seen. Exposure to acetic acid above 800 mM resulted in killing by necrosis. The occurrence of an acetic acid-induced active cell death process in Z. bailii reinforces the concept of a physiological role of the PCD in the normal yeast life cycle.

  10. Acid-inducible proton influx currents in the plasma membrane of murine osteoclast-like cells.

    PubMed

    Kuno, Miyuki; Li, Guangshuai; Moriura, Yoshie; Hino, Yoshiko; Kawawaki, Junko; Sakai, Hiromu

    2016-05-01

    Acidification of the resorption pits, which is essential for dissolving bone, is produced by secretion of protons through vacuolar H(+)-ATPases in the plasma membrane of bone-resorbing cells, osteoclasts. Consequently, osteoclasts face highly acidic extracellular environments, where the pH gradient across the plasma membrane could generate a force driving protons into the cells. Proton influx mechanisms during the acid exposure are largely unknown, however. In this study, we investigated extracellular-acid-inducible proton influx currents in osteoclast-like cells derived from a macrophage cell line (RAW264). Decreasing extracellular pH to <5.5 induced non-ohmic inward currents. The reversal potentials depended on the pH gradients across the membrane and were independent of concentrations of Na(+), Cl(-), and HCO3 (-), suggesting that they were carried largely by protons. The acid-inducible proton influx currents were not inhibited by amiloride, a widely used blocker for cation channels/transporters, or by 4,4'-diisothiocyanato-2,2'-stilbenesulfonate(DIDS) which blocks anion channels/transporters. Additionally, the currents were not significantly affected by V-ATPase inhibitors, bafilomycin A1 and N,N'-dicyclohexylcarbodiimide. Extracellular Ca(2+) (10 mM) did not affect the currents, but 1 mM ZnCl2 decreased the currents partially. The intracellular pH in the vicinity of the plasma membrane was dropped by the acid-inducible H(+) influx currents, which caused overshoot of the voltage-gated H(+) channels after removal of acids. The H(+) influx currents were smaller in undifferentiated, mononuclear RAW cells and were negligible in COS7 cells. These data suggest that the acid-inducible H(+) influx (H(+) leak) pathway may be an additional mechanism modifying the pH environments of osteoclasts upon exposure to strong acids.

  11. Autophagy Protects against Palmitic Acid-Induced Apoptosis in Podocytes in vitro.

    PubMed

    Jiang, Xu-Shun; Chen, Xue-Mei; Wan, Jiang-Min; Gui, Hai-Bo; Ruan, Xiong-Zhong; Du, Xiao-Gang

    2017-02-22

    Autophagy is a highly conserved degradation process that is involved in the clearance of proteins and damaged organelles to maintain intracellular homeostasis and cell integrity. Type 2 diabetes is often accompanied by dyslipidemia with elevated levels of free fatty acids (FFAs). Podocytes, as an important component of the filtration barrier, are susceptible to lipid disorders. The loss of podocytes causes proteinuria, which is involved in the pathogenesis of diabetic nephropathy. In the present study, we demonstrated that palmitic acid (PA) promoted autophagy in podocytes. We further found that PA increased the production of reactive oxygen species (ROS) in podocytes and that NAC (N-acetyl-cysteine), a potent antioxidant, significantly eliminated the excessive ROS and suppressed autophagy, indicating that the increased generation of ROS was associated with the palmitic acid-induced autophagy in podocytes. Moreover, we also found that PA stimulation decreased the mitochondrial membrane potential in podocytes and induced podocyte apoptosis, while the inhibition of autophagy by chloroquine (CQ) enhanced palmitic acid-induced apoptosis accompanied by increased ROS generation, and the stimulation of autophagy by rapamycin (Rap) remarkably suppressed palmitic acid-induced ROS generation and apoptosis. Taken together, these in vitro findings suggest that PA-induced autophagy in podocytes is mediated by ROS production and that autophagy plays a protective role against PA-induced podocyte apoptosis.

  12. Protective effect of hispidulin on kainic acid-induced seizures and neurotoxicity in rats.

    PubMed

    Lin, Tzu Yu; Lu, Cheng Wei; Wang, Su Jane; Huang, Shu Kuei

    2015-05-15

    Hispidulin is a flavonoid compound which is an active ingredient in a number of traditional Chinese medicinal herbs, and it has been reported to inhibit glutamate release. The purpose of this study was to investigate whether hispidulin protects against seizures induced by kainic acid, a glutamate analog with excitotoxic properties. The results indicated that intraperitoneally administering hispidulin (10 or 50mg/kg) to rats 30 min before intraperitoneally injecting kainic acid (15 mg/kg) increased seizure latency and decreased seizure score. In addition, hispidulin substantially attenuated kainic acid-induced hippocampal neuronal cell death, and this protective effect was accompanied by the suppression of microglial activation and the production of proinflammatory cytokines such as interleukin-1β, interleukin-6, and tumor necrosis factor-α in the hippocampus. Moreover, hispidulin reduced kainic acid-induced c-Fos expression and the activation of mitogen-activated protein kinases in the hippocampus. These data suggest that hispidulin has considerable antiepileptic, neuroprotective, and antiinflammatory effects on kainic acid-induced seizures in rats.

  13. Autophagy Protects against Palmitic Acid-Induced Apoptosis in Podocytes in vitro

    PubMed Central

    Jiang, Xu-shun; Chen, Xue-mei; Wan, Jiang-min; Gui, Hai-bo; Ruan, Xiong-zhong; Du, Xiao-gang

    2017-01-01

    Autophagy is a highly conserved degradation process that is involved in the clearance of proteins and damaged organelles to maintain intracellular homeostasis and cell integrity. Type 2 diabetes is often accompanied by dyslipidemia with elevated levels of free fatty acids (FFAs). Podocytes, as an important component of the filtration barrier, are susceptible to lipid disorders. The loss of podocytes causes proteinuria, which is involved in the pathogenesis of diabetic nephropathy. In the present study, we demonstrated that palmitic acid (PA) promoted autophagy in podocytes. We further found that PA increased the production of reactive oxygen species (ROS) in podocytes and that NAC (N-acetyl-cysteine), a potent antioxidant, significantly eliminated the excessive ROS and suppressed autophagy, indicating that the increased generation of ROS was associated with the palmitic acid-induced autophagy in podocytes. Moreover, we also found that PA stimulation decreased the mitochondrial membrane potential in podocytes and induced podocyte apoptosis, while the inhibition of autophagy by chloroquine (CQ) enhanced palmitic acid-induced apoptosis accompanied by increased ROS generation, and the stimulation of autophagy by rapamycin (Rap) remarkably suppressed palmitic acid-induced ROS generation and apoptosis. Taken together, these in vitro findings suggest that PA-induced autophagy in podocytes is mediated by ROS production and that autophagy plays a protective role against PA-induced podocyte apoptosis. PMID:28225005

  14. Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats.

    PubMed

    Kumar, Hariom; Sharma, Bhupesh

    2016-01-01

    Autism is a neurodevelopment disorder. One percent worldwide population suffers with autism and males suffer more than females. Microglia plays an important role in neurodevelopment, neuropsychiatric and neurodegenerative disorders. The present study has been designed to investigate the role of minocycline in prenatal valproic acid induced autism in rats. Animals with prenatal valproic acid have reduced social interaction (three chamber social behaviour apparatus), spontaneous alteration (Y-Maze), exploratory activity (Hole board test), intestinal motility, serotonin levels (both in prefrontal cortex and ileum) and prefrontal cortex mitochondrial complex activity (complexes I, II, IV). Furthermore, prenatal valproic acid treated animals have shown an increase in locomotion (actophotometer), anxiety (elevated plus maze), brain oxidative stress (thiobarbituric acid reactive species, glutathione, catalase), nitrosative stress (nitrite/nitrate), inflammation (both in brain and ileum myeloperoxidase activity), calcium and blood brain barrier permeability. Treatment with minocycline significantly attenuated prenatal valproic acid induced reduction in social interaction, spontaneous alteration, exploratory activity intestinal motility, serotonin levels and prefrontal cortex mitochondrial complex activity. Furthermore, minocycline has also attenuated prenatal valproic acid induced increase in locomotion, anxiety, brain oxidative and nitrosative stress, inflammation, calcium and blood brain barrier permeability. Thus, it may be concluded that prenatal valproic acid has induced autistic behaviour, biochemistry and blood brain barrier impairment in animals, which were significantly attenuated by minocycline. Minocycline should be explored further for its therapeutic benefits in autism.

  15. A role for sodium and chloride in kainic acid-induced beading of inhibitory interneuron dendrites.

    PubMed

    Al-Noori, S; Swann, J W

    2000-01-01

    Excitotoxic injury of the dendrites of inhibitory interneurons could lead to decreases in their synaptic activation and explain subsequent local circuit hyperexcitability and epilepsy. A hallmark of dendrotoxicity, at least in principal neurons of the hippocampus and cortex, is focal or varicose swellings of dendritic arbors. In experiments reported here, transient (1h) exposure of hippocampal explant cultures to kainic acid produced marked focal swellings of the dendrites of parvalbumin-immunoreactive pyramidal basket cells in a highly reproducible and dose-dependent manner. At 5mM kainic acid, more than half of the immunopositive apical dendrites in area CA(1) had a beaded appearance. However, the somal volumes of these cells were unaltered by the same treatment. The presence of focal swellings was reversible with kainate washout and was not accompanied by interneuronal cell death. In contrast, exposure to much higher concentrations (300mM) of kainic acid resulted in the total loss of parvalbumin-positive interneurons from explants. Surprisingly, kainic acid-induced dendritic beading does not appear to be mediated by extracellular calcium. Beading was unaltered in the presence of N-methyl-D-aspartate receptor antagonists, the L-type calcium channel antagonist, nimodipine, cadmium, or by removing extracellular calcium. However, blockade of voltage-gated sodium channels by either tetrodotoxin or lidocaine abolished dendritic beading, while the activation of existing voltage-gated sodium channels by veratridine mimicked the kainic acid-induced dendritic beading. Finally, the removal of extracellular chloride prevented the kainic acid-induced dendritic beading.Thus, we suggest that the movement of Na(+) and Cl(-), rather than Ca(2+), into cells underlies the focal swellings of interneuron dendrites in hippocampus.

  16. Calcium Uptake via Mitochondrial Uniporter Contributes to Palmitic Acid-induced Apoptosis in Mouse Podocytes.

    PubMed

    Yuan, Zeting; Cao, Aili; Liu, Hua; Guo, Henjiang; Zang, Yingjun; Wang, Yi; Wang, Yunman; Wang, Hao; Yin, Peihao; Peng, Wen

    2017-02-09

    Podocytes are component cells of the glomerular filtration barrier, and their loss by apoptosis is the main cause of proteinuria that leads to diabetic nephropathy (DN). Therefore, insights into podocyte apoptosis mechanism would allow a better understanding of DN pathogenesis and thus help develop adequate therapeutic strategies. Here, we investigated the molecular mechanism of palmitic acid-inhibited cell death in mouse podocytes, and found that palmitic acid increased cell death in a dose- and time-dependent manner. Palmitic acid induces apoptosis in podocytes through up-regulation of cytosolic and mitochondrial Ca(2+) , mitochondrial membrane potential (MMP), cytochrome c release and depletion of endoplasmic reticulum (ER) Ca(2+) , The intracellular calcium chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N, N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), partially prevented this up-regulation whereas 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-triphosphate receptor (IP3R) inhibitor; dantrolene, a ryanodine receptor (RyR) inhibitor; and 4,4'-diisothiocyanatostibene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, had no effect. Interestingly, ruthenium red and Ru360, both inhibitors of the mitochondrial Ca(2+) uniporter (MCU), blocked palmitic acid-induced mitochondrial Ca(2+) elevation, cytochrome c release from mitochondria to cytosol, and apoptosis. siRNA to MCU markedly reduced curcumin-induced apoptosis. These data indicate that Ca(2+) uptake via mitochondrial uniporter contributes to palmitic acid-induced apoptosis in mouse podocytes. This article is protected by copyright. All rights reserved.

  17. Clavulanic acid induces penile erection and yawning in male rats: comparison with apomorphine.

    PubMed

    Sanna, Fabrizio; Melis, Maria Rosaria; Angioni, Laura; Argiolas, Antonio

    2013-02-01

    The beta-lactamase inhibitor clavulanic acid induced penile erection and yawning in a dose dependent manner when given intraperitoneally (IP, 0.05-5mg/kg), perorally (OS, 0.1-5mg/kg) and intracereboventricularly (ICV, 0.01-5 μg/rat) to male rats. The effect resembles that of the dopamine receptor agonist apomorphine given subcutaneously (SC) (0.02-0.25mg/kg), although the responses of the latter followed a U inverted dose-response curve, disappearing at doses higher than 0.1mg/kg. Clavulanic acid responses were reduced by about 55% by haloperidol, a dopamine D2 receptor antagonist (0.1mg/kg IP), and by d(CH(2))(5)Tyr(Me)(2)-Orn(8)-vasotocin, an oxytocin receptor antagonist (2 μg/rat ICV), both given 15 min before clavulanic acid. A higher reduction of clavulanic acid responses (more than 80%) was also found with morphine, an opioid receptor agonist (5mg/kg IP), and with mianserin, a serotonin 5HT(2c) receptor antagonist (0.2mg/kg SC). In contrast, no reduction was found with naloxone, an opioid receptor antagonist (1mg/kg IP). The ability of haloperidol, d(CH(2))(5)Tyr(Me)(2)-Orn(8)-vasotocin and morphine to reduce clavulanic acid induced penile erection and yawning suggests that clavulanic acid induces these responses, at least in part, by increasing central dopaminergic neurotransmission. Dopamine in turn activates oxytocinergic neurotransmission and centrally released oxytocin induces penile erection and yawning. However, since both penile erection and yawning episodes were reduced not only by the blockade of central dopamine and oxytocin receptors and by the stimulation of opioid receptors, which inhibits oxytocinergic neurotransmission, but also by mianserin, an increase of central serotonin neurotransmission is also likely to participate in these clavulanic acid responses.

  18. Lipopolysaccharide Stimulates Butyric Acid-Induced Apoptosis in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Kurita-Ochiai, Tomoko; Fukushima, Kazuo; Ochiai, Kuniyasu

    1999-01-01

    We previously reported that butyric acid, an extracellular metabolite from periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we examined the ability of butyric acid to induce apoptosis in peripheral blood mononuclear cells (PBMC) and the effect of bacterial lipopolysaccharide (LPS) on this apoptosis. Butyric acid significantly inhibited the anti-CD3 monoclonal antibody- and concanavalin A-induced proliferative responses in a dose-dependent fashion. This inhibition of PBMC growth by butyric acid depended on apoptosis in vitro. It was characterized by internucleosomal DNA digestion and revealed by gel electrophoresis followed by a colorimetric DNA fragmentation assay to occur in a concentration-dependent fashion. Butyric acid-induced PBMC apoptosis was accompanied by caspase-3 protease activity but not by caspase-1 protease activity. LPS potentiated butyric acid-induced PBMC apoptosis in a dose-dependent manner. Flow-cytometric analysis revealed that LPS increased the proportion of sub-G1 cells and the number of late-stage apoptotic cells induced by butyric acid. Annexin V binding experiments with fractionated subpopulations of PBMC in flow cytometory revealed that LPS accelerated the butyric acid-induced CD3+-T-cell apoptosis followed by similar levels of both CD4+- and CD8+-T-cell apoptosis. The addition of LPS to PBMC cultures did not cause DNA fragmentation, suggesting that LPS was unable to induce PBMC apoptosis directly. These data suggest that LPS, in combination with butyric acid, potentiates CD3+ PBMC T-cell apoptosis and plays a role in the apoptotic depletion of CD4+ and CD8+ cells. PMID:9864191

  19. The acid-induced folded state of Sac7d is the native state.

    PubMed Central

    Bedell, J. L.; McCrary, B. S.; Edmondson, S. P.; Shriver, J. W.

    2000-01-01

    Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt (McCrary BS, Bedell J. Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203-224). Both near-ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid- and salt-induced folded states have a native fold and are not molten globular. 1H,15N heteronuclear single quantum coherence NMR spectra confirm that the native, acid-, and salt-induced folded states are essentially identical. The most significant differences in amide 1H and 15N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through-bond inductive effects. The 1H NMR chemical shifts of protons affected by ring currents in the hydrophobic core of the acid- and salt-induced folded states are identical to those observed in the native. The radius of gyration of the acid-induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X-ray scattering. We conclude that acid-induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the native state. The folding of Sac7d as a function of pH and anion concentration is summarized with a phase diagram that is similar to those observed for other proteins that undergo acid-induced folding except that the A-state is encompassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid. PMID:11106160

  20. TRIM25 Identification in the Chinese Goose: Gene Structure, Tissue Expression Profiles, and Antiviral Immune Responses In Vivo and In Vitro

    PubMed Central

    Zhou, Hao; Wang, Anqi; Sun, Lipei; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Yang, Qiao; Wu, Ying; Sun, Kunfeng; Chen, Xiaoyue

    2016-01-01

    The retinoic acid-inducible gene I (RIG-I) and the RIG-I-like receptor (RLR) protein play a critical role in the interferon (IFN) response during RNA virus infection. The tripartite motif containing 25 proteins (TRIM25) was reported to modify caspase activation and RIG-I recruitment domains (CARDs) via ubiquitin. These modifications allow TRIM25 to interact with mitochondrial antiviral signaling molecules (MAVs) and form CARD-CARD tetramers. Goose TRIM25 was cloned from gosling lungs, which possess a 1662 bp open reading flame (ORF). This ORF encodes a predicted 554 amino acid protein consisting of a B-box domain, a coiled-coil domain, and a PRY/SPRY domain. The protein sequence has 89.25% sequence identity with Anas platyrhynchos TRIM25, 78.57% with Gallus gallus TRIM25, and 46.92% with Homo sapiens TRIM25. TRIM25 is expressed in all gosling and adult goose tissues examined. QRT-PCR revealed that goose TRIM25 transcription could be induced by goose IFN-α, goose IFN-γ, and goose IFN-λ, as well as a35 s polyinosinic-polycytidylic acid (poly(I:C)), oligodeoxynucleotides 2006 (ODN 2006), and resiquimod (R848) in vitro; however, it is inhibited in H9N2 infected goslings for unknown reasons. These data suggest that goose TRIM25 might play a positive role in the regulation of the antiviral immune response. PMID:27995135

  1. TRIM25 Identification in the Chinese Goose: Gene Structure, Tissue Expression Profiles, and Antiviral Immune Responses In Vivo and In Vitro.

    PubMed

    Wei, Yunan; Zhou, Hao; Wang, Anqi; Sun, Lipei; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Yang, Qiao; Wu, Ying; Sun, Kunfeng; Chen, Xiaoyue; Cheng, Anchun; Chen, Shun

    2016-01-01

    The retinoic acid-inducible gene I (RIG-I) and the RIG-I-like receptor (RLR) protein play a critical role in the interferon (IFN) response during RNA virus infection. The tripartite motif containing 25 proteins (TRIM25) was reported to modify caspase activation and RIG-I recruitment domains (CARDs) via ubiquitin. These modifications allow TRIM25 to interact with mitochondrial antiviral signaling molecules (MAVs) and form CARD-CARD tetramers. Goose TRIM25 was cloned from gosling lungs, which possess a 1662 bp open reading flame (ORF). This ORF encodes a predicted 554 amino acid protein consisting of a B-box domain, a coiled-coil domain, and a PRY/SPRY domain. The protein sequence has 89.25% sequence identity with Anas platyrhynchos TRIM25, 78.57% with Gallus gallus TRIM25, and 46.92% with Homo sapiens TRIM25. TRIM25 is expressed in all gosling and adult goose tissues examined. QRT-PCR revealed that goose TRIM25 transcription could be induced by goose IFN-α, goose IFN-γ, and goose IFN-λ, as well as a35 s polyinosinic-polycytidylic acid (poly(I:C)), oligodeoxynucleotides 2006 (ODN 2006), and resiquimod (R848) in vitro; however, it is inhibited in H9N2 infected goslings for unknown reasons. These data suggest that goose TRIM25 might play a positive role in the regulation of the antiviral immune response.

  2. Evolutionary conservation of molecular structure and antiviral function of a viral receptor, LGP2, in amphioxus Branchiostoma japonicum.

    PubMed

    Liu, Shousheng; Liu, Yuanyuan; Yang, Shuangshuang; Huang, Youhua; Qin, Qiwei; Zhang, Shicui

    2015-12-01

    RIG-I-like (where RIG-I is retinoic acid inducible gene I) receptor LGP2 (where LGP2 is laboratory of genetics and physiology) is an important intracellular receptor that recognizes viral RNAs in innate immunity, but its origin and evolution remains unknown. Here we clearly demonstrate the presence of a RIG-I-like receptor, BjLGP2, in the basal chordate amphioxus. It is predominantly expressed in the hepatic caecum and hindgut, and is upregulated following challenge with poly(I:C). BjLGP2 is distributed in the cytoplasm of both grouper spleen and flounder gill (FG) cells, and the recombinant BjLGP2 interacts with poly(I:C). BjLGP2 can enhance the expression of IFN and IFN-inducible genes in FG cells upon poly(I:C) challenge. It also significantly induces the expression of the antiviral genes ifn-i and Mx as well as the signal transduction relevant genes MAVS, NF-κB, and IRF-3 in FG cells upon lymphocystis disease virus challenge. Moreover, BjLGP2 inhibits the replication of lymphocystis disease virus in FG cells and the gene transcription of Singapore grouper iridovirus in grouper spleen cells. This is the first report showing that a LGP2 protein in invertebrate species (amphioxus) is structurally conserved and plays an antiviral role similar to that of vertebrate LGP2 proteins.

  3. [MAVS protein and its interactions with hepatitis A, B and C viruses].

    PubMed

    Wyżewski, Zbigniew; Gregorczyk, Karolina P; Struzik, Justyna; Niemiałtowski, Marek; Szulc-Dąbrowska, Lidia

    2016-01-26

    Mitochondrial antiviral signaling protein (MAVS) transmits activation signal of type I interferon (IFN) gene transcription in the molecular intracellular pathway, which depends on the protein encoded by retinoic acid inducible gene I (RIG-I) or melanoma differentiation-associated protein-5 (MDA-5). MAVS, as a signal molecule, performs an essential function in the development of an antiviral immune response. The molecule of MAVS consists of two domains: the N-terminal domain and the C-terminal domain. The N-terminal end of MAVS contains the caspase activation and recruitment domain (CARD). CARD is responsible for MAVS interaction with RIG-I and MDA-5, which act as cytosolic sensors detecting foreign viral genetic material in the host cell. After binding to viral RNA, RIG-I or MDA-5 activates MAVS and transmits the signal of IFN type I gene expression. The C-terminal transmembrane domain (TM) of MAVS anchors the protein to the outer mitochondrial membrane. In this paper interactions between MAVS and hepatitis virus type A (HAV), type B (HBV) and type C (HCV) are presented. Mechanisms of indirect activation of MAVS by viral DNA and RNA, as well as the strategies of HAV, HBV and HCV for blocking of the intracellular signaling pathway at the level of MAVS, are described.

  4. Role of hepatocyte S6K1 in palmitic acid-induced endoplasmic reticulum stress, lipotoxicity, insulin resistance and in oleic acid-induced protection.

    PubMed

    Pardo, Virginia; González-Rodríguez, Águeda; Muntané, Jordi; Kozma, Sara C; Valverde, Ángela M

    2015-06-01

    The excess of saturated free fatty acids, such as palmitic acid, that induces lipotoxicity in hepatocytes, has been implicated in the development of non-alcoholic fatty liver disease also associated with insulin resistance. By contrast, oleic acid, a monounsaturated fatty acid, attenuates the effects of palmitic acid. We evaluated whether palmitic acid is directly associated with both insulin resistance and lipoapoptosis in mouse and human hepatocytes and the impact of oleic acid in the molecular mechanisms that mediate both processes. In human and mouse hepatocytes palmitic acid at a lipotoxic concentration triggered early activation of endoplasmic reticulum (ER) stress-related kinases, induced the apoptotic transcription factor CHOP, activated caspase 3 and increased the percentage of apoptotic cells. These effects concurred with decreased IR/IRS1/Akt insulin pathway. Oleic acid suppressed the toxic effects of palmitic acid on ER stress activation, lipoapoptosis and insulin resistance. Besides, oleic acid suppressed palmitic acid-induced activation of S6K1. This protection was mimicked by pharmacological or genetic inhibition of S6K1 in hepatocytes. In conclusion, this is the first study highlighting the activation of S6K1 by palmitic acid as a common and novel mechanism by which its inhibition by oleic acid prevents ER stress, lipoapoptosis and insulin resistance in hepatocytes.

  5. Comparative neuroprotective profile of statins in quinolinic acid induced neurotoxicity in rats.

    PubMed

    Kalonia, Harikesh; Kumar, Puneet; Kumar, Anil

    2011-01-01

    A possible neuroprotective role has been recently suggested for 3H3MGCoA reductase inhibitors (statins). Here, we sought to determine neuroprotective effect of statins in quinolinic acid induced neurotoxicity in rats. Rats were surgically administered quinolinic acid and treated with Atorvastatin (10, 20 mg/kg), simvastatin (15, 30 mg/kg) and fluvastatin (5, 10 mg/kg) once daily up to 3 weeks. Atorvastatin (10, 20 mg/kg), simvastatin (30 mg/kg) and fluvastatin (10 mg/kg) treatment significantly attenuated the quinolinic acid induced behavioral (locomotor activity, rotarod performance and beam walk test), biochemical (lipid peroxidation, nitrite concentration, SOD and catalase), mitochondrial enzyme complex alterations in rats suggesting their free radical scavenging potential. Additionally, atorvastatin (10, 20 mg/kg), simvastatin (30 mg/kg) and fluvastatin (10 mg/kg) significantly decrease the TNF-α level and striatal lesion volume in quinolinic acid treated animals indicating their anti-inflammatory effects. In comparing the protective effect of different statins, atorvastatin is effective at both the doses while simvastatin and fluvastatins at respective lower doses were not able to produce the protective effect in quinolinic acid treated animals. These modulations can account, at least partly, for the beneficial effect of statins in our rodent model of striatal degeneration. Our findings show that statins could be explored as possible neuroprotective agents for neurodegenerative disorders such as HD.

  6. Obestatin Accelerates the Healing of Acetic Acid-Induced Colitis in Rats

    PubMed Central

    Matuszyk, Aleksandra; Ceranowicz, Piotr; Warzecha, Zygmunt; Cieszkowski, Jakub; Bonior, Joanna; Jaworek, Jolanta; Kuśnierz-Cabala, Beata; Konturek, Peter; Ambroży, Tadeusz; Dembiński, Artur

    2016-01-01

    Obestatin, a 23-amino acid peptide derived from the proghrelin, has been shown to exhibit some protective and therapeutic effects in the gut. The aim of present study was to determine the effect of obestatin administration on the course of acetic acid-induced colitis in rats. Materials and Methods. Studies have been performed on male Wistar rats. Colitis was induced by a rectal enema with 3.5% acetic acid solution. Obestatin was administered intraperitoneally twice a day at a dose of 8 nmol/kg, starting 24 h after the induction of colitis. Seven or 14 days after the induction of colitis, the healing rate of the colon was evaluated. Results. Treatment with obestatin after induction of colitis accelerated the healing of colonic wall damage and this effect was associated with a decrease in the colitis-evoked increase in mucosal activity of myeloperoxidase and content of interleukin-1β. Moreover, obestatin administration significantly reversed the colitis-evoked decrease in mucosal blood flow and DNA synthesis. Conclusion. Administration of exogenous obestatin exhibits therapeutic effects in the course of acetic acid-induced colitis and this effect is related, at least in part, to the obestatin-evoked anti-inflammatory effect, an improvement of local blood flow, and an increase in cell proliferation in colonic mucosa. PMID:26798415

  7. Obestatin Accelerates the Healing of Acetic Acid-Induced Colitis in Rats.

    PubMed

    Matuszyk, Aleksandra; Ceranowicz, Piotr; Warzecha, Zygmunt; Cieszkowski, Jakub; Bonior, Joanna; Jaworek, Jolanta; Kuśnierz-Cabala, Beata; Konturek, Peter; Ambroży, Tadeusz; Dembiński, Artur

    2016-01-01

    Obestatin, a 23-amino acid peptide derived from the proghrelin, has been shown to exhibit some protective and therapeutic effects in the gut. The aim of present study was to determine the effect of obestatin administration on the course of acetic acid-induced colitis in rats. Materials and Methods. Studies have been performed on male Wistar rats. Colitis was induced by a rectal enema with 3.5% acetic acid solution. Obestatin was administered intraperitoneally twice a day at a dose of 8 nmol/kg, starting 24 h after the induction of colitis. Seven or 14 days after the induction of colitis, the healing rate of the colon was evaluated. Results. Treatment with obestatin after induction of colitis accelerated the healing of colonic wall damage and this effect was associated with a decrease in the colitis-evoked increase in mucosal activity of myeloperoxidase and content of interleukin-1β. Moreover, obestatin administration significantly reversed the colitis-evoked decrease in mucosal blood flow and DNA synthesis. Conclusion. Administration of exogenous obestatin exhibits therapeutic effects in the course of acetic acid-induced colitis and this effect is related, at least in part, to the obestatin-evoked anti-inflammatory effect, an improvement of local blood flow, and an increase in cell proliferation in colonic mucosa.

  8. Exogenous Ghrelin Accelerates the Healing of Acetic Acid-Induced Colitis in Rats.

    PubMed

    Matuszyk, Aleksandra; Ceranowicz, Piotr; Warzecha, Zygmunt; Cieszkowski, Jakub; Ceranowicz, Dagmara; Gałązka, Krystyna; Bonior, Joanna; Jaworek, Jolanta; Bartuś, Krzysztof; Gil, Krzysztof; Olszanecki, Rafał; Dembiński, Artur

    2016-09-01

    Previous studies have shown that ghrelin reduces colonic inflammation induced by trinitrobenzene sulfonic acid and dextran sodium sulfate. In the present study we determined the effect of treatment with ghrelin on the course of acetic acid-induced colitis in rats. Rectal administration of 3% acetic acid solution led to induction of colitis in all animals. Damage of the colonic wall was accompanied by an increase in mucosal concentration of pro-inflammatory interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), as well mucosal activity of myeloperoxidase. Moreover, induction of colitis led to a reduction in colonic blood flow and DNA synthesis. Administration of ghrelin after induction of colitis led to faster regeneration of the colonic wall and reduction in colonic levels of IL-1β, TNF-α, and myeloperoxidase. In addition, treatment with ghrelin improved mucosal DNA synthesis and blood flow. Our study disclosed that ghrelin exhibits a strong anti-inflammatory and healing effect in acetic acid-induced colitis. Our current observation in association with previous findings that ghrelin exhibits curative effect in trinitrobenzene sulfonic acid- and dextran sodium sulfate-induced colitis suggest that therapeutic effect of ghrelin in the colon is universal and independent of the primary cause of colitis.

  9. Salicylic acid induces mitochondrial injury by inhibiting ferrochelatase heme biosynthesis activity.

    PubMed

    Gupta, Vipul; Liu, Shujie; Ando, Hideki; Ishii, Ryohei; Tateno, Shumpei; Kaneko, Yuki; Yugami, Masato; Sakamoto, Satoshi; Yamaguchi, Yuki; Nureki, Osamu; Handa, Hiroshi

    2013-12-01

    Salicylic acid is a classic nonsteroidal anti-inflammatory drug. Although salicylic acid also induces mitochondrial injury, the mechanism of its antimitochondrial activity is not well understood. In this study, by using a one-step affinity purification scheme with salicylic acid-immobilized beads, ferrochelatase (FECH), a homodimeric enzyme involved in heme biosynthesis in mitochondria, was identified as a new molecular target of salicylic acid. Moreover, the cocrystal structure of the FECH-salicylic acid complex was determined. Structural and biochemical studies showed that salicylic acid binds to the dimer interface of FECH in two possible orientations and inhibits its enzymatic activity. Mutational analysis confirmed that Trp301 and Leu311, hydrophobic amino acid residues located at the dimer interface, are directly involved in salicylic acid binding. On a gel filtration column, salicylic acid caused a shift in the elution profile of FECH, indicating that its conformational change is induced by salicylic acid binding. In cultured human cells, salicylic acid treatment or FECH knockdown inhibited heme synthesis, whereas salicylic acid did not exert its inhibitory effect in FECH knockdown cells. Concordantly, salicylic acid treatment or FECH knockdown inhibited heme synthesis in zebrafish embryos. Strikingly, the salicylic acid-induced effect in zebrafish was partially rescued by FECH overexpression. Taken together, these findings illustrate that FECH is responsible for salicylic acid-induced inhibition of heme synthesis, which may contribute to its antimitochondrial and anti-inflammatory function. This study establishes a novel aspect of the complex pharmacological effects of salicylic acid.

  10. Deoxycholic and chenodeoxycholic bile acids induce apoptosis via oxidative stress in human colon adenocarcinoma cells.

    PubMed

    Ignacio Barrasa, Juan; Olmo, Nieves; Pérez-Ramos, Pablo; Santiago-Gómez, Angélica; Lecona, Emilio; Turnay, Javier; Antonia Lizarbe, M

    2011-10-01

    The continuous exposure of the colonic epithelium to high concentrations of bile acids may exert cytotoxic effects and has been related to pathogenesis of colon cancer. A better knowledge of the mechanisms by which bile acids induce toxicity is still required and may be useful for the development of new therapeutic strategies. We have studied the effect of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) treatments in BCS-TC2 human colon adenocarcinoma cells. Both bile acids promote cell death, being this effect higher for CDCA. Apoptosis is detected after 30 min-2 h of treatment, as observed by cell detachment, loss of membrane asymmetry, internucleosomal DNA degradation, appearance of mitochondrial transition permeability (MPT), and caspase and Bax activation. At longer treatment times, apoptosis is followed in vitro by secondary necrosis due to impaired mitochondrial activity and ATP depletion. Bile acid-induced apoptosis is a result of oxidative stress with increased ROS generation mainly by activation of plasma membrane enzymes, such as NAD(P)H oxidases and, to a lower extent, PLA2. These effects lead to a loss of mitochondrial potential and release of pro-apoptotic factors to the cytosol, which is confirmed by activation of caspase-9 and -3, but not caspase-8. This initial apoptotic steps promote cleavage of Bcl-2, allowing Bax activation and formation of additional pores in the mitochondrial membrane that amplify the apoptotic signal.

  11. Polyunsaturated Branched-Chain Fatty Acid Geranylgeranoic Acid Induces Unfolded Protein Response in Human Hepatoma Cells

    PubMed Central

    Iwao, Chieko; Shidoji, Yoshihiro

    2015-01-01

    The acyclic diterpenoid acid geranylgeranoic acid (GGA) has been reported to induce autophagic cell death in several human hepatoma-derived cell lines; however, the molecular mechanism for this remains unknown. In the present study, several diterpenoids were examined for ability to induce XBP1 splicing and/or lipotoxicity for human hepatoma cell lines. Here we show that three groups of diterpenoids emerged: 1) GGA, 2,3-dihydro GGA and 9-cis retinoic acid induce cell death and XBP1 splicing; 2) all-trans retinoic acid induces XBP1 splicing but little cell death; and 3) phytanic acid, phytenic acid and geranylgeraniol induce neither cell death nor XBP1 splicing. GGA-induced ER stress/ unfolded protein response (UPR) and its lipotoxicity were both blocked by co-treatment with oleic acid. The blocking activity of oleic acid for GGA-induced XBP1 splicing was not attenuated by methylation of oleic acid. These findings strongly suggest that GGA at micromolar concentrations induces the so-called lipid-induced ER stress response/UPR, which is oleate-suppressive, and shows its lipotoxicity in human hepatoma cells. PMID:26186544

  12. Exogenous Ghrelin Accelerates the Healing of Acetic Acid-Induced Colitis in Rats

    PubMed Central

    Matuszyk, Aleksandra; Ceranowicz, Piotr; Warzecha, Zygmunt; Cieszkowski, Jakub; Ceranowicz, Dagmara; Gałązka, Krystyna; Bonior, Joanna; Jaworek, Jolanta; Bartuś, Krzysztof; Gil, Krzysztof; Olszanecki, Rafał; Dembiński, Artur

    2016-01-01

    Previous studies have shown that ghrelin reduces colonic inflammation induced by trinitrobenzene sulfonic acid and dextran sodium sulfate. In the present study we determined the effect of treatment with ghrelin on the course of acetic acid-induced colitis in rats. Rectal administration of 3% acetic acid solution led to induction of colitis in all animals. Damage of the colonic wall was accompanied by an increase in mucosal concentration of pro-inflammatory interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), as well mucosal activity of myeloperoxidase. Moreover, induction of colitis led to a reduction in colonic blood flow and DNA synthesis. Administration of ghrelin after induction of colitis led to faster regeneration of the colonic wall and reduction in colonic levels of IL-1β, TNF-α, and myeloperoxidase. In addition, treatment with ghrelin improved mucosal DNA synthesis and blood flow. Our study disclosed that ghrelin exhibits a strong anti-inflammatory and healing effect in acetic acid-induced colitis. Our current observation in association with previous findings that ghrelin exhibits curative effect in trinitrobenzene sulfonic acid- and dextran sodium sulfate-induced colitis suggest that therapeutic effect of ghrelin in the colon is universal and independent of the primary cause of colitis. PMID:27598133

  13. Ginkgolic acids induce neuronal death and activate protein phosphatase type-2C.

    PubMed

    Ahlemeyer, B; Selke, D; Schaper, C; Klumpp, S; Krieglstein, J

    2001-10-26

    The standardized extract from Ginkgo biloba (EGb 761) is used for the treatment of dementia. Because of allergenic and genotoxic effects, ginkgolic acids are restricted in EGb 761 to 5 ppm. The question arises whether ginkgolic acids also have neurotoxic effects. In the present study, ginkgolic acids caused death of cultured chick embryonic neurons in a concentration-dependent manner, in the presence and in the absence of serum. Ginkgolic acids-induced death showed features of apoptosis as we observed chromatin condensation, shrinkage of the nucleus and reduction of the damage by the protein synthesis inhibitor cycloheximide, demonstrating an active type of cell death. However, DNA fragmentation detected by the terminal-transferase-mediated ddUTP-digoxigenin nick-end labeling (TUNEL) assay and caspase-3 activation, which are also considered as hallmarks of apoptosis, were not seen after treatment with 150 microM ginkgolic acids in serum-free medium, a dose which increased the percentage of neurons with chromatin condensation and shrunken nuclei to 88% compared with 25% in serum-deprived, vehicle-treated controls. This suggests that ginkgolic acid-induced death showed signs of apoptosis as well as of necrosis. Ginkgolic acids specifically increased the activity of protein phosphatase type-2C, whereas other protein phosphatases such as protein phosphatases 1A, 2A and 2B, tyrosine phosphatase, and unspecific acid- and alkaline phosphatases were inhibited or remained unchanged, suggesting protein phosphatase 2C to play a role in the neurotoxic effect mediated by ginkgolic acids.

  14. ACE Gene I/D Polymorphism and Obesity in 1,574 Patients with Type 2 Diabetes Mellitus

    PubMed Central

    Wang, Min; Huang, Yan-Mei; Wang, Ying-Hui; Chen, Yin-Ling; Geng, Li-Jun

    2016-01-01

    Association between ACE gene I/D polymorphism and the risk of overweight/obesity remains controversial. We investigated the possible relationship between ACE gene I/D polymorphism and obesity in Chinese type 2 diabetes mellitus (T2DM) patients. In this study, obesity was defined as a body mass index (BMI) value ≥ 25 kg/m2 and subjects were classified into 4 groups (lean, normal, overweight, and obese). PCR (polymerase chain reaction) was used to detect the ACE gene I/D polymorphism in T2DM patients. Metabolic measurements including blood glucose, lipid profile, and blood pressure were obtained. Frequencies of the ACE genotypes (DD, ID, and II) were not significant among the 4 groups of BMI-defined patients (P = 0.679) while ACE II carriers showed higher systolic blood pressure (SBP) and pulse pressure (PP) (all P < 0.050). Hyperglycemia, hypertension, and dyslipidemia in these T2DM patients were found to be significantly associated with BMI. In conclusion, the relationship of ACE gene I/D polymorphism with obesity is insignificant in Chinese patients with T2DM. SBP and PP might be higher in the ACE II carriers than in the DD and ID carriers. PMID:28115791

  15. CA3 Synaptic Silencing Attenuates Kainic Acid-Induced Seizures and Hippocampal Network Oscillations123

    PubMed Central

    Yu, Lily M. Y.; Wintzer, Marie E.

    2016-01-01

    Abstract Epilepsy is a neurological disorder defined by the presence of seizure activity, manifest both behaviorally and as abnormal activity in neuronal networks. An established model to study the disorder in rodents is the systemic injection of kainic acid, an excitatory neurotoxin that at low doses quickly induces behavioral and electrophysiological seizures. Although the CA3 region of the hippocampus has been suggested to be crucial for kainic acid-induced seizure, because of its strong expression of kainate glutamate receptors and its high degree of recurrent connectivity, the precise role of excitatory transmission in CA3 in the generation of seizure and the accompanying increase in neuronal oscillations remains largely untested. Here we use transgenic mice in which CA3 pyramidal cell synaptic transmission can be inducibly silenced in the adult to demonstrate CA3 excitatory output is required for both the generation of epileptiform oscillatory activity and the progression of behavioral seizures. PMID:27022627

  16. Cell wall dynamics modulate acetic acid-induced apoptotic cell death of Saccharomyces cerevisiae

    PubMed Central

    Rego, António; Duarte, Ana M.; Azevedo, Flávio; Sousa, Maria J.; Côrte-Real, Manuela; Chaves, Susana R.

    2014-01-01

    Acetic acid triggers apoptotic cell death in Saccharomyces cerevisiae, similar to mammalian apoptosis. To uncover novel regulators of this process, we analyzed whether impairing MAPK signaling affected acetic acid-induced apoptosis and found the mating-pheromone response and, especially, the cell wall integrity pathways were the major mediators, especially the latter, which we characterized further. Screening downstream effectors of this pathway, namely targets of the transcription factor Rlm1p, highlighted decreased cell wall remodeling as particularly important for acetic acid resistance. Modulation of cell surface dynamics therefore emerges as a powerful strategy to increase acetic acid resistance, with potential application in industrial fermentations using yeast, and in biomedicine to exploit the higher sensitivity of colorectal carcinoma cells to apoptosis induced by acetate produced by intestinal propionibacteria. PMID:28357256

  17. Heat shock protein 70-dependent protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells.

    PubMed

    Qin, Ying; Naito, Yuji; Handa, Osamu; Hayashi, Natsuko; Kuki, Aiko; Mizushima, Katsura; Omatsu, Tatsushi; Tanimura, Yuko; Morita, Mayuko; Adachi, Satoko; Fukui, Akifumi; Hirata, Ikuhiro; Kishimoto, Etsuko; Nishikawa, Taichiro; Uchiyama, Kazuhiko; Ishikawa, Takeshi; Takagi, Tomohisa; Yagi, Nobuaki; Kokura, Satoshi; Yoshikawa, Toshikazu

    2011-11-01

    Protection of the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs including acetylsalicylic acid is a critical issue in the field of gastroenterology. Polaprezinc an anti-ulcer drug, consisting of zinc and L-carnosine, provides gastric mucosal protection against various irritants. In this study, we investigated the protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of the RIE1 rat intestinal epithelial cell line. Confluent rat intestinal epithelial cells were incubated with 70 µM polaprezinc for 24 h, and then stimulated with or without 15 mM acetylsalicylic acid for a further 15 h. Subsequent cellular viability was quantified by fluorometric assay based on cell lysis and staining. Acetylsalicylic acid-induced cell death was also qualified by fluorescent microscopy of Hoechst33342 and propidium iodide. Heat shock proteins 70 protein expression after adding polaprezinc or acetylsalicylic acid was assessed by western blotting. To investigate the role of Heat shock protein 70, Heat shock protein 70-specific small interfering RNA was applied. Cell viability was quantified by fluorometric assay based on cell lysis and staining and apoptosis was analyzed by fluorescence-activated cell sorting. We found that acetylsalicylic acid significantly induced apoptosis of rat intestinal epithelial cells in a dose- and time-dependent manner. Polaprezinc significantly suppressed acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells at its late phase. At the same time, polaprezinc increased Heat shock protein 70 expressions of rat intestinal epithelial cells in a time-dependent manner. However, in Heat shock protein 70-silenced rat intestinal epithelial cells, polaprezinc could not suppress acetylsalicylic acid -induced apoptosis at its late phase. We conclude that polaprezinc-increased Heat shock protein 70 expression might be an important mechanism by which polaprezinc suppresses acetylsalicylic

  18. Neuroprotective effects of butterbur and rough aster against kainic Acid-induced oxidative stress in mice.

    PubMed

    Oh, Sang Hee; Sok, Dai-Eun; Kim, Mee Ree

    2005-01-01

    The separate and combined neuroprotective effects of rough aster (Aster scaber) and butterbur (Petasite japonicus) extracts against oxidative damage in the brain of mice challenged with kainic acid were examined by comparing behavioral changes and biochemical parameters of oxidative stress. Rough aster butanol extract (400 mg/kg) and/or butterbur butanol extract (150 or 400 mg/kg) were administered to male ICR mice, 6-8 weeks old, through a gavage for 4 days consecutively, and on day 4, kainic acid (50 mg/kg) was administered intraperitoneally. Compared with the vehicle-treated control, no significant changes in body and brain weight were observed in mice administered rough aster or butterbur butanol extract. Administration of kainic acid only, causing a lethality of approximately 54%, resulted in a significant decrease of total glutathione level and increase of thiobarbituric acid-reactive substances (TBARS) value in brain tissue. The administration of butterbur or rough aster extract (400 mg/kg) decreased the lethality (50%) of kainic acid to 25%, alleviated the behavioral signs of neurotoxicity, restored the cytosolic glutathione level of brain homogenate to approximately 80% (P < .05), and reduced kainic acid-induced increases in TBARS values. In contrast to no significant neuroprotection by butterbur extract at a low dose (150 mg/kg), the combination of rough aster extract and butterbur extract reduced the lethality to 12.5%. Moreover, the combination delayed the onset time of behavioral signs by twofold, and significantly preserved the level of cytosolic glutathione peroxidase and glutathione reductase activities. However, the other biochemical parameters were not altered significantly by the combination. Thus, the combination of two vegetable extracts significantly increased the neuroprotective action against kainic acid-induced neurotoxicity. Based on these findings, the combination of butterbur extract and rough aster extract contains a functional agent or

  19. The Ayurvedic drug, Ksheerabala, ameliorates quinolinic acid-induced oxidative stress in rat brain.

    PubMed

    Swathy, S S; Indira, M

    2010-01-01

    One of the mechanisms of neurotoxicity is the induction of oxidative stress. There is hardly any cure for neurotoxicity in modern medicine, whereas many drugs in Ayurveda possess neuroprotective effects; however, there is no scientific validation for these drugs. Ksheerabala is an ayurvedic drug which is used to treat central nervous system disorders, arthritis, and insomnia. The aim of our study was to evaluate the effect of Ksheerabala on quinolinic acid-induced toxicity in rat brain. The optimal dose of Ksheerabala was found from a dose escalation study, wherein it was found that Ksheerabala showed maximum protection against quinolinic acid-induced neurotoxicity at a dose of 15 microL/100 g body weight/day, which was selected for further experiments. Four groups of female albino rats were maintained for 21 days as follows: 1. Control group, 2. Quinolinic acid (55 microg/100 g body weight), 3. Ksheerabala (15 microL/100 g body weight), 4. Ksheerabala (15 microL/100 g body weight) + Quinolinic acid (55 microg/100 g body weight). At the end of the experimental period, levels of lipid peroxidation products, protein carbonyls, and activities of scavenging enzymes were analyzed. The results revealed that quinolinic acid intake caused enhanced lipid and protein peroxidation as evidenced by increased levels of peroxidation products such as malondialdehyde, hydroperoxide, conjugated dienes, and protein carbonyls. On the other hand, the activities of scavenging enzymes such as catalase, superoxide dismutase (SOD), glutathione peroxidase, and glutathione reductase as well as the concentration of glutathione were reduced. On coadminstration of Ksheerabala along with quinolinic acid, the levels of all the biochemical parameters were restored to near-normal levels, indicating the protective effect of the drug. These results were reinforced by histopathological studies.

  20. Proteomic investigation into betulinic acid-induced apoptosis of human cervical cancer HeLa cells.

    PubMed

    Xu, Tao; Pang, Qiuying; Zhou, Dong; Zhang, Aiqin; Luo, Shaman; Wang, Yang; Yan, Xiufeng

    2014-01-01

    Betulinic acid is a pentacyclic triterpenoid that exhibits anticancer functions in human cancer cells. This study provides evidence that betulinic acid is highly effective against the human cervical cancer cell line HeLa by inducing dose- and time-dependent apoptosis. The apoptotic process was further investigated using a proteomics approach to reveal protein expression changes in HeLa cells following betulinic acid treatment. Proteomic analysis revealed that there were six up- and thirty down-regulated proteins in betulinic acid-induced HeLa cells, and these proteins were then subjected to functional pathway analysis using multiple analysis software. UDP-glucose 6-dehydrogenase, 6-phosphogluconate dehydrogenase decarboxylating, chain A Horf6-a novel human peroxidase enzyme that involved in redox process, was found to be down-regulated during the apoptosis process of the oxidative stress response pathway. Consistent with our results at the protein level, an increase in intracellular reactive oxygen species was observed in betulinic acid-treated cells. The proteins glucose-regulated protein and cargo-selection protein TIP47, which are involved in the endoplasmic reticulum pathway, were up-regulated by betulinic acid treatment. Meanwhile, 14-3-3 family proteins, including 14-3-3β and 14-3-3ε, were down-regulated in response to betulinic acid treatment, which is consistent with the decrease in expression of the target genes 14-3-3β and 14-3-3ε. Furthermore, it was found that the antiapoptotic bcl-2 gene was down-regulated while the proapoptotic bax gene was up-regulated after betulinic acid treatment in HeLa cells. These results suggest that betulinic acid induces apoptosis of HeLa cells by triggering both the endoplasmic reticulum pathway and the ROS-mediated mitochondrial pathway.

  1. Enhancement of Interferon Induction by ORF3 Product of Hepatitis E Virus

    PubMed Central

    Nan, Yuchen; Ma, Zexu; Wang, Rong; Yu, Ying; Kannan, Harilakshmi; Fredericksen, Brenda

    2014-01-01

    ABSTRACT Hepatitis E virus (HEV) causes both the endemic and epidemic spread of acute hepatitis in many parts of the world. HEV open reading frame 3 (ORF3) encodes a 13-kDa multifunctional protein (vp13) that is essential for HEV infection of animals. The exact role of vp13 in HEV infection remains unclear. In this study, vp13 was found to enhance interferon (IFN) production induced by poly(I · C), a synthetic analog of double-stranded RNA. Poly(I · C) treatment induced a higher level of IFN-β mRNA in HeLa cells stably expressing vp13 than in control cells. Using a luciferase reporter construct driven by the IFN-β promoter, we demonstrated that vp13 enhanced retinoic acid-inducible gene I (RIG-I)-dependent luciferase expression. This enhancement was found to be due to both an increased level of RIG-I protein and its activation. The levels of both endogenous and exogenous RIG-I were increased by vp13 by extension of the half-life of RIG-I. Additionally, vp13 interacts with the RIG-I N-terminal domain and enhances its K63-linked ubiquitination, which is essential for RIG-I activation. Analysis of vp13 deletion constructs suggested that the C-terminal domain of vp13 was essential for the enhancement of RIG-I signaling. In HEV-infected hepatoma cells, wild-type HEV led to a higher level of RIG-I and more poly(I · C)-induced IFN-β expression than did ORF3-null mutants. Analysis of vp13 from four HEV genotypes showed that vp13 from genotype I and III strains boosted RIG-I signaling, while vp13 from genotype II and IV strains had a minimal effect. These results indicate that vp13 enhances RIG-I signaling, which may play a role in HEV invasion. IMPORTANCE Hepatitis E virus (HEV) is a significant pathogen causing hepatitis in many parts of the world, yet it is understudied compared with other viral hepatitis pathogens. Here we found that the HEV open reading frame 3 product, vp13, enhances interferon induction stimulated by a synthetic analog of double-stranded RNA

  2. Enhanced neutralising antibody response to bovine viral diarrhoea virus (BVDV) induced by DNA vaccination in calves.

    PubMed

    R El-Attar, Laila M; Thomas, Carole; Luke, Jeremy; A Williams, James; Brownlie, Joe

    2015-07-31

    DNA vaccination is effective in inducing potent immunity in mice; however it appears to be less so in large animals. Increasing the dose of DNA plasmid to activate innate immunity has been shown to improve DNA vaccine adaptive immunity. Retinoic acid-inducible gene I (RIG-I) is a critical cytoplasmic double-stranded RNA pattern receptor required for innate immune activation in response to viral infection. RIG-I recognise viral RNA and trigger antiviral response, resulting in type I interferon (IFN) and inflammatory cytokine production. In an attempt to enhance the antibody response induced by BVDV DNA in cattle, we expressed BVDV truncated E2 (E2t) and NS3 codon optimised antigens from antibiotic free-plasmid vectors expressing a RIG-I agonist and designated either NTC E2t(co) and NTC NS3(co). To evaluate vaccine efficacy, groups of five BVDV-free calves were intramuscularly injected three times with NTC E2t(co) and NTC NS3(co) vaccine plasmids individually or in combination. Animals vaccinated with our (previously published) conventional DNA vaccines pSecTag/E2 and pTriExNS3 and plasmids expressing RIG-I agonist only presented both the positive and mock-vaccine groups. Our results showed that vaccines coexpressing E2t with a RIG-I agonist induced significantly higher E2 antigen specific antibody response (p<0.05). Additionally, E2t augmented the immune response to NS3 when the two vaccines were delivered in combination. Despite the lack of complete protection, on challenge day 4/5 calves vaccinated with NTC E2t(co) alone or NTC E2t(co) plus NTC NS3(co) had neutralising antibody titres exceeding 1/240 compared to 1/5 in the mock vaccine control group. Based on our results we conclude that co-expression of a RIG-I agonist with viral antigen could enhance DNA vaccine potency in cattle.

  3. DIBROMOACETIC ACID-INDUCED ELEVATIONS OF ESTRADIOL IN THE CYCLING AND OVARIECTOMOZED/ESTRADIOL-IMPLANTED FEMALE RAT

    EPA Science Inventory

    Goldman, JM and Murr, AS. Dibromoacetic Acid-induced Elevations of Estradiol in Both Cycling and Ovariectomized / Estradiol-implanted Female Rats

    ABSTRACT
    Haloacetic acids are one of the principal classes of disinfection by-products generated by the chlorination of mun...

  4. DIBROMOACETIC ACID-INDUCED ELEVATIONS IN CIRCULATING ESTRADIOL: EFFECTS IN BOTH CYCLING AND OVARIECTOMIZED/STEROID-PRIMED FEMALE RATS

    EPA Science Inventory

    RTD-03-031
    Goldman, JM and Murr, AS. Dibromoacetic Acid-induced Elevations in Circulating Estradiol: Effects in Both Cycling and Ovariectomized/Steroid-primed Female Rats. Reproductive Toxicology (in press).

    Abstract

    Oral exposures to high concentrations of th...

  5. Transcriptional analysis of the acid-inducible asr gene in enterobacteria.

    PubMed

    Seputiene, Vaida; Suziedelis, Kestutis; Normark, Staffan; Melefors, Ojar; Suziedeliene, Edita

    2004-09-01

    We show here that transcription of the asr gene in Escherichia coli, Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae and Enterobacter cloacae is strongly dependent on the acidification level of the growth medium, with maximal induction at pH 4.0-4.5 as determined by Northern hybridization analysis. Previous gene array analyses have also shown that asr is the most acid-induced gene in the E. coli genome. Sequence alignment of the asr promoters from different enterobacterial species identified a highly conserved region located at position -70 to -30 relative to the asr transcriptional start site. By deletion of various segments of this region in the E. coli asr promoter it was shown that sequences upstream from the -40 position were important for induction. Transcription from the E. coli asr promoter was demonstrated to be growth-phase-dependent and to require the alternative sigma factor RpoS (sigma(S)) in stationary phase. Transcription of the asr gene was also found to be subject to negative control by the nucleoid protein H-NS.

  6. CCN1 is critical for acid-induced esophageal epithelial cell transformation.

    PubMed

    Modak, Cristina; Mouazzen, Wasim; Narvaez, Reinier; Reavis, Kevin M; Chai, Jianyuan

    2010-02-19

    CCN1 is a matricellular protein involved in both wound healing and cancer cell invasion. Increased CCN1 expression has been associated with the development of Barrett's esophagus and the increased risk of progression to esophageal adenocarcinoma. In both cases, acid reflux is a major contributor. Low pH has been shown to induce CCN1 gene expression in esophageal epithelial cells. Here we demonstrated that both CCN1 and low pH could cause esophageal epithelial cell transformation, including loss of E-cadherin, disruption of cell-cell junctions, and expression of mesenchymal markers. Furthermore, knockdown of CCN1 through RNA interference sufficiently attenuated acid-driven cell phenotypic changes, while over-expression of CCN1 exacerbated these effects, indicating a critical role of CCN1 in acid-induced esophageal epithelial cell transformation. Given the pivotal role of low pH in gastro-esophageal reflux disease and its progression towards esophageal adenocarcinoma, our study identified CCN1 as a key molecule mediating this process.

  7. Acid-induced gelation behavior of casein/whey protein solutions assessed by oscillatory rheology.

    PubMed

    Sadeghi, Mahboubeh; Madadlou, Ashkan; Khosrowshahi, Asghar; Mohammadifar, Mohammadamin

    2014-09-01

    Gelation process of acid-induced casein gels was studied using response surface method (RSM). Ratio of casein to whey proteins, incubation and heating temperatures were independent variables. Final storage modulus (G') measured 200 min after the addition of glucono-δ-lactone and the gelation time i.e. the time at which G' of gels became greater than 1 Pa were the parameters studied. Incubation temperature strongly affected both parameters. The higher the incubation temperature, the lower was the G' and the shorter the gelation time. Increased heating temperature however, increased the G' but again shortened the gelation time. Increase in G' was attributed to the formation of disulphide cross-linkages between denatured whey proteins and casein chains; whilst the latter was legitimized by considering the higher isoelectric pH of whey proteins. Maximum response (G' = 268.93 Pa) was obtained at 2.7 % w/w, 25 °C and 90 °C for casein content, incubation and heating temperatures, respectively.

  8. Folic acid induces salicylic acid-dependent immunity in Arabidopsis and enhances susceptibility to Alternaria brassicicola.

    PubMed

    Wittek, Finni; Kanawati, Basem; Wenig, Marion; Hoffmann, Thomas; Franz-Oberdorf, Katrin; Schwab, Wilfried; Schmitt-Kopplin, Philippe; Vlot, A Corina

    2015-08-01

    Folates are essential for one-carbon transfer reactions in all organisms and contribute, for example, to de novo DNA synthesis. Here, we detected the folate precursors 7,8-dihydropteroate (DHP) and 4-amino-4-deoxychorismate (ADC) in extracts from Arabidopsis thaliana plants by Fourier transform ion cyclotron resonance-mass spectrometry. The accumulation of DHP, but not ADC, was induced after infection of plants with Pseudomonas syringae delivering the effector protein AvrRpm1. Application of folic acid or the DHP precursor 7,8-dihydroneopterin (DHN) enhanced resistance in Arabidopsis to P. syringae and elevated the transcript accumulation of the salicylic acid (SA) marker gene pathogenesis-related1 in both the treated and systemic untreated leaves. DHN- and folic acid-induced systemic resistance was dependent on SA biosynthesis and signalling. Similar to SA, folic acid application locally enhanced Arabidopsis susceptibility to the necrotrophic fungus Alternaria brassicicola. Together, the data associate the folic acid pathway with innate immunity in Arabidopsis, simultaneously activating local and systemic SA-dependent resistance to P. syringae and suppressing local resistance to A. brassicicola.

  9. Acupuncture suppresses kainic acid-induced neuronal death and inflammatory events in mouse hippocampus.

    PubMed

    Kim, Seung-Tae; Doo, Ah-Reum; Kim, Seung-Nam; Kim, Song-Yi; Kim, Yoon Young; Kim, Jang-Hyun; Lee, Hyejung; Yin, Chang Shik; Park, Hi-Joon

    2012-09-01

    The administration of kainic acid (KA) causes seizures and produces neurodegeneration in hippocampal CA3 pyramidal cells. The present study investigated a possible role of acupuncture in reducing hippocampal cell death and inflammatory events, using a mouse model of kainic acid-induced epilepsy. Male C57BL/6 mice received acupuncture treatments at acupoint HT8 or in the tail area bilaterally once a day for 2 days and again immediately after an intraperitoneal injection of KA (30 mg/kg). HT8 is located on the palmar surface of the forelimbs, between the fourth and fifth metacarpal bones. Twenty-four hours after the KA injection, neuronal cell survival, the activations of microglia and astrocytes, and mRNA expression of two proinflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), were measured in the hippocampus. Acupuncture stimulation at HT8, but not in the tail area, significantly reduced the KA-induced seizure, neuron death, microglial and astrocyte activations, and IL-1β mRNA expression in the hippocampus. The acupuncture stimulation also decreased the mRNA expression of TNF-α, but it was not significant. These results indicate that acupuncture at HT8 can inhibit hippocampal cell death and suppress KA-induced inflammatory events, suggesting a possible role for acupuncture in the treatment of epilepsy.

  10. Ethanol promotes saturated fatty acid-induced hepatoxicity through endoplasmic reticulum (ER) stress response.

    PubMed

    Yi, Hong-Wei; Ma, Yu-Xiang; Wang, Xiao-Ning; Wang, Cui-Fen; Lu, Jian; Cao, Wei; Wu, Xu-Dong

    2015-04-01

    Serum palmitic acid (PA), a type of saturated fatty acid, causes lipid accumulation and induces toxicity in hepatocytes. Ethanol (EtOH) is metabolized by the liver and induces hepatic injury and inflammation. Herein, we analyzed the effects of EtOH on PA-induced lipotoxicity in the liver. Our results indicated that EtOH aggravated PA-induced apoptosis and lipid accumulation in primary rat hepatocytes in dose-dependent manner. EtOH intensified PA-caused endoplasmic reticulum (ER) stress response in vitro and in vivo, and the expressions of CHOP, ATF4, and XBP-1 in nucleus were significantly increased. EtOH also increased PA-caused cleaved caspase-3 in cytoplasm. In wild type and CHOP(-/-) mice treated with EtOH and high fat diet (HFD), EtOH worsened the HFD-induced liver injury and dyslipidemia, while CHOP knockout blocked toxic effects of EtOH and PA. Our study suggested that targeting UPR-signaling pathways is a promising, novel approach to reducing EtOH and saturated fatty acid-induced metabolic complications.

  11. The saturated fatty acid, palmitic acid, induces anxiety-like behavior in mice

    PubMed Central

    Moon, Morgan L.; Joesting, Jennifer J.; Lawson, Marcus A.; Chiu, Gabriel S.; Blevins, Neil A.; Kwakwa, Kristin A.; Freund, Gregory G.

    2014-01-01

    Objectives Excess fat in the diet can impact neuropsychiatric functions by negatively affecting cognition, mood and anxiety. We sought to show that the free fatty acid (FFA), palmitic acid, can cause adverse biobehaviors in mice that lasts beyond an acute elevation in plasma FFAs. Methods Mice were administered palmitic acid or vehicle as a single intraperitoneal (IP) injection. Biobehaviors were profiled 2 and 24 hrs after palmitic acid treatment. Quantification of dopamine (DA), norepinephrine (NE), serotonin (5-HT) and their major metabolites was performed in cortex, hippocampus and amygdala. FFA concentration was determined in plasma. Relative fold change in mRNA expression of unfolded protein response (UPR)-associated genes was determined in brain regions. Results In a dose-dependent fashion, palmitic acid rapidly reduced mouse locomotor activity by a mechanism that did not rely on TLR4, MyD88, IL-1, IL-6 or TNFα but was dependent on fatty acid chain length. Twenty-four hrs after palmitic acid administration mice exhibited anxiety-like behavior without impairment in locomotion, food intake, depressive-like behavior or spatial memory. Additionally, the serotonin metabolite 5-HIAA was increased by 33% in the amygdala 24 hrs after palmitic acid treatment. Conclusions Palmitic acid induces anxiety-like behavior in mice while increasing amygdala-based serotonin metabolism. These effects occur at a time point when plasma FFA levels are no longer elevated. PMID:25016520

  12. Palmitoleic acid induces the cardiac mitochondrial membrane permeability transition despite the presence of L-carnitine.

    PubMed

    Oyanagi, Eri; Uchida, Masataka; Miyakawa, Takeshi; Miyachi, Motohiko; Yamaguchi, Hidetaka; Nagami, Kuniatsu; Utsumi, Kozo; Yano, Hiromi

    Although palmitoleic acid (C16:1) is associated with arrhythmias, and increases in an age-dependent matter, the effects of L-carnitine, which is essential for the transport of long-chain fatty acids into the mitochondria, are unclear. It has been postulated that L-carnitine may attenuate palmitate (C16:0)-induced mitochondrial dysfunction and the apoptosis of cardiomyocytes. The aim of this study was to elucidate the activity of L-carnitine in the prevention of the palmitoleic acid-induced mitochondrial membrane permeability transition and cytochrome c release using isolated cardiac mitochondria from rats. Palmitoleoyl-CoA-induced mitochondrial respiration was not accelerated by L-carnitine treatment, and this respiration was slightly inhibited by oligomycin, which is an inhibitor of ATP synthase. Despite pretreatment with L-carnitine, the mitochondrial membrane potential decreased and mitochondrial swelling was induced by palmitoleoyl-CoA. In the presence of a combination of L-carnitine and tiron, a free radical scavenger, there was attenuated mitochondrial swelling and cytochrome c release following palmitoleoyl-CoA treatment. We concluded that palmitoleic acid, but not palmitate, induces the cardiac mitochondrial membrane permeability transition despite the presence of L-carnitine.

  13. Bile acid induced colonic irritation stimulates intracolonic nitric oxide release in humans.

    PubMed Central

    Casellas, F; Mourelle, M; Papo, M; Guarner, F; Antolin, M; Armengol, J R; Malagelada, J R

    1996-01-01

    AIM--To measure the intracolonic release of nitric oxide end products (nitrates plus nitrites) and eicosanoids in response to intraluminal irritation with deoxycholic acid (DCA). PATIENTS--Seven patients with irritable bowel syndrome. METHODS--The left colon was perfused with a solution with or without 3 mM deoxycholic acid. Aspirates were assayed for eicosanoids by specific radioimmuno-assay, and for nitrates plus nitrites by the Griess reaction. To confirm that stimulated colonic mucosa can produce nitric oxide (NO), ancillary studies were performed in vitro using samples of normal mucosa obtained from five surgically resected colons. Samples were incubated for 30 minutes in Kreb's solution, 3 mM DCA or DCA with 1 mM L-nitro-arginine-methyl-ester (L-NAME) to inhibit the NO synthase. Finally, NO synthase activity was measured in five samples of human colonic mucosa. RESULTS--Intracolonic release of nitrates plus nitrites was basally undetectable in six of seven patients. Bile acid considerably increased the release of prostaglandin E2 and nitrates plus nitrites (p < 0.01). By contrast, no increase in thromboxane and leukotriene was seen. In vitro mucosal incubation with DCA increased the production of NO synthase products, which was blocked by L-NAME. Activity of Ca+2 independent NO synthase was detectable in four of five samples of human colonic mucosa. CONCLUSION--The human colonic mucosa responds to bile acid induced irritation by a surge in NO generation via NO synthase. PMID:8707118

  14. Saturated phosphatidic acids mediate saturated fatty acid-induced vascular calcification and lipotoxicity.

    PubMed

    Masuda, Masashi; Miyazaki-Anzai, Shinobu; Keenan, Audrey L; Okamura, Kayo; Kendrick, Jessica; Chonchol, Michel; Offermanns, Stefan; Ntambi, James M; Kuro-O, Makoto; Miyazaki, Makoto

    2015-10-26

    Recent evidence indicates that saturated fatty acid-induced (SFA-induced) lipotoxicity contributes to the pathogenesis of cardiovascular and metabolic diseases; however, the molecular mechanisms that underlie SFA-induced lipotoxicity remain unclear. Here, we have shown that repression of stearoyl-CoA desaturase (SCD) enzymes, which regulate the intracellular balance of SFAs and unsaturated FAs, and the subsequent accumulation of SFAs in vascular smooth muscle cells (VSMCs), are characteristic events in the development of vascular calcification. We evaluated whether SMC-specific inhibition of SCD and the resulting SFA accumulation plays a causative role in the pathogenesis of vascular calcification and generated mice with SMC-specific deletion of both Scd1 and Scd2. Mice lacking both SCD1 and SCD2 in SMCs displayed severe vascular calcification with increased ER stress. Moreover, we employed shRNA library screening and radiolabeling approaches, as well as in vitro and in vivo lipidomic analysis, and determined that fully saturated phosphatidic acids such as 1,2-distearoyl-PA (18:0/18:0-PA) mediate SFA-induced lipotoxicity and vascular calcification. Together, these results identify a key lipogenic pathway in SMCs that mediates vascular calcification.

  15. Effect of galactose on acid induced molten globule state of Soybean Agglutinin: Biophysical approach

    NASA Astrophysics Data System (ADS)

    Alam, Parvez; Naseem, Farha; Abdelhameed, Ali Saber; Khan, Rizwan Hasan

    2015-11-01

    In the present study the formation of molten globule-like unfolding intermediate Soybean Agglutinin (SBA) in acidic pH range has been established with the help of acrylamide quenching, intrinsic fluorescence, ANS fluorescence measurement, far UV CD and dynamic light scattering measurement. A marked increase in ANS fluorescence was observed at pH 2.2. Ksv of acrylamide quenching was found to be higher at pH 2.2 than that of native SBA at pH 7. Far UV CD spectra of pH induced state suggest that SBA shows significant retention of secondary structure closure to native. Hydrodynamic radius of SBA at pH 2.2 was found be more as compared to native state and also in other pH induced states. Further we checked the effect of galactose on the molten globule state of SBA. This study suggests that SBA exist as molten globule at pH 2.2 and this study will help in acid induced molten globule state of other proteins.

  16. Pulmonary vasoconstriction in oleic acid induced lung injury. A morphometric study.

    PubMed Central

    Grotjohan, H. P.; van der Heijde, R. M.; Wagenvoort, C. A.; Wagenvoort, N.; Versprille, A.

    1993-01-01

    Distribution and severity of active vasoconstriction of muscular pulmonary arteries were morphometrically assessed in anaesthetized, paralysed and mechanically ventilated pigs with respiratory distress, induced by oleic acid. Vasoconstriction was deduced from the medial thickness which was measured and expressed as a percentage of external diameter. Six pigs received oleic acid (0.12 +/- 0.07 ml/kg), dissolved 1:1 in 96% alcohol, in multiple injections of 0.1 ml. Six pigs were used as controls. After the oleic acid injections a stable hypoxaemia (PaO2 = 57 +/- 8 mmHg, at an inspiratory oxygen fraction of 0.6) and pulmonary hypertension (mean Ppa = 36 +/- 2 mmHg) were obtained for several hours. Electron microscopy revealed swelling of endothelial cells with signs of degeneration. Medial thickness was far greater in the oleic acid group than in the control group; overall mean values were 8.1 +/- 3.2 and 3.8 +/- 1.7% respectively (P < 0.001). Arteries with prominent vasoconstriction were lying in clusters. This pattern was the same in dependent and non-dependent regions. We concluded that in oleic acid induced respiratory distress active vasoconstriction of muscular pulmonary arteries is an important factor in the development of pulmonary hypertension. Besides vasoconstriction, endothelial swelling and intravascular clotting may contribute to the development of pulmonary hypertension. Images Figure 1 Figure 2 Figure 3 PMID:8398807

  17. Tanshinone IIA Protects Against Folic Acid-Induced Acute Kidney Injury.

    PubMed

    Jiang, Chunming; Zhu, Wei; Shao, Qiuyuan; Yan, Xiang; Jin, Bo; Zhang, Miao; Xu, Biao

    2016-01-01

    Tanshinone IIA is a diterpene extracted from Salvia miltiorrhiza, a popular and safe herb medicine that has been widely used in China and other Asian countries. Previous studies have demonstrated the pleiotropic effects of Tanshinone IIA on many disease treatments via its antitoxicity, anti-inflammation, anti-oxidative stress, as well as antifibrosis activities. However, its effect on acute kidney injury (AKI) has not been fully investigated. Here, we show for the first time that systemic administration of Tanshinone IIA can lead to improved kidney function in folic acid-induced kidney injury mice. In the acute phase of AKI, Tanshinone IIA attenuated renal tubular epithelial injury, as determined by histologic changes and the detection of Neutrophil gelatinase-associated lipocalin (NGAL) in the kidney and urine. Additionally, Tanshinone IIA treatment resulted in elevated proliferating cell nuclear antigen (PCNA) expression and decreased inflammatory cells infiltration as well as chemokine expression, suggesting that Tanshinone IIA promoted renal repair following AKI and inhibited local inflammatory response in the injured kidney. This led to decreased long-term fibrosis in the injured kidney, characterized by less accumulation of fibronectin and collagen I in tubulointerstitium. Taken together, these results suggest that Tanshinone IIA may represent a potential approach for AKI treatment.

  18. Targeting oxidative stress attenuates malonic acid induced Huntington like behavioral and mitochondrial alterations in rats.

    PubMed

    Kalonia, Harikesh; Kumar, Puneet; Kumar, Anil

    2010-05-25

    Objective of the present study was to explore the possible role of oxidative stress in the malonic acid induced behavioral, biochemical and mitochondrial alterations in rats. In the present study, unilateral single injections of malonic acid at different doses (1.5, 3 and 6 micromol) were made into the ipsilateral striatum in rats. Behavioral parameters were accessed on 1st, 7th and 14th day post malonic acid administration. Oxidative stress parameters and mitochondrial enzyme functions were assessed on day 14 after behavioral observations. Ipsilateral striatal malonic acid (3 and 6 micromol) administration significantly reduced body weight, locomotor activity, motor coordination and caused oxidative damage (lipid peroxidation, nitrite, superoxide dismutase, catalase and glutathione) in the striatum as compared to sham treated animal. Mitochondrial enzyme complexes and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolinium bromide) activity were significantly inhibited by malonic acid. Vitamin E treatment (50 and 100 mg/kg, p.o.) significantly reversed the various behavioral, biochemical and mitochondrial alterations in malonic acid treated animals. Our findings show that targeting oxidative stress by vitamin E in malonic acid model, results in amelioration of behavioral and mitochondrial alterations are linked to inhibition of oxidative damage. Based upon these finding present study hypothesize that protection exerted by vitamin E on behavioral, mitochondrial markers indicates the possible preservation of the functional status of the striatal neurons by targeting the deleterious actions of oxidative stress.

  19. Tiagabine treatment in kainic acid induced cerebellar lesion of dystonia rat model

    PubMed Central

    Wang, Tsui-chin; Ngampramuan, Sukonthar; Kotchabhakdi, Naiphinich

    2016-01-01

    Dystonia is a neurological disorder characterized by excessive involuntary muscle contractions that lead to twisting movements. The exaggerated movements have been studied and have implicated basal ganglia as the point of origin. In more recent studies, the cerebellum has also been identified as the possible target of dystonia, in the search for alternative treatments. Tiagabine is a selective GABA transporter inhibitor, which blocks the reuptake and recycling of GABA. The study of GABAergic drugs as an alternative treatment for cerebellar induced dystonia has not been reported. In our study, tiagabine was i.p. injected into kainic acid induced, cerebellar dystonic adult rats, and the effects were compared with non-tiagabine injected and sham-operated groups. Beam walking apparatus, telemetric electromyography (EMG) recording, and histological verification were performed to confirm dystonic symptoms in the rats on post-surgery treatment. Involuntary dystonic spasm was observed with repetitive rigidity, and twisting movements in the rats were also confirmed by a high score on the dystonic scoring and a high amplitude on the EMG data. The rats with tiagabine treatment were scored based on motor amelioration assessed via beam walking. The result of this study suggests and confirms that low dose of kainic acid microinjection is sufficient to induce dystonia from the cerebellar vermis. In addition, from the results of the EMG recording and the behavioral assessment through beam walking, tiagabine is demonstrated as being effective in reducing dystonic spasm and may be a possible alternative therapeutic drug in the treatment of dystonia. PMID:28337103

  20. Tachykinin inhibition of acid-induced gastric hyperaemia in the rat.

    PubMed Central

    Heinemann, A.; Jocic, M.; Herzeg, G.; Holzer, P.

    1996-01-01

    1. Primary afferent neurones releasing the vasodilator, calcitonin gene-related peptide, mediate the gastric hyperaemic response to acid back-diffusion. The tachykinins neurokinin A (NKA) and substance P (SP) are located in the same neurones and are co-released with calcitonin gene-related peptide. In this study we investigated the effect and possible role of tachykinins in the acid-evoked gastric vasodilatation in urethane-anaesthetized rats. 2. Gastric acid back-diffusion, induced by perfusing the stomach with 15% ethanol in the presence of 0.05 M HCl, increased gastric mucosal blood flow by 60-90%, as determined by the hydrogen clearance technique. NKA and SP (0.14-3.78 nmol min-1 kg-1, infused intra-aortically) inhibited the gastric mucosal hyperaemic response to acid back-diffusion in a dose-dependent manner, an effect that was accompanied by aggravation of ethanol/acid-induced macroscopic haemorrhagic lesions. 3. The inhibitory effect of NKA (1.26 nmol min-1 kg-1) on the acid-induced gastric mucosal vasodilatation was prevented by the tachykinin NK2 receptor antagonists, MEN 10,627 (200 nmol kg-1) but left unaltered by the NK1 receptor antagonist, SR 140,333 (300 nmol kg-1) and the mast-cell stabilizer, ketotifen (4.6 mumol kg-1). 4. Under basal conditions, with 0.05 M HCl being perfused through the stomach, NKA (1.26 nmol min-1 kg-1) reduced gastric mucosal blood flow by about 25%, an effect that was abolished by SR 140,333 but not MEN 10,627 or ketotifen. 5. SR 140,333, MEN 10,627 or ketotifen had no significant effect on basal gastric mucosal blood flow nor did they modify the gastric mucosal hyperaemic reaction to acid back-diffusion. 6. The effect of NKA (1.26 nmol min-1 kg-1) in causing vasoconstriction and inhibiting the vasodilator response to acid back-diffusion was also seen when blood flow in the left gastric artery was measured with the ultrasonic transit time shift technique. 7. Arginine vasopressin (AVP, 0.1 nmol min-1 kg-1) induced gastric

  1. Pistacia lentiscus resin regulates intestinal damage and inflammation in trinitrobenzene sulfonic acid-induced colitis.

    PubMed

    Gioxari, Aristea; Kaliora, Andriana C; Papalois, Apostolos; Agrogiannis, George; Triantafillidis, John K; Andrikopoulos, Nikolaos K

    2011-11-01

    Mastic (Pistacia lentiscus) of the Anacardiaceae family has exhibited anti-inflammatory and antioxidant properties in patients with Crohn's disease. This study was based on the hypothesis that mastic inhibits intestinal damage in inflammatory bowel disease, regulating inflammation and oxidative stress in intestinal epithelium. Four different dosages of P. lentiscus powder in the form of powder were administered orally to trinitrobenzene sulfonic acid-induced colitic rats. Eighty-four male Wistar rats were randomly assigned to seven groups: A, control; B, colitic; C-F, colitic rats daily supplemented with P. lentiscus powder at (C) 50 mg/kg, (D) 100 mg/kg, (E) 200 mg/kg, and (F) 300 mg/kg of body weight; and G, colitic rats treated daily with cortisone (25 μg/kg of body weight). Colonic damage was assessed microscopically. The cytokines tumor necrosis factor-α, intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-6, IL-8, and IL-10 and malonaldehyde were measured in colonic specimens. Results were expressed as mean ± SE values. Histological amelioration of colitis (P≤.001) and significant differences in colonic indices occurred after 3 days of treatment. Daily administration of 100 mg of P. lentiscus powder/kg of body weight decreased all inflammatory cytokines (P≤.05), whereas 50 mg of P. lentiscus powder/kg of body weight and cortisone treatment reduced only ICAM-1 (P≤.05 and P≤.01, respectively). Malonaldehyde was significantly suppressed in all treated groups (P≤.01). IL-10 remained unchanged. Cytokines and malonaldehyde remained unaltered after 6 days of treatment. Thus P. lentiscus powder could possibly have a therapeutic role in Crohn's disease, regulating oxidant/antioxidant balance and modulating inflammation.

  2. Praeruptorin D and E attenuate lipopolysaccharide/hydrochloric acid induced acute lung injury in mice.

    PubMed

    Yu, Peng-Jiu; Li, Jing-Rong; Zhu, Zheng-Guang; Kong, Huan-Yu; Jin, Hong; Zhang, Jun-Yan; Tian, Yuan-Xin; Li, Zhong-Huang; Wu, Xiao-Yun; Zhang, Jia-Jie; Wu, Shu-Guang

    2013-06-15

    Acute lung injury is a life-threatening syndrome characterized by overwhelming lung inflammation and increased microvascular permeability, which causes a high mortality rate worldwide. The dry root of Peucedanum praeruptorum Dunn has been long used to treat respiratory diseases in China. In the present study, Praeruptorin A, C, D and E (PA, PC, PD and PE), four pyranocoumarins extracted from this herb, have been investigated for the pharmacological effects in experimental lung injury mouse models. In lipopolysaccharide (LPS) challenged mice, PA and PC did not show protective effect against lung injury at the dose of 80 mg/kg. However, PD and PE significantly inhibited the infiltration of activated polymorphonuclear leukocytes (PMNs) and decreased the levels of TNF-α and IL-6 in bronchoalveolar lavage fluid at the same dose. There was no statistically significant difference between PD and PE group. Further study demonstrated that PD and PE suppressed protein extravasations in bronchoalveolar lavage fluid, attenuated myeloperoxidase (MPO) activity and the pathological changes in the lung. Both PD and PE suppressed LPS induced Nuclear Factor-kappa B (NF-κB) pathway activation in the lung by decreasing the cytoplasmic loss of Inhibitor κB-α (IκB-α) protein and inhibiting the translocation of p65 from cytoplasm to nucleus. We also extended our study to acid-induced acute lung injury and found that these two compounds protected mice from hydrochloric acid (HCl)-induced lung injury by inhibiting PMNs influx, IL-6 release and protein exudation. Taken together, these results suggested that PD and PE might be useful in the therapy of lung injury.

  3. Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis

    SciTech Connect

    Woolbright, Benjamin L.; Dorko, Kenneth; Antoine, Daniel J.; Clarke, Joanna I.; Gholami, Parviz; Li, Feng; Kumer, Sean C.; Schmitt, Timothy M.; Forster, Jameson; Fan, Fang; Jenkins, Rosalind E.; Park, B. Kevin; Hagenbuch, Bruno; Olyaee, Mojtaba; Jaeschke, Hartmut

    2015-03-15

    Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. - Highlights: • Cholestatic liver injury is due to cytoplasmic bile acid accumulation in hepatocytes. • Primary human hepatocytes are resistant to BA-induced injury

  4. Human sweet taste receptor mediates acid-induced sweetness of miraculin.

    PubMed

    Koizumi, Ayako; Tsuchiya, Asami; Nakajima, Ken-ichiro; Ito, Keisuke; Terada, Tohru; Shimizu-Ibuka, Akiko; Briand, Loïc; Asakura, Tomiko; Misaka, Takumi; Abe, Keiko

    2011-10-04

    Miraculin (MCL) is a homodimeric protein isolated from the red berries of Richadella dulcifica. MCL, although flat in taste at neutral pH, has taste-modifying activity to convert sour stimuli to sweetness. Once MCL is held on the tongue, strong sweetness is sensed over 1 h each time we taste a sour solution. Nevertheless, no molecular mechanism underlying the taste-modifying activity has been clarified. In this study, we succeeded in quantitatively evaluating the acid-induced sweetness of MCL using a cell-based assay system and found that MCL activated hT1R2-hT1R3 pH-dependently as the pH decreased from 6.5 to 4.8, and that the receptor activation occurred every time an acid solution was applied. Although MCL per se is sensory-inactive at pH 6.7 or higher, it suppressed the response of hT1R2-hT1R3 to other sweeteners at neutral pH and enhanced the response at weakly acidic pH. Using human/mouse chimeric receptors and molecular modeling, we revealed that the amino-terminal domain of hT1R2 is required for the response to MCL. Our data suggest that MCL binds hT1R2-hT1R3 as an antagonist at neutral pH and functionally changes into an agonist at acidic pH, and we conclude this may cause its taste-modifying activity.

  5. Human sweet taste receptor mediates acid-induced sweetness of miraculin

    PubMed Central

    Koizumi, Ayako; Tsuchiya, Asami; Nakajima, Ken-ichiro; Ito, Keisuke; Terada, Tohru; Shimizu-Ibuka, Akiko; Briand, Loïc; Asakura, Tomiko; Misaka, Takumi; Abe, Keiko

    2011-01-01

    Miraculin (MCL) is a homodimeric protein isolated from the red berries of Richadella dulcifica. MCL, although flat in taste at neutral pH, has taste-modifying activity to convert sour stimuli to sweetness. Once MCL is held on the tongue, strong sweetness is sensed over 1 h each time we taste a sour solution. Nevertheless, no molecular mechanism underlying the taste-modifying activity has been clarified. In this study, we succeeded in quantitatively evaluating the acid-induced sweetness of MCL using a cell-based assay system and found that MCL activated hT1R2-hT1R3 pH-dependently as the pH decreased from 6.5 to 4.8, and that the receptor activation occurred every time an acid solution was applied. Although MCL per se is sensory-inactive at pH 6.7 or higher, it suppressed the response of hT1R2-hT1R3 to other sweeteners at neutral pH and enhanced the response at weakly acidic pH. Using human/mouse chimeric receptors and molecular modeling, we revealed that the amino-terminal domain of hT1R2 is required for the response to MCL. Our data suggest that MCL binds hT1R2-hT1R3 as an antagonist at neutral pH and functionally changes into an agonist at acidic pH, and we conclude this may cause its taste-modifying activity. PMID:21949380

  6. The cumulus cell layer protects the bovine maturing oocyte against fatty acid-induced lipotoxicity.

    PubMed

    Lolicato, Francesca; Brouwers, Jos F; de Lest, Chris H A van; Wubbolts, Richard; Aardema, Hilde; Priore, Paola; Roelen, Bernard A J; Helms, J Bernd; Gadella, Bart M

    2015-01-01

    Mobilization of fatty acids from adipose tissue during metabolic stress increases the amount of free fatty acids in blood and follicular fluid and is associated with impaired female fertility. In a previous report, we described the effects of the three predominant fatty acids in follicular fluid (saturated palmitate and stearate and unsaturated oleate) on oocyte maturation and quality. In the current study, the effects of elevated fatty acid levels on cumulus cells were investigated. In a dose-dependent manner, the three fatty acids induced lipid storage in cumulus cells accompanied by an enhanced immune labeling of perilipin-2, a marker for lipid droplets. Lipidomic analysis confirmed incorporation of the administered fatty acids into triglyceride, resulting in a 3- to 6-fold increase of triglyceride content. In addition, palmitate selectively induced ceramide formation, which has been implicated in apoptosis. Indeed, of the three fatty acids tested, palmitate induced reactive oxygen species formation, caspase 3 activation, and mitochondria deterioration, leading to degeneration of the cumulus cell layers. This effect could be mimicked by addition of the ceramide-C2 analog and could be inhibited by the ceramide synthase inhibitor fumonisin-B1. Interfering with the intactness of the cumulus cell layers, either by mechanical force or by palmitate treatment, resulted in enhanced uptake of lipids in the oocyte and increased radical formation. Our results show that cumulus cells act as a barrier, protecting oocytes from in vitro induced lipotoxic effects. We suggest that this protective function of the cumulus cell layers is important for the developmental competence of the oocyte. The relevance of our findings for assisted reproduction technologies is discussed.

  7. Behavior-associated Neuronal Activation After Kainic Acid-induced Hippocampal Neurotoxicity is Modulated in Time.

    PubMed

    Aguilar-Arredondo, Andrea; López-Hernández, Fernanda; García-Velázquez, Lizbeth; Arias, Clorinda; Zepeda, Angélica

    2017-02-01

    Kainic acid-induced (KA) hippocampal damage leads to neuronal death and further synaptic plasticity. Formation of aberrant as well as of functional connections after such procedure has been documented. However, the impact of such structural plasticity on cell activation along time after damage and in face of a behavioral demand has not been explored. We evaluated if the mRNA and protein levels of plasticity-related protein synaptophysin (Syp and SYP, respectively) and activity-regulated cytoskeleton-associated protein mRNA and protein levels (Arc and Arc, respectively) in the dentate gyrus were differentially modulated in time in response to a spatial-exploratory task after KA-induced hippocampal damage. In addition, we analyzed Arc+/NeuN+ immunopositive cells in the different experimental conditions. We infused KA intrahippocampally to young-adult rats and 10 or 30 days post-lesion (dpl) animals performed a hippocampus-activating spatial-exploratory task. Our results show that Syp mRNA levels significantly increase at 10dpl and return to control levels after 30dpl, whereas SYP protein levels are diminished at 10dpl, but significantly increase at 30dpl, as compared to 10dpl. Arc mRNA and protein levels are both increased at 30dpl as compared to sham. Also the number of NeuN+/Arc+ cells significantly increases at 30dpl in the group with a spatial-exploratory demand. These results provide information on the long-term modifications associated to structural plasticity and neuronal activation in the dentate gyrus after excitotoxic damage and in face of a spatial-exploratory behavior. Anat Rec, 300:425-432, 2017. © 2016 Wiley Periodicals, Inc.

  8. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase

    PubMed Central

    Ruisanchez, Éva; Dancs, Péter; Kerék, Margit; Németh, Tamás; Faragó, Bernadett; Balogh, Andrea; Patil, Renukadevi; Jennings, Brett L.; Liliom, Károly; Malik, Kafait U.; Smrcka, Alan V.; Tigyi, Gabor; Benyó, Zoltán

    2014-01-01

    Lysophosphatidic acid (LPA) has been implicated as a mediator of several cardiovascular functions, but its potential involvement in the control of vascular tone is obscure. Here, we show that both LPA (18:1) and VPC31143 (a synthetic agonist of LPA1–3 receptors) relax intact mouse thoracic aorta with similar Emax values (53.9 and 51.9% of phenylephrine-induced precontraction), although the EC50 of LPA- and VPC31143-induced vasorelaxations were different (400 vs. 15 nM, respectively). Mechanical removal of the endothelium or genetic deletion of endothelial nitric oxide synthase (eNOS) not only diminished vasorelaxation by LPA or VPC31143 but converted it to vasoconstriction. Freshly isolated mouse aortic endothelial cells expressed LPA1, LPA2, LPA4 and LPA5 transcripts. The LPA1,3 antagonist Ki16425, the LPA1 antagonist AM095, and the genetic deletion of LPA1, but not that of LPA2, abolished LPA-induced vasorelaxation. Inhibition of the phosphoinositide 3 kinase–protein kinase B/Akt pathway by wortmannin or MK-2206 failed to influence the effect of LPA. However, pharmacological inhibition of phospholipase C (PLC) by U73122 or edelfosine, but not genetic deletion of PLCε, abolished LPA-induced vasorelaxation and indicated that a PLC enzyme, other than PLCε, mediates the response. In summary, the present study identifies LPA as an endothelium-dependent vasodilator substance acting via LPA1, PLC, and eNOS.—Ruisanchez, É., Dancs, P., Kerék, M., Németh, T., Faragó, B., Balogh, A., Patil, R., Jennings, B. L., Liliom, K., Malik, K. U., Smrcka, A. V., Tigyi, G., Benyó, Z. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase. PMID:24249637

  9. The influence of pretreatment with ghrelin on the development of acetic-acid-induced colitis in rats.

    PubMed

    Maduzia, D; Matuszyk, A; Ceranowicz, D; Warzecha, Z; Ceranowicz, P; Fyderek, K; Galazka, K; Dembinski, A

    2015-12-01

    Ghrelin has been primarily shown to exhibit protective and therapeutic effect in the gut. Pretreatment with ghrelin inhibits the development of acute pancreatitis and accelerates pancreatic recovery in the course of this disease. In the stomach, ghrelin reduces gastric mucosal damage induced by ethanol, stress or alendronate, as well as accelerates the healing of acetic acid-induced gastric and duodenal ulcer. The aim of present studies was to investigate the effect of pretreatment with ghrelin on the development of acetic acid-induced colitis. Studies have been performed on male Wistar rats. Animals were treated intraperitoneally with saline (control) or ghrelin (4, 8 or 16 nmol/kg/dose). Saline or ghrelin was given twice: 8 and 1 h before induction of colitis. Colitis was induced by a rectal enema with 1 ml of 4% solution of acetic acid and the severity of colitis was assessed 1 or 24 hours after induction of inflammation. Rectal administration of acetic acid induced colitis in all animals. Damage of colonic wall was seen at the macroscopic and microscopic level. This effect was accompanied by a reduction in colonic blood flow and mucosal DNA synthesis. Moreover, induction of colitis significantly increased mucosal concentration of pro-inflammatory interleukin-1β (IL-1β), activity of myeloperoxidase and concentration of malondialdehyde (MDA). Mucosal activity of superoxide dismutase (SOD) was reduced. Pretreatment with ghrelin reduced the area and grade of mucosal damage. This effect was accompanied by an improvement of blood flow, DNA synthesis and SOD activity in colonic mucosa. Moreover, ghrelin administration reduced mucosal concentration of IL-1β and MDA, as well as decreased mucosal activity of myeloperoxidase. Administration of ghrelin protects the large bowel against the development of the acetic acid-induced colitis and this effect seems to be related to the ghrelin-evoked anti-inflammatory and anti-oxidative effects.

  10. Effect of CMC Molecular Weight on Acid-Induced Gelation of Heated WPI-CMC Soluble Complex.

    PubMed

    Huan, Yan; Zhang, Sha; Vardhanabhuti, Bongkosh

    2016-02-01

    Acid-induced gelation properties of heated whey protein isolate (WPI) and carboxymethylcellulose (CMC) soluble complex were investigated as a function of CMC molecular weight (270, 680, and 750 kDa) and concentrations (0% to 0.125%). Heated WPI-CMC soluble complex with 6% protein was made by heating biopolymers together at pH 7.0 and 85 °C for 30 min and diluted to 5% protein before acid-induced gelation. Acid-induced gel formed from heated WPI-CMC complexes exhibited increased hardness and decreased water holding capacity with increasing CMC concentrations but gel strength decreased at higher CMC content. The highest gel strength was observed with CMC 750 k at 0.05%. Gels with low CMC concentration showed homogenous microstructure which was independent of CMC molecular weight, while increasing CMC concentration led to microphase separation with higher CMC molecular weight showing more extensive phase separation. When heated WPI-CMC complexes were prepared at 9% protein the acid gels showed improved gel hardness and water holding capacity, which was supported by the more interconnected protein network with less porosity when compared to complexes heated at 6% protein. It is concluded that protein concentration and biopolymer ratio during complex formation are the major factors affecting gel properties while the effect of CMC molecular weight was less significant.

  11. Glucocorticoids modulate amino acid-induced translation initiation in human skeletal muscle.

    PubMed

    Liu, Zhenqi; Li, Guolian; Kimball, Scot R; Jahn, Linda A; Barrett, Eugene J

    2004-08-01

    Amino acids are unique anabolic agents in that they nutritively signal to mRNA translation initiation and serve as substrates for protein synthesis in skeletal muscle. Glucocorticoid excess antagonizes the anabolic action of amino acids on protein synthesis in laboratory animals. To examine whether excessive glucocorticoids modulate mixed amino acid-signaled translation initiation in human skeletal muscle, we infused an amino acid mixture (10% Travasol) systemically to 16 young healthy male volunteers for 6 h in the absence (n = 8) or presence (n = 8) of glucocorticoid excess (dexamethasone 2 mg orally every 6 h for 3 days). Vastus lateralis muscles were biopsied before and after amino acid infusion, and the phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1), ribosomal protein S6 kinase (p70(S6K)), and eIF2alpha and the guanine nucleotide exchange activity of eIF2B were measured. Systemic infusion of mixed amino acids significantly stimulated the phosphorylation of 4E-BP1 (P < 0.04) and p70(S6K) (P < 0.001) and the dephosphorylation of eIF2alpha (P < 0.003) in the control group. Dexamethasone treatment did not alter the basal phosphorylation state of 4E-BP1, p70(S6K), or eIF2alpha; however, it abrogated the stimulatory effect of amino acid infusion on the phosphorylation of 4E-BP1 (P = 0.31) without affecting amino acid-induced phosphorylation of p70(S6K) (P = 0.002) or dephosphorylation of eIF2alpha (P = 0.003). Neither amino acid nor dexamethasone treatment altered the guanine nucleotide exchange activity of eIF2B. We conclude that changes of amino acid concentrations within the physiological range stimulate mRNA translation by enhancing the binding of mRNA to the 43S preinitiation complex, and the activity of p70(S6K) and glucocorticoid excess blocks the former action in vivo in human skeletal muscle.

  12. Repeated citalopram administration counteracts kainic acid-induced spreading of PSA-NCAM-immunoreactive cells and loss of reelin in the adult mouse hippocampus.

    PubMed

    Jaako, Külli; Aonurm-Helm, Anu; Kalda, Anti; Anier, Kaili; Zharkovsky, Tamara; Shastin, Dmitri; Zharkovsky, Alexander

    2011-09-01

    Systemic or intracerebral administration of kainic acid in rodents induces neuronal death followed by a cascade of neuroplastic changes in the hippocampus. Kainic acid-induced neuroplasticity is evidenced by alterations in hippocampal neurogenesis, dispersion of the granule cell layer and re-organisation of mossy fibres. Similar abnormalities are observed in patients with temporal lobe epilepsy and, therefore, kainic acid-induced hippocampal neuroplasticity might mimic pathological mechanisms leading to the formation of 'epileptic brain' in patients with temporal lobe epilepsy. Previous studies have demonstrated that selective serotonin re-uptake inhibitor antidepressants might reduce the severity of seizures in epileptic patients and reduce neuronal death in laboratory animal models of kainic acid-induced neurotoxicity. In the present study, we investigated whether kainic acid-induced neuroplasticity in mice is modulated by the repeated administration of citalopram, a selective serotonin reuptake inhibitor. We found that at the histopathological level, repeated citalopram treatment counteracted the kainic acid-induced neuronal loss and dispersion of young granule neurons expressing the polysialylated neural cell adhesion molecule within the granule cell layer of the hippocampus. Citalopram also counteracted the downregulation of reelin on both mRNA and protein levels induced by kainic acid administration. Our findings indicate that repeated administration of citalopram is able to prevent kainic acid-induced abnormal brain plasticity and thereby prevent the formation of an epileptic phenotype.

  13. Hot Pepper (Capsicum spp.) protects brain from sodium nitroprusside- and quinolinic acid-induced oxidative stress in vitro.

    PubMed

    Oboh, G; Rocha, J B T

    2008-06-01

    One practical way through which free radical-mediated neurodegenerative diseases could be prevented is through the consumption of food rich in antioxidants. The ability of aqueous extracts of ripe and unripe Capsicum annum, Tepin (CAT) and Capsicum chinese, Habanero (CCH) to prevent lipid peroxidation induced by sodium nitroprusside and quinolinic acid in rat brain in vitro is assessed in this study. The aqueous extract of the peppers were prepared (1 g/20 mL). Incubating rat brain homogenates with pro-oxidant (7 microM sodium nitroprusside [222.5%] and 1 mM quinolinic acid [217.4%]) caused a significant increase (P < .05) in lipid peroxidation in rat brain homogenates. However, the aqueous extract of the peppers (4.2-16.8 mg/mL) caused a significant decrease (P < .05) in the lipid peroxidation in a dose-dependent manner. However, unripe CAT (92.5-55.2%) caused the highest inhibition of sodium nitroprusside-induced lipid peroxidation, while unripe CCH caused the least inhibition (161.0-102.1%). Furthermore, unripe CAT and CCH peppers had a significantly higher (P < .05) inhibitory effect on quinolinic acid-induced lipid peroxidation in rat brain than the ripe pepper (CAT and CCH). Therefore, the protection of the brain tissues by hot pepper depends on the total phenol content in sodium nitroprusside-induced lipid peroxidation, while ripening would reduce the protective properties of hot pepper against quinolinic acid-induced lipid peroxidation. However, unripe CAT has the highest protective properties against sodium nitroprusside- and quinolinic acid-induced lipid peroxidation in rat brain.

  14. Attenuation of kainic acid-induced status epilepticus by inhibition of endocannabinoid transport and degradation in guinea pigs.

    PubMed

    Shubina, Liubov; Aliev, Rubin; Kitchigina, Valentina

    2015-03-01

    Status epilepticus (SE) is a medical emergency associated with a high rate of mortality if not treated promptly. Exogenous and endogenous cannabinoids have been shown to possess anticonvulsant properties both in vivo and in vitro. Here we study the influence of endocannabinoid metabolism on the development of kainic acid-induced SE in guinea pigs. For this purpose, the inhibitors of endocannabinoid transport, AM404, and enzymatic (fatty acid amide hydrolase) degradation, URB597, were applied. Cannabinoid CB1 receptor antagonist, AM251, was also tested. Animal behavior as well as local electric field potentials in four structures: medial septum, hippocampus, entorhinal cortex and amygdala were analyzed when AM404 (120nmol), URB597 (4.8nmol) or AM251 (20nmol) were administrated alone or together with 0.4μg of kainic acid. All substances were injected i.c.v. AM404, URB597 or AM251 administered alone did not alter markedly local field potentials of all four studied structures in the long-term compared with their basal activity. AM404 and URB597 significantly alleviated kainic acid-induced SE, decreasing behavioral manifestations, duration of seizure events and SE in general without changing the amplitude of local field potentials. AM251 did not produce distinct effects on SE in terms of our experimental paradigm. There was no apparent change of the seizure initiation pattern when kainic acid was coadministrated with AM404, URB597 or AM251. The present study provides electrophysiologic and behavioral evidences that inhibition of endocannabinoid metabolism plays a protective role against kainic acid-induced SE and may be employed for therapeutic purposes. Further investigations of the influences of cannabinoid-related compounds on SE genesis and especially epileptogenesis are required.

  15. Genetic parameters for rennet- and acid-induced coagulation properties in milk from Swedish Red dairy cows.

    PubMed

    Gustavsson, F; Glantz, M; Poulsen, N A; Wadsö, L; Stålhammar, H; Andrén, A; Lindmark Månsson, H; Larsen, L B; Paulsson, M; Fikse, W F

    2014-01-01

    Milk coagulation is an important processing trait, being the basis for production of both cheese and fermented products. There is interest in including technological properties of these products in the breeding goal for dairy cattle. The aim of the present study was therefore to estimate genetic parameters for milk coagulation properties, including both rennet- and acid-induced coagulation, in Swedish Red dairy cattle using genomic relationships. Morning milk samples and blood samples were collected from 395 Swedish Red cows that were selected to be as genetically unrelated as possible. Using a rheometer, milk samples were analyzed for rennet- and acid-induced coagulation properties, including gel strength (G'), coagulation time, and yield stress (YS). In addition to the technological traits, milk composition was analyzed. A binary trait was created to reflect that milk samples that had not coagulated 40min after rennet addition were considered noncoagulating milk. The cows were genotyped by using the Illumina BovineHD BeadChip (Illumina Inc., San Diego, CA). Almost 600,000 markers remained after quality control and were used to construct a matrix of genomic relationships among the cows. Multivariate models including fixed effects of herd, lactation stage, and parity were fitted using the ASReml software to obtain estimates of heritabilities and genetic and phenotypic correlations. Heritability estimates (h(2)) for G' and YS in rennet and acid gels were found to be high (h(2)=0.38-0.62) and the genetic correlations between rennet-induced and acid-induced coagulation properties were weak but favorable, with the exception of YSrennet with G'acid and YSacid, both of which were strong. The high heritability (h(2)=0.45) for milk coagulating ability expressed as a binary trait suggests that noncoagulation could be eliminated through breeding. Additionally, the results indicated that the current breeding objective could increase the frequency of noncoagulating milk and

  16. Helicases in antiviral immunity: dual properties as sensors and effectors

    PubMed Central

    Ahmad, Sadeem; Hur, Sun

    2015-01-01

    Many helicases have a unique ability to couple cognate RNA binding to ATP hydrolysis, which can induce a large conformational change that affects its interaction with RNA, position along RNA, or oligomeric state. A growing number of these helicases contribute to the innate immune system, either as sensors that detect foreign nucleic acids and/or as effectors that directly participate in the clearance of such foreign species. We here discuss a few examples, including retinoic acid-inducible gene-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and Dicer, focusing on their dual functions as both sensors and effectors. We will also discuss the closely related, but less understood helicases, laboratory of genetics and physiology 2 (LGP2) and Dicer-related helicase-1 and -3 (DRH-1 and -3). PMID:26410598

  17. Palmitic acid but not palmitoleic acid induces insulin resistance in a human endothelial cell line by decreasing SERCA pump expression.

    PubMed

    Gustavo Vazquez-Jimenez, J; Chavez-Reyes, Jesus; Romero-Garcia, Tatiana; Zarain-Herzberg, Angel; Valdes-Flores, Jesus; Manuel Galindo-Rosales, J; Rueda, Angelica; Guerrero-Hernandez, Agustin; Olivares-Reyes, J Alberto

    2016-01-01

    Palmitic acid is a negative regulator of insulin activity. At the molecular level, palmitic acid reduces insulin stimulated Akt Ser473 phosphorylation. Interestingly, we have found that incubation with palmitic acid of human umbilical vein endothelial cells induced a biphasic effect, an initial transient elevation followed by a sustained reduction of SERCA pump protein levels. However, palmitic acid produced a sustained inhibition of SERCA pump ATPase activity. Insulin resistance state appeared before there was a significant reduction of SERCA2 expression. The mechanism by which palmitic acid impairs insulin signaling may involve endoplasmic reticulum stress, because this fatty acid induced activation of both PERK, an ER stress marker, and JNK, a kinase associated with insulin resistance. None of these effects were observed by incubating HUVEC-CS cells with palmitoleic acid. Importantly, SERCA2 overexpression decreased the palmitic acid-induced insulin resistance state. All these results suggest that SERCA pump might be the target of palmitic acid to induce the insulin resistance state in a human vascular endothelial cell line. Importantly, these data suggest that HUVEC-CS cells respond to palmitic acid-exposure with a compensatory overexpression of SERCA pump within the first hour, which eventually fades out and insulin resistance prevails.

  18. Allicin Alleviates Inflammation of Trinitrobenzenesulfonic Acid-Induced Rats and Suppresses P38 and JNK Pathways in Caco-2 Cells

    PubMed Central

    Li, Chen; Lun, Weijian; Zhao, Xinmei; Lei, Shan; Guo, Yandong; Ma, Jiayi

    2015-01-01

    Background. Allicin has anti-inflammatory, antioxidative and proapoptotic properties. Aims. To evaluate the effects and investigate the mechanism of allicin on trinitrobenzenesulfonic acid-induced colitis, specifically with mesalazine or sulfasalazine. Methods. 80 rats were divided equally into 8 groups: control; trinitrobenzenesulfonic acid; allicin prevention; allicin; mesalazine; sulfasalazine; allicin + sulfasalazine, and mesalazine + allicin. Systemic and colonic inflammation parameters were analysed. In addition, protein and culture medium of Caco-2 cells treated with various concentrations of IL-1β or allicin were collected for investigation of IL-8, NF-κB p65 P38, ERK, and JNK. One-way ANOVA and Kruskal-Wallis H test were used for parametric and nonparametric tests, respectively. Results. Allicin reduced the body weight loss of trinitrobenzenesulfonic acid-induced rats, histological score, serum TNF-α and IL-1β levels, and colon IL-1β mRNA level and induced serum IL-4 level, particularly in combination with mesalazine. In addition, 1 ng/mL IL-1β stimulated the P38, ERK, and JNK pathways, whereas pretreatment with allicin depressed this phenomenon, except for the ERK pathway. Conclusions. The inflammation induced by trinitrobenzenesulfonic acid is mitigated significantly by allicin treatment, particularly combined with mesalazine. Allicin inhibits the P38 and JNK pathways and the expression of NF-κB which explained the potential anti-inflammatory mechanisms of allicin. PMID:25729217

  19. Protective effect of Agave americana Linn. leaf extract in acetic acid-induced ulcerative colitis in rats

    PubMed Central

    Mannasaheb, Basheerahmed A.A.; Kulkarni, Preeti V.; Sangreskopp, Mashood Ahmed; Savant, Chetan; Mohan, Anjana

    2015-01-01

    Introduction: Natural plants always provide core compounds for new drug development. In the present life and food style, inflammatory bowel disease has become common and needs a lead compound for its drug development. Aim: To evaluate the effect of Agave americana Linn. leaf extract in acetic acid-induced ulcerative colitis in rats based on its traditional anti-inflammatory use. Materials and Methods: Male Wistar rats were pretreated with A. americana leaf extract in the dose of 200 and 400 mg/kg p.o. daily for 7 days. On 8th day, 2 ml of 4% v/v acetic acid in saline was instilled into rats’ rectum. Prednisolone was used as standard drug and it was administered on the day of acetic acid instillation and continued for 3 days. Extract treatment was continued till 11th day. Body weight, ulcer score, colonic muscle contraction, antioxidant activity and histopathology were studied. Statistical analysis was performed using Parametric one-way analysis of variance followed by Tukey's posttest. Results: A. americana have retained total body weight significantly (P < 0.01) and decreased colon weight/length ratio. Extract have shown a significant decrease (P < 0.001) in ulcer scores, myeloperoxidase, lipid peroxidase activity. Further, extract have shown significant improvement in colonic muscle contraction, histopathology of colon etc., which is comparable with standard drug. Conclusion: A. americana possess protective effect against acetic acid-induced colitis in rats. PMID:26730148

  20. Nicotinic acid induces secretion of prostaglandin D2 in human macrophages: an in vitro model of the niacin flush.

    PubMed

    Meyers, C Daniel; Liu, Paul; Kamanna, Vaijinath S; Kashyap, Moti L

    2007-06-01

    Nicotinic acid is a safe, broad-spectrum lipid agent shown to prevent cardiovascular disease, yet its widespread use is limited by the prostaglandin D2 (PGD2) mediated niacin flush. Previous research suggests that nicotinic acid-induced PGD2 secretion is mediated by the skin, but the exact cell type remains unclear. We hypothesized that macrophages are a source of nicotinic acid-induced PGD2 secretion and performed a series of experiments to confirm this. Nicotinic acid (0.1-3 mM) induced PGD2 secretion in cultured human macrophages, but not monocytes or endothelial cells. The PGD2 secretion was dependent on the concentration of nicotinic acid and the time of exposure. Nicotinuric acid, but not nicotinamide, also induced PGD2 secretion. Pre-incubation of the cells with aspirin (100 microM) entirely prevented the nicotinic acid effects on PGD2 secretion. The PGD2 secreting effects of nicotinic acid were additive to the effects of the calcium ionophore A23187 (6 microM), but were independent of extra cellular calcium. These findings, combined with recent in vivo work, provide evidence that macrophages play a significant role in mediating the niacin flush and may lead to better strategies to eliminate this limiting side effect.

  1. Oral administration of omega-7 palmitoleic acid induces satiety and the release of appetite-related hormones in male rats.

    PubMed

    Yang, Zhi-Hong; Takeo, Jiro; Katayama, Masashi

    2013-06-01

    We have analyzed the effect of palmitoleic acid on short-term food intake in male rats. Administration of omega-7 palmitoleic acid by oral gavage significantly decreased food intake compared to palmitic acid, omega-9 oleic acid, or a vehicle control. Palmitoleic acid exhibited a dose-dependent effect in this context and did not cause general malaise. A triglyceride form of palmitoleate also decreased food intake, whereas olive oil, which is rich in oleic acid, did not. Palmitoleic acid accumulated within the small intestine in a dose-dependent fashion and elevated levels of the satiety hormone cholecystokinin (CCK). Both protein and mRNA levels of CCK were affected in this context. The suppression of food intake by palmitoleic acid was attenuated by intravenous injection of devazepide, a selective peripheral CCK receptor antagonist. Palmitoleic acid did not alter the expression of peroxisome proliferator-activated receptor alpha (PPARα) target genes, and a PPARα antagonist did not affect palmitoleic acid-induced satiety. This suggests that the PPARα pathway might not be involved in suppressing food intake in response to palmitoleic acid. We have shown that orally administered palmitoleic acid induced satiety, enhanced the release of satiety hormones in rats.

  2. Evidence for the involvement of GPR40 and NADPH oxidase in palmitic acid-induced superoxide production and insulin secretion.

    PubMed

    Graciano, Maria Fernanda; Valle, Maíra Mello; Curi, Rui; Carpinelli, Angelo Rafael

    2013-01-01

    G protein coupled receptor 40 (GPR40) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex have been shown to be involved in the fatty acid amplification of glucose-stimulated insulin secretion (GSIS). The effect of palmitic acid on superoxide production and insulin secretion by INS-1E cells and the possible involvement of GPR40 and NADPH oxidase in these processes were examined in this study. Cells were incubated during 1 h with palmitic acid in low and high glucose concentrations, a GPR40 agonist (GW9508) and inhibitors of NADPH oxidase (diphenyleneiodonium, DPI) and PKC (calphostin C). GW9508 induced superoxide production at 2.8 and 5.6 mM glucose concentrations and stimulated insulin secretion at 16.7 mM glucose concentration involving both PKC and NADPH oxidase activation. Palmitic acid induced superoxide production through NADPH oxidase and GPR40-dependent pathways and the stimulation of insulin secretion in the presence of a high glucose concentration was reduced by knockdown of GPR40 using siRNA. Our results suggest that palmitic acid induces superoxide production and potentiates GSIS through NADPH oxidase and GPR40 pathways in pancreatic ? cells.

  3. Effect of capsaicin and cimetidine on the healing of acetic acid induced gastric ulceration in the rat.

    PubMed Central

    Kang, J Y; Teng, C H; Chen, F C

    1996-01-01

    BACKGROUND: Capsaicin protects the gastric mucosa against experimental injury while capsaicin desensitisation reduces the rate of gastric ulcer healing. The effect of exogenous capsaicin on gastric ulcer healing has not to date been reported. AIM/METHOD: To investigate the effect of capsaicin, cimetidine, and in combination, given intragastrically in the healing of acetic acid induced chronic gastric ulcer in the rat. Treatment started immediately after ulcer induction. RESULTS: At the end of one week, capsaicin, cimetidine, and in combination increased ulcer healing but the effect of combined treatment was less than that of capsaicin alone. In an in vivo gastric chamber preparation, capsaicin increased, while cimetidine decreased, gastric mucosal blood flow measured by laser Doppler flowmetry. A dose response effect in reduction of gastric mucosal blood flow could be demonstrated for cimetidine. The gastric hyperaemic effect of capsaicin was blunted by prior administration of cimetidine. In contrast, capsaicin had no effect on gastric acid secretion and its addition to cimetidine did not affect the acid suppressant effect of the latter. CONCLUSIONS: Capsaicin promotes the healing of acetic acid induced gastric ulcer, probably by its gastric hyperaemic effect. Although cimetidine also promotes ulcer healing due to its inhibitory effect on acid secretion it may have an antagonistic effect on the gastric ulcer healing effect of capsaicin by virtue of inhibition of gastric hyperaemia. PMID:8984019

  4. MTHFR gene C677T mutation and ACE gene I/D polymorphism in Turkish patients with osteoarthritis.

    PubMed

    Inanir, Ahmet; Yigit, Serbulent; Tural, Sengul; Cecen, Osman; Yildirim, Eren

    2013-01-01

    Osteoarthritis is a degenerative joint disorder resulting in destruction of articular cartilage, osteophyte formation, and subchondral bone sclerosis. In recent years, numerous genetic factors have been identified and implicated in osteoarthritis. The aim of the current study was to examine the influence of methylenetetrahydrofolate reductase (MTHFR) gene C677T mutation and angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) variations on the risk of osteoarthritis. Genomic DNA is obtained from 421 persons (221 patients with osteoarthritis and 200 healthy controls). ACE gene I/D polymorphism genotypes were determined using polymerase chain reaction using I and D allele-specific primers. The MTHFR C677T mutation was analyzed by polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) methods. We found significant difference between the groups with respect to both ACE and MTHFR genotype distributions (p< 0.001, p< 0.001 respectively). Our study suggests that ACE gene DD genotype and MTHFR gene CC genotype could be used as genetic markers in osteoarthritis in Turkish study populations.

  5. Mumps virus-induced innate immune responses in mouse Sertoli and Leydig cells.

    PubMed

    Wu, Han; Shi, Lili; Wang, Qing; Cheng, Lijing; Zhao, Xiang; Chen, Qiaoyuan; Jiang, Qian; Feng, Min; Li, Qihan; Han, Daishu

    2016-01-18

    Mumps virus (MuV) infection frequently causes orchitis and impairs male fertility. However, the mechanisms underlying the innate immune responses to MuV infection in the testis have yet to be investigated. This study showed that MuV induced innate immune responses in mouse Sertoli and Leydig cells through TLR2 and retinoic acid-inducible gene I (RIG-I) signaling, which result in the production of proinflammatory cytokines and chemokines, including TNF-α, IL-6, MCP-1, CXCL10, and type 1 interferons (IFN-α and IFN-β). By contrast, MuV did not induce the cytokine production in male germ cells. In response to MuV infection, Sertoli cells produced higher levels of proinflammatory cytokines and chemokines but lower levels of type 1 IFNs than Leydig cells did. The MuV-induced cytokine production by Sertoli and Leydig cells was significantly reduced by the knockout of TLR2 or the knockdown of RIG-I signaling. The local injection of MuV into the testis triggered the testicular innate immune responses in vivo. Moreover, MuV infection suppressed testosterone synthesis by Leydig cells. This is the first study examining the innate immune responses to MuV infection in testicular cells. The results provide novel insights into the mechanisms underlying the MuV-induced innate immune responses in the testis.

  6. Mumps virus-induced innate immune responses in mouse Sertoli and Leydig cells

    PubMed Central

    Wu, Han; Shi, Lili; Wang, Qing; Cheng, Lijing; Zhao, Xiang; Chen, Qiaoyuan; Jiang, Qian; Feng, Min; Li, Qihan; Han, Daishu

    2016-01-01

    Mumps virus (MuV) infection frequently causes orchitis and impairs male fertility. However, the mechanisms underlying the innate immune responses to MuV infection in the testis have yet to be investigated. This study showed that MuV induced innate immune responses in mouse Sertoli and Leydig cells through TLR2 and retinoic acid-inducible gene I (RIG-I) signaling, which result in the production of proinflammatory cytokines and chemokines, including TNF-α, IL-6, MCP-1, CXCL10, and type 1 interferons (IFN-α and IFN-β). By contrast, MuV did not induce the cytokine production in male germ cells. In response to MuV infection, Sertoli cells produced higher levels of proinflammatory cytokines and chemokines but lower levels of type 1 IFNs than Leydig cells did. The MuV-induced cytokine production by Sertoli and Leydig cells was significantly reduced by the knockout of TLR2 or the knockdown of RIG-I signaling. The local injection of MuV into the testis triggered the testicular innate immune responses in vivo. Moreover, MuV infection suppressed testosterone synthesis by Leydig cells. This is the first study examining the innate immune responses to MuV infection in testicular cells. The results provide novel insights into the mechanisms underlying the MuV-induced innate immune responses in the testis. PMID:26776505

  7. Functional Roles of Pattern Recognition Receptors That Recognize Virus Nucleic Acids in Human Adipose-Derived Mesenchymal Stem Cells

    PubMed Central

    Wang, Fangchao; Yang, Can; Liu, Guoyan; Song, Xiangfeng

    2016-01-01

    Human adipose-derived mesenchymal stem cells (hAD-MSCs) are mesenchymal stem cells with the capability to modulate immune responses. Evidence showing that hAD-MSCs could mediate innate immune responses through pattern recognition receptors (PRRs) is increasing. However, the roles of PRRs in regulating the innate sensing of virus nucleic acids (RNA and DNA) in hAD-MSCs have not yet been investigated. This study focused on the abundant expression of PRRs, including Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I), which recognize viral RNA, and gamma-interferon inducible protein 16 (IFI16), which recognizes viral DNA in hAD-MSCs. Poly(I:C), a synthetic dsRNA analogy, activated TLR3 and RIG-I and induced the expression of type I interferons (IFN-α/β) and antivirus proteins, including IFN-stimulating gene 15, 2′5′-oligoadenylate synthetase, and Mx GTPase 1 in hAD-MSCs, which were attenuated by the knockdown of each TLR3 or RIG-I. Synthetic herpes simplex viral DNA (HSV60) activated IFI16 and induced the expression of IFN-α/β and antivirus proteins in hAD-MSCs, which were inhibited by the knockdown of IFI16. Both poly(I:C) and HSV60 induced the expression of IFN-α/β through the phosphorylation of IFN-regulatory factor 3. All these results indicated that PRRs recognizing virus nucleic acids were expressed and can mediate antivirus responses in hAD-MSCs. PMID:28105439

  8. Functional Roles of Pattern Recognition Receptors That Recognize Virus Nucleic Acids in Human Adipose-Derived Mesenchymal Stem Cells.

    PubMed

    Yu, Lili; Xu, Yongtao; Wang, Fangchao; Yang, Can; Liu, Guoyan; Song, Xiangfeng

    2016-01-01

    Human adipose-derived mesenchymal stem cells (hAD-MSCs) are mesenchymal stem cells with the capability to modulate immune responses. Evidence showing that hAD-MSCs could mediate innate immune responses through pattern recognition receptors (PRRs) is increasing. However, the roles of PRRs in regulating the innate sensing of virus nucleic acids (RNA and DNA) in hAD-MSCs have not yet been investigated. This study focused on the abundant expression of PRRs, including Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I), which recognize viral RNA, and gamma-interferon inducible protein 16 (IFI16), which recognizes viral DNA in hAD-MSCs. Poly(I:C), a synthetic dsRNA analogy, activated TLR3 and RIG-I and induced the expression of type I interferons (IFN-α/β) and antivirus proteins, including IFN-stimulating gene 15, 2'5'-oligoadenylate synthetase, and Mx GTPase 1 in hAD-MSCs, which were attenuated by the knockdown of each TLR3 or RIG-I. Synthetic herpes simplex viral DNA (HSV60) activated IFI16 and induced the expression of IFN-α/β and antivirus proteins in hAD-MSCs, which were inhibited by the knockdown of IFI16. Both poly(I:C) and HSV60 induced the expression of IFN-α/β through the phosphorylation of IFN-regulatory factor 3. All these results indicated that PRRs recognizing virus nucleic acids were expressed and can mediate antivirus responses in hAD-MSCs.

  9. Yeast acetic acid-induced programmed cell death can occur without cytochrome c release which requires metacaspase YCA1.

    PubMed

    Guaragnella, Nicoletta; Bobba, Antonella; Passarella, Salvatore; Marra, Ersilia; Giannattasio, Sergio

    2010-01-04

    To investigate the role of cytochrome c (cyt c) release in yeast acetic acid-induced programmed cell death (AA-PCD), wild type (wt) and cells lacking metacaspase (Deltayca1), cytochrome c (Deltacyc1,7) and both (Deltacyc1,7Deltayca1) were compared for AA-PCD occurrence, hydrogen peroxide (H(2)O(2)) production and caspase activity. AA-PCD occurs in Deltacyc1,7 and Deltacyc1,7Deltayca1 cells slower than in wt, but similar to that in Deltayca1 cells, in which no cytochrome c release occurs. Both H(2)O(2) production and caspase activation occur in these cells with early and extra-activation in Deltacyc1,7 cells. We conclude that alternative death pathways can be activated in yeast AA-PCD, one dependent on cyt c release, which requires YCA1, and the other(s) independent on it.

  10. Combinatorial localized dissolution analysis: Application to acid-induced dissolution of dental enamel and the effect of surface treatments.

    PubMed

    Parker, Alexander S; Al Botros, Rehab; Kinnear, Sophie L; Snowden, Michael E; McKelvey, Kim; Ashcroft, Alexander T; Carvell, Mel; Joiner, Andrew; Peruffo, Massimo; Philpotts, Carol; Unwin, Patrick R

    2016-08-15

    A combination of scanning electrochemical cell microscopy (SECCM) and atomic force microscopy (AFM) is used to quantitatively study the acid-induced dissolution of dental enamel. A micron-scale liquid meniscus formed at the end of a dual barrelled pipette, which constitutes the SECCM probe, is brought into contact with the enamel surface for a defined period. Dissolution occurs at the interface of the meniscus and the enamel surface, under conditions of well-defined mass transport, creating etch pits that are then analysed via AFM. This technique is applied to bovine dental enamel, and the effect of various treatments of the enamel surface on acid dissolution (1mM HNO3) is studied. The treatments investigated are zinc ions, fluoride ions and the two combined. A finite element method (FEM) simulation of SECCM mass transport and interfacial reactivity, allows the intrinsic rate constant for acid-induced dissolution to be quantitatively determined. The dissolution of enamel, in terms of Ca(2+) flux ( [Formula: see text] ), is first order with respect to the interfacial proton concentration and given by the following rate law: [Formula: see text] , with k0=0.099±0.008cms(-1). Treating the enamel with either fluoride or zinc ions slows the dissolution rate, although in this model system the partly protective barrier only extends around 10-20nm into the enamel surface, so that after a period of a few seconds dissolution of modified surfaces tends towards that of native enamel. A combination of both treatments exhibits the greatest protection to the enamel surface, but the effect is again transient.

  11. Involvement of cyclooxygenase-1, prostaglandin E2 and EP1 receptors in acid-induced HCO3- secretion in stomach.

    PubMed

    Takeuchi, K; Aihara, E; Sasaki, Y; Nomura, Y; Ise, F

    2006-12-01

    We investigated the cyclooxygenase (COX) isoforms as well as prostaglandin E receptor EP subtypes responsible for acid-induced gastric HCO(3)(-) secretion in rats and EP receptor-knockout (-/-) mice. Under urethane anesthesia, a chambered stomach (in the presence of omeprazole) was perfused with saline, and HCO(3)(-) secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. Mucosal acidification was achieved by exposing the stomach for 10 min to 50 or 100 mM HCl. Acidification of the mucosa increased the secretion of HCO(3)(-) in the stomach of both rats and WT mice, in an indomethacin-inhibitable manner. The acid-induced gastric HCO(3)(-) secretion was inhibited by prior administration of indomethacin and SC-560 but not rofecoxib in rats and mice. Acidification increased the PGE(2) content of the rat stomach, and this response was significantly attenuated by indomethacin and SC-560 but not rofecoxib. This response was also attenuated by ONO-8711 (EP1 antagonist) but not AE3-208 (EP4 antagonist) in rats and disappeared in EP1 (-/-) but not EP3 (-/-) mice. PGE(2) increased gastric HCO(3)(-) secretion in both rats and WT mice, and this action was inhibited by ONO-8711 and disappeared in EP1 (-/-) but not EP3 (-/-) mice. These results support a mediator role for endogenous PGs in the gastric response induced by mucosal acidification and clearly indicate that the enzyme responsible for production of PGs in this process is COX-1. They further show that the presence of EP1 receptors is essential for the increase in the secretion of HCO(3)(-) in response to mucosal acidification in the stomach.

  12. Galantamine potentiates the protective effect of rofecoxib and caffeic acid against intrahippocampal Kainic acid-induced cognitive dysfunction in rat.

    PubMed

    Kumar, Anil; Prakash, Atish; Pahwa, Deeksha

    2011-05-30

    Role of neuroinflammatory mediators particularly cyclooxygenase (COX), lipoxygenase (LOX), have been well suggested in the pathophysiology of neurodegenerative disorders. Rofecoxib is a selective cyclooxygenase 2 enzymes belongs to non-steroidal anti-inflammatory drug, commonly called as coxibs. Whereas, caffeic acid (3,4-dihydroxycinnamic acid) is one of the natural phenolic compounds and reported to inhibit 5-lipoxygenase (5-LOX) activity as one of mechanisms. Present study has been designed to investigate the effects of rofecoxib, caffeic acid and its potentiation by galantamine against intrahippocampal kainic acid-induced cognitive impairment, oxidative damage and mitochondrial respiratory enzyme alterations in rats. Kainic acid (KA) was administrated in the hippocampus region of rat brain. Various behavioral (locomotor activity and memory performances were assessed by using actophotometer and Morris water maze respectively) followed by oxidative stress, mitochondrial enzyme complex were assessed. Intrahippocampal administration of KA significantly impaired locomotor activity, memory performance, mitochondrial enzyme complexes and caused oxidative stress as compared to sham treatment. Rofecoxib (5 and 10mg/kg), caffeic acid (5 and 10mg/kg), Gal (2.5 and 5mg/kg) treatment for 14 days significantly improved locomotor activity, memory retention and oxidative defense (as evidenced by decrease lipid peroxidation, nitrite, increased superoxide dismutase activity and redox ratio) in hippocampus. Besides, alterations in the levels of mitochondrial enzymes and acetylcholine esterase enzyme were significantly restored by rofecoxib and caffeic acid as compared to control. Further, combination of rofecoxib (5mg/kg) with caffeic acid (5mg/kg) and lower dose of gal (2.5mg/kg) with rofecoxib (5mg/kg) treatments significantly potentiated their protective effect which was significant as compared to their effect per se. The results of the present study suggest that galantamine

  13. Olodaterol attenuates citric acid-induced cough in naïve and ovalbumin-sensitized and challenged guinea pigs.

    PubMed

    Wex, Eva; Bouyssou, Thierry

    2015-01-01

    Excessive coughing is a common feature of airway diseases. Different G-protein coupled receptors, including β2-adrenergic receptors (β2-AR), have been implicated in the molecular mechanisms underlying the cough reflex. However, the potential antitussive property of β2-AR agonists in patients with respiratory disease is a matter of ongoing debate. The aim of our study was to test the efficacy of the long-acting β2-AR agonist olodaterol with regard to its antitussive property in a pre-clinical model of citric acid-induced cough in guinea pigs and to compare the results to different clinically relevant β2-AR agonists. In our study β2-AR agonists were intratracheally administered, as dry powder, into the lungs of naïve or ovalbumin-sensitized guinea pigs 15 minutes prior to induction of cough by exposure to citric acid. Cough events were counted over 15 minutes during the citric acid exposure. Olodaterol dose-dependently inhibited the number of cough events in naïve and even more potently and with a greater maximal efficacy in ovalbumin-sensitized guinea pigs (p < 0.01). Formoterol and salmeterol showed a trend towards reducing cough. On the contrary, indacaterol demonstrated pro-tussive properties as it significantly increased the number of coughs, both in naïve and ovalbumin-sensitized animals (p < 0.001). In conclusion, olodaterol, at doses eliciting bronchodilation, showed antitussive properties in a model of citric acid-induced cough in naïve and ovalbumin-sensitized guinea pigs. This is in agreement with pre-clinical and clinical studies showing antitussive efficacy of β2-AR agonists. Indacaterol increased the number of coughs in this model, which concurs with clinical data where a transient cough has been observed after indacaterol inhalation. While the antitussive properties of β2-AR agonists can be explained by their ability to lead to the cAMP-induced hyperpolarization of the neuron membrane thereby inhibiting sensory nerve activation and the

  14. Leucine-Rich Repeat Kinase 2 Modulates Retinoic Acid-Induced Neuronal Differentiation of Murine Embryonic Stem Cells

    PubMed Central

    Schulz, Cathrin; Paus, Marie; Frey, Katharina; Schmid, Ramona; Kohl, Zacharias; Mennerich, Detlev; Winkler, Jürgen; Gillardon, Frank

    2011-01-01

    Background Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson's disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment. Methodology/Principal Findings In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/− cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture. Conclusion/Significance Parkinson's disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases. PMID:21695257

  15. The effect of nedocromil sodium, sodium cromoglycate and codeine phosphate on citric acid-induced cough in dogs.

    PubMed Central

    Jackson, D. M.

    1988-01-01

    1. The effects of nedocromil sodium, sodium cromoglycate and codeine phosphate on citric acid-induced cough have been studied in conscious tracheostomised dogs. 2. Nedocromil sodium (approximately 15 mg given as an aerosol) and codeine phosphate (5 mg kg-1, i.v.) significantly increased the time to the first cough when dogs were challenged with citric acid aerosol. The mean number of coughs in the initial period of coughing fell after treatment of dogs with nedocromil sodium or with codeine phosphate, but this reduction in mean cough number was not statistically significant. 3. Neither sodium cromoglycate (approximately 15 mg given as an aerosol) nor saline had significant effect on a citric acid challenge. 4. It is concluded that nedocromil sodium, but not sodium cromoglycate, possesses an anti-tussive action that may result from inhibition of sensory nerve activity in the lung. Nedocromil sodium may prove useful in the treatment of unproductive cough in situations where the use of a centrally-acting antitussive is undesirable. PMID:2836011

  16. Biocontrol agents-mediated suppression of oxalic acid induced cell death during Sclerotinia sclerotiorum-pea interaction.

    PubMed

    Jain, Akansha; Singh, Akanksha; Singh, Surendra; Sarma, Birinchi Kumar; Singh, Harikesh Bahadur

    2015-05-01

    Oxalic acid (OA) is an important pathogenic factor during early Sclerotinia sclerotiorum-host interaction and might work by reducing hydrogen peroxide production (H2 O2 ). In the present investigation, oxalic acid-induced cell death in pea was studied. Pea plants treated with biocontrol agents (BCAs) viz., Pseudomonas aeruginosa PJHU15, Bacillus subtilis BHHU100, and Trichoderma harzianum TNHU27 either singly and/or in consortium acted on S. sclerotiorum indirectly by enabling plants to inhibit the OA-mediated suppression of oxidative burst via induction of H2 O2 . Our results showed that BCA treated plants upon treatment with culture filtrate of the pathogen, conferred the resistance via. significantly decreasing relative cell death of pea against S. sclerotiorum compared to control plants without BCA treatment but treated with the culture filtrate of the pathogen. The results obtained from the present study indicate that the microbes especially in consortia play significant role in protection against S. sclerotiorum by modulating oxidative burst and partially enhancing tolerance by increasing the H2 O2 generation, which is otherwise suppressed by OA produced by the pathogen.

  17. Rheological and physical properties of camel and cow milk gels enriched with phosphate and calcium during acid-induced gelation.

    PubMed

    Kamal, Mohammad; Foukani, Mohammed; Karoui, Romdhane

    2017-02-01

    The rheological properties of acid-induced coagulation of camel and cow milk gels following the addition of calcium chloride (CaCl2) and hydrogen phosphate dehydrate (Na2HPO4*2H2O) were investigated using a dynamic low amplitude oscillatory rheology. For a considered condition, the final values of storage modulus (G') and loss modulus (G″) of camel milk gels were significantly lower than those of cow milk gels. The increase of the added CaCl2 levels improved significantly the gelation properties of camel and cow milk gels, since a reduction in the gelation time and an increase in the gel firmness were observed. Following the addition of Na2HPO4*2H2O at 10 and 20 mM, no significant effect on the gelation rate and the firmness of camel milk gels was observed, while, a significant decrease in the gelation rate and firmness were observed for cow milk gels.

  18. Protective Effect of Cod (Gadus macrocephalus) Skin Collagen Peptides on Acetic Acid-Induced Gastric Ulcer in Rats.

    PubMed

    Niu, Huina; Wang, Zhicong; Hou, Hu; Zhang, Zhaohui; Li, Bafang

    2016-07-01

    This research was performed to explore the protective effect of cod skin collagen peptides (CCP) on gastric ulcer induced by acetic acid. The CCP were fractionated into low molecular CCP (LMCCP, Mw < 3 kDa) and high molecular CCP (HMCCP, Mw > 3 kDa). In HMCCP and LMCCP, glycine of accounted for about one-third of the total amino acids without cysteine and tryptophan, and hydrophobic amino acids accounted for about 50%. After 21 d CCP treatment (60 or 300 mg/kg, p.o./daily), the healing effects on acetic acid-induced gastric ulcers were evaluated by macroscopic measure, microscopic measure, and immune histochemistry. Moreover, the expression levels of the growth factors, such as vascular endothelial growth factor, epidermal growth factor, transforming growth factor β1 (TGFβ1), and the heat shock protein 70 (HSP70) was detected. The results showed that both LMCCP and HMCCP could significantly decrease the ulcer areas and promote the healing of the lesions. They also could improve the levels of hexosamine, glutathione, superoxide dismutase, and glutathione peroxidase, and reduce the content of malondialdehyde and inducible nitric oxide synthase. In addition, the expression level of TGFβ1 gene and HSP70 mRNA was significantly improved by the treatment. It suggested that CCP could be able to improve symptoms of gastric ulcer and probably be used in the treatment of gastric ulcer.

  19. Ellagic acid induces novel and atypical PKC isoforms and promotes caspase-3 dependent apoptosis by blocking energy metabolism.

    PubMed

    Mishra, Sudha; Vinayak, Manjula

    2014-01-01

    Antioxidant ellagic acid is a herbal polyphenolic compound shown to possess growth-inhibiting and apoptotic activities in cancer. Protein kinase C (PKC) plays an important role in cell proliferation, apoptosis, and differentiation. Apoptosis of tumor cells is induced by inactivation of glycolytic enzyme of anaerobic metabolism, lactate dehydrogenase (LDH)-A, and by activating apoptotic protein caspase-3 via PKCδ. The present study aims to analyze the role of ellagic acid on regulation of novel and atypical isozymes of PKC to modulate apoptosis and anaerobic metabolism to prevent lymphoma growth as its role on classical PKCs is reported earlier. Expression of novel and atypical isozymes of PKC, activity of PKCδ, expression and activity of caspase-3, and LDH-A have been analyzed. Expression is measured by RT-PCR, activities of PKCδ as level of its catalytic fragment, caspase-3 as level of its p17 fragment, and LDH-A by specific staining. Lymphoma bearing mice were treated with 3 different doses of ellagic acid. The treatment enhanced expression of all novel and atypical PKCs, activity and expression of caspase-3, and activity of PKCδ but decreased activity and expression of LDH-A. Our results suggest that ellagic acid induces apoptosis via novel and atypical PKCs in association with caspase-3 and induces cancer cell death by blocking the energy metabolism.

  20. Vitamin C (ascorbic acid) induced hydroxyl radical formation in copper contaminated household drinking water: role of bicarbonate concentration.

    PubMed

    Jansson, Patric J; Asplund, Klara U M; Mäkelä, Johanna C; Lindqvist, Christer; Nordström, Tommy

    2003-08-01

    We have previously shown that Vitamin C (ascorbic acid) can trigger hydroxyl radical formation in copper contaminated household drinking water. We report here that the capacity of ascorbic acid to catalyze hydroxyl radical generation in the drinking water samples is strongly dependent on the bicarbonate concentration (buffer capacity and pH) of the samples. We found that at least 50 mg/l bicarbonate was required in the water samples to maintain the pH over 5.0 after ascorbic acid addition. At this pH, that is higher than the pKa1 4.25 of ascorbic acid, a hydroxyl radical generating redox cycling reaction involving the mono-anion of vitamin C and copper could take place. The ascorbic acid induced hydroxyl radical generating reaction could easily be mimicked in Milli-Q water by supplementing the water with copper and bicarbonate. Our results demonstrate that ascorbic acid can induce a pH dependent hydroxyl radical generating reaction in copper contaminated household tap water that is buffered with bicarbonate. The impact of consuming ascorbic acid together with copper and bicarbonate containing drinking water on human health is discussed.

  1. Swelling-activated and arachidonic acid-induced currents are TREK-1 in rat bladder smooth muscle cells

    PubMed Central

    Fukasaku, Mitsuko; Kimura, Junko; Yamaguchi, Osamu

    2016-01-01

    Abstract Using the perforated patch voltage clamp, we investigated swelling-activated ionic channels (SACs) in rat urinary bladder smooth muscle cells. Hypo-osmotic (60%) bath solution increased a membrane current which was inhibited by the SAC inhibitor, gadolinium. The reversal potential of the hypotonicity-induced current shifted in the positive direction by increasing external K+ concentration. The hypotonicity-induced current was inhibited by extracellular acidic pH, phorbol ester and forskolin. These pharmacological properties are identical to those of arachidonic acid-induced current present in these cells, suggesting the presence of TREK-1, a four-transmembrane two pore domain K+ channel. Using RT-PCR we screened rat bladder smooth muscles and cerebellum for expression of TREK-1, TREK-2 and TRAAK mRNAs. Only TREK-1 mRNA was expressed in the bladder, while all three were expressed in the cerebellum. We conclude that a mechanosensitive K+ channel is present in rat bladder myocytes, which is activated by arachidonic acid and most likely is TREK-1. This K+ channel may have an important role in the regulation of bladder smooth muscle tone during urine storage. PMID:26911303

  2. Swelling-activated and arachidonic acid-induced currents are TREK-1 in rat bladder smooth muscle cells.

    PubMed

    Fukasaku, Mitsuko; Kimura, Junko; Yamaguchi, Osamu

    2016-06-08

    Using the perforated patch voltage clamp, we investigated swelling-activated ionic channels (SACs) in rat urinary bladder smooth muscle cells. Hypo-osmotic (60%) bath solution increased a membrane current which was inhibited by the SAC inhibitor, gadolinium. The reversal potential of the hypotonicity-induced current shifted in the positive direction by increasing external K(+) concentration. The hypotonicity-induced current was inhibited by extracellular acidic pH, phorbol ester and forskolin. These pharmacological properties are identical to those of arachidonic acid-induced current present in these cells, suggesting the presence of TREK-1, a four-transmembrane two pore domain K(+) channel. Using RT-PCR we screened rat bladder smooth muscles and cerebellum for expression of TREK-1, TREK-2 and TRAAK mRNAs. Only TREK-1 mRNA was expressed in the bladder, while all three were expressed in the cerebellum. We conclude that a mechanosensitive K(+) channel is present in rat bladder myocytes, which is activated by arachidonic acid and most likely is TREK-1. This K(+) channel may have an important role in the regulation of bladder smooth muscle tone during urine storage.

  3. Phenylbutyric acid induces the cellular senescence through an Akt/p21{sup WAF1} signaling pathway

    SciTech Connect

    Kim, Hag Dong; Jang, Chang-Young; Choe, Jeong Min; Sohn, Jeongwon; Kim, Joon

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Phenylbutyric acid induces cellular senescence. Black-Right-Pointing-Pointer Phenylbutyric acid activates Akt kinase. Black-Right-Pointing-Pointer The knockdown of PERK also can induce cellular senescence. Black-Right-Pointing-Pointer Akt/p21{sup WAF1} pathway activates in PERK knockdown induced cellular senescence. -- Abstract: It has been well known that three sentinel proteins - PERK, ATF6 and IRE1 - initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that can reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21{sup WAF1} induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21{sup WAF1} pathway by PERK inhibition.

  4. The phosphatase inhibitor okadaic acid induces AQP2 translocation independently from AQP2 phosphorylation in renal collecting duct cells.

    PubMed

    Valenti, G; Procino, G; Carmosino, M; Frigeri, A; Mannucci, R; Nicoletti, I; Svelto, M

    2000-06-01

    Phosphorylation by kinases and dephosphorylation by phosphatase markedly affect the biological activity of proteins involved in intracellular signaling. In this study we investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on water permeability properties and on aquaporin2 (AQP2) translocation in AQP2-transfected renal CD8 cells. In CD8 cells both forskolin alone and okadaic acid alone increased the osmotic water permeability coefficient P(f) by about 4- to 5-fold. In intact cells, in vivo phosphorylation studies revealed that forskolin stimulation resulted in a threefold increase in AQP2 phosphorylation. In contrast, okadaic acid treatment promoted only a 60% increase in AQP2 phosphorylation which was abolished when this treatment was performed in the presence of 1 microM H89, a specific protein kinase A (PKA) inhibitor. Nevertheless, in this latter condition, confocal microscopy analysis revealed that AQP2 translocated and fused to the apical membrane. Okadaic acid-induced AQP2 translocation was dose dependent having its maximal effect at a concentration of 1 microM. In conclusion, our results clearly indicate that okadaic acid exerts a full forskolin-like effect independent from AQP2 phosphorylation. Thus AQP2 phosphorylation is not essential for water channel translocation in renal cells, indicating that different pathways might exist leading to AQP2 apical insertion and increase in P(f).

  5. Attenuation of Folic Acid-Induced Renal Inflammatory Injury in Platelet-Activating Factor Receptor-Deficient Mice

    PubMed Central

    Doi, Kent; Okamoto, Koji; Negishi, Kousuke; Suzuki, Yoshifumi; Nakao, Akihide; Fujita, Toshiro; Toda, Akiko; Yokomizo, Takehiko; Kita, Yoshihiro; Kihara, Yasuyuki; Ishii, Satoshi; Shimizu, Takao; Noiri, Eisei

    2006-01-01

    Platelet-activating factor (PAF), a potent lipid mediator with various biological activities, plays an important role in inflammation by recruiting leukocytes. In this study we used platelet-activating factor receptor (PAFR)-deficient mice to elucidate the role of PAF in inflammatory renal injury induced by folic acid administration. PAFR-deficient mice showed significant amelioration of renal dysfunction and pathological findings such as acute tubular damage with neutrophil infiltration, lipid peroxidation observed with antibody to 4-hydroxy-2-hexenal (day 2), and interstitial fibrosis with macrophage infiltration associated with expression of monocyte chemoattractant protein-1 and tumor necrosis factor-α in the kidney (day 14). Acute tubular damage was attenuated by neutrophil depletion using a monoclonal antibody (RB6-8C5), demonstrating the contribution of neutrophils to acute phase injury. Macrophage infiltration was also decreased when treatment with a PAF antagonist (WEB2086) was started after acute phase. In vitro chemotaxis assay using a Boyden chamber demonstrated that PAF exhibits a strong chemotactic activity for macrophages. These results indicate that PAF is involved in pathogenesis of folic acid-induced renal injury by activating neutrophils in acute phase and macrophages in chronic interstitial fibrosis. Inhibiting the PAF pathway might be therapeutic to kidney injury from inflammatory cells. PMID:16651609

  6. Nucleotide sequence and spatial expression pattern of a drought- and abscisic Acid-induced gene of tomato.

    PubMed

    Plant, A L; Cohen, A; Moses, M S; Bray, E A

    1991-11-01

    The nucleotide sequence of le16, a tomato (Lycopersicon esculentum Mill.) gene induced by drought stress and regulated by abscisic acid specifically in aerial vegetative tissue, is presented. The single open reading frame contained within the gene has the capacity to encode a polypeptide of 12.7 kilodaltons and is interrupted by a small intron. The predicted polypeptide is rich in leucine, glycine, and alanine and has an isoelectric point of 8.7. The amino terminus is hydrophobic and characteristic of signal sequences that target polypeptides for export from the cytoplasm. There is homology (47.2% identity) between the amino terminus of the LE 16 polypeptide and the corresponding amino terminal domain of the maize phospholipid transfer protein. le16 was expressed in drought-stressed leaf, petiole, and stem tissue and to a much lower extent in the pericarp of mature green tomato fruit and developing seeds. No expression was detected in the pericarp of red fruit or in drought-stressed roots. Expression of le16 was also induced in leaf tissue by a variety of other abiotic stresses including polyethylene glycol-mediated water deficit, salinity, cold stress, and heat stress. None of these stresses or direct applications of abscisic acid induced the expression of le16 in the roots of the same plants. The unique expression characteristics of this gene indicates that novel regulatory mechanisms, in addition to endogenous abscisic acid, are involved in controlling gene expression.

  7. Hydroalcoholic extract of Brazilian red propolis exerts protective effects on acetic acid-induced ulcerative colitis in a rodent model.

    PubMed

    Barbosa Bezerra, Gislaine; de Menezes de Souza, Luana; Dos Santos, Adailma Santana; de Almeida, Grace Kelly Melo; Souza, Marília Trindade Santana; Santos, Sandra Lauton; Aparecido Camargo, Enilton; Dos Santos Lima, Bruno; de Souza Araújo, Adriano Antunes; Cardoso, Juliana Cordeiro; Gomes, Silvana Vieira Floresta; Gomes, Margarete Zanardo; de Albuquerque, Ricardo Luiz Cavalcanti

    2017-01-01

    Ulcerative colitis (UC) is a common intestinal inflammatory disease with an etiology that is not well understood. Although the anti-inflammatory and anti-oxidant effects of the hydroalcoholic extract of Brazilian red propolis (HERP) have been reported in various experimental models, its protective effect in models of UC have not been evaluated. The purpose of this study was to investigate the chemopreventive effect of hydroalcoholic extract of Brazilian red propolis (HERP) in acetic acid-induced colitis (AAIC) using a rodent model. The HERP was chemically characterised by HPLC/DAD analyses. Male rats were randomly assigned into four groups: sham, vehicle (with AAIC, treated with vehicle), P10 (with AAIC, treated with 10mg/kg HERP), and P100 (with AAIC, treated with 100mg/kg HERP). Treatments were performed for 7days, and colitis was induced on day seven. Animals were euthanized 24h after colitis induction and body weight, colon length, gross and histological scores, malondialdehyde (MDA) and myeloperoxidase (MPO) concentrations in colon tissue, and the immunohistochemical expression of inducible nitric oxide synthase (iNOS) were assessed. The major compounds found in HERP were liquiritigenin (68.8mg/g), formononetin (54.29mg/g), biochanin A (30.97mg/g), and daidzein (19.90mg/g). Rats treated with 10mg/kg HERP demonstrated significant decreases in MPO concentrations, gross and histological scores of tissue damage, and iNOS expression (p<0.05). Similarly, rats treated with 100mg/kg HERP demonstrated significant decreases in MPO levels (p<0.05) and histological scores of tissue damage (p<0.05). The results of this study indicate that oral administration of HERP attenuates AAIC in rats, which may be due to anti-inflammatory effects related to iNOS inhibition.

  8. Chronic activity wheel running reduces the severity of kainic acid-induced seizures in the rat: possible role of galanin.

    PubMed

    Reiss, J I; Dishman, R K; Boyd, H E; Robinson, J K; Holmes, P V

    2009-04-17

    Studies in both humans and rodents suggest that exercise can be neuroprotective, but the mechanisms by which this occurs are still poorly understood. Three weeks of voluntary, physical activity in rats upregulates prepro-galanin messenger RNA levels in the locus coeruleus. Galanin is a neuropeptide extensively coexisting with norepinephrine that decreases neuronal hyperexcitability both in vivo and in vitro. Thus, exercise may diminish neural hyperexcitability through a galaninergic mechanism. The current experiments tested whether voluntary activity wheel running would protect against kainic acid-evoked seizures and whether galaninergic signaling is a necessary factor in this protection. In experiment 1, rats were given access to running wheels or remained sedentary for three weeks. After this period, rats received an intraperitoneal (i.p.) injection of 0, 7, 10 or 14 mg/kg kainic acid. Exercise decreased the severity of or eliminated seizure behaviors and hippocampal c-fos expression induced by kainic acid. In experiment 2, exercising or sedentary rats were injected intracerebroventricularly (i.c.v.) with 0.2 or 0.4 microg of kainic acid following either an injection of M-40 (a galanin receptor antagonist) or saline. Exercise decreased kainic acid-induced seizures at the 0.2 microg dose, and M-40 (6 nmol) decreased this effect. In contrast, there were no detectable differences between exercising and sedentary rats in behavior at the 0.4 microg dose. The results suggest that the protective effects of exercise against seizures are at least partially mediated by regulation of neural excitability through a process involving galanin.

  9. Mitochondrial proteomics of the acetic acid - induced programmed cell death response in a highly tolerant Zygosaccharomyces bailii - derived hybrid strain

    PubMed Central

    Guerreiro, Joana F.; Sampaio-Marques, Belém; Soares, Renata; Coelho, Ana V.; Leão, Cecília; Ludovico, Paula; Sá-Correia, Isabel

    2016-01-01

    Very high concentrations of acetic acid at low pH induce programmed cell death (PCD) in both the experimental model Saccharomyces cerevisiae and in Zygosaccharomyces bailii, the latter being considered the most problematic acidic food spoilage yeast due to its remarkable intrinsic resistance to this food preservative. However, while the mechanisms underlying S. cerevisiae PCD induced by acetic acid have been previously examined, the corresponding molecular players remain largely unknown in Z. bailii. Also, the reason why acetic acid concentrations known to be necrotic for S. cerevisiae induce PCD with an apoptotic phenotype in Z. bailii remains to be elucidated. In this study, a 2-DE-based expression mitochondrial proteomic analysis was explored to obtain new insights into the mechanisms involved in PCD in the Z. bailii derived hybrid strain ISA1307. This allowed the quantitative assessment of expression of protein species derived from each of the parental strains, with special emphasis on the processes taking place in the mitochondria known to play a key role in acetic acid - induced PCD. A marked decrease in the content of proteins involved in mitochondrial metabolism, in particular, in respiratory metabolism (Cor1, Rip1, Lpd1, Lat1 and Pdb1), with a concomitant increase in the abundance of proteins involved in fermentation (Pdc1, Ald4, Dld3) was registered. Other differentially expressed identified proteins also suggest the involvement of the oxidative stress response, protein translation, amino acid and nucleotide metabolism, among other processes, in the PCD response. Overall, the results strengthen the emerging concept of the importance of metabolic regulation of yeast PCD. PMID:28357336

  10. Glia activation and cytokine increase in rat hippocampus by kainic acid-induced status epilepticus during postnatal development.

    PubMed

    Rizzi, Massimo; Perego, Carlo; Aliprandi, Marisa; Richichi, Cristina; Ravizza, Teresa; Colella, Daniele; Velískŏvá, Jana; Moshé, Solomon L; De Simoni, M Grazia; Vezzani, Annamaria

    2003-12-01

    In adult rats, status epilepticus (SE) induces cytokine production by glia especially when seizures are associated with neuronal injury. This suggests that cytokines may play a role in seizure-induced neuronal damage. As SE-induced injury is age-specific, we used rats of different ages (with distinct susceptibilities to seizure-induced neuronal injury) to elucidate the role of cytokines in this process. Thus, we investigated the activation of microglia and astrocytes, induction of cytokines, and hippocampal neuronal injury 4 and 24 h following kainic acid-induced SE in postnatal day (PN) 9, 15, and 21 rats. At PN9, there was little activation of microglia and astrocytes at any time point studied. Interleukin-1beta (IL), tumor necrosis factor-alpha (TNF), and IL-6 or the naturally occurring IL-1 receptor antagonist (Ra) mRNA expression did not increase. No evidence of cell injury has been detected. At PN15, immunostaining of microglia and astrocytes was enhanced, but only IL-1beta mRNA expression was increased. These changes were observed 4 h after SE. Scattered injured neurons in CA3 and subiculum, but not in any other region, were present 24 h following SE. At PN21, immunostaining of microglia and astrocytes and the mRNA expression of all cytokines studied was significantly increased already 4 h after SE. At 24 h, many injured neurons were present in CA1 and CA3 regions and in 40% of rats in other forebrain areas. These data show that (i) the pattern of glia activation and cytokine gene transcription induced by SE is age-dependent and (ii) neuronal injury in the hippocampus occurs only when cytokines are induced and their synthesis precedes the appearance of neuronal damage. Thus, cytokine expression in immature brain is associated specifically with cell injury rather than with seizures per se, suggesting that proinflammatory cytokines may contribute to the occurence of SE-induced hippocampal damage.

  11. Subchronic treatment of donepezil rescues impaired social, hyperactive, and stereotypic behavior in valproic acid-induced animal model of autism.

    PubMed

    Kim, Ji-Woon; Seung, Hana; Kwon, Kyung Ja; Ko, Mee Jung; Lee, Eun Joo; Oh, Hyun Ah; Choi, Chang Soon; Kim, Ki Chan; Gonzales, Edson Luck; You, Jueng Soo; Choi, Dong-Hee; Lee, Jongmin; Han, Seol-Heui; Yang, Sung Min; Cheong, Jae Hoon; Shin, Chan Young; Bahn, Geon Ho

    2014-01-01

    Autism spectrum disorder (ASD) is a group of pervasive developmental disorders with core symptoms such as sociability deficit, language impairment, and repetitive/restricted behaviors. Although worldwide prevalence of ASD has been increased continuously, therapeutic agents to ameliorate the core symptoms especially social deficits, are very limited. In this study, we investigated therapeutic potential of donepezil for ASD using valproic acid-induced autistic animal model (VPA animal model). We found that prenatal exposure of valproic acid (VPA) induced dysregulation of cholinergic neuronal development, most notably the up-regulation of acetylcholinesterase (AChE) in the prefrontal cortex of affected rat and mouse offspring. Similarly, differentiating cortical neural progenitor cell in culture treated with VPA showed increased expression of AChE in vitro. Chromatin precipitation experiments revealed that acetylation of histone H3 bound to AChE promoter region was increased by VPA. In addition, other histone deacetyalse inhibitors (HDACIs) such as trichostatin A and sodium butyrate also increased the expression of AChE in differentiating neural progenitor cells suggesting the essential role of HDACIs in the regulation of AChE expression. For behavioral analysis, we injected PBS or donepezil (0.3 mg/kg) intraperitoneally to control and VPA mice once daily from postnatal day 14 all throughout the experiment. Subchronic treatment of donepezil improved sociability and prevented repetitive behavior and hyperactivity of VPA-treated mice offspring. Taken together, these results provide evidence that dysregulation of ACh system represented by the up-regulation of AChE may serve as an effective pharmacological therapeutic target against autistic behaviors in VPA animal model of ASD, which should be subjected for further investigation to verify the clinical relevance.

  12. Priming by Hexanoic Acid Induce Activation of Mevalonic and Linolenic Pathways and Promotes the Emission of Plant Volatiles

    PubMed Central

    Llorens, Eugenio; Camañes, Gemma; Lapeña, Leonor; García-Agustín, Pilar

    2016-01-01

    Hexanoic acid (Hx) is a short natural monocarboxylic acid present in some fruits and plants. Previous studies reported that soil drench application of this acid induces effective resistance in tomato plants against Botrytis cinerea and Pseudomonas syringae and in citrus against Alternaria alternata and Xanthomonas citri. In this work, we performed an in deep study of the metabolic changes produced in citrus by the application of Hx in response to the challenge pathogen A. alternata, focusing on the response of the plant. Moreover, we used 13C labeled hexanoic to analyze its behavior inside the plants. Finally, we studied the volatile emission of the treated plants after the challenge inoculation. Drench application of 13C labeled hexanoic demonstrated that this molecule stays in the roots and is not mobilized to the leaves, suggesting long distance induction of resistance. Moreover, the study of the metabolic profile showed an alteration of more than 200 molecules differentially induced by the application of the compound and the inoculation with the fungus. Bioinformatics analysis of data showed that most of these altered molecules could be related with the mevalonic and linolenic pathways suggesting the implication of these pathways in the induced resistance mediated by Hx. Finally, the application of this compound showed an enhancement of the emission of 17 volatile metabolites. Taken together, this study indicates that after the application of Hx this compound remains in the roots, provoking molecular changes that may trigger the defensive response in the rest of the plant mediated by changes in the mevalonic and linolenic pathways and enhancing the emission of volatile compounds, suggesting for the first time the implication of mevalonic pathway in response to hexanoic application. PMID:27148319

  13. Retinoic Acid Inducible Gene 1 Protein (RIG1)-Like Receptor Pathway Is Required for Efficient Nuclear Reprogramming.

    PubMed

    Sayed, Nazish; Ospino, Frank; Himmati, Farhan; Lee, Jieun; Chanda, Palas; Mocarski, Edward S; Cooke, John P

    2017-03-09

    We have revealed a critical role for innate immune signaling in nuclear reprogramming to pluripotency, and in the nuclear reprogramming required for somatic cell transdifferentiation. Activation of innate immune signaling causes global changes in the expression and activity of epigenetic modifiers to promote epigenetic plasticity. In our previous articles, we focused on the role of toll-like receptor 3 (TLR3) in this signaling pathway. Here, we define the role of another innate immunity pathway known to participate in response to viral RNA, the retinoic acid-inducible gene 1 receptor (RIG-1)-like receptor (RLR) pathway. This pathway is represented by the sensors of viral RNA, RIG-1, LGP2, and melanoma differentiation-associated protein 5 (MDA5). We first found that TLR3 deficiency only causes a partial inhibition of nuclear reprogramming to pluripotency in mouse tail-tip fibroblasts, which motivated us to determine the contribution of RLR. We found that knockdown of interferon beta promoter stimulator 1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming induced by retroviral or by modified messenger RNA expression of Oct 4, Sox2, KLF4, and c-MYC (OSKM). Importantly, a double knockdown of both RLR and TLR3 pathway led to a further decrease in induced pluripotent stem cell (iPSC) colonies suggesting an additive effect of both these pathways on nuclear reprogramming. Furthermore, in murine embryonic fibroblasts expressing a doxycycline (dox)-inducible cassette of the genes encoding OSKM, an RLR agonist increased the yield of iPSCs. Similarly, the RLR agonist enhanced nuclear reprogramming by cell permeant peptides of the Yamanaka factors. Finally, in the dox-inducible system, RLR activation promotes activating histone marks in the promoter region of pluripotency genes. To conclude, innate immune signaling mediated by RLR plays a critical role in nuclear reprogramming. Manipulation of innate immune signaling may facilitate

  14. Niflumic acid-induced increase in potassium currents in frog motor nerve terminals: effects on transmitter release.

    PubMed

    Miralles, F; Marsal, J; Peres, J; Solsona, C

    1996-04-01

    The actions of the nonsteroidal antiinflammatory drug niflumic acid were studied on frog neuromuscular preparations by conventional electrophysiological techniques. Niflumic acid reduced the amplitude and increased the latency of endplate potentials in a concentration-dependent manner. Neuromuscular junctions pretreated with niflumic acid (0.05-0.5 mM) showed much less depression than control when they were stimulated with trains of impulses. Inhibition of acetylcholine release was reverted by raising the extracellular Ca(2+) concentration but not by simply washing out the preparations with niflumic acid-free solutions. Pretreatment with indomethacin (0.1 mM), another nonsteroidal antiinflammatory drug, did not affect the niflumic acid-induced inhibition of evoked responses. Niflumic acid (0.1 mM) did not change the amplitude of miniature endplate potentials and had a dual action on the frequency of miniatures: it decreased their frequency at 0.1 mM whereas it produced an enormous increase in the rate of spontaneous discharge at 0.5 mM. Niflumic acid (0.1 - 1 mM) reversibly increased the amplitude and affected the kinetics of presynaptic voltage-activated K+ current and Ca(2+)-activated K(+) current in a concentration-dependent manner. Niflumic acid (0.1 - 1 mM) irreversibly decreased the amplitude and reversibly affected the kinetics of the nodal Na(+) current. Indomethacin (0.1 mM) had no effect on presynaptic currents. In conclusion, niflumic acid reduces acetylcholine release by increasing presynaptic K+ currents. This may shorten the depolarizing phase of the presynaptic action potential and may reduce the entry of Ca(2+) with each impulse.

  15. Arachidonic acid-induced Ca2+ sensitization of smooth muscle contraction through activation of Rho-kinase.

    PubMed

    Araki, S; Ito, M; Kureishi, Y; Feng, J; Machida, H; Isaka, N; Amano, M; Kaibuchi, K; Hartshorne, D J; Nakano, T

    2001-02-01

    Arachidonic acid activates isolated Rho-kinase and contracts permeabilized smooth muscle fibres. Various assays were carried out to examine the mechanism of this activation. Native Rho-kinase was activated 5-6 times by arachidonic acid but an N-terminal, constitutively-active fragment of Rho-kinase, expressed as a glutathione-S-transferase (GST) fusion protein and including the catalytic subunit (GST-Rho-kinase-CAT), was not. GST-Rho-kinase-CAT was inhibited by a C-terminal fragment of Rho-kinase and arachidonic acid removed this inhibition. These results suggest that the C-terminal part of Rho-kinase, containing the RhoA binding site and the pleckstrin homology domain, acts as an autoinhibitor. It is suggested further that activation by arachidonic acid is due to its binding to the autoinhibitory region and subsequent release from the catalytic site. Arachidonic acid, at concentrations greater than 30 microM, increases force in alpha-toxin-permeabilized femoral artery but not in Triton X-100-skinned fibres. The content of Rho-kinase in the latter was lower than in alpha-toxin-treated or intact fibres. The arachidonic acid-induced contraction was not observed at a pCa above 8.0 and was inhibited by Y-27632 and wortmannin, inhibitors of Rho-kinase and myosin light-chain kinase (MLCK), respectively. The activation of Rho-kinase and subsequent phosphorylation of the myosin phosphatase target subunit inhibits myosin phosphatase and increases myosin phosphorylation.

  16. Stability of the Acetic Acid-Induced Bladder Irritation Model in Alpha Chloralose-Anesthetized Female Cats

    PubMed Central

    Kullmann, F. Aura; Wells, Grace I.; Langdale, Christopher L.; Zheng, Jihong; Thor, Karl B.

    2013-01-01

    Time- and vehicle-related variability of bladder and urethral rhabdosphincter (URS) activity as well as cardiorespiratory and blood chemistry values were examined in the acetic acid-induced bladder irritation model in α-chloralose-anesthetized female cats. Additionally, bladder and urethra were evaluated histologically using Mason trichrome and toluidine blue staining. Urodynamic, cardiovascular and respiratory parameters were collected during intravesical saline infusion followed by acetic acid (0.5%) to irritate the bladder. One hour after starting acetic acid infusion, a protocol consisting of a cystometrogram, continuous infusion-induced rhythmic voiding contractions, and a 5 min “quiet period” (bladder emptied without infusion) was precisely repeated every 30 minutes. Administration of vehicle (saline i.v.) occurred 15 minutes after starting each of the first 7 cystometrograms and duloxetine (1mg/kg i.v.) after the 8th. Acetic acid infusion into the bladder increased URS-EMG activity, bladder contraction frequency, and decreased contraction amplitude and capacity, compared to saline. Bladder activity and URS activity stabilized within 1 and 2 hours, respectively. Duloxetine administration significantly decreased bladder contraction frequency and increased URS-EMG activity to levels similar to previous reports. Cardiorespiratory parameters and blood gas levels remained consistent throughout the experiment. The epithelium of the bladder and urethra were greatly damaged and edema and infiltration of neutrophils in the lamina propria of urethra were observed. These data provide an ample evaluation of the health of the animals, stability of voiding function and appropriateness of the model for testing drugs designed to evaluate lower urinary tract as well as cardiovascular and respiratory systems function. PMID:24040064

  17. Functional and cellular characterization of human Retinoic Acid Induced 1 (RAI1) mutations associated with Smith-Magenis Syndrome

    PubMed Central

    2010-01-01

    Background Smith-Magenis Syndrome is a contiguous gene syndrome in which the dosage sensitive gene has been identified: the Retinoic Acid Induced 1 (RAI1). Little is known about the function of human RAI1. Results We generated the full-length cDNA of the wild type protein and five mutated forms: RAI1-HA 2687delC, RAI1-HA 3103delC, RAI1 R960X, RAI1-HA Q1562R, and RAI1-HA S1808N. Four of them have been previously associated with SMS clinical phenotype. Molecular weight, subcellular localization and transcription factor activity of the wild type and mutant forms were studied by western blot, immunofluorescence and luciferase assays respectively. The wild type protein and the two missense mutations presented a higher molecular weight than expected, localized to the nucleus and activated transcription of a reporter gene. The frameshift mutations generated a truncated polypeptide with transcription factor activity but abnormal subcellular localization, and the same was true for the 1-960aa N-terminal half of RAI1. Two different C-terminal halves of the RAI1 protein (1038aa-end and 1229aa-end) were able to localize into the nucleus but had no transactivation activity. Conclusion Our results indicate that transcription factor activity and subcellular localization signals reside in two separate domains of the protein and both are essential for the correct functionality of RAI1. The pathogenic outcome of some of the mutated forms can be explained by the dissociation of these two domains. PMID:20738874

  18. The Healing Effect of Teucrium polium in Acetic Acid-Induced Ulcerative Colitis in the Dog as an Animal Model

    PubMed Central

    Mehrabani, Davood; Bahrami, Faranak; Hosseini, Seyed Vahid; Ashraf, Mohammad Javad; Tanideh, Nader; Rezaianzadeh, Abbas; Amini, Masoud; Amini, Afshin

    2012-01-01

    BACKGROUND Inflammatory bowel diseases (IBD), which include ulcerative colitis (UC) and Crohn’s disease (CD), are debilitating and chronic disorders with unpredictable courses and complicated treatment measures. Therefore, an efficient treatment protocol seems necessary as therapeutic prophylaxis for these disorders. This study aims to determine the healing effect of Teucrium polium (T. polium) in acetic acid-induced UC in an experimental dog model. METHODS From September to December 2010, eight male (20-25 kg) crossbred dogs were used for induction of UC by 6% acetic acid, transrectally. After one week, three biopsies (10, 20 and 30 cm proximal to the anal verge) were taken from the colon of each animal for histological studies. In the presence of UC, 400 mg/kg/day of T. polium extract was administered orally and transrectally (via enema) for 30 days in six of the dogs. The remaining two dogs were used as controls and did not receive T. polium. Multiple biopsies were taken 7, 14, and 30 days after discontinuation of T. polium in the same manner as before treatment. RESULTS After administration of acetic acid, we noted the presence of multiple ulcers, diffuse inflammation, PMN infiltration in the lamina propria, glandular destruction and goblet cell depletion. Treatment with T. polium restored the colonic architecture with an increased number of healthy cells and a reduction in inflammatory cells. Damage of the surface epithelial cells and mucosal layer of the lumen were reversed, which lead to faster ulcer healing. CONCLUSION T. polium may be a treatment choice for UC and can broaden the current therapy options for UC. PMID:24829634

  19. Distribution of interstitial cells of Cajal in the bladders of fetal rats with retinoic acid induced myelomeningocele

    PubMed Central

    Tekin, Ali; Karakuş, Osman Zeki; Hakgüder, Gülce; Ateş, Oğuz; Özer, Erdener; Olguner, Mustafa; Akgür, Feza Miraç

    2016-01-01

    Objective Myelomeningocele (MMC) is one of the most common reason of neurogenic bladder dysfunction in children. Although neurogenic bladder dysfunction occurrence is related with bladder innervation, also there are some changes seen in the smooth muscle and neural cells of the bladder. Interstitial cells of Cajal (ICC) are the pacemaker cells found in organs with peristaltic activity. Although it has been shown that ICC are diminished in the rat urinary bladder with traumatic spinal cord injury, there is no data about ICC in fetal rat bladders with MMC. This study has been conducted to investigate the ICC in the bladders of fetal rats with retinoic acid induced MMC. Materials and methods Time dated pregnant Wistar albino rats were divided into 3 groups. In MMC group, dams were fed with gavage solution containing 60 mg/kg all-trans retinoic acid dissolved in olive oil on 10. embryologic day. Sham group animals were fed only olive oil. Control group dams were fed with standard rat chow. Fetuses were delivered by cesarean section and harvested on 22. embryologic day. MMC was identified by observing MMC sacs at the back of the fetuses. Distribution of ICCs were evaluated using immunohistochemical staining. Results ICCs were found in all groups, which have the same morphological features that had been described earlier in the gastrointestinal tract and the bladder. The density of the ICC in the MMC group was found to be significantly decreased when compared with the control and the sham groups (p<0.05). Conclusion The density of the ICC in the urinary bladder decreased in the neurogenic bladder developed in MMC. PMID:27909623

  20. The restrained expression of NF-kB in renal tissue ameliorates folic acid induced acute kidney injury in mice.

    PubMed

    Kumar, Dev; Singla, Surinder K; Puri, Veena; Puri, Sanjeev

    2015-01-01

    The Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) represent family of structurally-related eukaryotic transcription factors which regulate diverse array of cellular processes including immunological responses, inflammation, apoptosis, growth & development. Increased expression of NF-kB has often been seen in many diverse diseases, suggesting the importance of genomic deregulation to disease pathophysiology. In the present study we focused on acute kidney injury (AKI), which remains one of the major risk factor showing a high rate of mortality and morbidity. The pathology associated with it, however, remains incompletely known though inflammation has been reported to be one of the major risk factor in the disease pathophysiology. The role of NF-kB thus seemed pertinent. In the present study we show that high dose of folic acid (FA) induced acute kidney injury (AKI) characterized by elevation in levels of blood urea nitrogen & serum creatinine together with extensive tubular necrosis, loss of brush border and marked reduction in mitochondria. One of the salient observations of this study was a coupled increase in the expression of renal, relA, NF-kB2, and p53 genes and proteins during folic acid induced AKI (FA AKI). Treatment of mice with NF-kB inhibitor, pyrrolidine dithio-carbamate ammonium (PDTC) lowered the expression of these transcription factors and ameliorated the aberrant renal function by decreasing serum creatinine levels. In conclusion, our results suggested that NF-kB plays a pivotal role in maintaining renal function that also involved regulating p53 levels during FA AKI.

  1. Stability of the acetic acid-induced bladder irritation model in alpha chloralose-anesthetized female cats.

    PubMed

    Kullmann, F Aura; Wells, Grace I; Langdale, Christopher L; Zheng, Jihong; Thor, Karl B

    2013-01-01

    Time- and vehicle-related variability of bladder and urethral rhabdosphincter (URS) activity as well as cardiorespiratory and blood chemistry values were examined in the acetic acid-induced bladder irritation model in α-chloralose-anesthetized female cats. Additionally, bladder and urethra were evaluated histologically using Mason trichrome and toluidine blue staining. Urodynamic, cardiovascular and respiratory parameters were collected during intravesical saline infusion followed by acetic acid (0.5%) to irritate the bladder. One hour after starting acetic acid infusion, a protocol consisting of a cystometrogram, continuous infusion-induced rhythmic voiding contractions, and a 5 min "quiet period" (bladder emptied without infusion) was precisely repeated every 30 minutes. Administration of vehicle (saline i.v.) occurred 15 minutes after starting each of the first 7 cystometrograms and duloxetine (1mg/kg i.v.) after the 8(th). Acetic acid infusion into the bladder increased URS-EMG activity, bladder contraction frequency, and decreased contraction amplitude and capacity, compared to saline. Bladder activity and URS activity stabilized within 1 and 2 hours, respectively. Duloxetine administration significantly decreased bladder contraction frequency and increased URS-EMG activity to levels similar to previous reports. Cardiorespiratory parameters and blood gas levels remained consistent throughout the experiment. The epithelium of the bladder and urethra were greatly damaged and edema and infiltration of neutrophils in the lamina propria of urethra were observed. These data provide an ample evaluation of the health of the animals, stability of voiding function and appropriateness of the model for testing drugs designed to evaluate lower urinary tract as well as cardiovascular and respiratory systems function.

  2. Protective Effect of the Methanolic Extract of Malva parviflora L. leaves on Acetic Acid-induced Ulcerative Colitis in Rats

    PubMed Central

    Dugani, Aisha; Dakhil, Bushra; Treesh, Soad

    2016-01-01

    Background/Aims: Inflammatory bowel disease (IBD) is a general term describing chronic, idiopathic relapsing, inflammatory conditions of the gastrointestinal tract of unknown etiology. Previous studies have indicated that Malva parviflora leaf extract possesses anti-inflammatory, antioxidant, and antiulcerogenic activity. activity. This work aimed to investigatee the anti-inflammatory effect of the methanolic (MEMP) and aqueous (AEMP) extracts of M. parviflora leaves on acetic acid-induced colitis in rats. Materials and Methods: 42 male Wistar albino rats were divided into seven groups (n = 6). Group I: Normal saline control group with no colitis; Group II: Acetic acid colitis group; Group III: 100 mg/kg/5 d MEMP; Group IV: 200 mg/kg/5 d.MEMP; Group V: 100 mg/kg/5 d AEMP; Group VI: 200 mg/kg/5 d AEMP; Group VII: Prednisolone group (2 mg/kg/5 d). Treatments were followed by induction of colitis using intrarectal instillation of 2 mL of 4% acetic acid. Colon damage was evaluated macroscopically (spleen weight/body weight, colon weight/length ratio) and the histological changes were also recorded. Results: The results of this study showed that acetic acid caused severe inflammation of the colon and a significant increase in spleen weight/body weight, and an increase in colon weight/length ratio compared with normal control group. Pretreatment with MEMP and AEMP for 5 days followed by induction of colitis resulted in a significant attenuation of spleen weight and colon weight/length ratio compared with acetic acid control group. Methanolic extract provided better anticolitic effect than aqueous extract; the effect was prominent at the dose of 200 mg/kg. Histopathological findings confirmed the protective effect of the MEMP. Conclusion: In conclusion, MEMP could ameliorate mucosal damage in experimentally induced colitis when given orally. PMID:27184642

  3. Palmitic acid-induced apoptosis in pancreatic β-cells is increased by liver X receptor agonist and attenuated by eicosapentaenoate.

    PubMed

    Liang, Huasheng; Zhong, Yuhua; Zhou, Shaobi; Li, Qingdi Quentin

    2011-01-01

    Saturated fatty acids are implicated in the development of diabetes via the impairment of pancreatic islet β-cell viability and function. Liver X receptors (LXRs) and eicosapentaenoate (EPA) are known regulators of fatty acid metabolism. However, their roles in the pathogenesis of diabetes remain incompletely understood. The aim of this study was to determine the effects of EPA and the LXR agonist T0901317 on saturated fatty acid (palmitic acid)-induced apoptosis in the insulinoma β-cell line INS-1, a model for insulin-secreting β-cells. T0901317 significantly promoted palmitic acid-induced apoptotic cell death in the INS-1 cells. Consistent with these results, caspase-3 activity and BAX and sterol regulatory element binding protein-1c (SREBP-1c) mRNA levels were markedly increased in INS-1 cells co-administered palmitic acid and T0901317. The production of reactive oxygen species was considerably higher in the cells cultured concurrently with T0901317 and palmitic acid than in the cells incubated with either agent alone. EPA treatment attenuated the cellular death promoted by palmitic acid and T0901317 in the INS-1 cells, disclosing a possible mediating mechanism involving the inhibition of SREBP-1c. Finally, T0901317 up-regulated the palmitic acid-induced expression of p27(KIP1), transforming growth factor beta 1, and SMAD3 proteins in INS-1 cells. These results demonstrate that palmitic acid-induced apoptosis in β-cells is enhanced by T0901317 via the activation of LXRs and is blocked by EPA via the inhibition of SREBP-1c, suggesting that the regulation of lipogenesis and lipotoxicity affecting pancreatic β-cell viability and insulin production may be a unique strategy for diabetes therapy.

  4. Palmitic acid induces interleukin-1β secretion via NLRP3 inflammasomes and inflammatory responses through ROS production in human placental cells.

    PubMed

    Shirasuna, Koumei; Takano, Hiroki; Seno, Kotomi; Ohtsu, Ayaka; Karasawa, Tadayoshi; Takahashi, Masafumi; Ohkuchi, Akihide; Suzuki, Hirotada; Matsubara, Shigeki; Iwata, Hisataka; Kuwayama, Takehito

    2016-08-01

    Maternal obesity, a major risk factor for adverse pregnancy complications, results in inflammatory cytokine release in the placenta. Levels of free fatty acids are elevated in the plasma of obese human. These fatty acids include obesity-related palmitic acids, which is a major saturated fatty acid, that promotes inflammatory responses. Increasing evidence indicates that nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasomes mediate inflammatory responses induced by endogenous danger signals. We hypothesized that inflammatory responses associated with gestational obesity cause inflammation. To test this hypothesis, we investigated the effect of palmitic acid on the activation of NLRP3 inflammasomes and inflammatory responses in a human Sw.71 trophoblast cell line. Palmitic acid stimulated caspase-1 activation and markedly increased interleukin (IL)-1β secretion in Sw.71 cells. Treatment with a caspase-1 inhibitor diminished palmitic acid-induced IL-1β release. In addition, NLRP3 and caspase-1 genome editing using a CRISPR/Cas9 system in Sw.71 cells suppressed IL-1β secretion, which was stimulated by palmitic acid. Moreover, palmitic acid stimulated caspase-3 activation and inflammatory cytokine secretion (e.g., IL-6 and IL-8). Palmitic acid-induced cytokine secretion were dependent on caspase-3 activation. In addition, palmitic acid-induced IL-1β, IL-6, and IL-8 secretion was depended on reactive oxygen species (ROS) generation. In conclusion, palmitic acid caused activation of NLRP3 inflammasomes and inflammatory responses, inducing IL-1β, IL-6, and IL-8 secretion, which is associated with ROS generation, in human Sw.71 placental cells. We suggest that obesity-related palmitic acid induces placental inflammation, resulting in association with pregnancy complications.

  5. PAR-2 activation enhances weak acid-induced ATP release through TRPV1 and ASIC sensitization in human esophageal epithelial cells.

    PubMed

    Wu, Liping; Oshima, Tadayuki; Shan, Jing; Sei, Hiroo; Tomita, Toshihiko; Ohda, Yoshio; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

    2015-10-15

    Esophageal visceral hypersensitivity has been proposed to be the pathogenesis of heartburn sensation in nonerosive reflux disease. Protease-activated receptor-2 (PAR-2) is expressed in human esophageal epithelial cells and is believed to play a role in inflammation and sensation. PAR-2 activation may modulate these responses through adenosine triphosphate (ATP) release, which is involved in transduction of sensation and pain. The transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs) are both acid-sensitive nociceptors. However, the interaction among these molecules and the mechanisms of heartburn sensation are still not clear. We therefore examined whether ATP release in human esophageal epithelial cells in response to acid is modulated by TRPV1 and ASICs and whether PAR-2 activation influences the sensitivity of TRPV1 and ASICs. Weak acid (pH 5) stimulated the release of ATP from primary human esophageal epithelial cells (HEECs). This effect was significantly reduced after pretreatment with 5-iodoresiniferatoxin (IRTX), a TRPV1-specific antagonist, or with amiloride, a nonselective ASIC blocker. TRPV1 and ASIC3 small interfering RNA (siRNA) transfection also decreased weak acid-induced ATP release. Pretreatment of HEECs with trypsin, tryptase, or a PAR-2 agonist enhanced weak acid-induced ATP release. Trypsin treatment led to the phosphorylation of TRPV1. Acid-induced ATP release enhancement by trypsin was partially blocked by IRTX, amiloride, or a PAR-2 antagonist. Conversely, acid-induced ATP release was augmented by PAR-2 activation through TRPV1 and ASICs. These findings suggested that the pathophysiology of heartburn sensation or esophageal hypersensitivity may be associated with the activation of PAR-2, TRPV1, and ASICs.

  6. Human myeloblastic leukemia cells (HL-60) express a membrane receptor for estrogen that signals and modulates retinoic acid-induced cell differentiation

    SciTech Connect

    Kauss, M. Ariel; Reiterer, Gudrun; Bunaciu, Rodica P.; Yen, Andrew

    2008-10-01

    Estrogen receptors are historically perceived as nuclear ligand activated transcription factors. An estrogen receptor has now been found localized to the plasma membrane of human myeloblastic leukemia cells (HL-60). Its expression occurs throughout the cell cycle, progressively increasing as cells mature from G{sub 1} to S to G{sub 2}/M. To ascertain that the receptor functioned, the effect of ligands, including a non-internalizable estradiol-BSA conjugate and tamoxifen, an antagonist of nuclear estrogen receptor function, were tested. The ligands caused activation of the ERK MAPK pathway. They also modulated the effect of retinoic acid, an inducer of MAPK dependent terminal differentiation along the myeloid lineage in these cells. In particular the ligands inhibited retinoic acid-induced inducible oxidative metabolism, a functional marker of terminal myeloid cell differentiation. To a lesser degree they also diminished retinoic acid-induced earlier markers of cell differentiation, namely CD38 and CD11b. However, they did not regulate retinoic acid-induced G{sub 0} cell cycle arrest. There is thus a membrane localized estrogen receptor in HL-60 myeloblastic leukemia cells that can cause ERK activation and modulates the response of these cells to retinoic acid, indicating crosstalk between the membrane estrogen and retinoic acid evoked pathways relevant to propulsion of cell differentiation.

  7. Human myeloblastic leukemia cells (HL-60) express a membrane receptor for estrogen that signals and modulates retinoic acid-induced cell differentiation.

    PubMed

    Kauss, M Ariel; Reiterer, Gudrun; Bunaciu, Rodica P; Yen, Andrew

    2008-10-01

    Estrogen receptors are historically perceived as nuclear ligand activated transcription factors. An estrogen receptor has now been found localized to the plasma membrane of human myeloblastic leukemia cells (HL-60). Its expression occurs throughout the cell cycle, progressively increasing as cells mature from G(1) to S to G(2)/M. To ascertain that the receptor functioned, the effect of ligands, including a non-internalizable estradiol-BSA conjugate and tamoxifen, an antagonist of nuclear estrogen receptor function, were tested. The ligands caused activation of the ERK MAPK pathway. They also modulated the effect of retinoic acid, an inducer of MAPK dependent terminal differentiation along the myeloid lineage in these cells. In particular the ligands inhibited retinoic acid-induced inducible oxidative metabolism, a functional marker of terminal myeloid cell differentiation. To a lesser degree they also diminished retinoic acid-induced earlier markers of cell differentiation, namely CD38 and CD11b. However, they did not regulate retinoic acid-induced G(0) cell cycle arrest. There is thus a membrane localized estrogen receptor in HL-60 myeloblastic leukemia cells that can cause ERK activation and modulates the response of these cells to retinoic acid, indicating crosstalk between the membrane estrogen and retinoic acid evoked pathways relevant to propulsion of cell differentiation.

  8. Ovarian cancer G protein-coupled receptor 1 is involved in acid-induced apoptosis of endplate chondrocytes in intervertebral discs.

    PubMed

    Yuan, Feng-Lai; Wang, Hui-Ren; Zhao, Ming-Dong; Yuan, Wei; Cao, Lu; Duan, Ping-Guo; Jiang, Yun-Qi; Li, Xi-Lei; Dong, Jian

    2014-01-01

    Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown to be a receptor for protons. We investigated the role of proton-sensing G protein-coupled receptors in the apoptosis of endplate chondrocytes induced by extracellular acid. The expression of proton-sensing G protein-coupled receptors was examined in rat lumbar endplate chondrocytes. Knockdown of OGR1 was achieved by transfecting chondrocytes with specific short hairpin RNA (shRNA) for OGR1. Apoptotic changes were evaluated by DNA fragmentation ELISA, electron microscopy, and flow cytometry. Intracellular calcium ([Ca(2+) ]i) was analyzed with laser scanning confocal microscopy. The mechanism of OGR1 in acid-induced apoptosis of endplate chondrocytes was also investigated. We found that OGR1 was predominantly expressed in rat endplate chondrocytes, and its expression was highly upregulated in response to acidosis. Knocking down OGR1 with shRNAs effectively attenuated acid-induced apoptosis of endplate chondrocytes and increased [Ca(2+) ]i. Blocking OGR1-mediated [Ca(2+) ]i elevation inhibited acid-induced calcium-sensitive proteases such as calpain and calcineurin, and also inhibited the activation of Bid, Bad, and Caspase 3 and cleavage of poly (ADP-ribose) polymerase (PARP). OGR1-mediated [Ca(2+) ]i elevation has a crucial role in apoptosis of endplate chondrocytes by regulating activation of calcium-sensitive proteases and their downstream signaling.

  9. Sida rhomboidea.Roxb extract alleviates pathophysiological changes in experimental in vivo and in vitro models of high fat diet/fatty acid induced non-alcoholic steatohepatitis.

    PubMed

    Thounaojam, Menaka C; Jadeja, Ravirajsinh N; Dandekar, Deven S; Devkar, Ranjitsinh V; Ramachandran, A V

    2012-03-01

    The present study was aim to evaluate protective role of Sida rhomboidea.Roxb (SR) extract against high fat diet/fatty acid induced pathophysiological alterations in experimental model of non-alcoholic steatohepatitis (NASH). Effect of SR extract on plasma levels of markers of hepatic damage, plasma and hepatic lipids, mitochondrial oxidative stress, status of enzymatic and non-enzymatic antioxidants and histopathological changes in liver tissue were evaluated in high fat diet fed C57BL/6J mice. Also, the effect of SR supplementation on lipid accumulation, lipid peroxidation, cytotoxicity and cell viability were evaluated in oleic acid treated HepG2 cells. Supplementation of NASH mice with SR extract prevented high fat diet induced elevation in plasma marker enzymes of liver damage, plasma and hepatic lipids, mitochondrial oxidative stress and compromised enzymatic and non-enzymatic antioxidant status. Further, addition of SR extract to in vitro HepG2 cells minimized oleic acid induced lipid accumulation, higher lipid peroxidation, cytotoxicity and reduced cell viability. These in vivo and in vitro studies suggest that SR extract has the potential of preventing high fat/fatty acid induced NASH mainly due to its hypolipidemic and antioxidant activities.

  10. The acid-inducible asr gene in Escherichia coli: transcriptional control by the phoBR operon.

    PubMed

    Suziedeliené, E; Suziedélis, K; Garbenciūté, V; Normark, S

    1999-04-01

    Escherichia coli responds to external acidification (pH 4.0 to 5.0) by synthesizing a newly identified, approximately 450-nucleotide RNA component. At maximal levels of induction it is one of the most abundant small RNAs in the cell and is relatively stable bacterial RNA. The acid-inducible RNA was purified, and the gene encoding it, designated asr (for acid shock RNA), mapped at 35.98 min on the E. coli chromosome. Analysis of the asr DNA sequence revealed an open reading frame coding for a 111-amino-acid polypeptide with a deduced molecular mass of approximately 11.6 kDa. According to computer-assisted analysis, the predicted polypeptide contains a typical signal sequence of 30 amino acids and might represent either a periplasmic or an outer membrane protein. The asr gene cloned downstream from a T7 promoter was translated in vivo after transcription using a T7 RNA polymerase transcription system. Expression of a plasmid-encoded asr::lacZ fusion under a native asr promoter was reduced approximately 15-fold in a complex medium, such as Luria-Bertani medium, versus the minimal medium. Transcription of the chromosomal asr was abolished in the presence of a phoB-phoR (a two-component regulatory system, controlling the pho regulon inducible by phosphate starvation) deletion mutant. Acid-mediated induction of the asr gene in the Delta(phoB-phoR) mutant strain was restored by introduction of the plasmid with cloned phoB-phoR genes. Primer extension analysis of the asr transcript revealed a region similar to the Pho box (the consensus sequence found in promoters transcriptionally activated by the PhoB protein) upstream from the determined transcription start. The asr promoter DNA region was demonstrated to bind PhoB protein in vitro. We discuss our results in terms of how bacteria might employ the phoB-phoR regulatory system to sense an external acidity and regulate transcription of the asr gene.

  11. The Na+/H+ exchanger controls deoxycholic acid-induced apoptosis by a H+-activated, Na+-dependent ionic shift in esophageal cells.

    PubMed

    Goldman, Aaron; Chen, HwuDauRw; Khan, Mohammad R; Roesly, Heather; Hill, Kimberly A; Shahidullah, Mohammad; Mandal, Amritlal; Delamere, Nicholas A; Dvorak, Katerina

    2011-01-01

    Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na(+)/H(+) exchanger (NHE) and Na(+) influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM-0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na(+), subsequent loss of intracellular K(+), an increase of Ca(2+) and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na(+), K(+) and Ca(2+) changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na(+) levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na(+) concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na(+) influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis.

  12. Identification of Novel MAGE-G1-Interacting Partners in Retinoic Acid-Induced P19 Neuronal Differentiation Using SILAC-Based Proteomics

    PubMed Central

    Liu, Yong; Chen, Yujian; Lin, Shide; Yang, Shuguang; Liu, Shaojun

    2017-01-01

    MAGE-G1 is a protein plays role in the early process of neurogenesis. However, the fundamental roles MAGE-G1 played in neurogenesis have not yet been completely understood. Finding the partners MAGE-G1 interacting with will surely contribute to the function study of MAGE-G1. In this study, using Stable Isotope Labeling by Amino acids in Cell culture-immunoprecipitation quantitative proteomics, we screened the interacting proteins of MAGE-G1 during retinoic acid -induced neuronal differentiation of P19 cells and firstly found that FSCN1 and VIME were potential novel MAGE-G1-interacting proteins. Then, the interaction between overexpressed MAGE-G1 and FSCN1 or VIME was validated by GST-pull down assay in bacteria and by co-immunoprecipitation assay in COS7 cells. Endogenous co-immunoprecipitation assay further confirmed that MAGE-G1 interacted with FSCN1 or VIME in P19 cells after a 6-day retinoic acid-induced neuronal differentiation. Those results provide a functional linkage between MAGE-G1 and FSCN1 or VIME and may facilitate a better understanding of the fundamental aspects of MAGE-G1 during neurogenesis. PMID:28374796

  13. Beta-trace Protein as a new non-invasive immunological Marker for Quinolinic Acid-induced impaired Blood-Brain Barrier Integrity

    PubMed Central

    Baranyi, Andreas; Amouzadeh-Ghadikolai, Omid; Lewinski, Dirk von; Breitenecker, Robert J.; Stojakovic, Tatjana; März, Winfried; Robier, Christoph; Rothenhäusler, Hans-Bernd; Mangge, Harald; Meinitzer, Andreas

    2017-01-01

    Quinolinic acid, a macrophage/microglia-derived excitotoxin fulfills a plethora of functions such as neurotoxin, gliotoxin, and proinflammatory mediator, and it alters the integrity and cohesion of the blood-brain barrier in several pathophysiological states. Beta-trace protein (BTP), a monomeric glycoprotein, is known to indicate cerebrospinal fluid leakage. Thus, the prior aim of this study was to investigate whether BTP might non-invasively indicate quinolinic acid-induced impaired blood-brain barrier integrity. The research hypotheses were tested in three subsamples with different states of immune activation (patients with HCV-infection and interferon-α, patients with major depression, and healthy controls). BTP has also been described as a sensitive marker in detecting impaired renal function. Thus, the renal function has been considered. Our study results revealed highest quinolinic acid and highest BTP- levels in the subsample of patients with HCV in comparison with the other subsamples with lower or no immune activation (quinolinic acid: F = 21.027, p < 0.001 [ANOVA]; BTP: F = 6.792, p < 0.01 [ANOVA]). In addition, a two-step hierarchical linear regression model showed that significant predictors of BTP levels are quinolinic acid, glomerular filtration rate and age. The neurotoxin quinolinic acid may impair blood-brain barrier integrity. BTP might be a new non-invasive biomarker to indicate quinolinic acid-induced impaired blood-brain barrier integrity. PMID:28276430

  14. Use of Activated Carbon in Packaging to Attenuate Formaldehyde-Induced and Formic Acid-Induced Degradation and Reduce Gelatin Cross-Linking in Solid Dosage Forms.

    PubMed

    Colgan, Stephen T; Zelesky, Todd C; Chen, Raymond; Likar, Michael D; MacDonald, Bruce C; Hawkins, Joel M; Carroll, Sophia C; Johnson, Gail M; Space, J Sean; Jensen, James F; DeMatteo, Vincent A

    2016-07-01

    Formaldehyde and formic acid are reactive impurities found in commonly used excipients and can be responsible for limiting drug product shelf-life. Described here is the use of activated carbon in drug product packaging to attenuate formaldehyde-induced and formic acid-induced drug degradation in tablets and cross-linking in hard gelatin capsules. Several pharmaceutical products with known or potential vulnerabilities to formaldehyde-induced or formic acid-induced degradation or gelatin cross-linking were subjected to accelerated stability challenges in the presence and absence of activated carbon. The effects of time and storage conditions were determined. For all of the products studied, activated carbon attenuated drug degradation or gelatin cross-linking. This novel use of activated carbon in pharmaceutical packaging may be useful for enhancing the chemical stability of drug products or the dissolution stability of gelatin-containing dosage forms and may allow for the 1) extension of a drug product's shelf-life when the limiting attribute is a degradation product induced by a reactive impurity, 2) marketing of a drug product in hotter and more humid climatic zones than currently supported without the use of activated carbon, and 3) enhanced dissolution stability of products that are vulnerable to gelatin cross-linking.

  15. Synergic Interaction of Rifaximin and Mutaflor (Escherichia coli Nissle 1917) in the Treatment of Acetic Acid-Induced Colitis in Rats

    PubMed Central

    Warzecha, Zygmunt; Ceranowicz, Piotr; Dembiński, Marcin; Cieszkowski, Jakub; Bulanda, Małgorzata; Kuśnierz-Cabala, Beata; Gałązka, Krystyna; Konturek, Peter Christopher

    2016-01-01

    Background. Inflammatory bowel disease results from the dysregulation of immune response to environmental and microbial agents in genetically susceptible individuals. The aim of the present study was to examine the effect of rifaximin and/or Mutaflor (Escherichia coli Nissle 1917, EcN) administration on the healing of acetic acid-induced colitis. Methods. Colitis was induced in male Wistar rats by rectal enema with 3.5% acetic acid solution. Rifaximin (50 mg/kg/dose) and/or Mutaflor (109 CFU/dose) were given intragastrically once a day. The severity of colitis was assessed at the 8th day after induction of inflammation. Results. Treatment with rifaximin significantly accelerated the healing of colonic damage. This effect was associated with significant reversion of the acetic acid-evoked decrease in mucosal blood flow and DNA synthesis. Moreover, administration of rifaximin significantly reduced concentration of proinflammatory TNF-α and activity of myeloperoxidase in colonic mucosa. Mutaflor given alone was without significant effect on activity of colitis. In contrast, Mutaflor given in combination with rifaximin significantly enhanced therapeutic effect of rifaximin. Moreover, Mutaflor led to settle of the colon by EcN and this effect was augmented by pretreatment with rifaximin. Conclusion. Rifaximin and Mutaflor exhibit synergic anti-inflammatory and therapeutic effect in acetic acid-induced colitis in rats. PMID:27433160

  16. Acid-Induced Activation of the Urease Promoters Is Mediated Directly by the ArsRS Two-Component System of Helicobacter pylori

    PubMed Central

    Pflock, Michael; Kennard, Simone; Delany, Isabel; Scarlato, Vincenzo; Beier, Dagmar

    2005-01-01

    The nickel-containing enzyme urease is an essential colonization factor of the human gastric pathogen Helicobacter pylori which enables the bacteria to survive the low-pH conditions of the stomach. Transcription of the urease genes is positively controlled in response to increasing concentrations of nickel ions and acidic pH. Here we demonstrate that acid-induced transcription of the urease genes is mediated directly by the ArsRS two-component system. Footprint analyses identify binding sites of the phosphorylated ArsR response regulator within the ureA and ureI promoters. Furthermore, deletion of a distal upstream ArsR binding site of the ureA promoter demonstrates its role in acid-dependent activation of the promoter. In addition, acid-induced transcription of the ureA gene is unaltered in a nikR mutant, providing evidence that pH-responsive regulation and nickel-responsive regulation of the ureA promoter are mediated by independent mechanisms involving the ArsR response regulator and the NikR protein. PMID:16177315

  17. Synergic Interaction of Rifaximin and Mutaflor (Escherichia coli Nissle 1917) in the Treatment of Acetic Acid-Induced Colitis in Rats.

    PubMed

    Dembiński, Artur; Warzecha, Zygmunt; Ceranowicz, Piotr; Dembiński, Marcin; Cieszkowski, Jakub; Gosiewski, Tomasz; Bulanda, Małgorzata; Kuśnierz-Cabala, Beata; Gałązka, Krystyna; Konturek, Peter Christopher

    2016-01-01

    Background. Inflammatory bowel disease results from the dysregulation of immune response to environmental and microbial agents in genetically susceptible individuals. The aim of the present study was to examine the effect of rifaximin and/or Mutaflor (Escherichia coli Nissle 1917, EcN) administration on the healing of acetic acid-induced colitis. Methods. Colitis was induced in male Wistar rats by rectal enema with 3.5% acetic acid solution. Rifaximin (50 mg/kg/dose) and/or Mutaflor (10(9) CFU/dose) were given intragastrically once a day. The severity of colitis was assessed at the 8th day after induction of inflammation. Results. Treatment with rifaximin significantly accelerated the healing of colonic damage. This effect was associated with significant reversion of the acetic acid-evoked decrease in mucosal blood flow and DNA synthesis. Moreover, administration of rifaximin significantly reduced concentration of proinflammatory TNF-α and activity of myeloperoxidase in colonic mucosa. Mutaflor given alone was without significant effect on activity of colitis. In contrast, Mutaflor given in combination with rifaximin significantly enhanced therapeutic effect of rifaximin. Moreover, Mutaflor led to settle of the colon by EcN and this effect was augmented by pretreatment with rifaximin. Conclusion. Rifaximin and Mutaflor exhibit synergic anti-inflammatory and therapeutic effect in acetic acid-induced colitis in rats.

  18. Chicoric acid induces apoptosis in 3T3-L1 preadipocytes through ROS-mediated PI3K/Akt and MAPK signaling pathways.

    PubMed

    Xiao, Haifang; Wang, Jing; Yuan, Li; Xiao, Chunxia; Wang, Yutang; Liu, Xuebo

    2013-02-20

    Chicoric acid has been reported to possess various bioactivities. However, the antiobesity effects of chicoric acid remain poorly understood. In this study, we investigated the effects of chicoric acid on 3T3-L1 preadipocytes and its molecular mechanisms of apoptosis. Chicoric acid inhibited cell viability and induced apoptosis in 3T3-L1 preadipocytes which was characterized by chromatin condensation and poly ADP-ribose-polymerase (PARP) cleavage. Mitochondrial membrane potential (MMP) loss, Bax/Bcl-2 dysregulation, cytochrome c release, and caspase-3 activation were observed, indicating mitochondria-dependent apoptosis induced by chicoric acid. Furthermore, PI3K/Akt and MAPK (p38 MAPK, JNK, and ERK1/2) signaling pathways were involved in chicoric acid-induced apoptosis. The employment of protein kinase inhibitors LY294002, SB203580, SP600125, and U0126 revealed that PI3K/Akt signaling pathway interplayed with MAPK signaling pathways. Moreover, chicoric acid induced reactive oxygen species (ROS) generation. Pretreatment with the antioxidant N-acetylcysteine (NAC) significantly blocked cell death and changes of Akt and MAPK signalings induced by chicoric acid. In addition, chicoric acid down regulated HO-1 and COX-2 via the PI3K/Akt pathway.

  19. The contribution of a Ca(2+)-activated Cl(-) conductance to amino-acid-induced inward current responses of ciliated olfactory neurons of the rainbow trout.

    PubMed

    Sato, K; Suzuki, N

    2000-01-01

    To determine whether amino-acid-induced inward currents of ciliated olfactory receptor neurons (ORNs) in rainbow trout (Oncorhynchus mykiss) include a Ca(2+)-activated Cl(-) conductance, we first studied changes in reversal potential and the current/voltage relationships of the responses of ORNs to an amino acid mixture (l-alanine, l-arginine, l-glutamate and l-norvaline; all 10 mmol l(-)(1)) with different concentrations of Na(+) and Cl(-) in the perfusion and recording pipette solutions. We also examined the effects of six different Cl(-) channel blockers on the responses of ORNs using a conventional whole-cell voltage-clamp technique. The amino acid mixture and one blocker were applied focally to the cilia of ORNs using a double-barrelled micropipette and a pressure ejection system. The expected shifts in reversal potential, indicating the contribution of the Ca(2+)-activated Cl(-) conductance, occurred in both positive and negative directions depending on the external and internal Na(+) and Cl(-) concentrations. Niflumic acid, flufenamic acid, NPPB [5-nitro-2-(3-phenylpropylamino)-benzonate] and DCDPC (3', 5-dichlorodiphenylamine-2-carboxylate), at 0.5 mmol l(-)(1), reversibly blocked both the amino-acid-induced inward currents and the background activity in most ORNs. The effectiveness of these blocking agents varied from 77 to 91 % for ORNs perfused externally with standard Ringer's solution. SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonate), at 5.0 mmol l(-)(1), irreversibly inhibited the physiological response (100 % inhibition), whereas DIDS (4,4'-diisothiocyanatostilbene-2, 2'-disulphonate), at 5.0 mmol l(-)(1), had the smallest effect (45 %) of the inhibitors tested. The dose of niflumic acid inducing 50 % inhibition (IC(50)), determined specifically for the current component of the Ca(2+)-activated Cl(-) channels, was 70 micromol l(-)(1). Our results suggest that these blockers are not specific for Ca(2+)-activated Cl(-) channels and that

  20. Chicken cells sense influenza A virus infection through MDA5 and CARDIF signaling involving LGP2.

    PubMed

    Liniger, Matthias; Summerfield, Artur; Zimmer, Gert; McCullough, Kenneth C; Ruggli, Nicolas

    2012-01-01

    Avian influenza viruses (AIV) raise worldwide veterinary and public health concerns due to their potential for zoonotic transmission. While infection with highly pathogenic AIV results in high mortality in chickens, this is not necessarily the case in wild birds and ducks. It is known that innate immune factors can contribute to the outcome of infection. In this context, retinoic acid-inducible gene I (RIG-I) is the main cytosolic pattern recognition receptor known for detecting influenza A virus infection in mammalian cells. Chickens, unlike ducks, lack RIG-I, yet chicken cells do produce type I interferon (IFN) in response to AIV infection. Consequently, we sought to identify the cytosolic recognition elements in chicken cells. Chicken mRNA encoding the putative chicken analogs of CARDIF and LGP2 (chCARDIF and chLGP2, respectively) were identified. HT7-tagged chCARDIF was observed to associate with mitochondria in chicken DF-1 fibroblasts. The exogenous expression of chCARDIF, as well as of the caspase activation and recruitment domains (CARDs) of the chicken melanoma differentiation-associated protein 5 (chMDA5), strongly activated the chicken IFN-β (chIFN-β) promoter. The silencing of chMDA5, chCARDIF, and chIRF3 reduced chIFN-β levels induced by AIV, indicating their involvement in AIV sensing. As with mammalian cells, chLGP2 had opposing effects. While overexpression decreased the activation of the chIFN-β promoter, the silencing of endogenous chLGP2 reduced chIFN-β induced by AIV. We finally demonstrate that the chMDA5 signaling pathway is inhibited by the viral nonstructural protein 1. In conclusion, chicken cells, including DF-1 fibroblasts and HD-11 macrophage-like cells, employ chMDA5 for sensing AIV.

  1. Structural basis for Marburg virus VP35-mediated immune evasion mechanisms

    SciTech Connect

    Ramanan, Parameshwaran; Edwards, Megan R.; Shabman, Reed S.; Leung, Daisy W.; Endlich-Frazier, Ariel C.; Borek, Dominika M.; Otwinowski, Zbyszek; Liu, Gai; Huh, Juyoung; Basler, Christopher F.; Amarasinghe, Gaya K.

    2013-07-22

    Filoviruses, marburgvirus (MARV) and ebolavirus (EBOV), are causative agents of highly lethal hemorrhagic fever in humans. MARV and EBOV share a common genome organization but show important differences in replication complex formation, cell entry, host tropism, transcriptional regulation, and immune evasion. Multifunctional filoviral viral protein (VP) 35 proteins inhibit innate immune responses. Recent studies suggest double-stranded (ds)RNA sequestration is a potential mechanism that allows EBOV VP35 to antagonize retinoic-acid inducible gene-I (RIG-I) like receptors (RLRs) that are activated by viral pathogen–associated molecular patterns (PAMPs), such as double-strandedness and dsRNA blunt ends. Here, we show that MARV VP35 can inhibit IFN production at multiple steps in the signaling pathways downstream of RLRs. The crystal structure of MARV VP35 IID in complex with 18-bp dsRNA reveals that despite the similar protein fold as EBOV VP35 IID, MARV VP35 IID interacts with the dsRNA backbone and not with blunt ends. Functional studies show that MARV VP35 can inhibit dsRNA-dependent RLR activation and interferon (IFN) regulatory factor 3 (IRF3) phosphorylation by IFN kinases TRAF family member-associated NFkb activator (TANK) binding kinase-1 (TBK-1) and IFN kB kinase e (IKKe) in cell-based studies. We also show that MARV VP35 can only inhibit RIG-I and melanoma differentiation associated gene 5 (MDA5) activation by double strandedness of RNA PAMPs (coating backbone) but is unable to inhibit activation of RLRs by dsRNA blunt ends (end capping). In contrast, EBOV VP35 can inhibit activation by both PAMPs. Insights on differential PAMP recognition and inhibition of IFN induction by a similar filoviral VP35 fold, as shown here, reveal the structural and functional plasticity of a highly conserved virulence factor.

  2. Human respiratory syncytial virus nucleoprotein and inclusion bodies antagonize the innate immune response mediated by MDA5 and MAVS.

    PubMed

    Lifland, Aaron W; Jung, Jeenah; Alonas, Eric; Zurla, Chiara; Crowe, James E; Santangelo, Philip J

    2012-08-01

    Currently, the spatial distribution of human respiratory syncytial virus (hRSV) proteins and RNAs in infected cells is still under investigation, with many unanswered questions regarding the interaction of virus-induced structures and the innate immune system. Very few studies of hRSV have used subcellular imaging as a means to explore the changes in localization of retinoic-acid-inducible gene-I (RIG-I)-like receptors or the mitochondrial antiviral signaling (MAVS) protein, in response to the infection and formation of viral structures. In this investigation, we found that both RIG-I and melanoma differentiation-associated gene 5 (MDA5) colocalized with viral genomic RNA and the nucleoprotein (N) as early as 6 h postinfection (hpi). By 12 hpi, MDA5 and MAVS were observed within large viral inclusion bodies (IB). We used a proximity ligation assay (PLA) and determined that the N protein was in close proximity to MDA5 and MAVS in IBs throughout the course of the infection. Similar results were found with the transient coexpression of N and the phosphoprotein (P). Additionally, we demonstrated that the localization of MDA5 and MAVS in IBs inhibited the expression of interferon β mRNA 27-fold following Newcastle disease virus infection. From these data, we concluded that the N likely interacts with MDA5, is in close proximity to MAVS, and localizes these molecules within IBs in order to attenuate the interferon response. To our knowledge, this is the first report of a specific function for hRSV IBs and of the hRSV N protein as a modulator of the innate immune response.

  3. Structural basis for Marburg virus VP35-mediated immune evasion mechanisms.

    PubMed

    Ramanan, Parameshwaran; Edwards, Megan R; Shabman, Reed S; Leung, Daisy W; Endlich-Frazier, Ariel C; Borek, Dominika M; Otwinowski, Zbyszek; Liu, Gai; Huh, Juyoung; Basler, Christopher F; Amarasinghe, Gaya K

    2012-12-11

    Filoviruses, marburgvirus (MARV) and ebolavirus (EBOV), are causative agents of highly lethal hemorrhagic fever in humans. MARV and EBOV share a common genome organization but show important differences in replication complex formation, cell entry, host tropism, transcriptional regulation, and immune evasion. Multifunctional filoviral viral protein (VP) 35 proteins inhibit innate immune responses. Recent studies suggest double-stranded (ds)RNA sequestration is a potential mechanism that allows EBOV VP35 to antagonize retinoic-acid inducible gene-I (RIG-I) like receptors (RLRs) that are activated by viral pathogen-associated molecular patterns (PAMPs), such as double-strandedness and dsRNA blunt ends. Here, we show that MARV VP35 can inhibit IFN production at multiple steps in the signaling pathways downstream of RLRs. The crystal structure of MARV VP35 IID in complex with 18-bp dsRNA reveals that despite the similar protein fold as EBOV VP35 IID, MARV VP35 IID interacts with the dsRNA backbone and not with blunt ends. Functional studies show that MARV VP35 can inhibit dsRNA-dependent RLR activation and interferon (IFN) regulatory factor 3 (IRF3) phosphorylation by IFN kinases TRAF family member-associated NFkb activator (TANK) binding kinase-1 (TBK-1) and IFN kB kinase e (IKKe) in cell-based studies. We also show that MARV VP35 can only inhibit RIG-I and melanoma differentiation associated gene 5 (MDA5) activation by double strandedness of RNA PAMPs (coating backbone) but is unable to inhibit activation of RLRs by dsRNA blunt ends (end capping). In contrast, EBOV VP35 can inhibit activation by both PAMPs. Insights on differential PAMP recognition and inhibition of IFN induction by a similar filoviral VP35 fold, as shown here, reveal the structural and functional plasticity of a highly conserved virulence factor.

  4. Structural basis for Marburg virus VP35–mediated immune evasion mechanisms

    PubMed Central

    Ramanan, Parameshwaran; Edwards, Megan R.; Shabman, Reed S.; Leung, Daisy W.; Endlich-Frazier, Ariel C.; Borek, Dominika M.; Otwinowski, Zbyszek; Liu, Gai; Huh, Juyoung; Basler, Christopher F.; Amarasinghe, Gaya K.

    2012-01-01

    Filoviruses, marburgvirus (MARV) and ebolavirus (EBOV), are causative agents of highly lethal hemorrhagic fever in humans. MARV and EBOV share a common genome organization but show important differences in replication complex formation, cell entry, host tropism, transcriptional regulation, and immune evasion. Multifunctional filoviral viral protein (VP) 35 proteins inhibit innate immune responses. Recent studies suggest double-stranded (ds)RNA sequestration is a potential mechanism that allows EBOV VP35 to antagonize retinoic-acid inducible gene-I (RIG-I) like receptors (RLRs) that are activated by viral pathogen–associated molecular patterns (PAMPs), such as double-strandedness and dsRNA blunt ends. Here, we show that MARV VP35 can inhibit IFN production at multiple steps in the signaling pathways downstream of RLRs. The crystal structure of MARV VP35 IID in complex with 18-bp dsRNA reveals that despite the similar protein fold as EBOV VP35 IID, MARV VP35 IID interacts with the dsRNA backbone and not with blunt ends. Functional studies show that MARV VP35 can inhibit dsRNA-dependent RLR activation and interferon (IFN) regulatory factor 3 (IRF3) phosphorylation by IFN kinases TRAF family member-associated NFkb activator (TANK) binding kinase-1 (TBK-1) and IFN kB kinase e (IKKe) in cell-based studies. We also show that MARV VP35 can only inhibit RIG-I and melanoma differentiation associated gene 5 (MDA5) activation by double strandedness of RNA PAMPs (coating backbone) but is unable to inhibit activation of RLRs by dsRNA blunt ends (end capping). In contrast, EBOV VP35 can inhibit activation by both PAMPs. Insights on differential PAMP recognition and inhibition of IFN induction by a similar filoviral VP35 fold, as shown here, reveal the structural and functional plasticity of a highly conserved virulence factor. PMID:23185024

  5. Nonstructural protein 1{alpha} subunit-based inhibition of NF-{kappa}B activation and suppression of interferon-{beta} production by porcine reproductive and respiratory syndrome virus

    SciTech Connect

    Song Cheng; Krell, Peter; Yoo, Dongwan

    2010-11-25

    Induction of type I interferon (IFN-{alpha}/{beta}) is an early antiviral response of the host, and porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to downregulate the IFN response during infection in cells and pigs. We report that the PRRSV nonstructural protein 1{alpha} (Nsp1{alpha}) subunit of Nsp1 is a nuclear-cytoplasmic protein distributed to the nucleus and contains a strong suppressive activity for IFN-{beta} production that is mediated through the retinoic acid-inducible gene I (RIG-I) signaling pathway. Nsp1{alpha} suppressed the activation of nuclear factor (NF)-{kappa}B when stimulated with dsRNA or tumor necrosis factor (TNF)-{alpha}, and NF-{kappa}B suppression was RIG-I-dependent. The suppression of NF-{kappa}B activation was associated with the poor production of IFN-{beta} during PRRSV infection. The C-terminal 14 amino acids of the Nsp1{alpha} subunit were critical in maintaining immunosuppressive activity of Nsp1{alpha} for both IFN-{beta} and NF-{kappa}B, suggesting that the newly identified zinc finger configuration comprising of Met180 may be crucial for inhibitory activities. Nsp1{alpha} inhibited I{kappa}B phosphorylation and as a consequence NF-{kappa}B translocation to the nucleus was blocked, leading to the inhibition of NF-{kappa}B stimulated gene expression. Our results suggest that PRRSV Nsp1{alpha} is a multifunctional nuclear protein participating in the modulation of the host IFN system.

  6. Spleen Transcriptome Profile of Muscovy Ducklings in Response to Infection With Muscovy Duck Reovirus.

    PubMed

    Wang, Quanxi; Wu, Yijian; Cai, Yilong; Zhuang, Yubin; Xu, Lihui; Wu, Baocheng; Zhang, Yanding

    2015-06-01

    Muscovy duck reovirus (MDRV) causes high morbidity and mortality in ducklings. However, the molecular basis for pathogenesis of this virus remains poorly understood, and the complete genome sequence of Muscovy duck is lacking. Here we report the transcriptome profile of Muscovy ducks in response to MDRV infection. RNA sequencing technology was employed to obtain a representative complement of transcripts from the spleen of ducklings, and then differential gene expression was analyzed between MDRV-YB strain infected ducks and noninfected ducks. This analysis generated 65,536 unigenes. Of these, 6458 genes were found to be significantly differentially expressed between the infected and noninfected groups. The symptom and pathology of ducks infected with MDRV-YB was correlated with the greater proportion of decreased expression genes (4906) than increased expression (1552) level. Gene ontology analysis assigned differentially expressed genes to the categories: "biological processes," "cellular functions," and "molecular functions." Differentially expressed genes involved in the innate immune system were analyzed further, and 128 of these genes showed decreased expression and 86 showed increased expression between the infected and noninfected groups. These genes represented the Janus kinase-signal transducer and activator of transcription signaling pathway, and the retinoic acid-inducible gene I (RIG-I)-like and Toll-like receptor (TLR) signaling pathways and included interferon (IFN) α, IFNγ, interleukin 6, RIG-I, and TLR4. The data were verified by SYBR fluorescence quantitative polymerase chain reaction (SYBR-qPCR). Our findings offer new insight into the host immune response to MDRV infection.

  7. Yogurt containing Lactobacillus gasseri OLL 2716 (LG21 yogurt) accelerated the healing of acetic acid-induced gastric ulcer in rats.

    PubMed

    Uchida, Masayuki; Shimizu, Kimiko; Kurakazu, Keiko

    2010-01-01

    We have reported that LG21 yogurt containing Lactobacillus gasseri OLL 2716 (LG21 yogurt) inhibits the formation of HCl-induced acute gastric lesions through the generation of prostaglandin E₂. This study aimed to determine the role of viable Lactobacillus in the healing of acetic acid-induced chronic gastric ulcer. LG21 yogurt or γ-ray radiated LG21 yogurt was administered orally twice a day for 10 d at a dose of 5 ml/kg. LG21 yogurt significantly accelerated the healing of the ulcer, but γ-ray radiated LG21 yogurt did not. However, both yogurts significantly inhibited HCl-induced gastric erosive lesions and enhanced the generation of gastric mucosal prostaglandin E₂. From the above results, it was found that viable bacteria are needed to accelerate the healing of chronic gastric ulcer, but not to inhibit gastric lesions.

  8. Acid-induced crystallinity enhancement of graphite-like C3N3+xHy synthesized through a facile one-pot approach

    NASA Astrophysics Data System (ADS)

    Yin, Hao; Guo, Qixun; He, Dingzeng; Li, Juntao; Sun, Shigang

    2017-02-01

    Graphite-like C3N3+xHy with s-triazine rings as building blocks were synthesized through a facile one-pot approach. It is surprising that the degree of crystallinity of the synthesized sample at 330 °C (sample CNH-330) was remarkably enhanced by the dilute hydrochloric acid treatment. The mechanism of the acid-induced crystallinity enhancement was preliminarily studied. XRD, FTIR, SEM, photoluminescence spectra, elemental analysis, and XPS were performed to investigate the composition and structure of the obtained samples. The remarkable enhancement of the degree of crystallinity may be attributed to the ordered formation of ammonium-salt-like structure by the reaction of HCl with dbnd NH or sbnd NH2 in CNH-330.

  9. Analgesic and Anti-Inflammatory Properties of Gelsolin in Acetic Acid Induced Writhing, Tail Immersion and Carrageenan Induced Paw Edema in Mice

    PubMed Central

    Gupta, Ashok Kumar; Parasar, Devraj; Sagar, Amin; Choudhary, Vikas; Chopra, Bhupinder Singh; Garg, Renu; Ashish; Khatri, Neeraj

    2015-01-01

    Plasma gelsolin levels significantly decline in several disease conditions, since gelsolin gets scavenged when it depolymerizes and caps filamentous actin released in the circulation following tissue injury. It is well established that our body require/implement inflammatory and analgesic responses to protect against cell damage and injury to the tissue. This study was envisaged to examine analgesic and anti-inflammatory activity of exogenous gelsolin (8 mg/mouse) in mice models of pain and acute inflammation. Administration of gelsolin in acetic acid-induced writhing and tail immersion tests not only demonstrated a significant reduction in the number of acetic acid-induced writhing effects, but also exhibited an analgesic activity in tail immersion test in mice as compared to placebo treated mice. Additionally, anti-inflammatory function of gelsolin (8 mg/mouse) compared with anti-inflammatory drug diclofenac sodium (10 mg/kg)] was confirmed in the carrageenan injection induced paw edema where latter was measured by vernier caliper and fluorescent tomography imaging. Interestingly, results showed that plasma gelsolin was capable of reducing severity of inflammation in mice comparable to diclofenac sodium. Analysis of cytokines and histo-pathological examinations of tissue revealed administration of gelsolin and diclofenac sodium significantly reduced production of pro-inflammatory cytokines, TNF-α and IL-6. Additionally, carrageenan groups pretreated with diclofenac sodium or gelsolin showed a marked decrease in edema and infiltration of inflammatory cells in paw tissue. Our study provides evidence that administration of gelsolin can effectively reduce the pain and inflammation in mice model. PMID:26426535

  10. Protective Effect of Unsaturated Fatty Acids on Palmitic Acid-Induced Toxicity in Skeletal Muscle Cells is not Mediated by PPARδ Activation.

    PubMed

    Tumova, Jana; Malisova, Lucia; Andel, Michal; Trnka, Jan

    2015-10-01

    Unsaturated free fatty acids (FFA) are able to prevent deleterious effects of saturated FFA in skeletal muscle cells although the mechanisms involved are still not completely understood. FFA act as endogenous ligands of peroxisome proliferator-activated receptors (PPAR), transcription factors regulating the expression of genes involved in lipid metabolism. The aim of this study was to determine whether activation of PPARδ, the most common PPAR subtype in skeletal muscle, plays a role in mediating the protective effect of unsaturated FFA on saturated FFA-induced damage in skeletal muscle cells and to examine an impact on mitochondrial respiration. Mouse C2C12 myotubes were treated for 24 h with different concentrations of saturated FFA (palmitic acid), unsaturated FFA (oleic, linoleic and α-linolenic acid), and their combinations. PPARδ agonist GW501516 and antagonist GSK0660 were also used. Both mono- and polyunsaturated FFA, but not GW501516, prevented palmitic acid-induced cell death. Mono- and polyunsaturated FFA proved to be effective activators of PPARδ compared to saturated palmitic acid; however, in combination with palmitic acid their effect on PPARδ activation was blocked and stayed at the levels observed for palmitic acid alone. Unsaturated FFA at moderate physiological concentrations as well as GW501516, but not palmitic acid, mildly uncoupled mitochondrial respiration. Our results indicate that although unsaturated FFA are effective activators of PPARδ, their protective effect on palmitic acid-induced toxicity is not mediated by PPARδ activation and subsequent induction of lipid regulatory genes in skeletal muscle cells. Other mechanisms, such as mitochondrial uncoupling, may underlie their effect.

  11. Valproic acid increases conservative homologous recombination frequency and reactive oxygen species formation: a potential mechanism for valproic acid-induced neural tube defects.

    PubMed

    Defoort, Ericka N; Kim, Perry M; Winn, Louise M

    2006-04-01

    Valproic acid, a commonly used antiepileptic agent, is associated with a 1 to 2% incidence of neural tube defects when taken during pregnancy; however, the molecular mechanism by which this occurs has not been elucidated. Previous research suggests that valproic acid exposure leads to an increase in reactive oxygen species (ROS). DNA damage due to ROS can result in DNA double-strand breaks, which can be repaired through homologous recombination (HR), a process that is not error-free and can result in detrimental genetic changes. Because the developing embryo requires tight regulation of gene expression to develop properly, we propose that the loss or dysfunction of genes involved in embryonic development through aberrant HR may ultimately cause neural tube defects. To determine whether valproic acid induces HR, Chinese hamster ovary 3-6 cells, containing a neomycin direct repeat recombination substrate, were exposed to valproic acid for 4 or 24 h. A significant increase in HR after exposure to valproic acid (5 and 10 mM) for 24 h was observed, which seems to occur through a conservative HR mechanism. We also demonstrated that exposure to valproic acid (5 and 10 mM) significantly increased intracellular ROS levels, which were attenuated by preincubation with polyethylene glycol-conjugated (PEG)-catalase. A significant change in the ratio of 8-hydroxy-2'-deoxyguanosine/2'-de-oxyguanosine, a measure of DNA oxidation, was not observed after valproic acid exposure; however, preincubation with PEG-catalase significantly blocked the increase in HR. These data demonstrate that valproic acid increases HR frequency and provides a possible mechanism for valproic acid-induced neural tube defects.

  12. Involvement of p38 MAPK and Nrf2 in phenolic acid-induced P-form phenol sulfotransferase expression in human hepatoma HepG2 cells.

    PubMed

    Yeh, Chi-Tai; Yen, Gow-Chin

    2006-05-01

    Phenolic acids have significant biological and pharmacological properties and some have demonstrated remarkable ability to alter sulfate conjugation. However, the modulation mechanisms of phenolic acids on phenol sulfotransferase expression have not been described. In the present study, we investigated the effects of phenolic acids on the expression of the Phase II P-form of phenol sulfotransferase (PST-P) in human hepatoma HepG2 cells. RT-PCR and western blot data revealed that gallic acid induced increase in PST-P expression at the mRNA and protein levels, respectively. This induction was also marked by an increase in PST-P activity. Actinomycin D and cycloheximide inhibited gallic acid-responsive PST-P mRNA expression, indicating that gallic acid is a requirement for transcription and de novo protein synthesis. Transient transfection of HepG2 cells with a reporter plasmid of the upstream region of the human PST gene caused a significant increase in reporter gene activity after gallic acid exposure. Moreover, gallic acid increased the nuclear levels of Nrf2, a transcription factor governing antioxidant response element (ARE). Electrophoretic mobility shift assay showed increased binding of nuclear proteins to ARE consensus sequence after treatment with gallic acid. While investigating the signaling pathways responsible for PST-P induction, we observed that gallic acid activated the p38 mitogen-activated protein kinase (MAPK) pathway. SB203580, a specific inhibitor of p38 MAPK, abolished gallic acid-induced PST-P protein expression. Similarly, gallic acid also caused an accumulation of Nrf2. Moreover, the protective effects of gallic acid on tert-butyl hydroperoxide-induced toxicity was partially blocked by p38 MAPK and PST-P inhibitors, further demonstrating that gallic acid attenuates oxidative stress through a pathway that involves p38 MAPK and PST-P. These results indicate that gallic acid is a potent inducer of PST-P and that PST-P induction is responsible

  13. Assessment of the antinociceptive effects of pregabalin alone or in combination with morphine during acetic acid-induced writhing in mice.

    PubMed

    Shamsi Meymandi, Manzumeh; Keyhanfar, Fariborz

    2013-09-01

    Visceral pain currently represents one of the most important pain treatment challenges in clinical practice, and investigators across the world are continuously designing and conducting numerous studies in search of new analgesics and new combination therapies. The current study assessed the analgesic effects of saline, pregabalin (2, 5, 17, 50, 100, and 200 mg/kg, i.p.) and morphine (0.25, 0.5, 1, 3 and 5 mg/kg) alone or in combination on acetic-acid induced abdominal contractions in mice. The number of writhes and the inhibitory effects (as percentages, %E) were calculated as antinociception indexes. These indexes indicated that both pregabalin (Prg) and morphine (Mrp) produced dose-dependent antinociception. Pregabalin at 5 mg/kg (%E=32.5±4.0) or 2 mg/kg (%E=20.8±4.5) and morphine at 0.25 mg/kg (%E=20.2±7.8) and 0.5 mg/kg (%E=43.6±4.5) exhibited antinociceptive effects, and the combination of pregabalin and morphine produced significantly greater antinociceptive effects (%E=62.4±5.8 for Prg5+Mrp0.25; %E=71.7±4.8 for Prg5+Mrp0.5; and %E=54.1±4.0 for Prg2+Mrp0.25), although this enhancement was not observed when morphine was combined with 17 mg/kg pregabalin. Pre-treatment with 2 mg/kg (i.p.) naloxone did not affect increased analgesia when combined with these drugs. A dose-response curve was established for pregabalin at a fixed morphine dose and revealed that, at low doses, pregabalin dose-dependently enhanced the antinociceptive effects, while the opposite was true at high doses (17 and 25 mg/kg). In conclusion, pregabalin can produce levels of antinociception that are similar to those of morphine in acetic acid-induced viscero-somatic pain. The enhancement of antinociception produced by the co-administration of morphine and pregabalin is termed a supra-additive interaction and occurred at low doses but not at high doses. These findings militate for increased attention and caution in clinical settings.

  14. Molecular cloning and characterization of two novel retinoic acid-inducible orphan G-protein-coupled receptors (GPRC5B and GPRC5C).

    PubMed

    Robbins, M J; Michalovich, D; Hill, J; Calver, A R; Medhurst, A D; Gloger, I; Sims, M; Middlemiss, D N; Pangalos, M N

    2000-07-01

    Using homology searching of public databases with a metabotropic glutamate receptor sequence from Caenorhabditis elegans, two novel protein sequences (named RAIG-2 (HGMW-approved symbol GPRC5B) and RAIG-3 (HGMW-approved symbol GPRC5C) were identified containing seven putative transmembrane domains characteristic of G-protein-coupled receptors (GPCRs). RAIG-2 and RAIG-3 encode open reading frames of 403 and 442 amino acid polypeptides, respectively, and show 58% similarity to the recently identified retinoic acid-inducible gene-1 (RAIG-1, HGMW-approved symbol RAI3). Analysis of the three protein sequences places them within the type 3 GPCR family, which includes metabotropic glutamate receptors, GABA(B) receptors, calcium-sensing receptors, and pheromone receptors. However, in contrast to other type 3 GPCRs, RAIG-1, RAIG-2, and RAIG-3 have only short N-terminal domains. RAIG-2 and RAIG-3 cDNA sequences were cloned into the mammalian expression vector pcDNA3 with c-myc or HA epitope tags inserted at their N-termini, respectively. Transient transfection experiments in HEK239T cells using these constructs demonstrated RAIG-2 and RAIG-3 expression at the cell surface. Distribution profiles of mRNA expression obtained by semiquantitative Taq-Man PCR analysis showed RAIG-2 to be predominantly expressed in human brain areas and RAIG-3 to be predominantly expressed in peripheral tissues. In addition, expression of RAIG-2 and RAIG-3 mRNA was increased following treatment with all-trans-retinoic acid in a manner similar to that previously described for RAIG-1. Finally, RAIG-2 was mapped to chromosome 16p12 (D16S405-D16S3045) and RAIG-3 to chromosome 17q25 (D17S1352-D17S785). These results suggest that RAIG-1, RAIG-2, and RAIG-3 represent a novel family of retinoic acid-inducible receptors, most closely related to the type 3 GPCR subfamily, and provide further evidence for a linkage between retinoic acid and G-protein-coupled receptor signal transduction pathways.

  15. Manuka Honey Exerts Antioxidant and Anti-Inflammatory Activities That Promote Healing of Acetic Acid-Induced Gastric Ulcer in Rats

    PubMed Central

    Almasaudi, Saad B.; Al-Hindi, Rashad R.; Abdel-dayem, Umama A.; Ali, Soad S.; Saleh, Rasha M.; Al Jaouni, Soad K.

    2017-01-01

    Gastric ulcers are a major problem worldwide with no effective treatment. The objective of this study was to evaluate the use of manuka honey in the treatment of acetic acid-induced chronic gastric ulcers in rats. Different groups of rats were treated with three different concentrations of honey. Stomachs were checked macroscopically for ulcerative lesions in the glandular mucosa and microscopically for histopathological alterations. Treatment with manuka honey significantly reduced the ulcer index and maintained the glycoprotein content. It also reduced the mucosal myeloperoxidase activity, lipid peroxidation (MDA), and the inflammatory cytokines (TNF-α, IL-1β, and IL-6) as compared to untreated control group. In addition, honey-treated groups showed significant increase in enzymatic (GPx and SOD) and nonenzymatic (GSH) antioxidants besides levels of the anti-inflammatory cytokine IL-10. Flow cytometry studies showed that treatment of animals with manuka honey has normalized cell cycle distribution and significantly lowered apoptosis in gastric mucosa. In conclusion, the results indicated that manuka honey is effective in the treatment of chronic ulcer and preservation of mucosal glycoproteins. Its effects are due to its antioxidant and anti-inflammatory properties that resulted in a significant reduction of the gastric mucosal MDA, TNF-α, IL-1β, and IL-6 and caused an elevation in IL-10 levels. PMID:28250794

  16. Increase in α-tubulin modifications in the neuronal processes of hippocampal neurons in both kainic acid-induced epileptic seizure and Alzheimer’s disease

    PubMed Central

    Vu, Hang Thi; Akatsu, Hiroyasu; Hashizume, Yoshio; Setou, Mitsutoshi; Ikegami, Koji

    2017-01-01

    Neurodegeneration includes acute changes and slow-developing alterations, both of which partly involve common cellular machinery. During neurodegeneration, neuronal processes are impaired along with dysregulated post-translational modifications (PTMs) of cytoskeletal proteins. In neuronal processes, tubulin undergoes unique PTMs including a branched form of modification called glutamylation and loss of the C-terminal tyrosine residue and the penultimate glutamic acid residue forming Δ2-tubulin. Here, we investigated the state of two PTMs, glutamylation and Δ2 form, in both acute and slow-developing neurodegenerations, using a newly generated monoclonal antibody, DTE41, which had 2-fold higher affinity to glutamylated Δ2-tubulin, than to unmodified Δ2-tubulin. DTE41 recognised glutamylated Δ2-tubulin preferentially in immunostaining than in enzyme-linked immunosorbent assay and immunoblotting. In normal mouse brain, DTE41 stained molecular layer of the cerebellum as well as synapse-rich regions in pyramidal neurons of the cerebral cortex. In kainic acid-induced epileptic seizure, DTE41-labelled signals were increased in the hippocampal CA3 region, especially in the stratum lucidum. In the hippocampi of post-mortem patients with Alzheimer’s disease, intensities of DTE41 staining were increased in mossy fibres in the CA3 region as well as in apical dendrites of the pyramidal neurons. Our findings indicate that glutamylation on Δ2-tubulin is increased in both acute and slow-developing neurodegeneration. PMID:28067280

  17. Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid-induced ceramide biosynthesis in mice.

    PubMed

    Holland, William L; Bikman, Benjamin T; Wang, Li-Ping; Yuguang, Guan; Sargent, Katherine M; Bulchand, Sarada; Knotts, Trina A; Shui, Guanghou; Clegg, Deborah J; Wenk, Markus R; Pagliassotti, Michael J; Scherer, Philipp E; Summers, Scott A

    2011-05-01

    Obesity is associated with an enhanced inflammatory response that exacerbates insulin resistance and contributes to diabetes, atherosclerosis, and cardiovascular disease. One mechanism accounting for the increased inflammation associated with obesity is activation of the innate immune signaling pathway triggered by TLR4 recognition of saturated fatty acids, an event that is essential for lipid-induced insulin resistance. Using in vitro and in vivo systems to model lipid induction of TLR4-dependent inflammatory events in rodents, we show here that TLR4 is an upstream signaling component required for saturated fatty acid-induced ceramide biosynthesis. This increase in ceramide production was associated with the upregulation of genes driving ceramide biosynthesis, an event dependent of the activity of the proinflammatory kinase IKKβ. Importantly, increased ceramide production was not required for TLR4-dependent induction of inflammatory cytokines, but it was essential for TLR4-dependent insulin resistance. These findings suggest that sphingolipids such as ceramide might be key components of the signaling networks that link lipid-induced inflammatory pathways to the antagonism of insulin action that contributes to diabetes.

  18. Alkali- or acid-induced changes in structure, moisture absorption ability and deacetylating reaction of β-chitin extracted from jumbo squid (Dosidicus gigas) pens.

    PubMed

    Jung, Jooyeoun; Zhao, Yanyun

    2014-01-01

    Alkali- or acid-induced structural modifications in β-chitin from squid (Dosidicus gigas, d'Orbigny, 1835) pens and their moisture absorption ability (MAA) and deacetylating reaction were investigated and compared with α-chitin from shrimp shells. β-Chitin was converted into the α-form after 3h in 40% NaOH or 1-3 h in 40% HCl solution, and α-chitin obtained from NaOH treatment had higher MAA than had native α-chitin, due to polymorphic destructions. In contrast, induced α-chitin from acid treatment of β-chitin had few polymorphic modifications, showing no significant change (P>0.05) in MAA. β-Chitin was more susceptible to alkali deacetylation than was α-chitin, and required a lower concentration of NaOH and shorter reaction time. These results demonstrate that alkali- or acid-treated β-chitin retained high susceptibility toward solvents, which in turn resulted in good biological activity of β-chitosan for use as a natural antioxidant and antimicrobial substance or application as edible coatings and films for various food applications.

  19. Effect of dietary fibers on cholic acid induced cell proliferation in the colonic epithelium of C57BL/6J mice

    SciTech Connect

    Robblee, N.M.; Bruce, W.R.; Bird, R.P.

    1986-03-01

    It has been postulated that high fat diets promote tumorigenesis by increasing the level of secondary bile acids in the colonic lumen. Dietary fibers are thought to be protective perhaps through their interaction with bile acids. In the present study, animals were fed diets containing either 0%, 5%, or 10% cellulose (C), pectin (P), or wheat bran (WB). The diets were formulated to contain either 0% (control) or 0.2% cholic acid (test). After two weeks of dietary treatment the animals were injected with (/sup 3/H)-thymidine and their colons were processed for autoradiography. The number of labeled cells (LC) in the colonic crypts was determined. Among the control diets, 10%P induced a two-fold increase in the LC. All the test groups had significantly higher LC than in their controls. However, the C group excited a higher LC than the P or WB groups (5.2 +/- 0.8 vs 3.9 +/- 0.8 or 3.9 +/- 0.6). These results were substantiated by metaphase arrest technique. The authors results show that nonfermentable fiber does not alleviate bile acid induced cell proliferative activity in the colon whereas fermentable fibers will counteract the promotional effect of a high fat diet.

  20. Improvement of derivatized amino acid detection sensitivity in micellar electrokinetic capillary chromatography by means of acid-induced pH-mediated stacking technique.

    PubMed

    Dziomba, Szymon; Bekasiewicz, Adrian; Prahl, Adam; Bączek, Tomasz; Kowalski, Piotr

    2014-10-01

    Derivatization is a frequently used sample preparation procedure applicable to the enhancement of analyte detection sensitivity. Amino acids mostly require derivatization prior to electrophoretic or chromatographic analysis, especially if spectrophotometric detection is used. This study presents an on-line preconcentration technique for derivatized amino acids. The sensitivity of the method was improved by the utilization of the proposed acid-induced pH-mediated stacking mechanism. The method is demonstrated by preconcentration of amino acids labeled with 2,4-dinitrofluorobenzene. Use of optimized conditions for a large sample volume injection (40 s, 13.8 kPa) followed by electrokinetic injection of 0.1 M HCl (20 s, 10 kV) gave a 20- to 30-fold enhancement of sensitivity. The significance of the sweeping mechanism and pseudo-isotachophoresis for the on-line sample focusing and the influence of parameters on the preconcentration process were discussed. The applicability of the elaborated method was demonstrated using human urine samples.

  1. Evidence for genetic regulation of mRNA expression of the dosage-sensitive gene retinoic acid induced-1 (RAI1) in human brain

    PubMed Central

    Chen, Li; Tao, Yu; Song, Fan; Yuan, Xi; Wang, Jian; Saffen, David

    2016-01-01

    RAI1 (retinoic acid induced-1) is a dosage-sensitive gene that causes Smith-Magenis syndrome (SMS) when mutated or deleted and Potocki-Lupski Syndrome (PTLS) when duplicated, with psychiatric features commonly observed in both syndromes. How common genetic variants regulate this gene, however, is unknown. In this study, we found that RAI1 mRNA expression in Chinese prefrontal and temporal cortex correlate with genotypes of common single nucleotide polymorphisms (SNPs) located in the RAI1 5′-upstream region. Using genotype imputation, “R2-Δ2” analysis, and data from the RegulomeDB database, we identified SNPs rs4925102 and rs9907986 as possible regulatory variants, accounting for approximately 30–40% of the variance in RAI1 mRNA expression in both brain regions. Specifically, rs4925102 and rs9907986 are predicted to disrupt the binding of retinoic acid RXR-RAR receptors and the transcription factor DEAF1 (Deformed epidermal autoregulatory factor-1), respectively. Consistent with these predictions, we observed binding of RXRα and RARα to the predicted RAI1 target in chromatin immunoprecipitation assays. Retinoic acid is crucial for early development of the central neural system, and DEAF1 is associated with intellectual disability. The observation that a significant portion of RAI1 mRNA expression is genetically controlled raises the possibility that common RAI1 5′-region regulatory variants contribute more generally to psychiatric disorders. PMID:26743651

  2. Elucidation of Acid-induced Unfolding and Aggregation of Human Immunoglobulin IgG1 and IgG2 Fc

    PubMed Central

    Latypov, Ramil F.; Hogan, Sabine; Lau, Hollis; Gadgil, Himanshu; Liu, Dingjiang

    2012-01-01

    Understanding the underlying mechanisms of Fc aggregation is an important prerequisite for developing stable and efficacious antibody-based therapeutics. In our study, high resolution two-dimensional nuclear magnetic resonance (NMR) was employed to probe structural changes in the IgG1 Fc. A series of 1H-15N heteronuclear single-quantum correlation NMR spectra were collected between pH 2.5 and 4.7 to assess whether unfolding of CH2 domains precedes that of CH3 domains. The same pH range was subsequently screened in Fc aggregation experiments that utilized molecules of IgG1 and IgG2 subclasses with varying levels of CH2 glycosylation. In addition, differential scanning calorimetry data were collected over a pH range of 3–7 to assess changes in CH2 and CH3 thermostability. As a result, compelling evidence was gathered that emphasizes the importance of CH2 stability in determining the rate and extent of Fc aggregation. In particular, we found that Fc domains of the IgG1 subclass have a lower propensity to aggregate compared with those of the IgG2 subclass. Our data for glycosylated, partially deglycosylated, and fully deglycosylated molecules further revealed the criticality of CH2 glycans in modulating Fc aggregation. These findings provide important insights into the stability of Fc-based therapeutics and promote better understanding of their acid-induced aggregation process. PMID:22084250

  3. Effects of Abscisic Acid and Ethylene on the Gibberellic Acid-Induced Synthesis of α-Amylase by Isolated Wheat Aleurone Layers 1

    PubMed Central

    Varty, Keith; Arreguín, Barbarín L.; Gómez, Miguel T.; López, Pablo Jaime T.; Gómez, Miguel Angel L.

    1983-01-01

    Gibberellic acid-induced α-amylase synthesis in wheat aleurone layers (Triticum aestivum L. var Potam S-70) escaped from transcriptional control 30 h after addition of the hormone, as evidenced by the tissue's loss of susceptibility to cordycepin. Abscisic acid inhibited the accumulation of α-amylase activity when added to the tissue during this cordycepin-insensitive phase of enzyme induction. α-Amylase synthesis was not restored by the addition of cordycepin, indicating that the response to abscisic acid was not dependent upon the continuous synthesis of a short lived RNA. When ethylene was added simultaneously or some time after abscisic acid, the accumulation of α-amylase activity was sustained or quickly restored. The loss of susceptibility to cordycepin was completely prevented when aleurone layers were incubated with a combination of gibberellic and abscisic acids from the start of the induction period. This effect of abscisic acid was not reversed by ethylene. On the basis of these observations, it is suggested that abscisic acid inhibits both the transcription and translation of α-amylase mRNA, and that only the latter site of action is susceptible to reversal by ethylene. The rate of incorporation of [methyl-14C]choline into phospholipids was also inhibited by abscisic acid. Ethylene reversed this effect. The effects of abscisic acid and ethylene on phospholipid synthesis were not dependent upon the presence of gibberellic acid. No direct relationship was found between the control of α-amylase synthesis and membrane formation by abscisic acid and ethylene. PMID:16663284

  4. Hypoxia and retinoic acid-inducible NDRG1 expression is responsible for doxorubicin and retinoic acid resistance in hepatocellular carcinoma cells.

    PubMed

    Jung, Eun Uk; Yoon, Jung-Hwan; Lee, Youn-Jae; Lee, Jeong-Hoon; Kim, Bo Hyun; Yu, Su Jong; Myung, Sun Jung; Kim, Yoon Jun; Lee, Hyo-Suk

    2010-12-01

    Hypoxia may activate survival signals in cancer cells. Moreover, hypoxic cells are less sensitive than normoxic cells to doxorubicin cytotoxicity, a potent activator of the p53 tumor suppressor gene. N-myc downstream-regulated gene-1 (NDRG1) is a hypoxia- and retinoic acid-inducible protein, and has been previously implicated in carcinogenesis. As this protein is also a downstream target of p53 and hepatocellular carcinoma (HCC) cells frequently evidence resistance to retinoic acid (RA) cytotoxicity, we attempted to determine whether the suppression of NDRG1 expression may sensitize HCC cells to doxorubicin and/or RA cytotoxicity. HCC cells expressed NDRG1 protein, and the expression of this protein was hypoxia- and RA-inducible. Doxorubicin treatment induced HCC cell cytotoxicity via the activation of mitochondrial apoptotic signals, including caspase-9 activation. Hypoxic HCC cells are less sensitive to doxorubicin-induced apoptosis. The suppression of NDRG1 expression either by siRNA or flavopiridol sensitized hypoxic HCC cells to doxorubicin cytotoxicity, and this was attributed to more profound augmentation of JNK and caspase-9 activation. The suppression of NDRG1 expression also sensitized RA-resistant HCC cells to RA-induced apoptosis, and this sensitization was more apparent in hypoxic HCC cells than in normoxic cells. Glutaredoxin2 expression was down-regulated in NDRG1-suppressed HCC cells. These results show that hypoxia- and RA-inducible NDRG1 expression is responsible for doxorubicin and RA resistance in HCC cells. Thus, the selective interruption of NDRG1 signaling may prove to be therapeutically useful in HCCs, particularly in the advanced infiltrative type of tumors exposed to hypoxic environments.

  5. Gibberellic Acid-Induced Aleurone Layers Responding to Heat Shock or Tunicamycin Provide Insight into the N-Glycoproteome, Protein Secretion, and Endoplasmic Reticulum Stress1[W

    PubMed Central

    Barba-Espín, Gregorio; Dedvisitsakul, Plaipol; Hägglund, Per; Svensson, Birte; Finnie, Christine

    2014-01-01

    The growing relevance of plants for the production of recombinant proteins makes understanding the secretory machinery, including the identification of glycosylation sites in secreted proteins, an important goal of plant proteomics. Barley (Hordeum vulgare) aleurone layers maintained in vitro respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping effects on both the intracellular and secreted proteomes. Proteins in a total of 22 and 178 two-dimensional gel spots changing in intensity in extracellular and intracellular fractions, respectively, were identified by mass spectrometry. Among these are proteins with key roles in protein processing and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions. This represents major progress in characterization of the barley N-glycoproteome, since only four of these sites were previously described. Overall, these findings considerably advance knowledge of the plant protein secretion system in general and emphasize the versatility of the aleurone layer as a model system for studying plant protein secretion. PMID:24344171

  6. Molecular analysis of the Retinoic Acid Induced 1 gene (RAI1) in patients with suspected Smith-Magenis syndrome without the 17p11.2 deletion.

    PubMed

    Vilboux, Thierry; Ciccone, Carla; Blancato, Jan K; Cox, Gerald F; Deshpande, Charu; Introne, Wendy J; Gahl, William A; Smith, Ann C M; Huizing, Marjan

    2011-01-01

    Smith-Magenis syndrome (SMS) is a complex neurobehavioral disorder characterized by multiple congenital anomalies. The syndrome is primarily ascribed to a ∼3.7 Mb de novo deletion on chromosome 17p11.2. Haploinsufficiency of multiple genes likely underlies the complex clinical phenotype. RAI1 (Retinoic Acid Induced 1) is recognized as a major gene involved in the SMS phenotype. Extensive genetic and clinical analyses of 36 patients with SMS-like features, but without the 17p11.2 microdeletion, yielded 10 patients with RAI1 variants, including 4 with de novo deleterious mutations, and 6 with novel missense variants, 5 of which were familial. Haplotype analysis showed two major RAI1 haplotypes in our primarily Caucasian cohort; the novel RAI1 variants did not occur in a preferred haplotype. RNA analysis revealed that RAI1 mRNA expression was significantly decreased in cells of patients with the common 17p11.2 deletion, as well as in those with de novo RAI1 variants. Expression levels varied in patients with familial RAI1 variants and in non-17p11.2 deleted patients without identified RAI1 defects. No correlation between SNP haplotype and RAI1 expression was found. Two clinical features, ocular abnormalities and polyembolokoilomania (object insertion), were significantly correlated with decreased RAI1 expression. While not significantly correlated, the presence of hearing loss, seizures, hoarse voice, childhood onset of obesity and specific behavioral aspects and the absence of immunologic abnormalities and cardiovascular or renal structural anomalies, appeared to be specific for the de novo RAI1 subgroup. Recognition of the combination of these features will assist in referral for RAI1 analysis of patients with SMS-like features without detectable microdeletion of 17p11.2. Moreover, RAI1 expression emerged as a genetic target for development of therapeutic interventions for SMS.

  7. Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

    PubMed

    Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

    2013-10-01

    Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

  8. Increased beta-oxidation in muscle cells enhances insulin-stimulated glucose metabolism and protects against fatty acid-induced insulin resistance despite intramyocellular lipid accumulation.

    PubMed

    Perdomo, German; Commerford, S Renee; Richard, Ann-Marie T; Adams, Sean H; Corkey, Barbara E; O'Doherty, Robert M; Brown, Nicholas F

    2004-06-25

    Skeletal muscle insulin resistance may be aggravated by intramyocellular accumulation of fatty acid-derived metabolites that inhibit insulin signaling. We tested the hypothesis that enhanced fatty acid oxidation in myocytes should protect against fatty acid-induced insulin resistance by limiting lipid accumulation. L6 myotubes were transduced with adenoviruses encoding carnitine palmitoyltransferase I (CPT I) isoforms or beta-galactosidase (control). Two to 3-fold overexpression of L-CPT I, the endogenous isoform in L6 cells, proportionally increased oxidation of the long-chain fatty acids palmitate and oleate and increased insulin stimulation of [(14)C]glucose incorporation into glycogen by 60% while enhancing insulin-stimulated phosphorylation of p38MAPK. Incubation of control cells with 0.2 mm palmitate for 18 h caused accumulation of triacylglycerol, diacylglycerol, and ceramide (but not long-chain acyl-CoA) and decreased insulin-stimulated [(14)C]glucose incorporation into glycogen (60%), [(3)H]deoxyglucose uptake (60%), and protein kinase B phosphorylation (20%). In the context of L-CPT I overexpression, palmitate preincubation produced a relative decrease in insulin-stimulated incorporation of [(14)C]glucose into glycogen (60%) and [(3)H]deoxyglucose uptake (40%) but did not inhibit phosphorylation of protein kinase B. Due to the enhancement of insulin-stimulated glucose metabolism induced by L-CPT I overexpression itself, net insulin-stimulated incorporation of [(14)C]glucose into glycogen and [(3)H]deoxyglucose uptake in L-CPT I-transduced, palmitate-treated cells were significantly greater than in palmitate-treated control cells (71 and 75% greater, respectively). However, L-CPT I overexpression failed to decrease intracellular triacylglycerol, diacylglycerol, ceramide, or long-chain acyl-CoA. We propose that accelerated beta-oxidation in muscle cells exerts an insulin-sensitizing effect independently of changes in intracellular lipid content.

  9. Music application alleviates short-term memory impairments through increasing cell proliferation in the hippocampus of valproic acid-induced autistic rat pups.

    PubMed

    Lee, Sung-Min; Kim, Bo-Kyun; Kim, Tae-Woon; Ji, Eun-Sang; Choi, Hyun-Hee

    2016-06-01

    Autism is a neurodevelopmental disorder and this disorder shows impairment in reciprocal social interactions, deficits in communication, and restrictive and repetitive patterns of behaviors and interests. The effect of music on short-term memory in the view of cell proliferation in the hippocampus was evaluated using valproic acid-induced autistic rat pups. Animal model of autism was made by subcutaneous injection of 400-mg/kg valproic acid into the rat pups on the postnatal day 14. The rat pups in the music-applied groups were exposed to the 65-dB comfortable classic music for 1 hr once a day, starting postnatal day 15 and continued until postnatal day 28. In the present results, short-term memory was deteriorated by autism induction. The numbers of 5-bromo-2'-deoxyridine (BrdU)-positive, Ki-67-positive, and doublecortin (DCX)-positive cells in the hippocampal dentate gyrus were decreased by autism induction. Brain-derived neurotrophic factor (BDNF) and tyrosine kinase B (TrkB) expressions in the hippocampus were also suppressed in the autistic rat pups. Music application alleviated short-term memory deficits with enhancing the numbers of BrdU-positive, Ki-67-positive, and DCX-positive cells in the autistic rat pups. Music application also enhanced BDNF and TrkB expressions in the autistic rat pups. The present study show that application of music enhanced hippocampal cell proliferation and alleviated short-term memory impairment through stimulating BDNF-TrkB signaling in the autistic rat pups. Music can be suggested as the therapeutic strategy to overcome the autism-induced memory deficits.

  10. Music application alleviates short-term memory impairments through increasing cell proliferation in the hippocampus of valproic acid-induced autistic rat pups

    PubMed Central

    Lee, Sung-Min; Kim, Bo-Kyun; Kim, Tae-Woon; Ji, Eun-Sang; Choi, Hyun-Hee

    2016-01-01

    Autism is a neurodevelopmental disorder and this disorder shows impairment in reciprocal social interactions, deficits in communication, and restrictive and repetitive patterns of behaviors and interests. The effect of music on short-term memory in the view of cell proliferation in the hippocampus was evaluated using valproic acid-induced autistic rat pups. Animal model of autism was made by subcutaneous injection of 400-mg/kg valproic acid into the rat pups on the postnatal day 14. The rat pups in the music-applied groups were exposed to the 65-dB comfortable classic music for 1 hr once a day, starting postnatal day 15 and continued until postnatal day 28. In the present results, short-term memory was deteriorated by autism induction. The numbers of 5-bromo-2′-deoxyridine (BrdU)-positive, Ki-67-positive, and doublecortin (DCX)-positive cells in the hippocampal dentate gyrus were decreased by autism induction. Brain-derived neurotrophic factor (BDNF) and tyrosine kinase B (TrkB) expressions in the hippocampus were also suppressed in the autistic rat pups. Music application alleviated short-term memory deficits with enhancing the numbers of BrdU-positive, Ki-67-positive, and DCX-positive cells in the autistic rat pups. Music application also enhanced BDNF and TrkB expressions in the autistic rat pups. The present study show that application of music enhanced hippocampal cell proliferation and alleviated short-term memory impairment through stimulating BDNF-TrkB signaling in the autistic rat pups. Music can be suggested as the therapeutic strategy to overcome the autism-induced memory deficits. PMID:27419108

  11. A comparative study of the antitussive activity of levodropropizine and dropropizine in the citric acid-induced cough model in normal subjects.

    PubMed

    Fumagalli, G; Cordaro, C I; Vanasia, M; Balzarotti, C; Camusso, L; Caiazzo, G; Maghini, L; Mazzocchi, M; Zennaro, M

    1992-01-01

    Levodropropizine is the levo-rotatory (S)-enantiomer of dropropizine, a racemic non-opiate antitussive agent which has been used clinically for many years. Compared with the racemic drug, levodropropizine exhibits in animal models similar antitussive activity but considerably lower central nervous system (CNS) depressant effects. It is also less likely to cause sedation in treated patients. Since the comparative antitussive potency of the two drugs in clinical experimental models has not been evaluated, the authors performed a randomized, double blind, cross over investigation in which the effects of single oral doses (60 and 90 mg) of levodropropizine and dropropizine were assessed by using the citric acid-induced cough model in eight normal volunteers. Stimulation tests involved inhalation of individual cumulative doses of citric acid (6.3 to 53.3 mg) which at pre-study assessment had been found to induce reproducibly at least ten coughs over a 30 sec period. Each subject was studied by repeating the citric acid stimulation test four times (0 h, 1 h, 2 h and 6 h) on each of five different days separated by intervals of at least three days. In the absence of drug administration (control session), cough response to citric inhalation was remarkably reproducible throughout the 6 h period of observation. A marked and statistically significant reduction in cough response (to about one third--one sixth of the pre-drug values) was observed 1 h after intake for both compounds. At subsequent testing 2 h and 6 h after dosing, cough response was still depressed and did not differ significantly from that observed at 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Calcium-dependent nitric oxide production is involved in the cytoprotective properties of n-acetylcysteine in glycochenodeoxycholic acid-induced cell death in hepatocytes

    SciTech Connect

    Gonzalez-Rubio, Sandra; Linares, Clara I.; Bello, Rosario I.; Gonzalez, Raul; Ferrin, Gustavo; Hidalgo, Ana B.; Munoz-Gomariz, Elisa; Rodriguez, Blanca A.; Barrera, Pilar; Ranchal, Isidora; Duran-Prado, Mario; De la Mata, Manuel; Muntane, Jordi

    2010-01-15

    The intracellular oxidative stress has been involved in bile acid-induced cell death in hepatocytes. Nitric oxide (NO) exerts cytoprotective properties in glycochenodeoxycholic acid (GCDCA)-treated hepatocytes. The study evaluated the involvement of Ca{sup 2+} on the regulation of NO synthase (NOS)-3 expression during N-acetylcysteine (NAC) cytoprotection against GCDCA-induced cell death in hepatocytes. The regulation of Ca{sup 2+} pools (EGTA or BAPTA-AM) and NO (L-NAME or NO donor) production was assessed during NAC cytoprotection in GCDCA-treated HepG2 cells. The stimulation of Ca{sup 2+} entrance was induced by A23187 in HepG2. Cell death, Ca{sup 2+} mobilization, NOS-1, -2 and -3 expression, AP-1 activation, and NO production were evaluated. GCDCA reduced intracellular Ca{sup 2+} concentration and NOS-3 expression, and enhanced cell death in HepG2. NO donor prevented, and L-NAME enhanced, GCDCA-induced cell death. The reduction of Ca{sup 2+} entry by EGTA, but not its release from intracellular stores by BAPTA-AM, enhanced cell death in GCDCA-treated cells. The stimulation of Ca{sup 2+} entrance by A23187 reduced cell death and enhanced NOS-3 expression in GCDCA-treated HepG2 cells. The cytoprotective properties of NAC were related to the recovery of intracellular Ca{sup 2+} concentration, NOS-3 expression and NO production induced by GCDCA-treated HepG2 cells. The increase of NO production by Ca{sup 2+}-dependent NOS-3 expression during NAC administration reduces cell death in GCDCA-treated hepatocytes.

  13. The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces apoptosis, down-regulates the CXCR4 chemokine receptor and impairs migration of chronic lymphocytic leukemia cells

    PubMed Central

    Stamatopoulos, Basile; Meuleman, Nathalie; De Bruyn, Cécile; Delforge, Alain; Bron, Dominique; Lagneaux, Laurence

    2010-01-01

    Background Chronic lymphocytic leukemia is a neoplastic disorder that arises largely as a result of defective apoptosis leading to chemoresistance. Stromal cell-derived factor-1 and its receptor, CXCR4, have been shown to play an important role in chronic lymphocytic leukemia cell trafficking and survival. Design and Methods Since histone acetylation is involved in the modulation of gene expression, we evaluated the effects of suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, on chronic lymphocytic leukemia cells and in particular on cell survival, CXCR4 expression, migration, and drug sensitization. Results Here, we showed that treatment with suberoylanilide hydroxamic acid (20 μM) for 48 hours induced a decrease in chronic lymphocytic leukemia cell viability via apoptosis (n=20, P=0.0032). Using specific caspase inhibitors, we demonstrated the participation of caspases-3, -6 and -8, suggesting an activation of the extrinsic pathway. Additionally, suberoylanilide hydroxamic acid significantly decreased CXCR4 mRNA (n=10, P=0.0010) and protein expression (n=40, P<0.0001). As a result, chronic lymphocytic leukemia cell migration in response to stromal cell-derived factor-1 (n=23, P<0.0001) or through bone marrow stromal cells was dramatically impaired. Consequently, suberoylanilide hydroxamic acid reduced the protective effect of the microenvironment and thus sensitized chronic lymphocytic leukemia cells to chemotherapy such as fludarabine. Conclusions In conclusion, suberoylanilide hydroxamic acid induces apoptosis in chronic lymphocytic leukemia cells via the extrinsic pathway and down-regulates CXCR4 expression leading to decreased cell migration. Suberoylanilide hydroxamic acid in combination with other drugs represents a promising therapeutic approach to inhibiting migration, chronic lymphocytic leukemia cell survival and potentially overcoming drug resistance. PMID:20145270

  14. Downregulation of microRNA-451 in non-alcoholic steatohepatitis inhibits fatty acid-induced proinflammatory cytokine production through the AMPK/AKT pathway.

    PubMed

    Hur, Wonhee; Lee, Joon Ho; Kim, Sung Woo; Kim, Jung-Hee; Bae, Si Hyun; Kim, Minhyung; Hwang, Daehee; Kim, Young Seok; Park, Taesun; Um, Soo-Jong; Song, Byoung-Joon; Yoon, Seung Kew

    2015-07-01

    Mechanisms associated with the progression of non-alcoholic fatty liver disease (NAFLD) remain unclear. We attempted to identify the pattern of altered gene expression at different time points in a high fat diet (HFD)-induced NAFLD mouse model. The early up-regulated genes are mainly involved in the innate immune responses, while the late up-regulated genes represent the inflammation processes. Although recent studies have shown that microRNAs play important roles in hepatic metabolic functions, the pivotal role of microRNAs in the progression of NAFLD is not fully understood. We investigated the functions of miR-451, which was identified as a target gene in the inflammatory process in NAFLD. miR-451 expression was significantly decreased in the palmitate (PA)-exposed HepG2 cells and in liver tissues of HFD-induced non-alcoholic steatohepatitis (NASH) mice. Its decreased expressions were also observed in liver specimens of NASH patients. In vitro analysis of the effect of miR-451 on proinflammatory cytokine provided evidence for negative regulation of PA-induced interleukin (IL)-8 and tumor necrosis factor-alpha (TNF-α) production. Furthermore, miR-451 over-expression inhibited translocation of the PA-induced NF-κB p65 subunit into the nucleus. Our result showed that Cab39 is a direct target of miRNA-451 in steatotic cells. Further study showed that AMPK activated through Cab39 inhibits NF-κB transactivation induced in steatotic HepG2 cells. miR-451 over-expression in steatotic cells significantly suppressed PA-induced inflammatory cytokine. These results provide new insights into the negative regulation of miR-451 in fatty acid-induced inflammation via the AMPK/AKT pathway and demonstrate potential therapeutic applications for miR-451 in preventing the progression from simple steatosis to severely advanced liver disease.

  15. Blockage of epithelial to mesenchymal transition and upregulation of let 7b are critically involved in ursolic acid induced apoptosis in malignant mesothelioma cell

    PubMed Central

    Sohn, Eun Jung; Won, Gunho; Lee, Jihyun; Yoon, Sang Wook; Lee, Ilho; Kim, Hee Jeong; Kim, Sung-Hoon

    2016-01-01

    Malignant pleural mesothelioma (MPN), which is caused by asbestos exposure, is one of aggressive lung tumors. In the present study, we elucidated the anti-tumor mechanism of ursolic acid in malignant mesotheliomas. Ursolic acid significantly exerted cytotoxicity in a time and dose dependent manner in H28, H2452 and MSTO-211H mesothelioma cells and inhibited cell proliferation by colony formation assay in a dose-dependent fashion. Also, ursolic acid treatment accumulated the sub-G1 population, attenuated the expression of procapase 9, cyclin D1, pAKT, p-glycogen synthase kinase 3-alpha/beta (pGSK3α/β), β-catenin and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) and also cleaved caspase 3 and poly (ADP-ribose) polymerase (PARP) in mesothelioma cells. Furthermore, ursolic acid treatment blocked epithelial and mesenchymal transition (EMT) molecules by activating E-cadherin as an epithelial marker and attenuating Vimentin, and Twist as mesenchymal molecules. Interestingly, miRNA array revealed that 23 miRNAs (>2 folds) including let-7b and miRNA3613-5p, miRNA134 and miRNA196b were significantly upregulated while 33 miRNAs were downregulated in ursolic acid treated H2452 cells. Furthermore, overexpression of let 7b using let-7b mimics enhanced the antitumor effect of ursolic acid to attenuate the expression of procaspases 3, pro-PARP, pAKT, β-catenin and Twist and increase sub-G1 accumulation in H2452 mesothelioma cells. Overall, our findings suggest that ursolic acid induces apoptosis via inhibition of EMT and activation of let7b in mesothelioma cells as a potent chemotherapeutic agent for treatment of malignant mesotheliomas. PMID:28090191

  16. Blockage of epithelial to mesenchymal transition and upregulation of let 7b are critically involved in ursolic acid induced apoptosis in malignant mesothelioma cell.

    PubMed

    Sohn, Eun Jung; Won, Gunho; Lee, Jihyun; Yoon, Sang Wook; Lee, Ilho; Kim, Hee Jeong; Kim, Sung-Hoon

    2016-01-01

    Malignant pleural mesothelioma (MPN), which is caused by asbestos exposure, is one of aggressive lung tumors. In the present study, we elucidated the anti-tumor mechanism of ursolic acid in malignant mesotheliomas. Ursolic acid significantly exerted cytotoxicity in a time and dose dependent manner in H28, H2452 and MSTO-211H mesothelioma cells and inhibited cell proliferation by colony formation assay in a dose-dependent fashion. Also, ursolic acid treatment accumulated the sub-G1 population, attenuated the expression of procapase 9, cyclin D1, pAKT, p-glycogen synthase kinase 3-alpha/beta (pGSK3α/β), β-catenin and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) and also cleaved caspase 3 and poly (ADP-ribose) polymerase (PARP) in mesothelioma cells. Furthermore, ursolic acid treatment blocked epithelial and mesenchymal transition (EMT) molecules by activating E-cadherin as an epithelial marker and attenuating Vimentin, and Twist as mesenchymal molecules. Interestingly, miRNA array revealed that 23 miRNAs (>2 folds) including let-7b and miRNA3613-5p, miRNA134 and miRNA196b were significantly upregulated while 33 miRNAs were downregulated in ursolic acid treated H2452 cells. Furthermore, overexpression of let 7b using let-7b mimics enhanced the antitumor effect of ursolic acid to attenuate the expression of procaspases 3, pro-PARP, pAKT, β-catenin and Twist and increase sub-G1 accumulation in H2452 mesothelioma cells. Overall, our findings suggest that ursolic acid induces apoptosis via inhibition of EMT and activation of let7b in mesothelioma cells as a potent chemotherapeutic agent for treatment of malignant mesotheliomas.

  17. Rai14 (retinoic acid induced protein 14) is involved in regulating f-actin dynamics at the ectoplasmic specialization in the rat testis*.

    PubMed

    Qian, Xiaojing; Mruk, Dolores D; Cheng, C Yan

    2013-01-01

    Rai14 (retinoic acid induced protein 14) is an actin binding protein first identified in the liver, highly expressed in the placenta, the testis, and the eye. In the course of studying actin binding proteins that regulate the organization of actin filament bundles in the ectoplasmic specialization (ES), a testis-specific actin-rich adherens junction (AJ) type, Rai14 was shown to be one of the regulatory proteins at the ES. In the rat testis, Rai14 was found to be expressed by Sertoli and germ cells, structurally associated with actin and an actin cross-linking protein palladin. Its expression was the highest at the ES in the seminiferous epithelium of adult rat testes, most notably at the apical ES at the Sertoli-spermatid interface, and expressed stage-specifically during the epithelial cycle in stage VII-VIII tubules. However, Rai14 was also found at the basal ES near the basement membrane, associated with the blood-testis barrier (BTB) in stage VIII-IX tubules. A knockdown of Rai14 in Sertoli cells cultured in vitro by RNAi was found to perturb the Sertoli cell tight junction-permeability function in vitro, mediated by a disruption of F-actin, which in turn led to protein mis-localization at the Sertoli cell BTB. When Rai14 in the testis in vivo was knockdown by RNAi, defects in spermatid polarity and adhesion, as well as spermatid transport were noted mediated via changes in F-actin organization and mis-localization of proteins at the apical ES. In short, Rai14 is involved in the re-organization of actin filaments in Sertoli cells during the epithelial cycle, participating in conferring spermatid polarity and cell adhesion in the testis.

  18. Increased expression of retinoic acid-induced gene 1 in the dorsolateral prefrontal cortex in schizophrenia, bipolar disorder, and major depression

    PubMed Central

    Haybaeck, Johannes; Postruznik, Magdalena; Miller, Christine L; Dulay, Jeannette R; Llenos, Ida C; Weis, Serge

    2015-01-01

    Background Retinoids regulate gene expression in different cells and tissues at the transcriptional level. Retinoic acid transcriptionally regulates downstream regulatory molecules, including enzymes, transcription factors, cytokines, and cytokine receptors. Animal models indicate an involvement of retinoid signaling pathways in the regulation of synaptic plasticity and learning, especially in the hippocampus. Retinoic acid-inducible or induced gene 1 (RAI-1) is induced during neuronal differentiation, and was associated with the severity of the phenotype and response to medication in schizophrenic patients. Methods In the present study, we used immunohistochemistry to investigate the expression of RAI-1 in 60 brains from the Stanley Neuropathology Consortium (15 cases each from controls and from patients with schizophrenia, bipolar disorder, and major depression). Rating scores for density and intensity were determined in the dorsolateral prefrontal cortex. Results All four groups showed high interindividual variation. RAI-1-positive cells were identified as neurons and astrocytes. Significantly increased intensities in cortical neurons were noted in all three major psychiatric groups compared with controls. The density of RAI-1-positive neurons was increased (P=0.06) in schizophrenia and bipolar disorder. In bipolar disorder, RAI-1-positive astrocytes in gray matter showed a significantly increased intensity and compound value. Thus, a significant increase in the parameters measured was found in schizophrenia, bipolar disorder, and major depression. Conclusion Our study shows a significant increase in expression of RAI-1 in the brains from patients with schizophrenia, bipolar disorder, or major depression. The increased expression might reflect altered signaling pathways, like that for retinoic acid. The underlying mechanisms leading to the increased expression and its functional consequences are so far unknown, and remain to be investigated in future studies

  19. Domain-confined catalytic soot combustion over Co3O4 anchored on a TiO2 nanotube array catalyst prepared by mercaptoacetic acid induced surface-grafting.

    PubMed

    Ren, Jiale; Yu, Yifu; Dai, Fangfang; Meng, Ming; Zhang, Jing; Zheng, Lirong; Hu, Tiandou

    2013-12-21

    Herein, we introduce a specially designed domain-confined macroporous catalyst, namely, the Co3O4 nanocrystals anchored on a TiO2 nanotube array catalyst, which was synthesized by using the mercaptoacetic acid induced surface-grafting method. This catalyst exhibits much better performance for catalytic soot combustion than the conventional TiO2 powder supported one in gravitational contact mode (GMC).

  20. Non-Canonical Role of IKKα in the Regulation of STAT1 Phosphorylation in Antiviral Signaling

    PubMed Central

    Xing, Fei; Matsumiya, Tomoh; Shiba, Yuko; Hayakari, Ryo; Yoshida, Hidemi; Imaizumi, Tadaatsu

    2016-01-01

    Non-self RNA is recognized by retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), inducing type I interferons (IFNs). Type I IFN promotes the expression of IFN-stimulated genes (ISGs), which requires the activation of signal transducer and activator of transcription-1 (STAT1). We previously reported that dsRNA induced STAT1 phosphorylation via a type I IFN-independent pathway in addition to the well-known type I IFN-dependent pathway. IκB kinase α (IKKα) is involved in antiviral signaling induced by dsRNA; however, its role is incompletely understood. Here, we explored the function of IKKα in RLR-mediated STAT1 phosphorylation. Silencing of IKKα markedly decreased the level of IFN-β and STAT1 phosphorylation inHeH response to dsRNA. However, the inhibition of IKKα did not alter the RLR signaling-mediated dimerization of interferon responsive factor 3 (IRF3) or the nuclear translocation of nuclear factor-κB (NFκB). These results suggest a non-canonical role of IKKα in RLR signaling. Furthermore, phosphorylation of STAT1 was suppressed by IKKα knockdown in cells treated with a specific neutralizing antibody for the type I IFN receptor (IFNAR) and in IFNAR-deficient cells. Collectively, the dual regulation of STAT1 by IKKα in antiviral signaling suggests a role for IKKα in the fine-tuning of antiviral signaling in response to non-self RNA. PMID:27992555

  1. Cherry Valley Ducks Mitochondrial Antiviral-Signaling Protein-Mediated Signaling Pathway and Antiviral Activity Research

    PubMed Central

    Li, Ning; Hong, Tianqi; Li, Rong; Wang, Yao; Guo, Mengjiao; Cao, Zongxi; Cai, Yumei; Liu, Sidang; Chai, Tongjie; Wei, Liangmeng

    2016-01-01

    Mitochondrial antiviral-signaling protein (MAVS), an adaptor protein of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs)-mediated signal pathway, is involved in innate immunity. In this study, Cherry Valley duck MAVS (duMAVS) was cloned from the spleen and analyzed. duMAVS was determined to have a caspase activation and recruitment domain at N-terminal, followed by a proline-rich domain and a transmembrane domain at C-terminal. Quantitative real-time PCR indicated that duMAVS was expressed in all tissues tested across a broad expression spectrum. The expression of duMAVS was significantly upregulated after infection with duck Tembusu virus (DTMUV). Overexpression of duMAVS could drive the activation of interferon (IFN)-β, nuclear factor-κB, interferon regulatory factor 7, and many downstream factors (such as Mx, PKR, OAS, and IL-8) in duck embryo fibroblast cells. What is more, RNA interference further confirmed that duMAVS was an important adaptor for IFN-β activation. The antiviral assay showed that duMAVS could suppress the various viral replications (DTMUV, novel reovirus, and duck plague virus) at early stages of infection. Overall, these results showed that the main signal pathway mediated by duMAVS and it had a broad-spectrum antiviral ability. This research will be helpful to better understanding the innate immune system of ducks. PMID:27708647

  2. Cleavage of Interferon Regulatory Factor 7 by Enterovirus 71 3C Suppresses Cellular Responses

    PubMed Central

    Lei, Xiaobo; Xiao, Xia; Xue, Qinghua; Jin, Qi

    2013-01-01

    Enterovirus 71 (EV71) is a positive-stranded RNA virus which is capable of inhibiting innate immunity. Among virus-encoded proteins, the 3C protein compromises the type I interferon (IFN-I) response mediated by retinoid acid-inducible gene-I (RIG-I) or Toll-like receptor 3 that activates interferon regulatory 3 (IRF3) and IRF7. In the present study, we report that enterovirus 71 downregulates IRF7 through the 3C protein, which inhibits the function of IRF7. When expressed in mammalian cells, the 3C protein mediates cleavage of IRF7 rather than that of IRF3. This process is insensitive to inhibitors of caspase, proteasome, lysosome, and autophagy. H40D substitution in the 3C active site abolishes its activity, whereas R84Q or V154S substitution in the RNA binding motif has no effect. Furthermore, 3C-mediated cleavage occurs at the Q189-S190 junction within the constitutive activation domain of IRF7, resulting in two cleaved IRF7 fragments that are incapable of activating IFN expression. Ectopic expression of wild-type IRF7 limits EV71 replication. On the other hand, expression of the amino-terminal domain of IRF7 enhances EV71 infection, which correlates with its ability to interact with and inhibit IRF3. These results suggest that control of IRF7 by the 3C protein may represent a viral mechanism to escape cellular responses. PMID:23175366

  3. PPM1A silences cytosolic RNA sensing and antiviral defense through direct dephosphorylation of MAVS and TBK1.

    PubMed

    Xiang, Weiwen; Zhang, Qian; Lin, Xia; Wu, Shiying; Zhou, Yao; Meng, Fansen; Fan, Yunyun; Shen, Tao; Xiao, Mu; Xia, Zongping; Zou, Jian; Feng, Xin-Hua; Xu, Pinglong

    2016-07-01

    Cytosolic RNA sensing is a prerequisite for initiation of innate immune response against RNA viral pathogens. Signaling through RIG-I (retinoic acid-inducible gene I)-like receptors (RLRs) to TBK1 (Tank-binding kinase 1)/IKKε (IκB kinase ε) kinases is transduced by mitochondria-associated MAVS (mitochondrial antiviral signaling protein). However, the precise mechanism of how MAVS-mediated TBK1/IKKε activation is strictly controlled still remains obscure. We reported that protein phosphatase magnesium-dependent 1A (PPM1A; also known as PP2Cα), depending on its catalytic ability, dampened the RLR-IRF3 (interferon regulatory factor 3) axis to silence cytosolic RNA sensing signaling. We demonstrated that PPM1A was an inherent partner of the TBK1/IKKε complex, targeted both MAVS and TBK1/IKKε for dephosphorylation, and thus disrupted MAVS-driven formation of signaling complex. Conversely, a high level of MAVS can dissociate the TBK1/PPM1A complex to override PPM1A-mediated inhibition. Loss of PPM1A through gene ablation in human embryonic kidney 293 cells and mouse primary macrophages enabled robustly enhanced antiviral responses. Consequently, Ppm1a(-/-) mice resisted to RNA virus attack, and transgenic zebrafish expressing PPM1A displayed profoundly increased RNA virus vulnerability. These findings identify PPM1A as the first known phosphatase of MAVS and elucidate the physiological function of PPM1A in antiviral immunity on whole animals.

  4. Insights into the Antiviral Immunity against Grass Carp (Ctenopharyngodon idella) Reovirus (GCRV) in Grass Carp

    PubMed Central

    2015-01-01

    Global fish production from aquaculture has rapidly grown over the past decades, and grass carp shares the largest portion. However, hemorrhagic disease caused by grass carp reovirus (GCRV) results in tremendous loss of grass carp (Ctenopharyngodon idella) industry. During the past years, development of molecular biology and cellular biology technologies has promoted significant advances in the understanding of the pathogen and the immune system. Immunoprophylaxis based on stimulation of the immune system of fish has also got some achievements. In this review, authors summarize the recent progresses in basic researches on GCRV; viral nucleic acid sensors, high-mobility group box proteins (HMGBs); pattern recognition receptors (PRRs), Toll-like receptors (TLRs) and retinoic acid inducible gene I- (RIG-I-) like receptors (RLRs); antiviral immune responses induced by PRRs-mediated signaling cascades of type I interferon (IFN-I) and IFN-stimulated genes (ISGs) activation. The present review also notices the potential applications of molecule genetic markers. Additionally, authors discuss the current preventive and therapeutic strategies (vaccines, RNAi, and prevention medicine) and highlight the importance of innate immunity in long term control for grass carp hemorrhagic disease. PMID:25759845

  5. Mumps Virus Induces Protein-Kinase-R-Dependent Stress Granules, Partly Suppressing Type III Interferon Production

    PubMed Central

    Hashimoto, Shin; Yamamoto, Soh; Ogasawara, Noriko; Sato, Toyotaka; Yamamoto, Keisuke; Katoh, Hiroshi; Kubota, Toru; Shiraishi, Tsukasa; Kojima, Takashi; Himi, Tetsuo; Tsutsumi, Hiroyuki; Yokota, Shin-ichi

    2016-01-01

    Stress granules (SGs) are cytoplasmic granular aggregations that are induced by cellular stress, including viral infection. SGs have opposing antiviral and proviral roles, which depend on virus species. The exact function of SGs during viral infection is not fully understood. Here, we showed that mumps virus (MuV) induced SGs depending on activation of protein kinase R (PKR). MuV infection strongly induced interferon (IFN)-λ1, 2 and 3, and IFN-β through activation of IFN regulatory factor 3 (IRF3) via retinoic acid inducible gene-I (RIG-I) and the mitochondrial antiviral signaling (MAVS) pathway. MuV-induced IFNs were strongly upregulated in PKR-knockdown cells. MuV-induced SG formation was suppressed by knockdown of PKR and SG marker proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and T-cell-restricted intracellular antigen-1, and significantly increased the levels of MuV-induced IFN-λ1. However, viral titer was not altered by suppression of SG formation. PKR was required for induction of SGs by MuV infection and regulated type III IFN (IFN-λ1) mRNA stability. MuV-induced SGs partly suppressed type III IFN production by MuV; however, the limited suppression was not sufficient to inhibit MuV replication in cell culture. Our results provide insight into the relationship between SGs and IFN production induced by MuV infection. PMID:27560627

  6. Alteration of Antiviral Signalling by Single Nucleotide Polymorphisms (SNPs) of Mitochondrial Antiviral Signalling Protein (MAVS)

    PubMed Central

    Xing, Fei; Matsumiya, Tomoh; Hayakari, Ryo; Yoshida, Hidemi; Kawaguchi, Shogo; Takahashi, Ippei; Nakaji, Shigeyuki; Imaizumi, Tadaatsu

    2016-01-01

    Genetic variation is associated with diseases. As a type of genetic variation occurring with certain regularity and frequency, the single nucleotide polymorphism (SNP) is attracting more and more attention because of its great value for research and real-life application. Mitochondrial antiviral signalling protein (MAVS) acts as a common adaptor molecule for retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), which can recognize foreign RNA, including viral RNA, leading to the induction of type I interferons (IFNs). Therefore, MAVS is thought to be a crucial molecule in antiviral innate immunity. We speculated that genetic variation of MAVS may result in susceptibility to infectious diseases. To assess the risk of viral infection based on MAVS variation, we tested the effects of twelve non-synonymous MAVS coding-region SNPs from the National Center for Biotechnology Information (NCBI) database that result in amino acid substitutions. We found that five of these SNPs exhibited functional alterations. Additionally, four resulted in an inhibitory immune response, and one had the opposite effect. In total, 1,032 human genomic samples obtained from a mass examination were genotyped at these five SNPs. However, no homozygous or heterozygous variation was detected. We hypothesized that these five SNPs are not present in the Japanese population and that such MAVS variations may result in serious immune diseases. PMID:26954674

  7. Highlights of the advances in basic immunology in 2011

    PubMed Central

    Liu, Juan; Liu, Shuxun; Cao, Xuetao

    2012-01-01

    In this review, we summarize the major fundamental advances in immunological research reported in 2011. The highlights focus on the improved understanding of key questions in basic immunology, including the initiation and activation of innate responses as well as mechanisms for the development and function of various T-cell subsets. The research includes the identification of novel cytosolic RNA and DNA sensors as well as the identification of the novel regulators of the Toll-like receptor (TLR) and retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway. Moreover, remarkable advances have been made in the developmental and functional properties of innate lymphoid cells (ILCs). Helper T cells and regulatory T (Treg) cells play indispensable roles in orchestrating adaptive immunity. There have been exciting discoveries regarding the regulatory mechanisms of the development of distinct T-cell subsets, particularly Th17 cells and Treg cells. The emerging roles of microRNAs (miRNAs) in T cell immunity are discussed, as is the recent identification of a novel T-cell subset referred to as follicular regulatory T (TFR) cells. PMID:22522654

  8. Engagement of Fas on Macrophages Modulates Poly I:C induced cytokine production with specific enhancement of IP-10.

    PubMed

    Lyons, Caitriona; Fernandes, Philana; Fanning, Liam J; Houston, Aileen; Brint, Elizabeth

    2015-01-01

    Viral double-stranded RNA (dsRNA) is recognised by pathogen recognition receptors such as Toll-Like Receptor 3 (TLR3) and retinoic acid inducible gene-I (RIG-I), and results in cytokine and interferon production. Fas, a well characterised death receptor, has recently been shown to play a role in the inflammatory response. In this study we investigated the role of Fas in the anti-viral immune response. Stimulation of Fas on macrophages did not induce significant cytokine production. However, activation of Fas modified the response of macrophages to the viral dsRNA analogue poly I:C. In particular, poly I:C-induced IP-10 production was significantly enhanced. A similar augmentation of IP-10 by Fas was observed following stimulation with both poly A:U and Sendai virus. Fas activation suppressed poly I:C-induced phosphorylation of the MAP kinases p38 and JNK, while overexpression of the Fas adaptor protein, Fas-associated protein with death domain (FADD), activated AP-1 and inhibited poly I:C-induced IP-10 production. Consistent with an inhibitory role for AP-1 in IP-10 production, mutation of the AP-1 binding site on the IP-10 promoter resulted in augmented poly I:C-induced IP-10. These results demonstrate that engagement of the Fas receptor plays a role in modifying the innate immune response to viral RNA.

  9. Highlights of the advances in basic immunology in 2011.

    PubMed

    Liu, Juan; Liu, Shuxun; Cao, Xuetao

    2012-05-01

    In this review, we summarize the major fundamental advances in immunological research reported in 2011. The highlights focus on the improved understanding of key questions in basic immunology, including the initiation and activation of innate responses as well as mechanisms for the development and function of various T-cell subsets. The research includes the identification of novel cytosolic RNA and DNA sensors as well as the identification of the novel regulators of the Toll-like receptor (TLR) and retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway. Moreover, remarkable advances have been made in the developmental and functional properties of innate lymphoid cells (ILCs). Helper T cells and regulatory T (Treg) cells play indispensable roles in orchestrating adaptive immunity. There have been exciting discoveries regarding the regulatory mechanisms of the development of distinct T-cell subsets, particularly Th17 cells and Treg cells. The emerging roles of microRNAs (miRNAs) in T cell immunity are discussed, as is the recent identification of a novel T-cell subset referred to as follicular regulatory T (TFR) cells.

  10. MIP-T3 is a negative regulator of innate type I IFN response.

    PubMed

    Ng, Ming-Him James; Ho, Ting-Hin; Kok, Kin-Hang; Siu, Kam-Leung; Li, Jun; Jin, Dong-Yan

    2011-12-15

    TNFR-associated factor (TRAF) 3 is an important adaptor that transmits upstream activation signals to protein kinases that phosphorylate transcription factors to induce the production of type I IFNs, the important effectors in innate antiviral immune response. MIP-T3 interacts specifically with TRAF3, but its function in innate IFN response remains unclear. In this study, we demonstrated a negative regulatory role of MIP-T3 in type I IFN production. Overexpression of MIP-T3 inhibited RIG-I-, MDA5-, VISA-, TBK1-, and IKKε-induced transcriptional activity mediated by IFN-stimulated response elements and IFN-β promoter. MIP-T3 interacted with TRAF3 and perturbed in a dose-dependent manner the formation of functional complexes of TRAF3 with VISA, TBK1, IKKε, and IFN regulatory factor 3. Consistent with this finding, retinoic acid-inducible gene I- and TBK1-induced phosphorylation of IFN regulatory factor 3 was significantly diminished when MIP-T3 was overexpressed. Depletion of MIP-T3 facilitated Sendai virus-induced activation of IFN production and attenuated the replication of vesicular stomatitis virus. In addition, MIP-T3 was found to be dissociated from TRAF3 during the course of Sendai virus infection. Our findings suggest that MIP-T3 functions as a negative regulator of innate IFN response by preventing TRAF3 from forming protein complexes with critical downstream transducers and effectors.

  11. Parvalbumin interneurons and calretinin fibers arising from the thalamic nucleus reuniens degenerate in the subiculum after kainic acid-induced seizures.

    PubMed

    Drexel, M; Preidt, A P; Kirchmair, E; Sperk, G

    2011-08-25

    The subiculum is the major output area of the hippocampus. It is closely interconnected with the entorhinal cortex and other parahippocampal areas. In animal models of temporal lobe epilepsy (TLE) and in TLE patients it exerts increased network excitability and may crucially contribute to the propagation of limbic seizures. Using immunohistochemistry and in situ-hybridization we now investigated neuropathological changes affecting parvalbumin and calretinin containing neurons in the subiculum and other parahippocampal areas after kainic acid-induced status epilepticus. We observed prominent losses in parvalbumin containing interneurons in the subiculum and entorhinal cortex, and in the principal cell layers of the pre- and parasubiculum. Degeneration of parvalbumin-positive neurons was associated with significant precipitation of parvalbumin-immunoreactive debris 24 h after kainic acid injection. In the subiculum the superficial portion of the pyramidal cell layer was more severely affected than its deep part. In the entorhinal cortex, the deep layers were more severely affected than the superficial ones. The decrease in number of parvalbumin-positive neurons in the subiculum and entorhinal cortex correlated with the number of spontaneous seizures subsequently experienced by the rats. The loss of parvalbumin neurons thus may contribute to the development of spontaneous seizures. On the other hand, surviving parvalbumin neurons revealed markedly increased expression of parvalbumin mRNA notably in the pyramidal cell layer of the subiculum and in all layers of the entorhinal cortex. This indicates increased activity of these neurons aiming to compensate for the partial loss of this functionally important neuron population. Furthermore, calretinin-positive fibers terminating in the molecular layer of the subiculum, in sector CA1 of the hippocampus proper and in the entorhinal cortex degenerated together with their presumed perikarya in the thalamic nucleus reuniens. In

  12. Role of dioxygenase α-DOX2 and SA in basal response and in hexanoic acid-induced resistance of tomato (Solanum lycopersicum) plants against Botrytis cinerea.

    PubMed

    Angulo, Carlos; de la O Leyva, María; Finiti, Ivan; López-Cruz, Jaime; Fernández-Crespo, Emma; García-Agustín, Pilar; González-Bosch, Carmen

    2015-03-01

    Resistance of tomato (Solanum Lycopersicum) to the fungal pathogen Botrytis cinerea requires complex interplay between hormonal signalling. In this study, we explored the involvement of new oxylipins in the tomato basal and induced response to this necrotroph through the functional analysis of the tomato α-dioxygenase2 (α-DOX2)-deficient mutant divaricata. We also investigated the role of SA in the defence response against this necrotrophic fungus using SA-deficient tomato nahG plants. The plants lacking dioxigenase α-DOX2, which catalyses oxylipins production from fatty acids, were more susceptible to Botrytis, and hexanoic acid-induced resistance (Hx-IR) was impaired; hence α-DOX2 is required for both tomato defence and the enhanced protection conferred by natural inducer hexanoic acid (Hx) against B. cinerea. The divaricata plants accumulated less pathogen-induced callose and presented lower levels of jasmonic acid (JA) and 12-oxo-phytodienoic acid (OPDA) upon infection if compared to the wild type. Glutathion-S-transferase (GST) gene expression decreased and ROS production significantly increased in Botrytis-infected divaricata plants. These results indicate that absence of α-DOX2 influences the hormonal changes, oxidative burst and callose deposition that occur upon Botrytis infection in tomato. The study of SA-deficient nahG tomato plants showed that the plants with low SA levels displayed increased resistance to Botrytis, but were unable to display Hx-IR. This supports the involvement of SA in Hx-IR. NaghG plants displayed reduced callose and ROS accumulation upon infection and an increased GST expression. This reflects a positive relationship between SA and these defensive mechanisms in tomato. Finally, Hx boosted the pathogen-induced callose in nahG plants, suggesting that this priming mechanism is SA-independent. Our results support the involvement of the oxylipins pathway and SA in tomato response to Botrytis, probably through complex crosstalk of

  13. Conjugated linoleic acid-induced milk fat depression in lactating ewes is accompanied by reduced expression of mammary genes involved in lipid synthesis.

    PubMed

    Hussein, M; Harvatine, K H; Weerasinghe, W M P B; Sinclair, L A; Bauman, D E

    2013-06-01

    Conjugated linoleic acids (CLA) are produced during rumen biohydrogenation and exert a range of biological effects. The trans-10,cis-12 CLA isomer is a potent inhibitor of milk fat synthesis in lactating dairy cows and some aspects of the mechanism have been established. Conjugated linoleic acid-induced milk fat depression has also been observed in small ruminants and our objective was to examine the molecular mechanism in lactating ewes. Multiparous lactating ewes were fed a basal ration (0.55:0.45 concentrate-to-forage ratio; dry matter basis) and randomly allocated to 2 dietary CLA levels (n=8 ewes/treatment). Treatments were zero CLA (control) or 15 g/d of lipid-encapsulated CLA supplement containing cis-9,trans-11 and trans-10,cis-12 CLA isomers in equal proportions. Treatments were fed for 10 wk and the CLA supplement provided 1.5 g of trans-10,cis-12/d. No treatment effects were observed on milk yield or milk composition for protein or lactose at wk 10 of the study. In contrast, CLA treatment significantly decreased both milk fat percentage and milk fat yield (g/d) by about 23%. The de novo synthesized fatty acids (FA; C16) was increased (10%) for the CLA treatment. In agreement with the reduced de novo FA synthesis, mRNA abundance of acetyl-coenzyme A carboxylase α, FA synthase, stearoyl-CoA desaturase 1, and glycerol-3-phosphate acyltransferase 6 decreased by 25 to 40% in the CLA-treated group. Conjugated linoleic acid treatment did not significantly reduce the mRNA abundance of enzymes involved in NADPH production, but the mRNA abundance for sterol regulatory element-binding factor 1 and insulin-induced gene 1, genes involved in regulation of transcription of lipogenic enzymes, was decreased by almost 30 and 55%, respectively, with CLA treatment. Furthermore, mRNA abundance of lipoprotein lipase decreased by almost 40% due to CLA treatment

  14. Evolution of the DEAD box helicase family in chicken: chickens have no DHX9 ortholog.

    PubMed

    Sato, Haruko; Oshiumi, Hiroyuki; Takaki, Hiromi; Hikono, Hirokazu; Seya, Tsukasa

    2015-10-01

    Viral RNA represents a pattern molecule that can be recognized by RNA sensors in innate immunity. Humans and mice possess cytoplasmic DNA/RNA sensors for detecting viral replication. There are a number of DEAD (Asp-Glu-Ala-Asp; DExD/H) box-type helicases in mammals, among which retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA50) are indispensable for RNA sensing; however, they are functionally supported by a number of sensors that directly bind viral RNA or replicative RNA intermediates to convey signals to RIG-I and MDA5. Some DEAD box helicase members recognize DNA irrespective of the origin. These sensors transmit IFN-inducing signals through adaptors, including mitochondrial antiviral signaling. Viral double-stranded RNAs are reportedly sensed by the helicases DDX1, DDX21, DHX36, DHX9, DDX3, DDX41, LGP2 and DDX60, in addition to RIG-I and MDA5, and induce type I IFNs, thereby blocking viral replication. Humans and mice have all nucleic acid sensors listed here. In the RNA sensing system in chicken, it was found in the present study that most DEAD box helicases are conserved; however, DHX9 is genetically deficient in addition to reported RIG-I. Based on the current genome databases, similar DHX9 deficiency was observed in ducks and several other bird species. Because chicken, but not duck, was found to be deficient in RIG-I, the RNA-sensing system of chicken lacks RIG-I and DHX9 and is thus more fragile than that of duck or mammal. DHX9 may generally compensate for the function of RIG-I and deficiency of DHX9 possibly participates in exacerbations of viral infection such as influenza in chickens.

  15. Duox2 is required for the transcription of pattern recognition receptors in acute viral lung infection: An interferon-independent regulatory mechanism.

    PubMed

    Hong, Seung-No; Kim, Ji Young; Kim, Hanna; Kim, Dong-Young; Won, Tae-Bin; Han, Doo Hee; Rhee, Chae-Seo; Kim, Hyun Jik

    2016-10-01

    The innate immune response, which constitutes the first line of defense against influenza A virus (IAV) infection, is activated by pattern recognition receptors (PRRs) that recognize viral structures. We found that the PRRs, retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), which have been implicated as interferon (IFN)-stimulated genes, were dominantly responsible for the recognition of IAV in lungs of mice at 3 and 7 days post infection (dpi). Intranasal administration of IFNs enhanced RIG-I and MDA5 gene expression after IAV infection and mRNA levels of RIG-I and MDA5 were significantly reduced at 7 dpi in mice with neutralization of secreted IFNs. However, blockade of IFNs did not alter the transcription of RIG-I and MDA5 at 3 dpi. We studied the antiviral effect of Duox2 in vivo lung to elucidate the role of Duox2 in respiratory mucosa. RIG-I and MDA5 mRNA levels were induced to a lower extent in lungs of mice that were inoculated with Duox2 small hairpin RNA regardless of secreted IFNs at 3 dpi. We propose that Duox2 is responsible for IFN-independent signaling for induction of PRRs transcription and can control acute IAV lung infection at the beginning of infection.

  16. Neu1 sialidase and matrix metalloproteinase-9 cross-talk regulates nucleic acid-induced endosomal TOLL-like receptor-7 and -9 activation, cellular signaling and pro-inflammatory responses.

    PubMed

    Abdulkhalek, Samar; Szewczuk, Myron R

    2013-11-01

    The precise mechanism(s) by which intracellular TOLL-like receptors (TLRs) become activated by their ligands remains unclear. Here, we report a molecular organizational G-protein coupled receptor (GPCR) signaling platform to potentiate a novel mammalian neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP-9) cross-talk in alliance with neuromedin B GPCR, all of which form a tripartite complex with TLR-7 and -9. siRNA silencing Neu1, MMP-9 and neuromedin-B GPCR in RAW-blue macrophage cells significantly reduced TLR7 imiquimod- and TLR9 ODN1826-induced NF-κB (NF-κB-pSer(536)) activity. Tamiflu, specific MMP-9 inhibitor, neuromedin B receptor specific antagonist BIM23127, and the selective inhibitor of whole heterotrimeric G-protein complex BIM-46174 significantly block nucleic acid-induced TLR-7 and -9 MyD88 recruitment, NF-κB activation and proinflammatory TNFα and MCP-1 cytokine responses. For the first time, Neu1 clearly plays a central role in mediating nucleic acid-induced intracellular TLR activation, and the interactions involving NMBR-MMP9-Neu1 cross-talk constitute a novel intracellular TLR signaling platform that is essential for NF-κB activation and pro-inflammatory responses.

  17. Palmitic acid induces production of proinflammatory cytokines interleukin-6, interleukin-1β, and tumor necrosis factor-α via a NF-κB-dependent mechanism in HaCaT keratinocytes.

    PubMed

    Zhou, Bing-rong; Zhang, Jia-an; Zhang, Qian; Permatasari, Felicia; Xu, Yang; Wu, Di; Yin, Zhi-qiang; Luo, Dan

    2013-01-01

    To investigate whether palmitic acid can be responsible for the induction of inflammatory processes, HaCaT keratinocytes were treated with palmitic acid at pathophysiologically relevant concentrations. Secretion levels of interleukin-6 (IL-6), tumor necrosis factor- α (TNF- α), interleukin-1 β (IL-1 β), NF- κ B nuclear translocation, NF- κ B activation, Stat3 phosphorylation, and peroxisome proliferator-activated receptor alpha (PPAR α) mRNA and protein levels, as well as the cell proliferation ability were measured at the end of the treatment and after 24 hours of recovery. Pyrrolidine dithiocarbamate (PDTC, a selective chemical inhibitor of NF- κ B) and goat anti-human IL-6 polyclonal neutralizing antibody were used to inhibit NF- κ B activation and IL-6 production, respectively. Our results showed that palmitic acid induced an upregulation of IL-6, TNF- α , IL-1 β secretions, accompanied by NF- κ B nuclear translocation and activation. Moreover, the effect of palmitic acid was accompanied by PPAR α activation and Stat3 phosphorylation. Palmitic acid-induced IL-6, TNF- α , IL-1 β productions were attenuated by NF- κ B inhibitor PDTC. Palmitic acid was administered in amounts able to elicit significant hyperproliferation and can be attenuated by IL-6 blockage. These data demonstrate for the first time that palmitic acid can stimulate IL-6, TNF- α , IL-1 β productions in HaCaT keratinocytes and cell proliferation, thereby potentially contributing to acne inflammation and pilosebaceous duct hyperkeratinization.

  18. Knock-out of metacaspase and/or cytochrome c results in the activation of a ROS-independent acetic acid-induced programmed cell death pathway in yeast.

    PubMed

    Guaragnella, Nicoletta; Passarella, Salvatore; Marra, Ersilia; Giannattasio, Sergio

    2010-08-20

    To gain further insight into yeast acetic acid-induced programmed cell death (AA-PCD) we analyzed the effects of the antioxidant N-acetyl-L-cysteine (NAC) on cell viability, hydrogen peroxide (H(2)O(2)) production, DNA fragmentation, cytochrome c (cyt c) release and caspase-like activation in wild type (wt) and metacaspase and/or cyt c-lacking cells. We found that NAC prevents AA-PCD in wt cells, by scavenging H(2)O(2) and by inhibiting both cyt c release and caspase-like activation. This shows the occurrence of a reactive oxygen species (ROS)-dependent AA-PCD. Contrarily no NAC dependent change in AA-PCD of mutant cells was detectable, showing that a ROS-independent AA-PCD can also occur.

  19. Co-administration of α-lipoic acid and cyclosporine aggravates colon ulceration of acetic acid-induced ulcerative colitis via facilitation of NO/COX-2/miR-210 cascade.

    PubMed

    El-Gowelli, Hanan M; Saad, Evan I; Abdel-Galil, Abdel-Galil A; Ibrahim, Einas R

    2015-11-01

    In this work, α-lipoic acid and cyclosporine demonstrated significant protection against acetic acid-induced ulcerative colitis in rats. We proposed that α-lipoic acid and cyclosporine co-administration might modulate their individual effects. Induction of ulcerative colitis in rats was performed by intra-rectal acetic acid (5% v/v) administration for 3 consecutive days. Effects of individual or combined used of α-lipoic acid (35 mg/kg ip) or cyclosporine (5mg/kg sc) for 6 days starting 2 days prior to acetic acid were assessed. Acetic acid caused colon ulceration, bloody diarrhea and weight loss. Histologically, there was mucosal atrophy and inflammatory cells infiltration in submucosa, associated with depletion of colon reduced glutathione, superoxide dismutase and catalase activities and elevated colon malondialdehyde, serum C-reactive protein (C-RP) and tumor necrosis factor-α (TNF-α). Colon gene expression of cyclooxygenase-2 and miR-210 was also elevated. These devastating effects of acetic acid were abolished upon concurrent administration of α-lipoic acid. Alternatively, cyclosporine caused partial protection against acetic acid-induced ulcerative colitis. Cyclosporine did not restore colon reduced glutathione, catalase activity, serum C-RP or TNF-α. Unexpectedly, co-administration of α-lipoic acid and cyclosporine aggravated colon ulceration. Concomitant use of α-lipoic acid and cyclosporine significantly increased nitric oxide production, cyclooxygenase-2 and miR-210 gene expression compared to all other studied groups. The current findings suggest that facilitation of nitric oxide/cyclooxygenase-2/miR-210 cascade constitutes, at least partially, the cellular mechanism by which concurrent use of α-lipoic acid and cyclosporine aggravates colon damage. Collectively, the present work highlights the probable risk of using α-lipoic acid/cyclosporine combination in ulcerative colitis patients.

  20. Role of extracellular signal-regulated kinase in synaptic transmission and plasticity of a nociceptive input on capsular central amygdaloid neurons in normal and acid-induced muscle pain mice.

    PubMed

    Cheng, Sin-Jhong; Chen, Chien-Chang; Yang, Hsiu-Wen; Chang, Ya-Ting; Bai, Shin-Wen; Chen, Chih-Cheng; Yen, Chen-Tung; Min, Ming-Yuan

    2011-02-09

    Application of phorbol 12,13-diacetate (PDA) caused marked enhancement of synaptic transmission of nociceptive parabrachio-amygdaloid (PBA) input onto neurons of the capsular central amygdaloid (CeAC) nucleus. The potentiation of PBA-CeAC EPSCs by PDA involved a presynaptic protein kinase C (PKC)-dependent component and a postsynaptic PKC-extracellular-regulated kinase (ERK)-dependent component. NMDA glutamatergic receptor (NMDAR)-dependent long-term potentiation (LTP) of PBA-CeAC EPSCs, which was also dependent on the PKC-ERK signaling pathway, was induced by tetanus stimulation at 100 Hz. In slices from mice subjected to acid-induced muscle pain (AIMP), phosphorylated ERK levels in the CeAC increased, and PBA-CeAC synaptic transmission was postsynaptically enhanced. The enhanced PBA-CeAC synaptic transmission in AIMP mice shared common mechanisms with the postsynaptic potentiation effect of PDA and induction of NMDAR-dependent LTP by high-frequency stimulation in normal slices, both of which required ERK activation. Since the CeAC plays an important role in the emotionality of pain, enhanced synaptic function of nociceptive (PBA) inputs onto CeAC neurons might partially account for the supraspinal mechanisms underlying central sensitization.

  1. In Vitro Screening for Antihepatic Steatosis Active Components within Coptidis Rhizoma Alkaloids Extract Using Liver Cell Extraction with HPLC Analysis and a Free Fatty Acid-Induced Hepatic Steatosis HepG2 Cell Assay.

    PubMed

    Fan, Hui; Chen, Yuan-Yuan; Bei, Wei-Jian; Wang, Lai-You; Chen, Bao-Tian; Guo, Jiao

    2013-01-01

    A high-throughput method was developed and applied to screen for the active antihepatic steatosis components within Coptidis Rhizoma Alkaloids Extract (CAE). This method was a combination of two previously described assays: HepG2 cell extraction with HPLC analysis and a free fatty acid-induced (FFA) hepatic steatosis HepG2 cell assay. Two alkaloids within CAE, berberine and coptisine, were identified by HepG2 cell extraction with HPLC analysis as high affinity components for HepG2. These alkaloids were also determined to be active and potent compounds capable of lowering triglyceride (TG) accumulation in the FFA-induced hepatic steatosis HepG2 cell assay. This remarkable inhibition of TG accumulation (P < 0.01) by berberine and coptisine occurred at concentrations of 0.2  μ g/mL and 5.0 μ g/mL, respectively. At these concentrations, the effect seen was similar to that of a CAE at 100.0  μ g/mL. Another five alkaloids within CAE, palmatine, epiberberine, jateorhizine, columbamine, and magnoline, were found to have a lower affinity for cellular components from HepG2 cells and a lower inhibition of TG accumulation. The finding of two potent and active compounds within CAE indicates that the screening method we developed is a feasible, rapid, and useful tool for studying traditional Chinese medicines (TCMs) in treating hepatic steatosis.

  2. A functional C-terminal TRAF3-binding site in MAVS participates in positive and negative regulation of the IFN antiviral response.

    PubMed

    Paz, Suzanne; Vilasco, Myriam; Werden, Steven J; Arguello, Meztli; Joseph-Pillai, Deshanthe; Zhao, Tiejun; Nguyen, Thi Lien-Anh; Sun, Qiang; Meurs, Eliane F; Lin, Rongtuan; Hiscott, John

    2011-06-01

    Recognition of viral RNA structures by the cytosolic sensor retinoic acid-inducible gene-I (RIG-I) results in the activation of signaling cascades that culminate with the generation of the type I interferon (IFN) antiviral response. Onset of antiviral and inflammatory responses to viral pathogens necessitates the regulated spatiotemporal recruitment of signaling adapters, kinases and transcriptional proteins to the mitochondrial antiviral signaling protein (MAVS). We previously demonstrated that the serine/threonine kinase IKKε is recruited to the C-terminal region of MAVS following Sendai or vesicular stomatitis virus (VSV) infection, mediated by Lys63-linked polyubiquitination of MAVS at Lys500, resulting in inhibition of downstream IFN signaling (Paz et al, Mol Cell Biol, 2009). In this study, we demonstrate that C-terminus of MAVS harbors a novel TRAF3-binding site in the aa450-468 region of MAVS. A consensus TRAF-interacting motif (TIM), 455-PEENEY-460, within this site is required for TRAF3 binding and activation of IFN antiviral response genes, whereas mutation of the TIM eliminates TRAF3 binding and the downstream IFN response. Reconstitution of MAVS(-/-) mouse embryo fibroblasts with a construct expressing a TIM-mutated version of MAVS failed to restore the antiviral response or block VSV replication, whereas wild-type MAVS reconstituted antiviral inhibition of VSV replication. Furthermore, recruitment of IKKε to an adjacent C-terminal site (aa 468-540) in MAVS via Lys500 ubiquitination decreased TRAF3 binding and protein stability, thus contributing to IKKε-mediated shutdown of the IFN response. This study demonstrates that MAVS harbors a functional C-terminal TRAF3-binding site that participates in positive and negative regulation of the IFN antiviral response.

  3. Enterovirus 71 Protease 2Apro Targets MAVS to Inhibit Anti-Viral Type I Interferon Responses

    PubMed Central

    Wang, Bei; Xi, Xueyan; Lei, Xiaobo; Zhang, Xiaoyan; Cui, Sheng; Wang, Jianwei; Jin, Qi; Zhao, Zhendong

    2013-01-01

    Enterovirus 71 (EV71) is the major causative pathogen of hand, foot, and mouth disease (HFMD). Its pathogenicity is not fully understood, but innate immune evasion is likely a key factor. Strategies to circumvent the initiation and effector phases of anti-viral innate immunity are well known; less well known is whether EV71 evades the signal transduction phase regulated by a sophisticated interplay of cellular and viral proteins. Here, we show that EV71 inhibits anti-viral type I interferon (IFN) responses by targeting the mitochondrial anti-viral signaling (MAVS) protein—a unique adaptor molecule activated upon retinoic acid induced gene-I (RIG-I) and melanoma differentiation associated gene (MDA-5) viral recognition receptor signaling—upstream of type I interferon production. MAVS was cleaved and released from mitochondria during EV71 infection. An in vitro cleavage assay demonstrated that the viral 2A protease (2Apro), but not the mutant 2Apro (2Apro-110) containing an inactivated catalytic site, cleaved MAVS. The Protease-Glo assay revealed that MAVS was cleaved at 3 residues between the proline-rich and transmembrane domains, and the resulting fragmentation effectively inactivated downstream signaling. In addition to MAVS cleavage, we found that EV71 infection also induced morphologic and functional changes to the mitochondria. The EV71 structural protein VP1 was detected on purified mitochondria, suggesting not only a novel role for mitochondria in the EV71 replication cycle but also an explanation of how EV71-derived 2Apro could approach MAVS. Taken together, our findings reveal a novel strategy employed by EV71 to escape host anti-viral innate immunity that complements the known EV71-mediated immune-evasion mechanisms. PMID:23555247

  4. Arenavirus nucleoprotein targets interferon regulatory factor-activating kinase IKKε.

    PubMed

    Pythoud, Christelle; Rodrigo, W W Shanaka I; Pasqual, Giulia; Rothenberger, Sylvia; Martínez-Sobrido, Luis; de la Torre, Juan Carlos; Kunz, Stefan

    2012-08-01

    Arenaviruses perturb innate antiviral defense by blocking induction of type I interferon (IFN) production. Accordingly, the arenavirus nucleoprotein (NP) was shown to block activation and nuclear translocation of interferon regulatory factor 3 (IRF3) in response to virus infection. Here, we sought to identify cellular factors involved in innate antiviral signaling targeted by arenavirus NP. Consistent with previous studies, infection with the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) prevented phosphorylation of IRF3 in response to infection with Sendai virus, a strong inducer of the retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS) pathway of innate antiviral signaling. Using a combination of coimmunoprecipitation and confocal microscopy, we found that LCMV NP associates with the IκB kinase (IKK)-related kinase IKKε but that, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKKε interaction was highly conserved among arenaviruses from different clades. In LCMV-infected cells, IKKε colocalized with NP but not with MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKKε (IKBKE) and blocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our data identify IKKε as a novel target of arenavirus NP. Engagement of NP seems to sequester IKKε in an inactive complex. Considering the important functions of IKKε in innate antiviral immunity and other cellular processes, the NP-IKKε interaction likely plays a crucial role in arenavirus-host interaction.

  5. Effect of age on the pathogenesis of DHV-1 in Pekin ducks and on the innate immune responses of ducks to infection.

    PubMed

    Song, Cuiping; Yu, Shengqing; Duan, Yunbing; Hu, Yue; Qiu, Xvsheng; Tan, Lei; Sun, Yingjie; Wang, Mingshu; Cheng, Anchun; Ding, Chan

    2014-05-01

    Duck hepatitis virus (DHV) affects 1-week-old but not 3-week-old ducks, and it causes a more severe disease in the younger ducks. These differences may be partially due to the host response to DHV infection. In order to understand this difference, we characterized the pathobiology of and innate immune response to DHV infection in 1-day-old (1D) and 3-week-old (3 W) ducks. Viral RNA was detected in duck livers at 24, 36 and 72 h after inoculation with DHV at a dose of 10(3) LD50. Virus-induced pathology ranged from no clinical signs to severe disease and death, and it was more severe in the 1D ducks. Infection with DHV induced up-regulation of gene expression of Toll-like receptor (TLR)-7, TLR3, retinoic-acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), interleukin (IL)-6, interferon (IFN)-α, interferon-induced transmembrane protein 1 (IFITM1), interferon-stimulated gene 12 (ISG12), and 2'-5' oligoadenylate synthetase-like gene (OASL) in the livers of 3 W ducks. Of these, IL-6, OASL and ISG12 mRNA levels were more than 100-fold higher in infected 3 W ducks than in mock-infected ducks of the same age. These genes were induced much less in infected 1D ducklings. We present evidence that a lower level of viral replication in the hepatocytes of 3 W ducks, whose basal level of cytokines is higher than that in 1D ducklings, may be related to the strong innate immunity induced. From our data, we conclude that duck age plays an important role in the pathogenicity of and innate immune responses to DHV.

  6. HCV-Induced miR-21 Contributes to Evasion of Host Immune System by Targeting MyD88 and IRAK1

    PubMed Central

    Chen, Yanni; Chen, Junbo; Wang, Hui; Shi, Jingjing; Wu, Kailang; Liu, Shi; Liu, Yingle; Wu, Jianguo

    2013-01-01

    Upon recognition of viral components by pattern recognition receptors, such as the toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like helicases, cells are activated to produce type I interferon (IFN) and proinflammatory cytokines. These pathways are tightly regulated by the host to prevent an inappropriate cellular response, but viruses can modulate these pathways to proliferate and spread. In this study, we revealed a novel mechanism in which hepatitis C virus (HCV) evades the immune surveillance system to proliferate by activating microRNA-21 (miR-21). We demonstrated that HCV infection upregulates miR-21, which in turn suppresses HCV-triggered type I IFN production, thus promoting HCV replication. Furthermore, we demonstrated that miR-21 targets two important factors in the TLR signaling pathway, myeloid differentiation factor 88 (MyD88) and interleukin-1 receptor-associated kinase 1 (IRAK1), which are involved in HCV-induced type I IFN production. HCV-mediated activation of miR-21 expression requires viral proteins and several signaling components. Moreover, we identified a transcription factor, activating protein-1 (AP-1), which is partly responsible for miR-21 induction in response to HCV infection through PKCε/JNK/c-Jun and PKCα/ERK/c-Fos cascades. Taken together, our results indicate that miR-21 is upregulated during HCV infection and negatively regulates IFN-α signaling through MyD88 and IRAK1 and may be a potential therapeutic target for antiviral intervention. PMID:23633945

  7. Perflurooctanoic Acid Induces Developmental Cardiotoxicity in ...

    EPA Pesticide Factsheets

    Perfluorooctanoic acid (PFOA) is a widespread environmental contaminant that is detectable in serum of the general U.S. population. PFOA is a known developmental toxicant that induces mortality in mammalian embryos and is thought to induce toxicity via interaction with the peroxisome proliferator activated receptor alpha (PPAR_). As the cardiovascular system is crucial for embryonic survival, PFOA-induced effects on the heart may partially explain embryonic mortality. To assess impacts of PFOA exposure on the developing heart in an avian model, we used histopathology and immunohistochemical staining for myosin to assess morphological alterations in 19-day-old chicken embryo hearts after PFOA exposure. Additionally, echocardiography and cardiac myofibril ATPase activity assays were used to assess functional alterations in 1-day-old hatchling chickens following developmental PFOA exposure. Overall thinning and thinning of a dense layer of myosin in the right ventricular wall were observed in PFOA-exposed chicken embryo hearts. Alteration of multiple cardiac structural and functional parameters, including left ventricular wall thickness, left ventricular volume, heart rate, stroke volume, and ejection fraction were detected with echocardiography in the exposed hatchling chickens. Assessment of ATPase activity indicated that the ratio of cardiac myofibril calcium-independent ATPase activity to calcium-dependent ATPase activity was not affected, which suggests that d

  8. Tranexamic Acid-Induced Fixed Drug Eruption

    PubMed Central

    Matsumura, Natsuko; Hanami, Yuka; Yamamoto, Toshiyuki

    2015-01-01

    A 33-year-old male showed multiple pigmented patches on his trunk and extremities after he took tranexamic acid for common cold. He stated that similar eruptions appeared when he was treated with tranexamic acid for influenza 10 months before. Patch test showed positive results at 48 h and 72 h by 1% and 10% tranexamic acid at the lesional skin only. To our knowledge, nine cases of fixed drug eruption induced by tranexamic acid have been reported in Japan. Tranexamic acid is a safe drug and frequently used because of its anti-fibrinolytic and anti-inflammatory effects, but caution of inducing fixed drug eruption should be necessary. PMID:26288438

  9. Conjugated Linoleic Acid Induces Human Adipocyte Delipidation

    PubMed Central

    Brown, J. Mark; Boysen, Maria Sandberg; Chung, Soonkyu; Fabiyi, Olowatoyin; Morrison, Ron F.; Mandrup, Susanne; McIntosh, Michael K.

    2005-01-01

    Dietary conjugated linoleic acid (CLA) reduces body fat in animals and some humans. Here we show that trans-10, cis-12 CLA, but not cis-9, trans-11 CLA, when added to cultures of stromal vascular cells containing newly differentiated human adipocytes, caused a time-dependent decrease in triglyceride content, insulin-stimulated glucose and fatty acid uptake, incorporation into lipid, and oxidation compared with controls. In parallel, gene expression of peroxisome proliferator-activated receptor-γ and many of its downstream targets were diminished by trans-10, cis-12 CLA, whereas leptin gene expression was increased. Prior to changes in gene expression and metabolism, trans-10, cis-12 CLA caused a robust and sustained activation of mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK) signaling. Furthermore, the trans-10, cis-12 CLA-mediated activation of MEK/ERK could be attenuated by pretreatment with U0126 and pertussis toxin. In parallel, pretreatment with U0126 blocked the ability of trans-10, cis-12 CLA to alter gene expression and attenuate glucose and fatty acid uptake of the cultures. Intriguingly, the induction by CLA of MEK/ERK signaling was linked to hypersecretion of adipocytokines interleukin-6 and interleukin-8. Collectively, these data demonstrate for the first time that trans-10, cis-12 CLA decreases the triglyceride content of newly differentiated human adipocytes by inducing MEK/ERK signaling through the autocrine/paracrine actions of interleukins-6 and 8. PMID:15067015

  10. Tranexamic Acid-Induced Fixed Drug Eruption.

    PubMed

    Matsumura, Natsuko; Hanami, Yuka; Yamamoto, Toshiyuki

    2015-01-01

    A 33-year-old male showed multiple pigmented patches on his trunk and extremities after he took tranexamic acid for common cold. He stated that similar eruptions appeared when he was treated with tranexamic acid for influenza 10 months before. Patch test showed positive results at 48 h and 72 h by 1% and 10% tranexamic acid at the lesional skin only. To our knowledge, nine cases of fixed drug eruption induced by tranexamic acid have been reported in Japan. Tranexamic acid is a safe drug and frequently used because of its anti-fibrinolytic and anti-inflammatory effects, but caution of inducing fixed drug eruption should be necessary.

  11. Ursodeoxycholic acid induced generalized fixed drug eruption.

    PubMed

    Ozkol, Hatice Uce; Calka, Omer; Dulger, Ahmet Cumhur; Bulut, Gulay

    2014-09-01

    Fixed drug eruption (FDE) is a rare form of drug allergies that recur at the same cutaneous or mucosal site in every usage of drug. Single or multiple round, sharply demarcated and dusky red plaques appear soon after drug exposure. Ursodeoxycholic acid (UDCA: 3α,7β-dihydroxy-5β-cholanic acid) is used for the treatment of cholestatic liver diseases. Some side effects may be observed, such as diarrhea, dyspepsia, pruritus and headaches. We encountered only three cases of lichenoid reaction regarding the use of UDCA among previous studies. In this article, we reported a generalized FDE case related to UDCA intake in a 59-year-old male patient with cholestasis for the first time in the literature.

  12. Lysophosphatidic acid induces osteocyte dendrite outgrowth.

    PubMed

    Karagiosis, Sue A; Karin, Norman J

    2007-05-25

    Osteocytes elaborate an extensive mechanosensory network in bone matrix and communicate intercellularly via gap junctions established at dendrite termini. We developed a method to measure osteocyte dendritogenesis in vitro using a modified transwell assay and determined that the lipid growth factor lysophosphatidic acid (LPA) is a potent stimulator of dendrite outgrowth in MLO-Y4 osteocytes. The stimulatory effects were dose-dependent with maximal outgrowth observed within a physiological range of LPA. LPA-treated osteocytes exhibited distinct rearrangements of the actin cytoskeleton and a more stellate morphology than control cells. LPA also promoted osteocyte chemotaxis, suggesting a shared molecular mechanism between dendrite outgrowth and cell motility. The LPA-induced increase in dendrite formation was blocked by the specific LPA-receptor antagonist Ki16425 and by pertussis toxin. Bone cells in vivo encounter platelet-derived LPA in regions of bone damage, and we postulate that this lipid factor is important for re-establishing osteocyte connectivity during fracture repair.

  13. Anti-Inflammatory and Analgesic Effects of Pyeongwisan on LPS-Stimulated Murine Macrophages and Mouse Models of Acetic Acid-Induced Writhing Response and Xylene-Induced Ear Edema

    PubMed Central

    Oh, You-Chang; Jeong, Yun Hee; Cho, Won-Kyung; Ha, Jeong-Ho; Gu, Min Jung; Ma, Jin Yeul

    2015-01-01

    Pyeongwisan (PW) is an herbal medication used in traditional East Asian medicine to treat anorexia, abdominal distension, borborygmus and diarrhea caused by gastric catarrh, atony and dilatation. However, its effects on inflammation-related diseases are unknown. In this study, we investigated the biological effects of PW on lipopolysaccharide (LPS)-mediated inflammation in macrophages and on local inflammation in vivo. We investigated the biological effects of PW on the production of inflammatory mediators, pro-inflammatory cytokines and related products as well as the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in LPS-stimulated macrophages. Additionally, we evaluated the analgesic effect on the acetic acid-induced writhing response and the inhibitory activity on xylene-induced ear edema in mice. PW showed anti-inflammatory effects by inhibiting the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and interleukin-1β (IL-1β). In addition, PW strongly suppressed inducible nitric oxide synthase (iNOS), a NO synthesis enzyme, induced heme oxygenase-1 (HO-1) expression and inhibited NF-κB activation and MAPK phosphorylation. Also, PW suppressed TNF-α, IL-6 and IL-1β cytokine production in LPS-stimulated peritoneal macrophage cells. Furthermore, PW showed an analgesic effect on the writhing response and an inhibitory effect on mice ear edema. We demonstrated the anti-inflammatory effects and inhibitory mechanism in macrophages as well as inhibitory activity of PW in vivo for the first time. Our results suggest the potential value of PW as an inflammatory therapeutic agent developed from a natural substance. PMID:25569097

  14. Anti-inflammatory and analgesic effects of pyeongwisan on LPS-stimulated murine macrophages and mouse models of acetic acid-induced writhing response and xylene-induced ear edema.

    PubMed

    Oh, You-Chang; Jeong, Yun Hee; Cho, Won-Kyung; Ha, Jeong-Ho; Gu, Min Jung; Ma, Jin Yeul

    2015-01-06

    Pyeongwisan (PW) is an herbal medication used in traditional East Asian medicine to treat anorexia, abdominal distension, borborygmus and diarrhea caused by gastric catarrh, atony and dilatation. However, its effects on inflammation-related diseases are unknown. In this study, we investigated the biological effects of PW on lipopolysaccharide (LPS)-mediated inflammation in macrophages and on local inflammation in vivo. We investigated the biological effects of PW on the production of inflammatory mediators, pro-inflammatory cytokines and related products as well as the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in LPS-stimulated macrophages. Additionally, we evaluated the analgesic effect on the acetic acid-induced writhing response and the inhibitory activity on xylene-induced ear edema in mice. PW showed anti-inflammatory effects by inhibiting the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and interleukin-1β (IL-1β). In addition, PW strongly suppressed inducible nitric oxide synthase (iNOS), a NO synthesis enzyme, induced heme oxygenase-1 (HO-1) expression and inhibited NF-κB activation and MAPK phosphorylation. Also, PW suppressed TNF-α, IL-6 and IL-1β cytokine production in LPS-stimulated peritoneal macrophage cells. Furthermore, PW showed an analgesic effect on the writhing response and an inhibitory effect on mice ear edema. We demonstrated the anti-inflammatory effects and inhibitory mechanism in macrophages as well as inhibitory activity of PW in vivo for the first time. Our results suggest the potential value of PW as an inflammatory therapeutic agent developed from a natural substance.

  15. Stearic acid-induced cardiac lipotoxicity is independent of cellular lipid and is mitigated by the fatty acids oleic and capric acid but not by the PPAR agonist troglitazone.

    PubMed

    Rabkin, Simon W; Lodhia, Parth; Lodha, Parth

    2009-08-01

    The objective of this study was to examine the potential of stearic acid to induce cardiomyocyte cell death and the hypothesis that the amount of cellular lipid is a determinant of cell death. In cardiomyocytes from embryonic chick heart, stearic acid (SA) produced a significant (P < 0.001) concentration-dependent increase in cell death with an ED(50) of 71 microM. In contrast, capric (C10:0) or oleic acid (OA; C18:1), at < 200 microM, did not alter cell viability. Stearic acid-induced cell death was significantly reduced by OA and to a lesser extent by capric acid. Neither OA nor capric acid altered cell death produced by potassium cyanide and deoxyglucose. Stearic acid (100 microM) induced a significant (P < 0.05) twofold increase in cellular lipid as assessed by Nile blue and Sudan Black staining. A role for cellular lipid in cardiomyocyte death was excluded because OA increased cellular lipid, at concentrations that did not induce cell death; OA did not alter SA-induced cellular fat stores but reduced cell death; and the PPARgamma; agonist troglitazone at concentrations that reduced cellular lipid content did not alter cell death. High concentrations of troglitazone, however, induced cell death. In summary, SA is a potent inducer of cardiac cell death and intracellular lipid accumulation. The amount of intracellular lipid, however, is not a determinant of cardiomyocyte cell death. Troglitazone has potential cardiotoxicity at high doses but, at lower concentrations, does not prevent cardiac lipotoxicity, which can be completely prevented by low concentrations of oleic acid.

  16. Stimulation of large-conductance calcium-activated potassium channels inhibits neurogenic contraction of human bladder from patients with urinary symptoms and reverses acetic acid-induced bladder hyperactivity in rats.

    PubMed

    La Fuente, José M; Fernández, Argentina; Cuevas, Pedro; González-Corrochano, Rocío; Chen, Mao Xiang; Angulo, Javier

    2014-07-15

    We have analysed the effects of large-conductance calcium-activated potassium channel (BK) stimulation on neurogenic and myogenic contraction of human bladder from healthy subjects and patients with urinary symptoms and evaluated the efficacy of activating BK to relief bladder hyperactivity in rats. Bladder specimens were obtained from organ donors and from men with benign prostatic hyperplasia (BPH). Contractions elicited by electrical field stimulation (EFS) and carbachol (CCh) were evaluated in isolated bladder strips. in vivo cystometric recordings were obtained in anesthetized rats under control and acetic acid-induced hyperactive conditions. Neurogenic contractions of human bladder were potentiated by blockade of BK and small-conductance calcium-activated potassium channels (SK) but were unaffected by the blockade of intermediate calcium-activated potassium channels (IK). EFS-induced contractions were inhibited by BK stimulation with NS-8 or NS1619 or by SK/IK stimulation with NS309 (3µM). CCh-induced contractions were not modified by blockade or stimulation of BK, IK or SK. The anti-cholinergic agent, oxybutynin (0.3µM) inhibited either neurogenic or CCh-induced contractions. Neurogenic contractions of bladders from BPH patients were less sensitive to BK inhibition and more sensitive to BK activation than healthy bladders. The BK activator, NS-8 (5mg/kg; i.v.), reversed bladder hyperactivity induced by acetic acid in rats, while oxybutynin was ineffective. NS-8 did not significantly impact blood pressure or heart rate. BK stimulation specifically inhibits neurogenic contractions in patients with urinary symptoms and relieves bladder hyperactivity in vivo without compromising bladder contractile capacity or cardiovascular safety, supporting its potential therapeutic use for relieving bladder overactivity.

  17. Nonpeptide tachykinin receptor antagonists. III. SB 235375, a low central nervous system-penetrant, potent and selective neurokinin-3 receptor antagonist, inhibits citric acid-induced cough and airways hyper-reactivity in guinea pigs.

    PubMed

    Hay, Douglas W P; Giardina, Giuseppe A M; Griswold, Don E; Underwood, David C; Kotzer, Charles J; Bush, Brian; Potts, William; Sandhu, Punam; Lundberg, Dave; Foley, James J; Schmidt, Dulcie B; Martin, Lenox D; Kilian, David; Legos, Jeffrey J; Barone, Frank C; Luttmann, Mark A; Grugni, Mario; Raveglia, Luca F; Sarau, Henry M

    2002-01-01

    In this report the in vitro and in vivo pharmacological and pharmacokinetic profile of (-)-(S)-N-(alpha-ethylbenzyl)-3-(carboxymethoxy)-2-phenylquinoline-4-carboxamide (SB 235375), a low central nervous system (CNS)-penetrant, human neurokinin-3 (NK-3) receptor (hNK-3R) antagonist, is described. SB 235375 inhibited (125)I-[MePhe(7)]-neurokinin B (NKB) binding to membranes of Chinese hamster ovary (CHO) cells expressing the hNK-3R (CHO-hNK-3R) with a K(i) = 2.2 nM and antagonized competitively NKB-induced Ca(2+) mobilization in human embryonic kidney (HEK) 293 cells expressing the hNK-3R (HEK 293-hNK-3R) with a K(b) = 12 nM. SB 235375 antagonized senktide (NK-3R)-induced contractions in rabbit isolated iris sphincter (pA(2) = 8.1) and guinea pig ileal circular smooth muscles (pA(2) = 8.3). SB 235375 was selective for the hNK-3R compared with hNK-1 (K(i) > 100,000 nM) and hNK-2 receptors (K(i) = 209 nM), and was without effect, at 1 microM, in 68 other receptor, enzyme, and ion channel assays. Intravenous SB 235375 produced a dose-related inhibition of miosis induced by i.v. senktide in the rabbit (ED(50) of 0.56 mg/kg). Intraperitoneal SB 235375 (10-30 mg/kg) inhibited citric acid-induced cough and airways hyper-reactivity in guinea pigs. In mice oral SB 235375 (3-30 mg/kg) was without significant effect on the behavioral responses induced by intracerebral ventricular administration of senktide. Pharmacokinetic evaluation in the mouse and rat revealed that oral SB 235375 was well absorbed systemically but did not effectively cross the blood-brain barrier. The preclinical profile of SB 235375, encompassing high affinity, selectivity, oral activity, and low CNS penetration, suggests that it is an appropriate tool compound to define the pathophysiological roles of the NK-3Rs in the peripheral nervous system.

  18. Linoleic acid-induced expression of inducible nitric oxide synthase and cyclooxygenase II via p42/44 mitogen-activated protein kinase and nuclear factor-kappaB pathway in retinal pigment epithelial cells.

    PubMed

    Fang, I-Mo; Yang, Chang-Hao; Yang, Chung-May; Chen, Muh-Shy

    2007-11-01

    High linoleic acid (LA) intake is known to correlate with age-related macular degeneration (AMD), but the molecular mechanisms remain unclear. This study was conducted to investigate the effects of LA on expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase II (COX-2) and their associated signaling pathways in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were treated with different concentrations of LA. Expressions of iNOS and COX-2 were examined using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Concentrations of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in the culture medium were determined by enzyme-link immunosorbent assay (ELISA). Activation of p42/44, p38, JNK mitogen-activated protein kinase (MAPK) and nuclear factors (NF)-kappaB were evaluated by Western blot analysis and electrophoretic mobility shift assay (EMSA). We found that LA induced expression of iNOS and COX-2 in RPE cells at the mRNA and protein levels in a time-and dose-dependent manner. Upregulation of iNOS and COX-2 resulted in increased production of NO and PGE(2). Moreover, LA caused degradation of IkappaB and increased NF-kappaB DNA binding activity. Effects of LA-induced iNOS and COX-2 expression were inhibited by a NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC). LA activated p42/44, but not p38 or JNK MAPK. Inhibition of p42/44 activity by PD98059 significantly reduced LA-induced activation of NF-kappaB. Linoleic acid-induced expression of iNOS and COX-2 as well as PGE(2) and NO release in RPE cells were sequentially mediated through activation of p42/p44, MAPK, then NF-kappaB. These results may provide new insights into both mechanisms of LA action on RPE cells and pathogenesis of age-related macular degeneration.

  19. Synthetic RNAs Mimicking Structural Domains in the Foot-and-Mouth Disease Virus Genome Elicit a Broad Innate Immune Response in Porcine Cells Triggered by RIG-I and TLR Activation.

    PubMed

    Borrego, Belén; Rodríguez-Pulido, Miguel; Revilla, Concepción; Álvarez, Belén; Sobrino, Francisco; Domínguez, Javier; Sáiz, Margarita

    2015-07-17

    The innate immune system is the first line of defense against viral infections. Exploiting innate responses for antiviral, therapeutic and vaccine adjuvation strategies is being extensively explored. We have previously described, the ability of small in vitro RNA transcripts, mimicking the sequence and structure of different domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs), to trigger a potent and rapid innate immune response. These synthetic non-infectious molecules have proved to have a broad-range antiviral activity and to enhance the immunogenicity of an FMD inactivated vaccine in mice. Here, we have studied the involvement of pattern-recognition receptors (PRRs) in the ncRNA-induced innate response and analyzed the antiviral and cytokine profiles elicited in swine cultured cells, as well as peripheral blood mononuclear cells (PBMCs).

  20. Synthetic RNAs Mimicking Structural Domains in the Foot-and-Mouth Disease Virus Genome Elicit a Broad Innate Immune Response in Porcine Cells Triggered by RIG-I and TLR Activation

    PubMed Central

    Borrego, Belén; Rodríguez-Pulido, Miguel; Revilla, Concepción; Álvarez, Belén; Sobrino, Francisco; Domínguez, Javier; Sáiz, Margarita

    2015-01-01

    The innate immune system is the first line of defense against viral infections. Exploiting innate responses for antiviral, therapeutic and vaccine adjuvation strategies is being extensively explored. We have previously described, the ability of small in vitro RNA transcripts, mimicking the sequence and structure of different domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs), to trigger a potent and rapid innate immune response. These synthetic non-infectious molecules have proved to have a broad-range antiviral activity and to enhance the immunogenicity of an FMD inactivated vaccine in mice. Here, we have studied the involvement of pattern-recognition receptors (PRRs) in the ncRNA-induced innate response and analyzed the antiviral and cytokine profiles elicited in swine cultured cells, as well as peripheral blood mononuclear cells (PBMCs). PMID:26193305

  1. Nucleic acid sensing receptors in systemic lupus erythematosus: development of novel DNA- and/or RNA-like analogues for treating lupus

    PubMed Central

    Lenert, P

    2010-01-01

    Double-stranded (ds) DNA, DNA- or RNA-associated nucleoproteins are the primary autoimmune targets in SLE, yet their relative inability to trigger similar autoimmune responses in experimental animals has fascinated scientists for decades. While many cellular proteins bind non-specifically negatively charged nucleic acids, it was discovered only recently that several intracellular proteins are involved directly in innate recognition of exogenous DNA or RNA, or cytosol-residing DNA or RNA viruses. Thus, endosomal Toll-like receptors (TLR) mediate responses to double-stranded RNA (TLR-3), single-stranded RNA (TLR-7/8) or unmethylated bacterial cytosine (phosphodiester) guanine (CpG)-DNA (TLR-9), while DNA-dependent activator of IRFs/Z-DNA binding protein 1 (DAI/ZBP1), haematopoietic IFN-inducible nuclear protein-200 (p202), absent in melanoma 2 (AIM2), RNA polymerase III, retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) mediate responses to cytosolic dsDNA or dsRNA, respectively. TLR-induced responses are more robust than those induced by cytosolic DNA- or RNA- sensors, the later usually being limited to interferon regulatory factor 3 (IRF3)-dependent type I interferon (IFN) induction and nuclear factor (NF)-κB activation. Interestingly, AIM2 is not capable of inducing type I IFN, but rather plays a role in caspase I activation. DNA- or RNA-like synthetic inhibitory oligonucleotides (INH-ODN) have been developed that antagonize TLR-7- and/or TLR-9-induced activation in autoimmune B cells and in type I IFN-producing dendritic cells at low nanomolar concentrations. It is not known whether these INH-ODNs have any agonistic or antagonistic effects on cytosolic DNA or RNA sensors. While this remains to be determined in the future, in vivo studies have already shown their potential for preventing spontaneous lupus in various animal models of lupus. Several groups are exploring the possibility of translating these INH-ODNs into

  2. Epigenetic modifications in valproic acid-induced teratogenesis

    SciTech Connect

    Tung, Emily W.Y.; Winn, Louise M.

    2010-11-01

    Exposure to the anticonvulsant drug valproic acid (VPA) in utero is associated with a 1-2% increase in neural tube defects (NTDs), however the molecular mechanisms by which VPA induces teratogenesis are unknown. Previous studies demonstrated that VPA, a direct inhibitor of histone deacetylase, can induce histone hyperacetylation and other epigenetic changes such as histone methylation and DNA demethylation. The objective of this study was to determine if maternal exposure to VPA in mice has the ability to cause these epigenetic alterations in the embryo and thus contribute to its mechanism of teratogenesis. Pregnant CD-1 mice (GD 9.0) were administered a teratogenic dose of VPA (400 mg/kg, s.c.) and embryos extracted 1, 3, 6, and 24 h after injection. To assess embryonic histone acetylation and histone methylation, Western blotting was performed on whole embryo homogenates, as well as immunohistochemical staining on embryonic sections. To measure DNA methylation changes, the cytosine extension assay was performed. Results demonstrated that a significant increase in histone acetylation that peaked 3 h after VPA exposure was accompanied by an increase in histone methylation at histone H3 lysine 4 (H3K4) and a decrease in histone methylation at histone H3 lysine 9 (H3K9). Immunohistochemical staining revealed increased histone acetylation in the neuroepithelium, heart, and somites. A decrease in methylated histone H3K9 staining was observed in the neuroepithelium and somites, METHYLATED histone H3K4 staining was observed in the neuroepithelium. No significant differences in global or CpG island DNA methylation were observed in embryo homogenates. These results support the possibility that epigenetic modifications caused by VPA during early mouse organogenesis results in congenital malformations.

  3. A molecular basis for retinoic acid-induced axial truncation.

    PubMed

    Iulianella, A; Beckett, B; Petkovich, M; Lohnes, D

    1999-01-01

    Dietary deprivation and gene disruption studies clearly demonstrate that biologically active retinoids, such as retinoic acid (RA), are essential for numerous developmental programs. Similar ontogenic processes are also affected by retinoic acid excess, suggesting that the effects of retinoid administration reflect normal retinoid-dependent events. In the mouse, exogenous retinoic acid can induce both anterior (anencephaly, exencephaly) and posterior (spina bifida) neural tube defects depending on the developmental stage of treatment. Retinoic acid receptor gamma (RARgamma) mediates these effects on the caudal neural tube at 8.5 days postcoitum, as RARgamma-/- mice are completely resistant to spina bifida induced by retinoic acid at this stage. We therefore used this null mouse as a model to examine the molecular nature of retinoid-induced caudal neural tube defects by using a panel of informative markers and comparing their expression between retinoic acid-treated wild-type and RARgamma-/- embryos. Our findings indicate that treatment of wild-type embryos led to a rapid and significant decrease in the caudal expression of all mesodermal markers examined (e.g., brachyury, wnt-3a, cdx-4), whereas somite, neuroepithelial, notochord, floorplate, and hindgut markers were unaffected. RARgamma-/- mutants exhibited normal expression patterns for all markers examined, consistent with the notion that mesodermal defects underlie the etiology of retinoid-induced spina bifida. We also found that posterior somitic, but not caudal presomitic, embryonic tissues contained detectable bioactive retinoids, an observation which correlated with the ability of caudal explants to rapidly clear exogenous RA. Interestingly, transcripts encoding mP450RAI, a cytochrome P450, the product of which is believed to catabolize retinoic acid, were abundant in the retinoid-poor region of the caudal embryo. mP450RAI was rapidly induced by retinoic acid treatment in vivo, consistent with previous studies suggesting that it plays a critical role in retinoid signaling. These data suggest that nascent mesoderm is highly sensitive to retinoic acid and that mP450RAI serves to tightly regulate retinoid levels in the caudal embryo. These findings also raise the possibility that RA may play a role in the generation of posterior mesoderm derivatives in part by affecting brachyury expression.

  4. Ameliorative effects of phycocyanin against gibberellic acid induced hepatotoxicity.

    PubMed

    Hussein, Mohamed M A; Ali, Haytham A; Ahmed, Mona M

    2015-03-01

    Gibberellic acid (GA3) was used extensively unaware in agriculture in spite of its dangerous effects on human health. The current study was designed to investigate the ameliorative effects of the co-administration of phycocyanin with GA3 induced oxidative stress and histopathological changes in the liver. Forty male albino rats were randomly divided into four groups. Group I (control group) received normal saline for 6 weeks, Group II (GA3 treated group) received 3.85 mg/kg body weight GA3 once daily for 6 weeks, Group III (phycocyanin treated group) received Phycocyanin 200 mg/kg body weight/day for 6 weeks orally dissolved in distilled water and Group IV was treated with GA3 and phycocyanin at the same doses as groups 2 and 3. All treatments were given daily using intra-gastric intubation and continued for 6 weeks. Our results revealed significant downregulation of antioxidant enzyme activities and their mRNA levels (CAT, GPx and Cu-Zn, SOD) with marked elevation of liver enzymes and extensive fibrous connective tissue deposition with large biliary cells in hepatic tissue of GA3 treated rats, while treatment with phycocyanin improved the antioxidant defense system, liver enzymes and structural hepatocytes recovery in phycocyanin treated group with GA3. These data confirm the antioxidant potential of Phycocyanin and provide strong evidence to support the co-administration of Phycocyanin during using GA3.

  5. Ursolic acid induces neural regeneration after sciatic nerve injury

    PubMed Central

    Liu, Biao; Liu, Yan; Yang, Guang; Xu, Zemin; Chen, Jiajun

    2013-01-01

    In this study, we aimed to explore the role of ursolic acid in the neural regeneration of the injured sciatic nerve. BALB/c mice were used to establish models of sciatic nerve injury through unilateral sciatic nerve complete transection and microscopic anastomosis at 0.5 cm below the ischial tube-rosity. The successfully generated model mice were treated with 10, 5, or 2.5 mg/kg ursolic acid via intraperitoneal injection. Enzyme-linked immunosorbent assay results showed that serum S100 protein expression level gradually increased at 1–4 weeks after sciatic nerve injury, and significantly decreased at 8 weeks. As such, ursolic acid has the capacity to significantly increase S100 protein expression levels. Real-time quantitative PCR showed that S100 mRNA expression in the L4–6 segments on the injury side was increased after ursolic acid treatment. In addition, the muscular mass index in the soleus muscle was also increased in mice treated with ursolic acid. Toluidine blue staining revealed that the quantity and average diameter of myelinated nerve fibers in the injured sciatic nerve were significantly increased after treatment with ursolic acid. 10 and 5 mg/kg of ursolic acid produced stronger effects than 2.5 mg/kg of ursolic acid. Our findings indicate that ursolic acid can dose-dependently increase S100 expression and promote neural regeneration in BALB/c mice following sciatic nerve injury. PMID:25206561

  6. Epigenetic modifications in valproic acid-induced teratogenesis.

    PubMed

    Tung, Emily W Y; Winn, Louise M

    2010-11-01

    Exposure to the anticonvulsant drug valproic acid (VPA) in utero is associated with a 1-2% increase in neural tube defects (NTDs), however the molecular mechanisms by which VPA induces teratogenesis are unknown. Previous studies demonstrated that VPA, a direct inhibitor of histone deacetylase, can induce histone hyperacetylation and other epigenetic changes such as histone methylation and DNA demethylation. The objective of this study was to determine if maternal exposure to VPA in mice has the ability to cause these epigenetic alterations in the embryo and thus contribute to its mechanism of teratogenesis. Pregnant CD-1 mice (GD 9.0) were administered a teratogenic dose of VPA (400mg/kg, s.c.) and embryos extracted 1, 3, 6, and 24h after injection. To assess embryonic histone acetylation and histone methylation, Western blotting was performed on whole embryo homogenates, as well as immunohistochemical staining on embryonic sections. To measure DNA methylation changes, the cytosine extension assay was performed. Results demonstrated that a significant increase in histone acetylation that peaked 3h after VPA exposure was accompanied by an increase in histone methylation at histone H3 lysine 4 (H3K4) and a decrease in histone methylation at histone H3 lysine 9 (H3K9). Immunohistochemical staining revealed increased histone acetylation in the neuroepithelium, heart, and somites. A decrease in methylated histone H3K9 staining was observed in the neuroepithelium and somites, METHYLATED histone H3K4 staining was observed in the neuroepithelium. No significant differences in global or CpG island DNA methylation were observed in embryo homogenates. These results support the possibility that epigenetic modifications caused by VPA during early mouse organogenesis results in congenital malformations.

  7. Formic-acid-induced depolymerization of oxidized lignin to aromatics.

    PubMed

    Rahimi, Alireza; Ulbrich, Arne; Coon, Joshua J; Stahl, Shannon S

    2014-11-13

    Lignin is a heterogeneous aromatic biopolymer that accounts for nearly 30% of the organic carbon on Earth and is one of the few renewable sources of aromatic chemicals. As the most recalcitrant of the three components of lignocellulosic biomass (cellulose, hemicellulose and lignin), lignin has been treated as a waste product in the pulp and paper industry, where it is burned to supply energy and recover pulping chemicals in the operation of paper mills. Extraction of higher value from lignin is increasingly recognized as being crucial to the economic viability of integrated biorefineries. Depolymerization is an important starting point for many lignin valorization strategies, because it could generate valuable aromatic chemicals and/or provide a source of low-molecular-mass feedstocks suitable for downstream processing. Commercial precedents show that certain types of lignin (lignosulphonates) may be converted into vanillin and other marketable products, but new technologies are needed to enhance the lignin value chain. The complex, irregular structure of lignin complicates chemical conversion efforts, and known depolymerization methods typically afford ill-defined products in low yields (that is, less than 10-20wt%). Here we describe a method for the depolymerization of oxidized lignin under mild conditions in aqueous formic acid that results in more than 60wt% yield of low-molecular-mass aromatics. We present the discovery of this facile C-O cleavage method, its application to aspen lignin depolymerization, and mechanistic insights into the reaction. The broader implications of these results for lignin conversion and biomass refining are also considered.

  8. Fused liposome and acid induced method for liposome fusion

    SciTech Connect

    Huang, L.; Connor, J.

    1988-12-06

    This patent describes a method of fusing liposomes. It comprises: preparing a suspension of liposomes containing at least one lipid which has a tendency to form the inverted hexagonal phase and at least 20 mol percent of palmitoylhomocysteine; and in the absence of externally added divalent cations, proteins or other macromolecules, acidifying the liposome suspension to reduce the pH of the liposomes to below pH 7, such that at least about 20% of the liposomes fuse to one another.

  9. Nitrous acid induced damage in T7 DNA and phage

    SciTech Connect

    Scearce, L.M.; Masker, W.E.

    1986-05-01

    The response of bacteriophage T7 to nitrous acid damage was investigated. The T7 system allows in vitro mimicry of most aspects of in vivo DNA metabolism. Nitrous acid is of special interest since it has been previously shown to induce deletions and point mutations as well as novel adducts in DNA. T7 phage was exposed to 56 mM nitrous acid at pH 4.6 in vivo, causing a time dependent 98% decrease in survival for each 10 min duration of exposure to nitrous acid. These studies were extended to include examination of pure T7 DNA exposed in vitro to nitrous acid conditions identical to those used in the in vivo survival studies. The treated DNA was dialyzed to remove the nitrous acid and the DNA was encapsulated into empty phage heads. These in vitro packaged phage showed a survival curve analogous to the in vivo system. There was no change in survival when either in vitro or in vivo exposed phage were grown on wild type E. coli or on E. coli strains deficient in DNA repair due to mutations in DNA polymerase I, exonuclease III or a uvrA mutation. Survival was not increased when nitrous acid treated T7 were grown on E. coli induced for SOS repair. In vitro replication of nitrous acid treated DNA showed a time dependent decrease in the total amount of DNA synthesized.

  10. Docosahexaenoic Acid Induces Apoptosis in Primary Chronic Lymphocytic Leukemia Cells

    PubMed Central

    Gyan, Emmanuel; Tournilhac, Olivier; Halty, Christelle; Veyrat-Masson, Richard; Akil, Saïda; Berger, Marc; Hérault, Olivier; Callanan, Mary; Bay, Jacques-Olivier

    2015-01-01

    Chronic lymphocytic leukemia is an indolent disorder with an increased infectious risk remaining one of the main causes of death. Development of therapies with higher safety profile is thus a challenging issue. Docosahexaenoic acid (DHA, 22:6) is an omega-3 fatty acid, a natural compound of normal cells, and has been shown to display antitumor potency in cancer. We evaluated the potential in vitro effect of DHA in primary CLL cells. DHA induces high level of in vitro apoptosis compared to oleic acid in a dose-dependent and time-dependent manner. Estimation of IC50 was only of 4.813 µM, which appears lower than those reported in solid cancers. DHA is highly active on CLL cells in vitro. This observation provides a rationale for further studies aiming to understand its mechanisms of action and its potent in vivo activity. PMID:26734128

  11. Formic-acid-induced depolymerization of oxidized lignin to aromatics

    NASA Astrophysics Data System (ADS)

    Rahimi, Alireza; Ulbrich, Arne; Coon, Joshua J.; Stahl, Shannon S.

    2014-11-01

    Lignin is a heterogeneous aromatic biopolymer that accounts for nearly 30% of the organic carbon on Earth and is one of the few renewable sources of aromatic chemicals. As the most recalcitrant of the three components of lignocellulosic biomass (cellulose, hemicellulose and lignin), lignin has been treated as a waste product in the pulp and paper industry, where it is burned to supply energy and recover pulping chemicals in the operation of paper mills. Extraction of higher value from lignin is increasingly recognized as being crucial to the economic viability of integrated biorefineries. Depolymerization is an important starting point for many lignin valorization strategies, because it could generate valuable aromatic chemicals and/or provide a source of low-molecular-mass feedstocks suitable for downstream processing. Commercial precedents show that certain types of lignin (lignosulphonates) may be converted into vanillin and other marketable products, but new technologies are needed to enhance the lignin value chain. The complex, irregular structure of lignin complicates chemical conversion efforts, and known depolymerization methods typically afford ill-defined products in low yields (that is, less than 10-20wt%). Here we describe a method for the depolymerization of oxidized lignin under mild conditions in aqueous formic acid that results in more than 60wt% yield of low-molecular-mass aromatics. We present the discovery of this facile C-O cleavage method, its application to aspen lignin depolymerization, and mechanistic insights into the reaction. The broader implications of these results for lignin conversion and biomass refining are also considered.

  12. Viewpoints on Acid-Induced Inflammatory Mediators in Esophageal Mucosa

    PubMed Central

    Harnett, Karen M; Rieder, Florian; Behar, Jose

    2010-01-01

    We have focused on understanding the onset of gastroesophageal reflux disease by examining the mucosal response to the presence of acid in the esophageal lumen. Upon exposure to HCl, inflammation of the esophagus begins with activation of the transient receptor potential channel vanilloid subfamily member-1 (TRPV1) in the mucosa, and production of IL-8, substance P (SP), calcitonin gene related peptide (CGRP) and platelet activating factor (PAF). Production of SP and CGRP, but not PAF, is abolished by the neural blocker tetrodotoxin suggesting that SP and CGRP are neurally released and that PAF arises from non neural pathways. Epithelial cells contain TRPV1 receptor mRNA and protein and respond to HCl and to the TRPV1 agonist capsaicin with production of PAF. PAF, SP and IL-8 act as chemokines, inducing migration of peripheral blood leukocytes. PAF and SP activate peripheral blood leukocytes inducing the production of H2O2. In circular muscle, PAF causes production of IL-6, and IL-6 causes production of additional H2O2, through activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Among these, NADPH oxidase 5 cDNA is significantly up-regulated by exposure to PAF; H2O2 content of esophageal and lower esophageal sphincter circular muscle is elevated in human esophagitis, causing dysfunction of esophageal circular muscle contraction and reduction in esophageal sphincter tone. Thus esophageal keratinocytes, that constitute the first barrier to the refluxate, may also serve as the initiating cell type in esophageal inflammation, secreting inflammatory mediators and pro-inflammatory cytokines and affecting leukocyte recruitment and activity. PMID:21103419

  13. Lysophosphatidic acid-induced chemotaxis of bone cells.

    SciTech Connect

    Karagiosis, Sue A.; Masiello, Lisa M.; Bollinger, Nikki; Karin, Norm J.

    2006-07-01

    Lysophosphatidic acid (LPA) is a platelet-derived bioactive lipid that is postulated to regulate wound healing. LPA activates G protein-coupled receptors to induce Ca2+ signaling in MC3T3-E1 pre-osteoblasts, and is a potent chemotactic stimulus for these cells. Since bone fracture healing requires the migration of osteoblast progenitors, we postulate that LPA is among the factors that stimulate bone repair. UMR 106-01 cells, which express a more mature osteoblastic phenotype than MC3T3-E1 cells, did not migrate in response to LPA, although they express LPA receptors and exhibit LPA-induced Ca2+ signals. This suggests that LPA differentially induces pre-osteoblast chemotaxis, consistent with our hypothesis that LPA stimulates the motility of osteoblast progenitors during bone healing. LPA-stimulated MC3T3-E1 cells exhibit striking changes in morphology and F-actin architecture, and phosphatidylinositol-3 kinase (PI3K) is required for motility-associated cytoskeletal rearrangements in many cell types. We found a dose-dependent reduction in LPA-induced osteoblast migration when cells also were treated with the PI3K inhibitor, LY294002. Treatment of many cell types with LPA is associated with an autocrine/paracrine transactivation of the EGF receptor (EGFR) via shedding of surface-tethered EGFR ligands, a phenomenon often required for LPA-induced chemotaxis. MC3T3-E1 cells express multiple EGFR ligands (epigen, epiregulin, HB-EGF and amphiregulin) and migrated in response to EGF. However, while EGF-stimulated motility in MC3T3-E1 cells was blocked by an EGFR inhibitor, there was no significant effect on LPA-induced chemotaxis. Activation of MAP kinases is a hallmark of EGFR-mediated signaling, and EGF treatment of MC3T3-E1 cells led to a strong stimulation of ERK1/2 kinase. In contrast, LPA induced only a minor elevation in ERK activity. Thus, it is likely that the increase in ERK activity by LPA is related to cell proliferation associated with lipid treatment. We conclude that LPA-induced MC3T3-E1 cell chemotaxis requires PI3K-associated cytoskeletal changes, but not transactivation of the EGF receptor.

  14. Depressed phosphatidic acid-induced contractile activity of failing cardiomyocytes.

    PubMed

    Tappia, Paramjit S; Maddaford, Thane G; Hurtado, Cecilia; Panagia, Vincenzo; Pierce, Grant N

    2003-01-10

    The effects of phosphatidic acid (PA), a known inotropic agent, on Ca(2+) transients and contractile activity of cardiomyocytes in congestive heart failure (CHF) due to myocardial infarction were examined. In control cells, PA induced a significant increase (25%) in active cell shortening and Ca(2+) transients. The phospholipase C (PLC) inhibitor, 2-nitro-4-carboxyphenyl N,N-diphenylcarbonate, blocked the positive inotropic action induced by PA, indicating that PA induces an increase in contractile activity and Ca(2+) transients through stimulation of PLC. Conversely, in failing cardiomyocytes there was a loss of PA-induced increase in active cell shortening and Ca(2+) transients. PA did not alter resting cell length. Both diastolic and systolic [Ca(2+)] were significantly elevated in the failing cardiomyocytes. In vitro assessment of the cardiac sarcolemmal (SL) PLC activity revealed that the impaired failing cardiomyocyte response to PA was associated with a diminished stimulation of SL PLC activity by PA. Our results identify an important defect in the PA-PLC signaling pathway in failing cardiomyocytes, which may have significant implications for the depressed contractile function during CHF.

  15. Perflurooctanoic Acid Induces Developmental Cardiotoxicity in Chicken Embryos and Hatchlings

    EPA Science Inventory

    Perfluorooctanoic acid (PFOA) is a widespread environmental contaminant that is detectable in serum of the general U.S. population. PFOA is a known developmental toxicant that induces mortality in mammalian embryos and is thought to induce toxicity via interaction with the peroxi...

  16. Monomethylarsonous acid induces transformation of human bladder cells

    SciTech Connect

    Bredfeldt, Tiffany G.; Jagadish, Bhumasamudram; Eblin, Kylee E.; Mash, Eugene A.; Gandolfi, A. Jay . E-mail: gandolfi@pharmacy.arizona.edu

    2006-10-01

    Arsenic is a human bladder carcinogen. Arsenic is methylated to both monomethyl and dimethyl metabolites which have been detected in human urine. The trivalent methylated arsenicals are more toxic than inorganic arsenic. It is unknown if these trivalent methylated metabolites can directly cause malignant transformation in human cells. The goal of this study is determine if monomethylarsonous acid (MMA{sup III}) can induce malignant transformation in a human bladder urothelial cell line. To address this goal, a non-tumorigenic human urothelial cell line (UROtsa) was continuously exposed to 0.05 {mu}M MMA{sup III} for 52 weeks. Hyperproliferation was the first phenotypic change observed in exposed UROtsa (URO-MSC). After 12 weeks of exposure, doubling time had decreased from 42 h in unexposed control cells to 27 h in URO-MSC. Hyperproliferation continued to be a quality possessed by the URO-MSC cells after both 24 and 52 weeks of exposure to MMA{sup III}, which had a 40-50% reduction in doubling time. Throughout the 52-week exposure, URO-MSC cells retained an epithelial morphology with subtle morphological differences from control cells. 24 weeks of MMA{sup III} exposure was required to induce anchorage-independent growth as detected by colony formation in soft agar, a characteristic not found in UROtsa cells. To further substantiate that malignant transformation had occurred, URO-MSC cells were tested after 24 and 52 weeks of exposure to MMA{sup III} for the ability to form tumors in SCID mice. Enhanced tumorigenicity in SCID mouse xenografts was observed after 52 weeks of treatment with MMA{sup III}. These observations are the first demonstration of MMA{sup III}-induced malignant transformation in a human bladder urothelial cell line and provide important evidence that MMA{sup III} may be carcinogenic in human tissues.

  17. Valproic Acid Induces Antimicrobial Compound Production in Doratomyces microspores

    PubMed Central

    Zutz, Christoph; Bacher, Markus; Parich, Alexandra; Kluger, Bernhard; Gacek-Matthews, Agnieszka; Schuhmacher, Rainer; Wagner, Martin; Rychli, Kathrin; Strauss, Joseph

    2016-01-01

    One of the biggest challenges in public health is the rising number of antibiotic resistant pathogens and the lack of novel antibiotics. In recent years there is a rising focus on fungi as sources of antimicrobial compounds due to their ability to produce a large variety of bioactive compounds and the observation that virtually every fungus may still contain yet unknown so called “cryptic,” often silenced, compounds. These putative metabolites could include novel bioactive compounds. Considerable effort is spent on methods to induce production of these “cryptic” metabolites. One approach is the use of small molecule effectors, potentially influencing chromatin landscape in fungi. We observed that the supernatant of the fungus Doratomyces (D.) microsporus treated with valproic acid (VPA) displayed antimicrobial activity against Staphylococcus (S.) aureus and two methicillin resistant clinical S. aureus isolates. VPA treatment resulted in enhanced production of seven antimicrobial compounds: cyclo-(L-proline-L-methionine) (cPM), p-hydroxybenzaldehyde, cyclo-(phenylalanine-proline) (cFP), indole-3-carboxylic acid, phenylacetic acid (PAA) and indole-3-acetic acid. The production of the antimicrobial compound phenyllactic acid was exclusively detectable after VPA treatment. Furthermore three compounds, cPM, cFP, and PAA, were able to boost the antimicrobial activity of other antimicrobial compounds. cPM, for the first time isolated from fungi, and to a lesser extent PAA, are even able to decrease the minimal inhibitory concentration of ampicillin in MRSA strains. In conclusion we could show in this study that VPA treatment is a potent tool for induction of “cryptic” antimicrobial compound production in fungi, and that the induced compounds are not exclusively linked to the secondary metabolism. Furthermore this is the first discovery of the rare diketopiperazine cPM in fungi. Additionally we could demonstrate that cPM and PAA boost antibiotic activity against antibiotic resistant strains, suggesting a possible application in combinatorial antibiotic treatment against resistant pathogens. PMID:27148199

  18. Dimethylarsenic acid induces tetraploids in Chinese hamster cells

    SciTech Connect

    Endo, Ginji; Horiguchi, Shun'ichi ); Kuroda, Koichi; Okamoto, Akiyoshi )

    1992-01-01

    Arsenic has been documented as a human carcinogen of the skin and lungs. However, attempts to induce tumors in experimental animals with inorganoarsenic compounds have mostly failed except in a few studies in which animals were given arsenic trioxide by intratracheal instillation. Moreover, inorganoarsenics are either inactive or too weak to induce gene mutations in vitro. The mechanism of arsenic carcinogenicity has not yet been discovered. Most mammals including human are able to methylate inorganoarsenic compounds to methylarsonic acid and dimethylarsenic acid. However, the genotoxicity of organoarsenic compounds has hardly been examined. The authors therefore decided to study this genotoxicity, including the frequency of sister chromatid exchange (SCE) of nine organic and three inorganic arsenic compounds. Observation of the metaphases in the SCE test revealed that only DMA of the organo- and inorgano-arsenic compounds induces tetraploids and mitotic arrest. This indicates that the role of DMA may be important in arsenic genotoxicity and may give a clue to the carcinogenic mechanism of arsenic.

  19. Lysophosphatidic acid induced red blood cell aggregation in vitro.

    PubMed

    Kaestner, Lars; Steffen, Patrick; Nguyen, Duc Bach; Wang, Jue; Wagner-Britz, Lisa; Jung, Achim; Wagner, Christian; Bernhardt, Ingolf

    2012-10-01

    Under physiological conditions healthy RBCs do not adhere to each other. There are indications that RBCs display an intercellular adhesion under certain (pathophysiological) conditions. Therefore we investigated signaling steps starting with transmembrane calcium transport by means of calcium imaging. We found a lysophosphatidic acid (LPA) concentration dependent calcium influx with an EC(50) of 5 μM LPA. Downstream signaling was investigated by flow cytometry as well as by video-imaging comparing LPA induced with "pure" calcium mediated phosphatidylserine exposure and concluded the coexistence of two branches of the signaling pathway. Finally we performed force measurements with holographic optical tweezers (HOT): The intercellular adhesion of RBCs (aggregation) exceeds a force of 25 pN. These results support (i) earlier data of a RBC associated component in thrombotic events under certain pathophysiological conditions and (ii) the concept to use RBCs in studies of cellular adhesion behavior, especially in combination with HOT. The latter paves the way to use RBCs as model cells to investigate molecular regulation of cellular adhesion processes.

  20. Retinoic acid induces TGFbeta-dependent autocrine fibroblast growth.

    PubMed

    Fadloun, A; Kobi, D; Delacroix, L; Dembélé, D; Michel, I; Lardenois, A; Tisserand, J; Losson, R; Mengus, G; Davidson, I

    2008-01-17

    To evaluate the role of murine TFIID subunit TAF4 in activation of cellular genes by all-trans retinoic acid (T-RA), we have characterized the T-RA response of taf4(lox/-) and taf4(-/-) embryonic fibroblasts. T-RA regulates almost 1000 genes in taf4(lox/-) cells, but less than 300 in taf4(-/-) cells showing that TAF4 is required for T-RA regulation of most, but not all cellular genes. We further show that T-RA-treated taf4(lox/-) cells exhibit transforming growth factor (TGF)beta-dependent autocrine growth and identify a set of genes regulated by loss of TAF4 and by T-RA corresponding to key mediators of the TGFbeta signalling pathway. T-RA rapidly and potently induces expression of connective tissue growth factor (CTGF) via a conserved DR2 type response element in its proximal promoter leading to serum-free autocrine growth. These results highlight the role of TAF4 as a cofactor in the cellular response to T-RA and identify the genetic programme of a novel cross talk between the T-RA and TGFbeta pathways that leads to deregulated cell growth.

  1. Antiviral Efficacy and Host Innate Immunity Associated with SB 9200 Treatment in the Woodchuck Model of Chronic Hepatitis B.

    PubMed

    Korolowicz, Kyle E; Iyer, Radhakrishnan P; Czerwinski, Stefanie; Suresh, Manasa; Yang, Junming; Padmanabhan, Seetharamaiyer; Sheri, Anjaneyulu; Pandey, Rajendra K; Skell, Jeffrey; Marquis, Judith K; Kallakury, Bhaskar V; Tucker, Robin D; Menne, Stephan

    2016-01-01

    SB 9200, an oral prodrug of the dinucleotide SB 9000, is being developed for the treatment of chronic hepatitis B virus (HBV) infection and represents a novel class of antivirals. SB 9200 is thought to activate the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) resulting in interferon (IFN) mediated antiviral immune responses in virus-infected cells. Additionally, the binding of SB 9200 to these sensor proteins could also sterically block the ability of the viral polymerase to access pre-genomic RNA for nucleic acid synthesis. The immune stimulating and direct antiviral properties of SB 9200 were evaluated in woodchucks chronically infected with woodchuck hepatitis virus (WHV) by daily, oral dosing at 15 and 30 mg/kg for 12 weeks. Prolonged treatment resulted in 2.2 and 3.7 log10 reductions in serum WHV DNA and in 0.5 and 1.6 log10 declines in serum WHV surface antigen from pretreatment level with the lower or higher dose of SB 9200, respectively. SB 9200 treatment also resulted in lower hepatic levels of WHV nucleic acids and antigen and reduced liver inflammation. Following treatment cessation, recrudescence of viral replication was observed but with dose-dependent delays in viral relapse. The antiviral effects were associated with dose-dependent and long-lasting induction of IFN-α, IFN-β and IFN-stimulated genes in blood and liver, which correlated with the prolonged activation of the RIG-I/NOD2 pathway and hepatic presence of elevated RIG-I protein levels. These results suggest that in addition to a direct antiviral activity, SB 9200 induces antiviral immunity during chronic hepadnaviral infection via activation of the viral sensor pathway.

  2. Antiviral Efficacy and Host Innate Immunity Associated with SB 9200 Treatment in the Woodchuck Model of Chronic Hepatitis B

    PubMed Central

    Korolowicz, Kyle E.; Iyer, Radhakrishnan P.; Czerwinski, Stefanie; Suresh, Manasa; Yang, Junming; Padmanabhan, Seetharamaiyer; Sheri, Anjaneyulu; Pandey, Rajendra K.; Skell, Jeffrey; Marquis, Judith K.; Kallakury, Bhaskar V.; Tucker, Robin D.; Menne, Stephan

    2016-01-01

    SB 9200, an oral prodrug of the dinucleotide SB 9000, is being developed for the treatment of chronic hepatitis B virus (HBV) infection and represents a novel class of antivirals. SB 9200 is thought to activate the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) resulting in interferon (IFN) mediated antiviral immune responses in virus-infected cells. Additionally, the binding of SB 9200 to these sensor proteins could also sterically block the ability of the viral polymerase to access pre-genomic RNA for nucleic acid synthesis. The immune stimulating and direct antiviral properties of SB 9200 were evaluated in woodchucks chronically infected with woodchuck hepatitis virus (WHV) by daily, oral dosing at 15 and 30 mg/kg for 12 weeks. Prolonged treatment resulted in 2.2 and 3.7 log10 reductions in serum WHV DNA and in 0.5 and 1.6 log10 declines in serum WHV surface antigen from pretreatment level with the lower or higher dose of SB 9200, respectively. SB 9200 treatment also resulted in lower hepatic levels of WHV nucleic acids and antigen and reduced liver inflammation. Following treatment cessation, recrudescence of viral replication was observed but with dose-dependent delays in viral relapse. The antiviral effects were associated with dose-dependent and long-lasting induction of IFN-α, IFN-β and IFN-stimulated genes in blood and liver, which correlated with the prolonged activation of the RIG-I/NOD2 pathway and hepatic presence of elevated RIG-I protein levels. These results suggest that in addition to a direct antiviral activity, SB 9200 induces antiviral immunity during chronic hepadnaviral infection via activation of the viral sensor pathway. PMID:27552102

  3. Human gestation-associated tissues express functional cytosolic nucleic acid sensing pattern recognition receptors.

    PubMed

    Bryant, Aled H; Menzies, Georgina E; Scott, Louis M; Spencer-Harty, Samantha; Davies, Lleucu B; Smith, Rachel A; Jones, Ruth H; Thornton, Catherine A

    2017-03-13

    The role of viral infections in adverse pregnancy outcomes has gained interest in recent years. Innate immune pattern recognition receptors (PRRs) and their signalling pathways that yield a cytokine output in response to pathogenic stimuli have been postulated to link infection at the maternal-fetal interface and adverse pregnancy outcomes. The objective of this study was to investigate the expression and functional response of nucleic acid ligand responsive Toll-like receptors (TLR3, 7, 8 and 9), and retinoic acid-inducible gene 1 (RIG-I)-like receptors (RIG-I, MDA5 and LGP2) in human term gestation-associated tissues (placenta, choriodecidua and amnion) using an explant model. Immunohistochemistry revealed that these PRRs were expressed by the term placenta, choriodecidua and amnion. A statistically significant increase in interleukin (IL)-6 and/or IL-8 production in response to specific agonists for TLR3 (Poly(I:C); low and high molecular weight), TLR7 (Imiquimod), TLR8 (ssRNA40) and RIG-I/MDA5 (Poly(I:C)LyoVec) was observed; there was no response to a TLR9 (ODN21798) agonist. A hierarchical clustering approach was used to compare the response of each tissue type to the ligands studied and revealed that the placenta and choriodecidua generate a more similar IL-8 response, while the choriodecidua and amnion generate a more similar IL-6 response to nucleic acid ligands. These findings demonstrate that responsiveness via TLR3, TLR7, TLR8 and RIG-1/MDA5 is a broad feature of human term gestation-associated tissues with differential responses by tissue that might underpin adverse obstetric outcomes. This article is protected by copyright. All rights reserved.

  4. Innate Immunity Evasion by Enteroviruses: Insights into Virus-Host Interaction.

    PubMed

    Lei, Xiaobo; Xiao, Xia; Wang, Jianwei

    2016-01-15

    Enterovirus genus includes multiple important human pathogens, such as poliovirus, coxsackievirus, enterovirus (EV) A71, EV-D68 and rhinovirus. Infection with EVs can cause numerous clinical conditions including poliomyelitis, meningitis and encephalitis, hand-foot-and-mouth disease, acute flaccid paralysis, diarrhea, myocarditis and respiratory illness. EVs, which are positive-sense single-stranded RNA viruses, trigger activation of the host antiviral innate immune responses through pathogen recognition receptors such as retinoic acid-inducible gene (RIG-I)-likeand Toll-like receptors. In turn, EVs have developed sophisticated strategies to evade host antiviral responses. In this review, we discuss the interplay between the host innate immune responses and EV infection, with a primary focus on host immune detection and protection against EV infection and viral strategies to evade these antiviral immune responses.

  5. Organoselenium bis selenide attenuates 3-nitropropionic acid-induced neurotoxicity in rats.

    PubMed

    Bortolatto, Cristiani F; Jesse, Cristiano R; Wilhelm, Ethel A; Chagas, Pietro M; Nogueira, Cristina W

    2013-04-01

    This study was designed to evaluate the effects of bis selenide on Huntington disease (HD)-like signs induced by 3-nitropropionic acid (3-NP) in rats. To this aim, rats were treated for 4 days with bis selenide (5 or 20 mg/kg/day, per oral) 30 min before 3-NP (20 mg/kg/day, intraperitoneally). The body weight gain, locomotor activity, motor coordination, and biochemical parameters in striatal preparations were assessed 24 h after the last injection of 3-NP. The highest dose of bis selenide was effective in protecting against body weight loss and motor coordination deficit induced by 3-NP. The impairment of locomotor activity caused by 3-NP was abolished by bis selenide at both doses. Bis selenide (5 and 20 mg/kg) partially restored succinate dehydrogenase activity inhibited after 3-NP exposure. The dose of 20 mg/kg of bis selenide recovered partially δ-aminolevulinic acid dehydratase activity, and totally Na(+), K(+)-ATPase activity, two sulfhydryl enzymes sensitive to oxidizing agents, which had their activities inhibited by 3-NP. Also, 3-NP led to an increase in protein carbonyl levels and glutathione reductase activity and inhibited catalase activity-alterations that were reversed by bis selenide administration at both doses. The highest dose of bis selenide was effective against the increase of RS levels, the depletion of reduced glutathione content, and the inhibition of glutathione peroxidase activity induced by 3-NP. Bis selenide was not effective against inhibition of SOD activity caused by 3-NP. These findings demonstrate that bis selenide elicited protective effects against HD-like signs induced by 3-NP in rats.

  6. Rutin inhibits oleic acid induced lipid accumulation via reducing lipogenesis and oxidative stress in hepatocarcinoma cells.

    PubMed

    Wu, Cheng-Hsun; Lin, Ming-Cheng; Wang, Hsueh-Chun; Yang, Mon-Yuan; Jou, Ming-Jia; Wang, Chau-Jong

    2011-03-01

    Excessive lipid accumulation within liver has been proposed to cause obesity, hyperlipidemia, diabetes, and fatty liver disease. Rutin, a common dietary flavonoid that is consumed in fruits, vegetables, and plant-derived beverages, has various biological functions, including antioxidant, anti-inflammatory, and anticancer effects. However, a hypolipidemic effect of rutin on fatty liver disease has not been reported. In this study, we examined the effect of rutin on reducing lipid accumulation in hepatic cells. Hepatocytes were treated with oleic acid (OA) containing with or without rutin to observe the lipid accumulation by Nile red stain. The result showed rutin suppressed OA-induced lipid accumulation and increased adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activity in hepatocytes. The expression of critical molecule involved in lipid synthesis, sterol regulatory element binding proteins-1 (SREBP-1), was attenuated in rutin-treated cells. Moreover, long-term incubation of rutin inhibited the transcriptions of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR), glycerol-3-phosphate acyltransferase (GPAT), fatty acid synthase (FAS), and acetyl-coenzyme carboxylase (ACC). Besides, we also found out the antioxidative effect of rutin by increasing the expression of peroxisome proliferator-activated receptor (PPAR)-α and antioxidative enzymes. Taken together, our findings suggest rutin could attenuate lipid accumulation by decreasing lipogenesis and oxidative stress in hepatocyte.

  7. Nadroparin sodium activates Nrf2/HO-1 pathway in acetic acid-induced colitis in rats.

    PubMed

    Yalniz, Mehmet; Demirel, Ulvi; Orhan, Cemal; Bahcecioglu, Ibrahim Halil; Ozercan, Ibrahim Hanefi; Aygun, Cem; Tuzcu, Mehmet; Sahin, Kazim

    2012-06-01

    Effects of nadroparin sodium, a low molecular weight heparin, in colitis was investigated by analyzing proteins implicated in nuclear factor E2-related factor-2/heme oxygenase-1 (Nrf2/HO-1) and nuclear factor kappa B (NF-κB) pathways. Twenty-eight rats were used. Colitis was induced by acetic acid (AA). Nadroparin sodium was given to prevention and treatment groups in addition to AA. Colitis was assessed histologically and levels of proteins were analyzed with Western blot. Nadroparin not only prevented and ameliorated the AA-induced colitis histopathologically but also decreased expression of colon NF-κB, activator protein-1, cyclooxygenase-2, tumor necrosis factor-alpha, and IL-6, which were significantly increased in group AA compared to control. The accumulation of Nrf2 in nuclear fraction and HO-1 found low in group AA was increased with nadroparin (p < 0.05). The mean malondialdehyde level increased with AA and was decreased significantly with nadroparin prevention and treatment (p < 0.001). Nadroparin sodium has both protective and therapeutic effects against colonic inflammation via exerting anti-oxidative and anti-inflammatory effects by modulating Nrf2/HO-1 and NF-κB pathways.

  8. Lithocholic acid induces endoplasmic reticulum stress, autophagy and mitochondrial dysfunction in human prostate cancer cells

    PubMed Central

    Gafar, Ahmed A.; Draz, Hossam M.; Goldberg, Alexander A.; Bashandy, Mohamed A.; Bakry, Sayed; Khalifa, Mahmoud A.; AbuShair, Walid; Titorenko, Vladimir I.

    2016-01-01

    Lithocholic acid (LCA) is a secondary bile acid that is selectively toxic to human neuroblastoma, breast and prostate cancer cells, whilst sparing normal cells. We previously reported that LCA inhibited cell viability and proliferation and induced apoptosis and necrosis of androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cells. In the present study, we investigated the roles of endoplasmic reticulum (ER) stress, autophagy and mitochondrial dysfunction in the toxicity of LCA in PC-3 and autophagy deficient, androgen-independent DU-145 cells. LCA induced ER stress-related proteins, such as CCAAT-enhancer-binding protein homologous protein (CHOP), and the phosphorylation of eukaryotic initiation factor 2-alpha (p-eIF2α) and c-Jun N-terminal kinases (p-JNK) in both cancer cell-types. The p53 upregulated modulator of apoptosis (PUMA) and B cell lymphoma-like protein 11 (BIM) levels were decreased at overtly toxic LCA concentrations, although PUMA levels increased at lower LCA concentrations in both cell lines. LCA induced autophagy-related conversion of microtubule-associated proteins 1A/1B light chain 3B (LC3BI–LC3BII), and autophagy-related protein ATG5 in PC-3 cells, but not in autophagy-deficient DU-145 cells. LCA (>10 µM) increased levels of reactive oxygen species (ROS) concentration-dependently in PC-3 cells, whereas ROS levels were not affected in DU-145 cells. Salubrinal, an inhibitor of eIF2α dephosphorylation and ER stress, reduced LCA-induced CHOP levels slightly in PC-3, but not DU-145 cells. Salubrinal pre-treatment increased the cytotoxicity of LCA in PC-3 and DU-145 cells and resulted in a statistically significant loss of cell viability at normally non-toxic concentrations of LCA. The late-stage autophagy inhibitor bafilomycin A1 exacerbated LCA toxicity at subtoxic LCA concentrations in PC-3 cells. The antioxidant α-tocotrienol strongly inhibited the toxicity of LCA in PC-3 cells, but not in DU-145 cells. Collectively, although LCA induces autophagy and ER stress in PC-3 cells, these processes appear to be initially of protective nature and subsequently consequential to, but not critical for the ROS-mediated mitochondrial dysfunction and cytotoxicity of LCA. The full mechanism of LCA-induced mitochondrial dysfunction and cytotoxicity in the similarly sensitive DU-145 cells remains to be elucidated. PMID:27896021

  9. Dissecting the beta-aminobutyric acid-induced priming phenomenon in Arabidopsis.

    PubMed

    Ton, Jurriaan; Jakab, Gabor; Toquin, Valérie; Flors, Victor; Iavicoli, Annalisa; Maeder, Muriel N; Métraux, Jean-Pierre; Mauch-Mani, Brigitte

    2005-03-01

    Plants treated with the nonprotein amino acid beta-aminobutyric acid (BABA) develop an enhanced capacity to resist biotic and abiotic stresses. This BABA-induced resistance (BABA-IR) is associated with an augmented capacity to express basal defense responses, a phenomenon known as priming. Based on the observation that high amounts of BABA induce sterility in Arabidopsis thaliana, a mutagenesis screen was performed to select mutants impaired in BABA-induced sterility (ibs). Here, we report the isolation and subsequent characterization of three T-DNA-tagged ibs mutants. Mutant ibs1 is affected in a cyclin-dependent kinase-like protein, and ibs2 is defective in AtSAC1b encoding a polyphosphoinositide phosphatase. Mutant ibs3 is affected in the regulation of the ABA1 gene encoding the abscisic acid (ABA) biosynthetic enzyme zeaxanthin epoxidase. To elucidate the function of the three IBS genes in plant resistance, the mutants were tested for BABA-IR against the bacterium Pseudomonas syringae pv tomato, the oomycete Hyaloperonospora parasitica, and BABA-induced tolerance to salt. All three ibs mutants were compromised in BABA-IR against H. parasitica, although to a different extent. Whereas ibs1 was reduced in priming for salicylate (SA)-dependent trailing necrosis, mutants ibs2 and ibs3 were affected in the priming for callose deposition. Only ibs1 failed to express BABA-IR against P. syringae, which coincided with a defect in priming for SA-inducible PR-1 gene expression. By contrast, ibs2 and ibs3 showed reduced BABA-induced tolerance to salt, which correlated with an affected priming for ABA-inducible gene expression. For all three ibs alleles, the defects in BABA-induced sterility and BABA-induced protection against P. syringae, H. parasitica, and salt could be confirmed in independent mutants. The data presented here introduce three novel regulatory genes involved in priming for different defense responses.

  10. Mechanisms of Motility Change on Trinitrobenzenesulfonic Acid-Induced Colonic Inflammation in Mice

    PubMed Central

    Cheon, Gab Jin; Cui, Yuan; Yeon, Dong-Soo; Kwon, Seong-Chun

    2012-01-01

    Ulcerative colitis is an inflammatory bowel disease (IBD) characterized by recurrent episodes of colonic inflammation and tissue degeneration in human or animal models. The contractile force generated by the smooth muscle is significantly attenuated, resulting in altered motility leading to diarrhea or constipation in IBD. The aim of this study is to clarify the altered contractility of circular and longitudinal smooth muscle layers in proximal colon of trinitrobenzen sulfonic acid (TNBS)-induced colitis mouse. Colitis was induced by direct injection of TNBS (120 mg/kg, 50% ethanol) in proximal colon of ICR mouse using a 30 G needle anesthetized with ketamin (50 mg/kg), whereas animals in the control group were injected of 50% ethanol alone. In TNBS-induced colitis, the wall of the proximal colon is diffusely thickened with loss of haustration, and showed mucosal and mucular edema with inflammatory infiltration. The colonic inflammation is significantly induced the reduction of colonic contractile activity including spontaneous contractile activity, depolarization-induced contractility, and muscarinic acetylcholine receptor-mediated contractile response in circular muscle layer compared to the longitudinal muscle layer. The inward rectification of currents, especially, important to Ca2+ and Na+ influx-induced depolarization and contraction, was markedly reduced in the TNBS-induced colitis compared to the control. The muscarinic acetylcholine-mediated contractile responses were significantly attenuated in the circular and longitudinal smooth muscle strips induced by the reduction of membrane expression of canonical transient receptor potential (TRPC) channel isoforms from the proximal colon of the TNBS-induced colitis mouse than the control. PMID:23269907

  11. Hippocampal neuropathology of domoic acid-induced epilepsy in California sea lions (Zalophus californianus).

    PubMed

    Buckmaster, Paul S; Wen, Xiling; Toyoda, Izumi; Gulland, Frances M D; Van Bonn, William

    2014-05-01

    California sea lions (Zalophus californianus) are abundant human-sized carnivores with large gyrencephalic brains. They develop epilepsy after experiencing status epilepticus when naturally exposed to domoic acid. We tested whether sea lions previously exposed to DA (chronic DA sea lions) display hippocampal neuropathology similar to that of human patients with temporal lobe epilepsy. Hippocampi were obtained from control and chronic DA sea lions. Stereology was used to estimate numbers of Nissl-stained neurons per hippocampus in the granule cell layer, hilus, and pyramidal cell layer of CA3, CA2, and CA1 subfields. Adjacent sections were processed for somatostatin immunoreactivity or Timm-stained, and the extent of mossy fiber sprouting was measured stereologically. Chronic DA sea lions displayed hippocampal neuron loss in patterns and extents similar but not identical to those reported previously for human patients with temporal lobe epilepsy. Similar to human patients, hippocampal sclerosis in sea lions was unilateral in 79% of cases, mossy fiber sprouting was a common neuropathological abnormality, and somatostatin-immunoreactive axons were exuberant in the dentate gyrus despite loss of immunopositive hilar neurons. Thus, hippocampal neuropathology of chronic DA sea lions is similar to that of human patients with temporal lobe epilepsy.

  12. Inhibition of retinoic acid-induced skin irritation in calorie-restricted mice.

    PubMed

    Varani, James; Bhagavathula, Narasimharao; Aslam, Muhammad Nadeem; Fay, Kevin; Warner, Roscoe L; Hanosh, Andrew; Barron, Adam G; Miller, Richard A

    2008-01-01

    Mice on a calorie-restricted (CR) diet (total calories restricted to 70% of ad libitum; AL) for periods of time ranging from 3 to 18 months were examined for response to topical treatment with all-trans retinoic acid (RA). Daily application of a 0.1% solution of RA to the shaved skin of UM-HET3 mice on an AL diet produced a severe irritation that was evident by day 4, maximal at day 7-8 and still detectable at day 14. Skin irritation was characterized by redness, dryness, flaking and failure of the hair to grow at the treated site. In CR mice, the same treatment produced little detectable irritation. Animals were sacrificed at the end of the retinoid-treatment period (day 7 or day 14) and skin from these animals was examined histologically. In both AL and CR mice, a similar degree of epidermal hyperplasia was observed. Numerous inflammatory cells (mononuclear cells and granulocytes) were present in the skin of both groups. Occasional S100-positive cells (presumably Langerhans cells) were also observed in the epidermis of skin from both groups. S100-positive cells were also observed in the dermis. When skin from CR and AL mice was incubated in organ culture for 3 days (on day 7 after initiation of RA treatment), similar levels of four different pro-inflammatory cytokines were found in the conditioned medium. Soluble type I collagen levels were also similar. In contrast, the level of matrix metalloproteinase-9 was lower in the conditioned medium of skin from CR mice than in conditioned medium from skin cultures of AL mice. Taken together, these studies suggest that CR may provide a way to mitigate the irritation that normally accompanies RA treatment without compromising the beneficial effects of retinoid use. CR appears to exert a protective effect at the target tissue level rather than by a reduction in pro-inflammatory events, per se.

  13. Myrrh attenuates oxidative and inflammatory processes in acetic acid-induced ulcerative colitis

    PubMed Central

    Fatani, Amal Jamil; Alrojayee, Fatima Salih; Parmar, Mihir Yogeshkumar; Abuohashish, Hatem Mustafa; Ahmed, Mohammed Mahboobuddin; Al-Rejaie, Salim Salih

    2016-01-01

    The pathogenesis of ulcerative colitis (UC) has been associated with a weakened antioxidant capacity and increased inflammatory processes. Myrrh is traditionally used for the treatment of inflammatory diseases due to its antioxidant and anti-inflammatory properties. The present study aimed to evaluate the effects of myrrh on an experimental rat model of UC. UC was induced in rats using acetic acid (AA) after pre-treatment with myrrh (125, 250 or 500 mg/kg/day) or mesalazine (MES; 300 mg/kg/day) for 7 days. The levels of various inflammatory cytokines, prostaglandin E2 (PGE2) and nitric oxide (NO) in the rat colon tissues were assessed. In addition, the colonic levels of thiobarbituric acid reactive substances (TBARS) and non-protein sulfhydryl groups (NP-SH), as well as the activities of superoxide dismutase (SOD) and catalase (CAT), were estimated. Furthermore, total protein (TP) contents and the levels of DNA and RNA were measured, and histopathological changes in colonic tissues were analyzed. The results indicated that the levels of pro-inflammatory cytokines, PGE2, NO and TBARS were markedly increased. By contrast, the levels of interleukin-10, NP-SH, TP and nucleic acids, and the enzymatic activities of SOD and CAT were significantly decreased in the AA model group. In addition, pretreatment with myrrh and MES was able to attenuate the impaired oxidative stress response and upregulation of inflammatory biomarkers. Furthermore, the enzymatic activities of SOD and CAT were near to normal in the myrrh and MES pretreated groups. The ability of myrrh to protect against UC was further confirmed by histopathological analysis, and the high dose of myrrh exerted an effect comparable to MES. In conclusion, the results of the present study suggested that myrrh has potent therapeutic value in the amelioration of experimental colitis in laboratory animals by downregulating the expression of proinflammatory mediators and improving endogenous antioxidative activities. PMID:27446268

  14. Neuroprotective effect of caffeic acid phenethyl ester in 3-nitropropionic acid-induced striatal neurotoxicity

    PubMed Central

    Bak, Jia; Kim, Hee Jung; Kim, Seong Yun

    2016-01-01

    Caffeic acid phenethyl ester (CAPE), derived from honeybee hives, is a bioactive compound with strong antioxidant activity. This study was designed to test the neuroprotective effect of CAPE in 3-nitropropionic acid (3NP)-induced striatal neurotoxicity, a chemical model of Huntington's disease (HD). Initially, to test CAPE's antioxidant activity, a 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) antioxidant assay was employed, and CAPE showed a strong direct radical-scavenging eff ect. In addition, CAPE provided protection from 3NP-induced neuronal cell death in cultured striatal neurons. Based on these observations, the in vivo therapeutic potential of CAPE in 3NP-induced HD was tested. For this purpose, male C57BL/6 mice were repeatedly given 3NP to induce HD-like pathogenesis, and 30 mg/kg of CAPE or vehicle (5% dimethyl sulfoxide and 95% peanut oil) was administered daily. CAPE did not cause changes in body weight, but it reduced mortality by 29%. In addition, compared to the vehicle-treated group, robustly reduced striatal damage was observed in the CAPE-treated animals, and the 3NP-induced behavioral defi cits on the rotarod test were signifi cantly rescued after the CAPE treatment. Furthermore, immunohistochemical data showed that immunoreactivity to glial fibrillary acidic protein (GFAP) and CD45, markers for astrocyte and microglia activation, respectively, were strikingly reduced. Combined, these data unequivocally indicate that CAPE has a strong antioxidant eff ect and can be used as a potential therapeutic agent against HD. PMID:27162482

  15. Role of apoptosis in mediating salicylic acid-induced teratogenesis in vitro.

    PubMed

    Singh, Gyanendra; Sinha, Neeraj; Mahipag G, S N R

    2009-02-01

    Salicylic acid (SAL) is among the most commonly used drugs worldwide and is known to cause congenital malformations and fetal death in animals. In this study, the effect of SAL on major organogenesis period and the role of apoptosis in mediating congenital malformations have been carried out. In the present study, post-implantation rat embryos of day 11 were cultured for 24 h with various concentrations of SAL, i.e. 10, 100, and 1000 microg/ml cultures, respectively. The growth and developmental of each embryo was evaluated and compared with control ones for the presence of any malformations. The SAL decreased all growth and developmental parameters in a concentration-dependent manner, when compared with control. However, exposure to SAL at 10 microg/ml culture did not show any significant effect on embryonic growth and development. Parallel to this, flow cytometric analysis (cell cycle and annexin V binding) and DNA fragmentation assay were carried out followed by quantitation by 3'-OH labeling of cultured rat embryos to evaluate the role of apoptosis in bringing about SAL-induced teratogenesis. All results were found to be dose-dependent and an increase in apoptosis in embryonic tissues may be related to the increased risk of congenital malformations. The data suggested that apoptosis might be involved in mediating teratogenesis of SAL in vitro.

  16. All-trans retinoic acid induces oxidative phosphorylation and mitochondria biogenesis in adipocytes[S

    PubMed Central

    Tourniaire, Franck; Musinovic, Hana; Gouranton, Erwan; Astier, Julien; Marcotorchino, Julie; Arreguin, Andrea; Bernot, Denis; Palou, Andreu; Bonet, M. Luisa; Ribot, Joan; Landrier, Jean-François

    2015-01-01

    A positive effect of all-trans retinoic acid (ATRA) on white adipose tissue (WAT) oxidative and thermogenic capacity has been described and linked to an in vivo fat-lowering effect of ATRA in mice. However, little is known about the effects of ATRA on mitochondria in white fat. Our objective has been to characterize the effect of ATRA on mitochondria biogenesis and oxidative phosphorylation (OXPHOS) capacity in mature white adipocytes. Transcriptome analysis, oxygraphy, analysis of mitochondrial DNA (mtDNA), and flow cytometry-based analysis of mitochondria density were performed in mature 3T3-L1 adipocytes after 24 h incubation with ATRA (2 µM) or vehicle. Selected genes linked to mitochondria biogenesis and function and mitochondria immunostaining were analyzed in WAT tissues of ATRA-treated as compared with vehicle-treated mice. ATRA upregulated the expression of a large set of genes linked to mtDNA replication and transcription, mitochondrial biogenesis, and OXPHOS in adipocytes, as indicated by transcriptome analysis. Oxygen consumption rate, mtDNA content, and staining of mitochondria were increased in the ATRA-treated adipocytes. Similar results were obtained in WAT depots of ATRA-treated mice. We conclude that ATRA impacts mitochondria in adipocytes, leading to increased OXPHOS capacity and mitochondrial content in these cells. PMID:25914170

  17. Valproic acid induces apoptosis and cell cycle arrest in poorly differentiated thyroid cancer cells.

    PubMed

    Catalano, Maria G; Fortunati, Nicoletta; Pugliese, Mariateresa; Costantino, Lucia; Poli, Roberta; Bosco, Ornella; Boccuzzi, Giuseppe

    2005-03-01

    Poorly differentiated thyroid carcinoma is an aggressive human cancer that is resistant to conventional therapy. Histone deacetylase inhibitors are a promising class of drugs, acting as antiproliferative agents by promoting differentiation, as well as inducing apoptosis and cell cycle arrest. Valproic acid (VPA), a class I selective histone deacetylase inhibitor widely used as an anticonvulsant, promotes differentiation in poorly differentiated thyroid cancer cells by inducing Na(+)/I(-) symporter and increasing iodine uptake. Here, we show that it is also highly effective at suppressing growth in poorly differentiated thyroid cancer cell lines (N-PA and BHT-101). Apoptosis induction and cell cycle arrest are the underlying mechanisms of VPA's effect on cell growth. It induces apoptosis by activating the intrinsic pathway; caspases 3 and 9 are activated but not caspase 8. Cell cycle is selectively arrested in G(1) and is associated with the increased expression of p21 and the reduced expression of cyclin A. Both apoptosis and cell cycle arrest are induced by treatment with 1 mm VPA, a dose that promotes cell redifferentiation and that is slightly above the serum concentration reached in patients treated for epilepsy. These multifaceted properties make VPA of clinical interest as a new approach to treating poorly differentiated thyroid cancer.

  18. NDRG1 contributes to retinoic acid-induced differentiation of leukemic cells.

    PubMed

    Chen, Su; Han, Yu-Hui; Zheng, Ying; Zhao, Meng; Yan, Hua; Zhao, Qiao; Chen, Guo-Qiang; Li, Dao

    2009-08-01

    N-Myc downstream-regulated gene 1 (NDRG1) protein has been shown to be up-regulated during leukemic cell differentiation induced by some differentiation-inducing agents such as all-trans retinoic acid (ATRA). However, the potential role of up-regulated NDRG1 in the event is greatly unknown. In this work, we show that inducible NDRG1 expression can drive leukemic U937 cells to undergo differentiation, while the knock-down of NDRG1 expression by specific small interfering RNA significantly antagonizes ATRA-induced differentiation of leukemic cells, proposing the role of NDRG1 in leukemic cell differentiation. Furthermore, our work shows that CCAAT/enhancer-binding protein beta (C/EBPbeta) and PU.1, which are important hematopoiesis-related transcription factors, may act as downstream effectors of NDRG1 in leukemic cell differentiation. Taking together, this study provides direct evidence for the role of NDRG1 protein in myeloid leukemic cell differentiation.

  19. Kainic Acid-Induced Excitotoxicity Experimental Model: Protective Merits of Natural Products and Plant Extracts

    PubMed Central

    Mohd Sairazi, Nur Shafika; Sirajudeen, K. N. S.; Asari, Mohd Asnizam; Muzaimi, Mustapha; Mummedy, Swamy; Sulaiman, Siti Amrah

    2015-01-01

    Excitotoxicity is well recognized as a major pathological process of neuronal death in neurodegenerative diseases involving the central nervous system (CNS). In the animal models of neurodegeneration, excitotoxicity is commonly induced experimentally by chemical convulsants, particularly kainic acid (KA). KA-induced excitotoxicity in rodent models has been shown to result in seizures, behavioral changes, oxidative stress, glial activation, inflammatory mediator production, endoplasmic reticulum stress, mitochondrial dysfunction, and selective neurodegeneration in the brain upon KA administration. Recently, there is an emerging trend to search for natural sources to combat against excitotoxicity-associated neurodegenerative diseases. Natural products and plant extracts had attracted a considerable amount of attention because of their reported beneficial effects on the CNS, particularly their neuroprotective effect against excitotoxicity. They provide significant reduction and/or protection against the development and progression of acute and chronic neurodegeneration. This indicates that natural products and plants extracts may be useful in protecting against excitotoxicity-associated neurodegeneration. Thus, targeting of multiple pathways simultaneously may be the strategy to maximize the neuroprotection effect. This review summarizes the mechanisms involved in KA-induced excitotoxicity and attempts to collate the various researches related to the protective effect of natural products and plant extracts in the KA model of neurodegeneration. PMID:26793262

  20. Do pH and flavonoids influence hypochlorous acid-induced catalase inhibition and heme modification?

    PubMed

    Krych-Madej, Justyna; Gebicka, Lidia

    2015-09-01

    Hypochlorous acid (HOCl), highly reactive oxidizing and chlorinating species, is formed in the immune response to invading pathogens by the reaction of hydrogen peroxide with chloride catalyzed by the enzyme myeloperoxidase. Catalase, an important antioxidant enzyme, catalyzing decomposition of hydrogen peroxide to water and molecular oxygen, hampers in vitro HOCl formation, but is also one of the main targets for HOCl. In this work we have investigated HOCl-induced catalase inhibition at different pH, and the influence of flavonoids (catechin, epigallocatechin gallate and quercetin) on this process. It has been shown that HOCl-induced catalase inhibition is independent on pH in the range 6.0-7.4. Preincubation of catalase with epigallocatechin gallate and quercetin before HOCl treatment enhances the degree of catalase inhibition, whereas catechin does not affect this process. Our rapid kinetic measurements of absorption changes around the heme group have revealed that heme modification by HOCl is mainly due to secondary, intramolecular processes. The presence of flavonoids, which reduce active catalase intermediate, Compound I to inactive Compound II have not influenced the kinetics of HOCl-induced heme modification. Possible mechanisms of the reaction of hypochlorous acid with catalase are proposed and the biological consequences are discussed.

  1. Apigenin reduce lipoteichoic acid-induced inflammatory response in rat cardiomyoblast cells.

    PubMed

    Gutiérrez-Venegas, Gloria; González-Rosas, Zeltzin

    2017-02-01

    Infective endocarditis is caused by Streptococcus sanguinis present in dental plaque, which can induce inflammatory responses in the endocardium. The present study depicts research on the properties of apigenin in embryonic mouse heart cells (H9c2) treated with lipoteichoic acid (LTA) obtained from S. sanguinis. Interleukin-1β and cyclooxygenase (COX)-2 expression were detected by reverse transcriptase polymerase chain reaction. In addition, western blot assays and immuno-fluorescence staining were used to assess translocation of nuclear factor kappa beta (NF-κB), degradation of IκB, as well as activity of the mitogen activated protein kinases: extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK). Effect of apigenin on cell viability was equally assessed in other experimental series. Our results showed that apigenin blocked activation of ERK, JNK, and p38 in cardiomyocytes treated with LTA in a dose-dependent fashion. Moreover, apigenin showed no cytotoxic effects; it blocked NF-κB translocation and IκB degradation. Our findings suggested that apigenin possessed potential value in the treatment of infectious endocarditis.

  2. Myricetin blocks lipoteichoic acid-induced COX-2 expression in human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Luna, Oscar Alonso; Arreguín-Cano, Juan Antonio; Hernández-Bermúdez, Cristina

    2014-03-01

    Periodontitis is an infectious disease caused by microorganisms present in dental bacterial plaque. Lipoteichoic acid (LTA) is a component of the external membrane of Gram-positive bacteria. It causes septic shock. Ingested flavonoids have been reported to directly affect the regulation of cyclooxygenase-2 (COX-2) expression induced by bacterial toxins. In this study, we examined the effects of four flavonoids (luteolin, fisetin, morin and myricetin) on the activation of ERK1/2, p38 and AKT, and on the synthesis of COX-2 in human gingival fibroblasts treated with LTA from Streptococcus sanguinis. We found that luteolin and myricetin blocked AKT and p38 activation and that myricetin blocked LTA-induced COX-2 expression. The results of our study are important for elucidating the mechanism of action of flavonoid regulation of inflammatory responses.

  3. MicroRNA gene expression during retinoic acid-induced differentiation of human acute promyelocytic leukemia.

    PubMed

    Garzon, R; Pichiorri, F; Palumbo, T; Visentini, M; Aqeilan, R; Cimmino, A; Wang, H; Sun, H; Volinia, S; Alder, H; Calin, G A; Liu, C-G; Andreeff, M; Croce, C M

    2007-06-14

    MicroRNAs (miRNAs) are small non-coding RNAs of 19-25 nucleotides that are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. However, little is known about the role of miRNAs in granulopoiesis. Here, we report the expression of miRNAs in acute promyelocytic leukemia patients and cell lines during all-trans-retinoic acid (ATRA) treatment by using a miRNA microarrays platform and quantitative real time-polymerase chain reaction (qRT-PCR). We found upregulation of miR-15a, miR-15b, miR-16-1, let-7a-3, let-7c, let-7d, miR-223, miR-342 and miR-107, whereas miR-181b was downregulated. Among the upregulated miRNAs, miR-107 is predicted to target NFI-A, a gene that has been involved in a regulatory loop involving miR-223 and C/EBPa during granulocytic differentiation. Indeed, we have confirmed that miR-107 targets NF1-A. To get insights about ATRA regulation of miRNAs, we searched for ATRA-modulated transcription factors binding sites in the upstream genomic region of the let-7a-3/let-7b cluster and identified several putative nuclear factor-kappa B (NF-kappaB) consensus elements. The use of reporter gene assays, chromatin immunoprecipitation and site-directed mutagenesis revealed that one proximal NF-kappaB binding site is essential for the transactivation of the let-7a-3/let-7b cluster. Finally, we show that ATRA downregulation of RAS and Bcl2 correlate with the activation of known miRNA regulators of those proteins, let-7a and miR-15a/miR-16-1, respectively.

  4. Lipid Rafts Promote trans Fatty Acid-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

    PubMed

    Pan, Yao; Liu, Benxin; Deng, Zeyuan; Fan, Yawei; Li, Jing; Li, Hongyan

    2017-01-01

    The effects of two fatty acids, oleic acid (OLA) and elaidic acid (ELA) on normal human umbilical vein endothelial cells (HUVEC) and non-rafts HUVEC were investigated in this study. The expression levels of inflammatory cytokines (ICAM-1, VCAM-1 and IL-6) were analyzed. Western blot was used to analyze the expression levels of inflammation-related proteins (NF-κB, ERK1/2) and toll-like receptors 4 (TLR4). The results showed that the levels of nuclear translocation of NF-κB p65 and phosphorylated ERK1/2 were significantly decreased only in non-lipid rafts cells pretreated with trans fatty acid (TFA). The expression of TLR4 in the ELA-treated normal cells was higher than that in non-lipid rafts HUVEC. When the lipid rafts was destroyed by methyl-β-cyclodextrin, the levels of nuclear translocation of NF-κB p65, phosphorylated ERK1/2 and TLR4 were decreased significantly. Therefore, lipid rafts may be involved in TFA induced-inflammation in HUVEC through blocking the inflammatory signal pathway. Lipid rafts might be a platform for specific receptors such as TLR4 for TFA to activate the pro-inflammation on cell membranes.

  5. Oleanolic Acid Induces the Type III Secretion System of Ralstonia solanacearum

    PubMed Central

    Wu, Dousheng; Ding, Wei; Zhang, Yong; Liu, Xuejiao; Yang, Liang

    2015-01-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, can naturally infect a wide range of host plants. The type III secretion system (T3SS) is a major virulence determinant in this bacterium. Studies have shown that plant-derived compounds are able to inhibit or induce the T3SS in some plant pathogenic bacteria, though no specific T3SS inhibitor or inducer has yet been identified in R. solanacearum. In this study, a total of 50 different compounds were screened and almost half of them (22 of 50) significantly inhibited or induced the T3SS expression of R. solanacearum. Based on the strong induction activity on T3SS, the T3SS inducer oleanolic acid (OA) was chosen for further study. We found that OA induced the expression of T3SS through the HrpG-HrpB pathway. Some type III effector genes were induced in T3SS inducing medium supplemented with OA. In addition, OA targeted only the T3SS and did not affect other virulence determinants. Finally, we observed that induction of T3SS by OA accelerated disease progress on tobacco. Overall our results suggest that plant-derived compounds are an abundant source of R. solanacearum T3SS regulators, which could prove useful as tools to interrogate the regulation of this key virulence pathway. PMID:26732647

  6. Costs of jasmonic acid induced defense in aboveground and belowground parts of corn (Zea mays L.).

    PubMed

    Feng, Yuanjiao; Wang, Jianwu; Luo, Shiming; Fan, Huizhi; Jin, Qiong

    2012-08-01

    Costs of jasmonic acid (JA) induced plant defense have gained increasing attention. In this study, JA was applied continuously to the aboveground (AG) or belowground (BG) parts, or AG plus BG parts of corn (Zea mays L.) to investigate whether JA exposure in one part of the plant would affect defense responses in another part, and whether or not JA induced defense would incur allocation costs. The results indicated that continuous JA application to AG parts systemically affected the quantities of defense chemicals in the roots, and vice versa. Quantities of DIMBOA and total amounts of phenolic compounds in leaves or roots generally increased 2 or 4 wk after the JA treatment to different plant parts. In the first 2 wk after application, the increase of defense chemicals in leaves and roots was accompanied by a significant decrease of root length, root surface area, and root biomass. Four weeks after the JA application, however, no such costs for the increase of defense chemicals in leaves and roots were detected. Instead, shoot biomass and root biomass increased. The results suggest that JA as a defense signal can be transferred from AG parts to BG parts of corn, and vice versa. Costs for induced defense elicited by continuous JA application were found in the early 2 wk, while distinct benefits were observed later, i.e., 4 wk after JA treatment.

  7. Dietary linoleic acid-induced alterations in pro- and anti-nociceptive lipid autacoids

    PubMed Central

    Ringel, Amit; Majchrzak-Hong, Sharon F; Yang, Jun; Blanchard, Helene; Zamora, Daisy; Loewke, James D; Rapoport, Stanley I; Hibbeln, Joseph R; Davis, John M; Hammock, Bruce D; Taha, Ameer Y

    2016-01-01

    Background Chronic idiopathic pain syndromes are major causes of personal suffering, disability, and societal expense. Dietary n-6 linoleic acid has increased markedly in modern industrialized populations over the past century. These high amounts of linoleic acid could hypothetically predispose to physical pain by increasing the production of pro-nociceptive linoleic acid-derived lipid autacoids and by interfering with the production of anti-nociceptive lipid autacoids derived from n-3 fatty acids. Here, we used a rat model to determine the effect of increasing dietary linoleic acid as a controlled variable for 15 weeks on nociceptive lipid autacoids and their precursor n-6 and n-3 fatty acids in tissues associated with idiopathic pain syndromes. Results Increasing dietary linoleic acid markedly increased the abundance of linoleic acid and its pro-nociceptive derivatives and reduced the abundance of n-3 eicosapentaenoic acid and docosahexaenoic acid and their anti-nociceptive monoepoxide derivatives. Diet-induced changes occurred in a tissue-specific manner, with marked alterations of nociceptive lipid autacoids in both peripheral and central tissues, and the most pronounced changes in their fatty acid precursors in peripheral tissues. Conclusions The present findings provide biochemical support for the hypothesis that the high linoleic acid content of modern industrialized diets may create a biochemical susceptibility to develop chronic pain. Dietary linoleic acid lowering should be further investigated as part of an integrative strategy for the prevention and management of idiopathic pain syndromes. PMID:27030719

  8. Uncoupling protein 2 regulates palmitic acid-induced hepatoma cell autophagy.

    PubMed

    Lou, Jiaxin; Wang, Yunjiao; Wang, Xuejiang; Jiang, Ying

    2014-01-01

    Mitochondrial uncoupling protein 2 (UCP2) is suggested to have a role in the development of nonalcoholic steatohepatitis (NASH). However, the mechanism remains unclear. Autophagy is an important mediator of many pathological responses. This study aims to investigate the relationship between UCP2 and hepatoma cells autophagy in palmitic acid- (PA-) induced lipotoxicity. H4IIE cells were treated with palmitic acid (PA), and cell autophagy and apoptosis were examined. UCP2 expression, in association with LC3-II and caspase-3, which are indicators of cell autophagy and apoptosis, respectively,was measured. Results demonstrated that UCP2 was associated with autophagy during PA-induced hepatic carcinoma cells injury. Tests on reactive oxygen species (ROS) showed that UCP2 overexpression strongly decreases PA-induced ROS production and apoptosis. Conversely, UCP2 inhibition by genipin or UCP2 mRNA silencing enhances PA-induced ROS production and apoptosis. Autophagy partially participates in this progress. Moreover, UCP2 was associated with ATP synthesis during PA-induced autophagy. In conclusion, increasing UCP2 expression in hepatoma cells may contribute to cell autophagy and antiapoptotic as result of fatty acid injury. Our results may bring new insights for potential NASH therapies.

  9. Acid-induced gene expression in Helicobacter pylori: study in genomic scale by microarray.

    PubMed

    Ang, S; Lee, C Z; Peck, K; Sindici, M; Matrubutham, U; Gleeson, M A; Wang, J T

    2001-03-01

    To understand the RNA expression in response to acid stress of Helicobacter pylori in genomic scale, a microarray membrane containing 1,534 open reading frames (ORFs) from strain 26695 was used. Total RNAs of H. pylori under growth conditions of pH 7.2 and 5.5 were extracted, reverse transcribed into cDNA, and labeled with biotin. Each microarray membrane was hybridized with cDNA probe from the same strain under two different pH conditions and developed by a catalyzed reporter deposition method. Gene expression of all ORFs was measured by densitometry. Among the 1,534 ORFs, 53 ORFs were highly expressed (> or = 30% of rRNA control in densitometry ratios). There were 445 ORFs which were stably expressed (<30% of rRNA in densitometry) under both pH conditions without significant variation. A total of 80 ORFs had significantly increased expression levels at low pH, while expressions of 4 ORFs were suppressed under acidic condition. The remaining 952 ORFs were not detectable under either pH condition. These data were highly reproducible and comparable to those obtained by the RNA slot blot method. Our results suggest that microarray can be used in monitoring prokaryotic gene expression in genomic scale.

  10. Salicylic acid-induced abiotic stress tolerance and underlying mechanisms in plants

    PubMed Central

    Khan, M. Iqbal R.; Fatma, Mehar; Per, Tasir S.; Anjum, Naser A.; Khan, Nafees A.

    2015-01-01

    Abiotic stresses (such as metals/metalloids, salinity, ozone, UV-B radiation, extreme temperatures, and drought) are among the most challenging threats to agricultural system and economic yield of crop plants. These stresses (in isolation and/or combination) induce numerous adverse effects in plants, impair biochemical/physiological and molecular processes, and eventually cause severe reductions in plant growth, development and overall productivity. Phytohormones have been recognized as a strong tool for sustainably alleviating adverse effects of abiotic stresses in crop plants. In particular, the significance of salicylic acid (SA) has been increasingly recognized in improved plant abiotic stress-tolerance via SA-mediated control of major plant-metabolic processes. However, the basic biochemical/physiological and molecular mechanisms that potentially underpin SA-induced plant-tolerance to major abiotic stresses remain least discussed. Based on recent reports, this paper: (a) overviews historical background and biosynthesis of SA under both optimal and stressful environments in plants; (b) critically appraises the role of SA in plants exposed to major abiotic stresses; (c) cross-talks potential mechanisms potentially governing SA-induced plant abiotic stress-tolerance; and finally (d) briefly highlights major aspects so far unexplored in the current context. PMID:26175738

  11. Organic acids induce tolerance to zinc- and copper-exposed fungi under various growth conditions.

    PubMed

    Sazanova, Katerina; Osmolovskaya, Natalia; Schiparev, Sergey; Yakkonen, Kirill; Kuchaeva, Ludmila; Vlasov, Dmitry

    2015-04-01

    Heavy metals, Zn and Cu, in high concentration (2 mM for Zn and 0.5 mM for Cu) have some inhibiting effect on the growth of Aspergillus niger and Penicillium citrinum. Toxic effects of these metals considerably depend on cultivation conditions including nitrogen sources, pH of nutrient media, and its consistency (presence or absence of agar). In general, nitrate media provides less inhibiting effect on fungal growth under heavy metal exposure than ammonium-containing media. Adding of Zn in nitrate media induces oxalic acid production by fungi. Importance of oxalic acid production in detoxification of heavy metals is confirmed by the formation of Zn-containing crystals in fungal cultures. Cu bringing to the cultural media had no stimulating effect on oxalic acid production as well as no copper-containing crystals were observed. But proceeding from essential increase in oxalic acid production during a long-term fungi adaptation to Cu, it may be proposed that oxalic acid plays some functional role in Cu tolerance of fungi as well. It may be concluded that the role of organic acids and oxalate, in particular, in fungi tolerance and adaptation to heavy metals can be determined by the nature of the metal and its ability to form stable complexes with an acid anion. Stimulating effect of metals on acid production is not universal for all species of fungi and largely depends on metal concentration, nitrogen form in a medium, and other cultivation conditions.

  12. Identification of okadaic acid-induced phosphorylation events by a mass spectrometry approach

    SciTech Connect

    Hill, Jennifer J. . E-mail: Jennifer.Hill@nrc.gc.ca; Callaghan, Deborah A.; Ding Wen; Kelly, John F.; Chakravarthy, Balu R.

    2006-04-14

    Okadaic acid (OA) is a widely used small-molecule phosphatase inhibitor that is thought to selectively inhibit protein phosphatase 2A (PP2A). Multiple studies have demonstrated that PP2A activity is compromised in Brains of Alzheimer's disease patients. Thus, we set out to determine changes in phosphorylation that occur upon OA treatment of neuronal cells. Utilizing isotope-coded affinity tags and mass spectrometry analysis, we determined the relative abundance of proteins in a phosphoprotein enriched fraction from control and OA-treated primary cortical neurons. We identified many proteins whose phosphorylation state is regulated by OA, including glycogen synthase kinase 3{beta}, collapsin-response mediator proteins (DRP-2, DPYSL-5, and CRMP-4), and the B subunit of PP2A itself. Most interestingly, we have found that complexin 2, an important regulator of neurotransmitter release and synaptic plasticity, is phosphorylated at serine 93 upon OA treatment of neurons. This is First report of a phosphorylation site on complexin 2.

  13. Mitochondrial Toxin 3-Nitropropionic Acid Induces Cardiac and Neurotoxicity Differentially in Mice

    PubMed Central

    Gabrielson, Kathleen L.; Hogue, Barbara A.; Bohr, Vilhelm A.; Cardounel, A. J.; Nakajima, Waco; Kofler, Julia; Zweier, Jay L.; Rodriguez, E. Rene; Martin, Lee J.; de Souza-Pinto, Nadja C.; Bressler, Joseph

    2001-01-01

    We investigated the effects of 3-nitropropionic acid (3NPA), a previously characterized neurotoxin, in four strains of mice to better understand the molecular basis of variable host responses to this agent. Unexpectedly, we found significant cardiac toxicity that always accompanied the neurotoxicity in all strains of mice in acute and subacute/chronic toxicity testing. Caudate putamen infarction never occurred without cardiac toxicity. All mouse strains tested are sensitive to 3NPA although the C57BL/6 and BALB/c mice require more exposure than 129SVEMS and FVB/n mice. Cardiac toxicity alone was found in 50% of symptomatic mice tested and morphologically, the cardiac toxicity is characterized by diffuse swelling of cardiomyocytes and multifocal coagulative contraction band necrosis. In subacute to chronic exposure, atrial thrombosis, cardiac mineralization, cell loss, and fibrosis are combined with cardiomyocyte swelling and necrosis. Ultrastructurally, mitochondrial swelling occurs initially, followed by disruption of myofilaments. Biochemically, isolated heart mitochondria from the highly sensitive 129SVEMS mice have a significant reduction of succinate dehydrogenase activity, succinate oxygen consumption rates, and heart adenosine triphosphate after 3NPA treatment. The severity of morphological changes parallels the biochemical alterations caused by 3NPA, consistent with cardiac toxicity being a consequence of the effects of 3NPA on succinate dehydrogenase. These experiments show, for the first time, that 3NPA has important cardiotoxic effects as well as neurotoxic effects, and that cardiac toxicity possibly resulting from inhibition of the succinate dehydrogenase in heart mitochondria, contributes to the cause of death in 3NPA poisoning in acute and subacute/chronic studies in mice. PMID:11583977

  14. Palmitic acid induces central leptin resistance and impairs hepatic glucose and lipid metabolism in male mice.

    PubMed

    Cheng, Licai; Yu, Yinghua; Szabo, Alexander; Wu, Yizhen; Wang, Hongqin; Camer, Danielle; Huang, Xu-Feng

    2015-05-01

    The consumption of diets rich in saturated fat largely contributes to the development of obesity in modern societies. A diet high in saturated fats can induce inflammation and impair leptin signaling in the hypothalamus. However, the role of saturated fatty acids on hypothalamic leptin signaling, and hepatic glucose and lipid metabolism remains largely undiscovered. In this study, we investigated the effects of intracerebroventricular (icv) administration of a saturated fatty acid, palmitic acid (PA, C16:0), on central leptin sensitivity, hypothalamic leptin signaling, inflammatory molecules and hepatic energy metabolism in C57BL/6J male mice. We found that the icv administration of PA led to central leptin resistance, evidenced by the inhibition of central leptin's suppression of food intake. Central leptin resistance was concomitant with impaired hypothalamic leptin signaling (JAK2-STAT3, PKB/Akt-FOXO1) and a pro-inflammatory response (TNF-α, IL1-β, IL-6 and pIκBa) in the mediobasal hypothalamus and paraventricular hypothalamic nuclei. Furthermore, the pre-administration of icv PA blunted the effect of leptin-induced decreases in mRNA expression related to gluconeogenesis (G6Pase and PEPCK), glucose transportation (GLUT2) and lipogenesis (FAS and SCD1) in the liver of mice. Therefore, elevated central PA concentrations can induce pro-inflammatory responses and leptin resistance, which are associated with disorders of energy homeostasis in the liver as a result of diet-induced obesity.

  15. Delineating Normal from Diseased Brain by Aminolevulinic Acid-Induced Fluorescence

    NASA Astrophysics Data System (ADS)

    Stepp, Herbert; Stummer, Walter

    5-Aminolevulinic acid (5-ALA) as a precursor of protoporphyrin IX (PpIX) has been established as an orally applied drug to guide surgical resection of malignant brain tumors by exciting the red fluorescence of PpIX. The accumulation of PpIX in glioblastoma multiforme (GBM) is highly selective and provides excellent contrast to normal brain when using surgical microscopes with appropriately filtered light sources and cameras. The positive predictive value of fluorescent tissue is very high, enabling safe gross total resection of GBM and other brain tumors and improving prognosis of patients. Compared to other intraoperative techniques that have been developed with the aim of increasing the rate of safe gross total resections of malignant gliomas, PpIX fluorescence is considerably simpler, more cost effective, and comparably reliable. We present the basics of 5-ALA-based fluorescence-guided resection, and discuss the clinical results obtained for GBM and the experience with the fluorescence staining of other primary brain tumors and metastases as well as the results for spinal cord tumors. The phototoxicity of PpIX, increasingly used for photodynamic therapy of brain tumors, is mentioned briefly in this chapter.

  16. Okadaic acid-induced inhibition of B-50 dephosphorylation by presynaptic membrane-associated protein phosphatases.

    PubMed

    Han, Y F; Dokas, L A

    1991-10-01

    The neuronal tissue-specific protein kinase C (PKC) substrate B-50 can be dephosphorylated by endogenous protein phosphatases (PPs) in synaptic plasma membranes (SPMs). The present study characterizes membrane-associated B-50 phosphatase activity by using okadaic acid (OA) and purified 32P-labeled substrates. At a low concentration of [gamma-32P]ATP, PKC-mediated [32P]phosphate incorporation into B-50 in SPMs reached a maximal value at 30 s, followed by dephosphorylation. OA, added 30 s after the initiation of phosphorylation, partially prevented the dephosphorylation of B-50 at 2 nM, a dose that inhibits PP-2A. At the higher concentration of 1 microM, a dose of OA that inhibits PP-1 as well as PP-2A, a nearly complete blockade of B-50 dephosphorylation was seen. Heat-stable PP inhibitor-2 (I-2) also inhibited dephosphorylation of B-50. The effects of OA and I-2 on B-50 phosphatase activity were additive. Endogenous PP-1- and PP-2A-like activities in SPMs were also demonstrated by their capabilities of dephosphorylating [32P]phosphorylase a and [32P]casein. With these exogenous substrates, sensitivities of the membrane-bound phosphatases to OA and I-2 were found to be similar to those of purified forms of these enzymes. These results indicate that PP-1- and PP-2A-like enzymes are the major B-50 phosphatases in SPMs.

  17. Jasmonic acid-induced changes in Brassica oleracea affect oviposition preference of two specialist herbivores.

    PubMed

    Bruinsma, Maaike; Van Dam, Nicole M; Van Loon, Joop J A; Dicke, Marcel

    2007-04-01

    Jasmonic acid (JA) is a key hormone involved in plant defense responses. The effect of JA treatment of cabbage plants on their acceptability for oviposition by two species of cabbage white butterflies, Pieris rapae and P. brassicae, was investigated. Both butterfly species laid fewer eggs on leaves of JA-treated plants compared to control plants. We show that this is due to processes in the plant after JA treatment rather than an effect of JA itself. The oviposition preference for control plants is adaptive, as development time from larval hatch until pupation of P. rapae caterpillars was longer on JA-treated plants. Total glucosinolate content in leaf surface extracts was similar for control and treated plants; however, two of the five glucosinolates were present in lower amounts in leaf surface extracts of JA-treated plants. When the butterflies were offered a choice between the purified glucosinolate fraction isolated from leaf surface extracts of JA-treated plants and that from control plants, they did not discriminate. Changes in leaf surface glucosinolate profile, therefore, do not seem to explain the change in oviposition preference of the butterflies after JA treatment, suggesting that as yet unknown infochemicals are involved.

  18. Atenolol offers better protection than clonidine against cardiac injury in kainic acid-induced status epilepticus

    PubMed Central

    Read, M I; Harrison, J C; Kerr, D S; Sammut, I A

    2015-01-01

    Background and Purpose Status epilepticus is increasingly associated with cardiac injury in both clinical and animal studies. The current study examined ECG activity for up to 48 h following kainic acid (KA) seizure induction and compared the potential of atenolol and clonidine to attenuate this cardiac pathology. Experimental Approach Sprague-Dawley rats (male, 300–350 g) were implanted with ECG and electrocorticogram electrodes to allow simultaneous telemetric recordings of cardiac and cortical responses during and after KA-induced seizures. Animals were randomized into saline controls, and saline vehicle-, clonidine- or atenolol-pretreated KA groups. Key Results KA administration in the saline-pretreated group produced an immediate bradycardic response (maximal decrease of 28 ± 6%), coinciding with low-level seizure activity. As high-level seizure behaviours and EEG spiking increased, tachycardia also developed, with a maximum heart rate increase of 38 ± 7% coinciding with QTc prolongation and T wave elevation. Both clonidine and atenolol pretreatment attenuated seizure activity and reduced KA-induced changes in heart rate, QTc interval and T wave amplitude observed during both bradycardic and tachycardic phases in saline-pretreated KA animals. Clonidine, however, failed to reduce the power of EEG frequencies. Atenolol and to a lesser extent clonidine attenuated the cardiac hypercontraction band necrosis, inflammatory infiltration, and oedema at 48 h after KA, relative to the saline-KA group. Conclusions and Implications Severe seizure activity in this model was clearly associated with altered ECG activity and cardiac pathology. We suggest that modulation of sympathetic activity by atenolol provides a promising cardioprotective approach in status epilepticus. PMID:25765931

  19. Free Fatty Acids Induce Autophagy and LOX-1 Upregulation in Cultured Aortic Vascular Smooth Muscle Cells.

    PubMed

    Cheng, Cheng-I; Lee, Yueh-Hong; Chen, Po-Han; Lin, Yu-Chun; Chou, Ming-Huei; Kao, Ying-Hsien

    2016-11-05

    Elevation of free fatty acids (FFAs) is known to affect microvascular function and contribute to obesity-associated insulin resistance, hypertension, and microangiopathy. Proliferative and synthetic vascular smooth muscle cells (VSMCs) increase intimal thickness and destabilize atheromatous plaques. This study aimed to investigate whether saturated palmitic acid (PA) and monounsaturated oleic acid (OA) modulate autophagy activity, cell proliferation, and vascular tissue remodeling in an aortic VSMC cell line. Exposure to PA and OA suppressed growth of VSMCs without apoptotic induction, but enhanced autophagy flux with elevation of Beclin-1, Atg5, and LC3I/II. Cotreatment with autophagy inhibitors potentiated the FFA-suppressed VSMC growth and showed differential actions of PA and OA in autophagy flux retardation. Both FFAs upregulated lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) but only OA increased LDL uptake by VSMCs. Mechanistically, FFAs induced hyperphosphorylation of Akt, ERK1/2, JNK1/2, and p38 MAPK. All pathways, except OA-activated PI3K/Akt cascade, were involved in the LOX-1 upregulation, whereas blockade of PI3K/Akt and MEK/ERK cascades ameliorated the FFA-induced growth suppression on VSMCs. Moreover, both FFAs exhibited tissue remodeling effect through increasing MMP-2 and MMP-9 expression and their gelatinolytic activities, whereas high-dose OA significantly suppressed collagen type I expression. Conversely, siRNA-mediated LOX-1 knockdown significantly attenuated the OA-induced tissue remodeling effects in VSMCs. In conclusion, OA and PA enhance autophagy flux, suppress aortic VSMC proliferation, and exhibit vascular remodeling effect, thereby leading to the loss of VSMCs and interstitial ECM in vascular walls and eventually the instability of atheromatous plaques. J. Cell. Biochem. 9999: 1-13, 2016. © 2016 Wiley Periodicals, Inc.

  20. Valproic acid induces neuronal cell death through a novel calpain-dependent necroptosis pathway

    PubMed Central

    Bollino, Dominique; Balan, Irina; Aurelian, Laure

    2015-01-01

    A growing body of evidence indicates that valproic acid (VPA), a histone deacetylase (HDAC) inhibitor used to treat epilepsy and mood disorders, has HDAC-related and -unrelated neurotoxic activity, the mechanism of which is still poorly understood. We report that VPA induces neuronal cell death through an atypical calpain-dependent necroptosis pathway that initiates with downstream activation of c-Jun N-terminal kinase 1 (JNK1) and increased expression of receptor-interacting protein 1 (RIP-1) and is accompanied by cleavage and mitochondrial release/nuclear translocation of apoptosis-inducing-factor (AIF), mitochondrial release of Smac/DIABLO, and inhibition of the anti-apoptotic protein X-linked inhibitor of apoptosis (XIAP). Coinciding with AIF nuclear translocation, VPA induces phosphorylation of the necroptosis-associated histone H2A family member H2AX, which is known to contribute to lethal DNA degradation. These signals are inhibited in neuronal cells that express constitutively activated MEK/ERK and/or PI3-K/Akt survival pathways, allowing them to resist VPA-induced cell death. The data indicate that VPA has neurotoxic activity and identify a novel calpain-dependent necroptosis pathway that includes JNK1 activation and RIP-1 expression. PMID:25581256

  1. Polycomb recruitment attenuates retinoic acid-induced transcription of the bivalent NR2F1 gene.

    PubMed

    Laursen, Kristian B; Mongan, Nigel P; Zhuang, Yong; Ng, Mary M; Benoit, Yannick D; Gudas, Lorraine J

    2013-07-01

    Polycomb proteins play key roles in mediating epigenetic modifications that occur during cell differentiation. The Polycomb repressive complex 2 (PRC2) mediates the tri-methylation of histone H3 lysine 27 (H3K27me3). In this study, we identify a distinguishing feature of two classes of PRC2 target genes, represented by the Nr2F1 (Coup-TF1) and the Hoxa5 gene, respectively. Both genes are transcriptionally activated by all-trans retinoic acid (RA) and display increased levels of the permissive H3K9/K14ac and tri-methylated histone H3 lysine 4 epigenetic marks in response to RA. However, while in response to RA the PRC2 and H3K27me3 marks are greatly decreased at the Hoxa5 promoter, these marks are initially increased at the Nr2F1 promoter. Functional depletion of the essential PRC2 protein Suz12 by short hairpin RNA (shRNA) technology enhanced the RA-associated transcription of Nr2F1, Nr2F2, Meis1, Sox9 and BMP2, but had no effect on the Hoxa5, Hoxa1, Cyp26a1, Cyp26b1 and RARβ2 transcript levels in wild-type embryonic stem cells. We propose that PRC2 recruitment attenuates the RA-associated transcriptional activation of a subset of genes. Such a mechanism would permit the fine-tuning of transcriptional networks during differentiation.

  2. Quinolinic acid induces disrupts cytoskeletal homeostasis in striatal neurons. Protective role of astrocyte-neuron interaction.

    PubMed

    Pierozan, Paula; Ferreira, Fernanda; de Lima, Bárbara Ortiz; Pessoa-Pureur, Regina

    2015-02-01

    Quinolinic acid (QUIN) is an endogenous metabolite of the kynurenine pathway involved in several neurological disorders. Among the several mechanisms involved in QUIN-mediated toxicity, disruption of the cytoskeleton has been demonstrated in striatally injected rats and in striatal slices. The present work searched for the actions of QUIN in primary striatal neurons. Neurons exposed to 10 µM QUIN presented hyperphosphorylated neurofilament (NF) subunits (NFL, NFM, and NFH). Hyperphosphorylation was abrogated in the presence of protein kinase A and protein kinase C inhibitors H89 (20 μM) and staurosporine (10 nM), respectively, as well as by specific antagonists to N-methyl-D-aspartate (50 µM DL-AP5) and metabotropic glutamate receptor 1 (100 µM MPEP). Also, intra- and extracellular Ca(2+) chelators (10 µM BAPTA-AM and 1 mM EGTA, respectively) and Ca(2+) influx through L-type voltage-dependent Ca(2+) channel (10 µM verapamil) are implicated in QUIN-mediated effects. Cells immunostained for the neuronal markers βIII-tubulin and microtubule-associated protein 2 showed altered neurite/neuron ratios and neurite outgrowth. NF hyperphosphorylation and morphological alterations were totally prevented by conditioned medium from QUIN-treated astrocytes. Cocultured astrocytes and neurons interacted with one another reciprocally, protecting them against QUIN injury. Cocultured cells preserved their cytoskeletal organization and cell morphology together with unaltered activity of the phosphorylating system associated with the cytoskeleton. This article describes cytoskeletal disruption as one of the most relevant actions of QUIN toxicity in striatal neurons in culture with soluble factors secreted by astrocytes, with neuron-astrocyte interaction playing a role in neuroprotection.

  3. Three-dimensional optical tomographic brain imaging during kainic-acid-induced seizures in rats

    NASA Astrophysics Data System (ADS)

    Bluestone, Avraham Y.; Sakamoto, Kenichi; Hielscher, Andreas H.; Stewart, Mark

    2005-04-01

    In this study, we explored the potential of diffuse optical tomography for brain oximetry and describe our efforts towards imaging hemodynamic changes in rat brains during kainic-acid (KA) induced seizures. Using electrophysiological techniques we first showed that KA induces a pronounced transient hypotension in urethane anesthetized rats that is coincident with seizure activity beginning in ventral and spreading to dorsal hippocampus. We observed sustained increases in vagus and sympathetic activity during generalized limbic seizure activity, which alters blood pressure regulation and heart rhythms. Subsequently, we used optical tomographic methods to study KA induced seizures in anesthetized animals to better define the hemodynamic cerebral vascular response. We observed a lateralized increase in deoxyhemoglobin after KA injection at the time when the blood pressure (BP) was decreased. By contrast, injection of phenylephrine produced a symmetric global increase in total hemoglobin. These findings indicate that our instrument is sensitive to the local hemodynamics, both in response to a global increase in blood pressure (phenylephrine injection) and a lateralized decrease in oxyhemoglobin produced by an asymmetric response to KA; a response that may be critically important for severe autonomic nervous system alterations during seizures. The results of this study provide the impetus for combining complimentary modalities, imaging and electrophysiological, to ultimately gain a better understanding of the underlying physiology of seizure activity in the rat.

  4. PI3K/AKT and ERK regulate retinoic acid-induced neuroblastoma cellular differentiation

    SciTech Connect

    Qiao, Jingbo; Paul, Pritha; Lee, Sora; Qiao, Lan; Josifi, Erlena; Tiao, Joshua R.; Chung, Dai H.

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Retinoic acid (RA) induces neuroblastoma cells differentiation, which is accompanied by G0/G1 cell cycle arrest. Black-Right-Pointing-Pointer RA resulted in neuroblastoma cell survival and inhibition of DNA fragmentation; this is regulated by PI3K pathway. Black-Right-Pointing-Pointer RA activates PI3K and ERK1/2 pathway; PI3K pathway mediates RA-induced neuroblastoma cell differentiation. Black-Right-Pointing-Pointer Upregulation of p21 is necessary for RA-induced neuroblastoma cell differentiation. -- Abstract: Neuroblastoma, the most common extra-cranial solid tumor in infants and children, is characterized by a high rate of spontaneous remissions in infancy. Retinoic acid (RA) has been known to induce neuroblastoma differentiation; however, the molecular mechanisms and signaling pathways that are responsible for RA-mediated neuroblastoma cell differentiation remain unclear. Here, we sought to determine the cell signaling processes involved in RA-induced cellular differentiation. Upon RA administration, human neuroblastoma cell lines, SK-N-SH and BE(2)-C, demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Moreover, cell cycle arrest occurred in G1/G0 phase. The protein levels of cyclin-dependent kinase inhibitors, p21 and p27{sup Kip}, which inhibit cell proliferation by blocking cell cycle progression at G1/S phase, increased after RA treatment. Interestingly, RA promoted cell survival during the differentiation process, hence suggesting a potential mechanism for neuroblastoma resistance to RA therapy. Importantly, we found that the PI3K/AKT pathway is required for RA-induced neuroblastoma cell differentiation. Our results elucidated the molecular mechanism of RA-induced neuroblastoma cellular differentiation, which may be important for developing novel therapeutic strategy against poorly differentiated neuroblastoma.

  5. Fuzzy approach for improved recognition of citric acid induced piglet coughing from continuous registration

    NASA Astrophysics Data System (ADS)

    Van Hirtum, A.; Berckmans, D.

    2003-09-01

    A natural acoustic indicator of animal welfare is the appearance (or absence) of coughing in the animal habitat. A sound-database of 5319 individual sounds including 2034 coughs was collected on six healthy piglets containing both animal vocalizations and background noises. Each of the test animals was repeatedly placed in a laboratory installation where coughing was induced by nebulization of citric acid. A two-class classification into 'cough' or 'other' was performed by the application of a distance function to a fast Fourier spectral sound analysis. This resulted in a positive cough recognition of 92%. For the whole sound-database however there was a misclassification of 21%. As spectral information up to 10000 Hz is available, an improved overall classification on the same database is obtained by applying the distance function to nine frequency ranges and combining the achieved distance-values in fuzzy rules. For each frequency range clustering threshold is determined by fuzzy c-means clustering.

  6. Sulfuric acid-induced changes in the physiology and structure of the tracheobronchial airways

    SciTech Connect

    Gearhart, J.M.; Schlesinger, R.B.

    1989-02-01

    Sulfuric acid aerosols occur in the ambient particulate mode due to atmospheric conversion from sulfur dioxide (SO2). This paper describes the response of the rabbit tracheobronchial tree to daily exposures to sulfuric acid (H2SO4) aerosol, relating physiological and morphological parameters. Rabbits were exposed to filtered air (sham control) or to submicrometer-sized H2SO4 at 250 micrograms/m3 H2SO4, for 1 hr/day, 5 days/week, with sacrifices after 4, 8, and 12 months of acid (or sham) exposure; some rabbits were allowed a 3-month recovery after all exposures ended. H2SO4 produced a slowing of tracheobronchial mucociliary clearance during the first weeks of exposure; this change became significantly greater with continued exposures and did not improve after exposures ended. Airway hyperresponsiveness was evident by 4 months of acid exposure; the condition worsened by 8 months of exposure and appeared to stabilize after this time. Standard pulmonary mechanics parameters showed no significant trends with repeated acid exposure, except for a decline in dynamic lung compliance in animals exposed to acid for 12 months. Lung tissue samples obtained from exposed animals showed a shift toward a greater frequency of smaller airways compared to control, an increase in epithelial secretory cell density in smaller airways, and a shift from neutral to acidic glycoproteins in the secretory cells. The effect on airway diameter resolved after the exposures ceased, but the secretory cell response did not return to normal within the recovery period. No evidence of inflammatory cell infiltration was found due to H2SO4 exposure. Thus, significant alterations in the physiology of the tracheobronchial tree have been demonstrated due to repeated 1-hr exposures to a concentration of H2SO4 that is one-fourth the current 8-hr threshold limit value for exposure in the work environment.

  7. Bias-dependent amino-acid-induced conductance changes in short semi-metallic carbon nanotubes.

    PubMed

    Abadir, G B; Walus, K; Pulfrey, D L

    2010-01-08

    We study the interaction between short semi-metallic carbon nanotubes and different amino acids using molecular dynamics and ab initio (density functional theory/non-equilibrium Green's function) simulations. We identify two different mechanisms of nanotube conductance change upon adsorption of amino acids: one due to the change of the coordinates of the nanotube arising from van der Waals forces of interaction with the adsorbed amino acid; and one due to electrostatic interactions, which appear only in the case of charged amino acids. We also find that the transport mechanism and the changes in the conductance of the tube upon amino acid adsorption are bias dependent.

  8. Neuroprotective activity of L-theanine on 3-nitropropionic acid-induced neurotoxicity in rat striatum.

    PubMed

    Thangarajan, Sumathi; Deivasigamani, Asha; Natarajan, Suganya Sarumani; Krishnan, Prasanna; Mohanan, Sandhya Koombankallil

    2014-09-01

    The present study has been designed to investigate the protective effect of L-theanine against 3-nitropropionic acid (3-NP)-induced Huntington's disease (HD)-like symptoms in rats. The present experimental protocol design includes systemic 3-NP acid (10 mg/kg intraperitonially) treatment for 14 d. L-theanine (100 and 200 mg/kg) was given orally, once a day, 1 h before 3-NP acid treatment for 14 d. Body weight and behavioral parameters (Morris water maze, open field test (OFT), forced swim test (FST) and rotarod activity) were assessed on 1st, 5th, 10th and 15th day post-3-NP acid administration. Malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) levels and mitochondrial enzyme complex. Succinate dehydrogenase (SDH) were measured on the 15th day in the striatum. Systemic 3-NP acid treatment significantly reduced body weight, locomotor activity and oxidative defense. The mitochondrial enzyme activity was also significantly impaired in the striatum region in 3-NP acid-treated animals. L-theanine (100 and 200 mg/kg b.wt.) treatment significantly attenuated the impairment in behavioral, biochemical and mitochondrial enzyme activities as compared to the 3-NP acid-treated group. The results of the present study suggest that pretreatment with L-theanine significantly attenuated 3-NP induced oxidative stress and restored the decreased SOD, GSH, CAT and SDH activity. It also decreased the neuronal damage as evidenced by histopathological analysis of striatum. Based on the above study, it has been proved that L-theanine has neuroprotective activity against 3-NP induced neurotoxicity.

  9. Healing Acceleration of Acetic Acid-induced Colitis by Marigold (Calendula officinalis) in Male Rats

    PubMed Central

    Tanideh, Nader; Jamshidzadeh, Akram; Sepehrimanesh, Masood; Hosseinzadeh, Masood; Koohi-Hosseinabadi, Omid; Najibi, Asma; Raam, Mozhdeh; Daneshi, Sajad; Asadi-Yousefabad, Seyedeh-Leili

    2016-01-01

    Background/Aim: Ulcerative colitis (UC) is a type of chronic inflammatory bowel disease with unknown etiology. Several therapeutic strategies such as consumption of medicinal plants have been used for its treatment. The aim of this study was to evaluate healing effects of Calendula officinalis hydroalcoholic extract in experimentally induced UC in rat. Materials and Methods: Ninety-six rats, weighing 200 ± 20 g, were randomly divided into eight equal groups. UC induced by 3% acetic acid and oral doses of C. officinalis extract, 1500 and 3000 mg/kg, and enema (gel 10% and 20%) were given. Two groups as positive controls were given asacol (enema) and oral mesalamine. Negative control groups were given normal saline and base gel. On days 3 and 7, intestinal histopathology and weight changes, plus oxidative stress indices including malondialdehyde (MDA) level and myeloperoxidase (MPO) activity were assayed. Results: A significant increase in the body weight of rats was seen in the group given C. officinalis extract 3000 mg/kg orally, oral mesalamine, and 20% intracolonic gel form of marigold extract compared with negative control and base gel groups during the experimental period. Acute inflammation and granular atrophy after UC induction were resolved completely completely by both 20% intracolonic gel and 3000 mg/kg orally. An increase in MPO activity and a decrease in MDA level in response to oral and intracolonic gel form of C. officinalis were observed 3 and and 7 days after treatment (P < 0.05). Conclusion: Our results indicate that oral and enema forms of hydroalcoholic extract of C. officinalis can be offered as are potential therapeutic agents for UC induced in rats. PMID:26831607

  10. Abscisic Acid Induces Rapid Reductions in Mesophyll Conductance to Carbon Dioxide

    PubMed Central

    Sorrentino, Giuseppe; Haworth, Matthew; Wahbi, Said; Mahmood, Tariq; Zuomin, Shi; Centritto, Mauro

    2016-01-01

    The rate of photosynthesis (A) of plants exposed to water deficit is a function of stomatal (gs) and mesophyll (gm) conductance determining the availability of CO2 at the site of carboxylation within the chloroplast. Mesophyll conductance often represents the greatest impediment to photosynthetic uptake of CO2, and a crucial determinant of the photosynthetic effects of drought. Abscisic acid (ABA) plays a fundamental role in signalling and co-ordination of plant responses to drought; however, the effect of ABA on gm is not well-defined. Rose, cherry, olive and poplar were exposed to exogenous ABA and their leaf gas exchange parameters recorded over a four hour period. Application with ABA induced reductions in values of A, gs and gm in all four species. Reduced gm occurred within one hour of ABA treatment in three of the four analysed species; indicating that the effect of ABA on gm occurs on a shorter timescale than previously considered. These declines in gm values associated with ABA were not the result of physical changes in leaf properties due to altered turgor affecting movement of CO2, or caused by a reduction in the sub-stomatal concentration of CO2 (Ci). Increased [ABA] likely induces biochemical changes in the properties of the interface between the sub-stomatal air-space and mesophyll layer through the actions of cooporins to regulate the transport of CO2. The results of this study provide further evidence that gm is highly responsive to fluctuations in the external environment, and stress signals such as ABA induce co-ordinated modifications of both gs and gm in the regulation of photosynthesis. PMID:26862904

  11. Effects of dexpanthenol on acetic acid-induced colitis in rats

    PubMed Central

    Cagin, Yasir Furkan; Parlakpinar, Hakan; Vardi, Nigar; Polat, Alaadin; Atayan, Yahya; Erdogan, Mehmet Ali; Tanbek, Kevser

    2016-01-01

    While the pathogenesis of acetic acid (AA)-induced colitis is unclear, reactive oxygen species are considered to have a significant effect. The aim of the present study was to elucidate the therapeutic potential of dexpanthenol (Dxp) on the amelioration of colitis in rats. Group I (n=8; control group) was intrarectally administered 1 ml saline solution (0.9%); group II [n=8; AA] was administered 4% AA into the colon via the rectum as a single dose for three consecutive days; group III (n=8; AA + Dxp) was administered AA at the same dosage as group II from day 4, and a single dose of Dxp was administered intraperitoneally; and group IV (n=8; Dxp) was administered Dxp similarly to Group III. Oxidative stress and colonic damage were assessed via biochemical and histologic examination methods. AA treatment led to an increase in oxidative parameters and a decrease in antioxidant systems. Histopathological examination showed that AA treatment caused tissue injury and increased caspase-3 activity in the distal colon and triggered apoptosis. Dxp treatment caused biochemical and histopathological improvements, indicating that Dxp may have an anti-oxidant effect in colitis; therefore, Dxp may be a potential therapeutic agent for the amelioration of IBD. PMID:27882101

  12. Effects of dexpanthenol on acetic acid-induced colitis in rats.

    PubMed

    Cagin, Yasir Furkan; Parlakpinar, Hakan; Vardi, Nigar; Polat, Alaadin; Atayan, Yahya; Erdogan, Mehmet Ali; Tanbek, Kevser

    2016-11-01

    While the pathogenesis of acetic acid (AA)-induced colitis is unclear, reactive oxygen species are considered to have a significant effect. The aim of the present study was to elucidate the therapeutic potential of dexpanthenol (Dxp) on the amelioration of colitis in rats. Group I (n=8; control group) was intrarectally administered 1 ml saline solution (0.9%); group II [n=8; AA] was administered 4% AA into the colon via the rectum as a single dose for three consecutive days; group III (n=8; AA + Dxp) was administered AA at the same dosage as group II from day 4, and a single dose of Dxp was administered intraperitoneally; and group IV (n=8; Dxp) was administered Dxp similarly to Group III. Oxidative stress and colonic damage were assessed via biochemical and histologic examination methods. AA treatment led to an increase in oxidative parameters and a decrease in antioxidant systems. Histopathological examination showed that AA treatment caused tissue injury and increased caspase-3 activity in the distal colon and triggered apoptosis. Dxp treatment caused biochemical and histopathological improvements, indicating that Dxp may have an anti-oxidant effect in colitis; therefore, Dxp may be a potential therapeutic agent for the amelioration of IBD.

  13. Valproic acid-induced acute pancreatitis in pediatric age: case series and review of literature

    PubMed Central

    COFINI, M.; QUADROZZI, F.; FAVORITI, P.; FAVORITI, M.; COFINI, G.

    2015-01-01

    Valproic acid (VPA) is commonly prescribed medication for epilepsy, migraine and bipolar disorder. Although the common adverse effect associated with VPA are typically benign, less common adverse effect can occur; these include hepatotixicity, teratogenicity and acute pancreatitis (AP). VPA-induced pancreatitis does not depend on valproic acid serum level and may occur anytime after onset of therapy. Re-challenge with VPA is dangerous and should be avoided. The diagnosis of VPA-induced pancreatitis seems to be underestimated because of difficulties in determining the causative agent and the need for a retrospective re-evaluation of the causative factor. More of idiopathic pancreatitis should be a drug-induced pancreatitis. We report four cases of VPA-induced AP found in a group of 52 cases of AP in children come to our attention from January 2008 to December 2012. The aim of these reports is to point out our experience about clinical presentation, diagnosis, management, outcome in children with VPA-induced AP and review of literature. PMID:26712070

  14. Purified eicosapentaenoic acid induces prolonged survival of cardiac allografts and generates regulatory T cells.

    PubMed

    Iwami, D; Zhang, Q; Aramaki, O; Nonomura, K; Shirasugi, N; Niimi, M

    2009-06-01

    Fish oil, which is rich in eicosapentaenoic acid (EPA), has been found to have immunomodulatory effects. We examined whether administration of purified EPA affected survival of fully mismatched murine cardiac allografts. Hearts from C57BL/10 (H-2(b)) mice were transplanted into CBA (H-2(k)) recipients treated with one intraperitoneal dose of purified EPA the day of transplantation. Untreated CBA recipients and recipients given 0.1 g/kg of EPA rejected C57BL/10 hearts (median survival time [MST], 8 and 13 days, respectively). With a 1.0 g/kg dose of EPA, graft survival was markedly prolonged (MST >100 days). To determine whether regulatory cells were generated, naïve mice (secondary recipients) underwent adoptive transfer of splenocytes from EPA-treated primary recipients and cardiac allograft transplantation. Adoptive transfer of whole, CD4(+) and CD4(+)CD25(+) splenocytes from EPA-treated recipients induced indefinite survival in secondary recipients. Flow cytometry showed that the CD4(+)CD25(+) cells were Foxp3(+). In reverse transcriptase-polymerase chain reaction (RT-PCR) studies, the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA was upregulated by EPA treatment. A PPARgamma antagonist abrogated the prolongation of graft survival induced by EPA treatment (MST, 13 days). Thus, in our model, purified EPA induced prolonged survival of fully mismatched cardiac allografts and generated regulatory T cells dependent on PPARgamma activation.

  15. Lipotoxic effect of p21 on free fatty acid-induced steatosis in L02 cells.

    PubMed

    Wang, Jie-wei; Wan, Xing-yong; Zhu, Hua-tuo; Lu, Chao; Yu, Wei-lai; Yu, Chao-hui; Shen, Zhe; Li, You-ming

    2014-01-01

    Nonalcoholic fatty liver disease (NAFLD) is increasingly regarded as a hepatic manifestation of metabolic syndrome. Though with high prevalence, the mechanism is poorly understood. This study aimed to investigate the effects of p21 on free fatty acid (FFA)-induced steatosis in L02 cells. We therefore analyzed the L02 cells with MG132 and siRNA treatment for different expression of p21 related to lipid accumulation and lipotoxicity. Cellular total lipid was stained by Oil Red O, while triglyceride content, cytotoxicity assays, lipid peroxidation markers and anti-oxidation levels were measured by enzymatic kits. Treatment with 1 mM FFA for 48 hr induced magnificent intracellular lipid accumulation and increased oxidative stress in p21 overload L02 cells compared to that in p21 knockdown L02 cells. By increasing oxidative stress and peroxidation, p21 accelerates FFA-induced lipotoxic effect in L02 cells and might provide information about potentially new targets for drug development and treatments of NAFLD.

  16. Salicylic acid induced cysteine protease activity during programmed cell death in tomato plants.

    PubMed

    Kovács, Judit; Poór, Péter; Szepesi, Ágnes; Tari, Irma

    2016-06-01

    The hypersensitive response (HR), a type of programmed cell death (PCD) during biotic stress is mediated by salicylic acid (SA). The aim of this work was to reveal the role of proteolysis and cysteine proteases in the execution of PCD in response of SA. Tomato plants were treated with sublethal (0.1 mM) and lethal (1 mM) SA concentrations through the root system. Treatment with 1 mM SA increased the electrolyte leakage and proteolytic activity and reduced the total protein content of roots after 6 h, while the proteolytic activity did not change in the leaves and in plants exposed to 0.1 mM SA. The expression of the papain-type cysteine protease SlCYP1, the vacuolar processing enzyme SlVPE1 and the tomato metacaspase SlMCA1 was induced within the first three hours in the leaves and after 0.5 h in the roots in the presence of 1 mM SA but the transcript levels did not increase significantly at sublethal SA. The Bax inhibitor-1 (SlBI-1), an antiapoptotic gene was over-expressed in the roots after SA treatments and it proved to be transient in the presence of sublethal SA. Protease inhibitors, SlPI2 and SlLTC were upregulated in the roots by sublethal SA but their expression remained low at 1 mM SA concentration. It is concluded that in contrast to leaves the SA-induced PCD is associated with increased proteolytic activity in the root tissues resulting from a fast up-regulation of specific cysteine proteases and down-regulation of protease inhibitors.

  17. Ursolic Acid-Induced Elevation of Serum Irisin Augments Muscle Strength During Resistance Training in Men

    PubMed Central

    Bang, Hyun Seok; Seo, Dae Yun; Chung, Yong Min; Oh, Kyoung-Mo; Park, Jung Jun; Arturo, Figueroa; Jeong, Seung-Hun; Kim, Nari

    2014-01-01

    Ursolic acid (UA), a type of pentacyclic triterpenoid carboxylic acid purified from natural plants, can promote skeletal muscle development. We measured the effect of resistance training (RT) with/without UA on skeletal muscle development and related factors in men. Sixteen healthy male participants (age, 29.37±5.14 years; body mass index=27.13±2.16 kg/m2) were randomly assigned to RT (n=7) or RT with UA (RT+UA, n=9) groups. Both groups completed 8 weeks of intervention consisting of 5 sets of 26 exercises, with 10~15 repetitions at 60~80% of 1 repetition maximum and a 60~90-s rest interval between sets, performed 6 times/week. UA or placebo was orally ingested as 1 capsule 3 times/day for 8 weeks. The following factors were measured pre-and post-intervention: body composition, insulin, insulin-like growth factor-1 (IGF-1), irisin, and skeletal muscle strength. Body fat percentage was significantly decreased (p<0.001) in the RT+UA group, despite body weight, body mass index, lean body mass, glucose, and insulin levels remaining unchanged. IGF-1 and irisin were significantly increased compared with baseline levels in the RT+UA group (p<0.05). Maximal right and left extension (p<0.01), right flexion (p<0.05), and left flexion (p<0.001) were significantly increased compared with baseline levels in the RT+UA group. These findings suggest that UA-induced elevation of serum irisin may be useful as an agent for the enhancement of skeletal muscle strength during RT. PMID:25352765

  18. Mast cell mediators in citric acid-induced airway constriction of guinea pigs

    SciTech Connect

    Lin, C.-H.; Lai, Y.-L. . E-mail: tiger@ha.mc.ntu.edu.tw

    2005-08-15

    We demonstrated previously that mast cells play an important role in citric acid (CA)-induced airway constriction. In this study, we further investigated the underlying mediator(s) for this type of airway constriction. At first, to examine effects caused by blocking agents, 67 young Hartley guinea pigs were divided into 7 groups: saline + CA; methysergide (serotonin receptor antagonist) + CA; MK-886 (leukotriene synthesis inhibitor) + CA; mepyramine (histamine H{sub 1} receptor antagonist) + CA; indomethacin (cyclooxygenase inhibitor) + CA; cromolyn sodium (mast cell stabilizer) + CA; and compound 48/80 (mast cell degranulating agent) + CA. Then, we tested whether leukotriene C{sub 4} (LTC{sub 4}) or histamine enhances CA-induced airway constriction in compound 48/80-pretreated guinea pigs. We measured dynamic respiratory compliance (Crs) and forced expiratory volume in 0.1 s (FEV{sub 0.1}) during either baseline or recovery period. In addition, we detected histamine level, an index of pulmonary mast cell degranulation, in bronchoalveolar lavage (BAL) samples. Citric acid aerosol inhalation caused decreases in Crs and FEV{sub 0.1}, indicating airway constriction in the control group. This airway constriction was significantly attenuated by MK-886, mepyramine, cromolyn sodium, and compound 48/80, but not by either methysergide or indomethacin. Both LTC{sub 4} and histamine infusion significantly increased the magnitude of CA-induced airway constriction in compound 48/80-pretreated guinea pigs. Citric acid inhalation caused significant increase in histamine level in the BAL sample, which was significantly suppressed by compound 48/80. These results suggest that leukotrienes and histamine originating from mast cells play an important role in CA inhalation-induced noncholinergic airway constriction.

  19. Mast cells in citric acid-induced cough of guinea pigs

    SciTech Connect

    Lai, Y.-L. . E-mail: tiger@ha.mc.ntu.edu.tw; Lin, T.-Y.

    2005-01-01

    It was demonstrated previously that mast cells play an important role in citric acid (CA)-induced airway constriction. To investigate the role of mast cells in CA-induced cough, three experiments were carried out in this study. In the first experiment, 59 guinea pigs were employed and we used compound 48/80 to deplete mast cells, cromolyn sodium to stabilize mast cells, MK-886 to inhibit leukotriene synthesis, pyrilamine to antagonize histamine H{sub 1} receptor, methysergide to antagonize serotonin receptor, and indomethacin to inhibit cyclooxygenase. In the second experiment, 56 compound 48/80-pretreated animals were divided into two parts; the first one was used to test the role of exogenous leukotriene (LT) C{sub 4}, while the second one to test the role of exogenous histamine in CA-induced cough. Each animal with one of the above pretreatments was exposed sequentially to saline (baseline) and CA (0.6 M) aerosol, each for 3 min. Then, cough was recorded for 12 min using a barometric body plethysmograph. In the third experiment, the activation of mast cells upon CA inhalation was investigated by determining arterial plasma histamine concentration in 17 animals. Exposure to CA induced a marked increase in cough number. Compound 48/80, cromolyn sodium, MK-886 and pyrilamine, but not indomethacin or methysergide, significantly attenuated CA-induced cough. Injection of LTC{sub 4} or histamine caused a significant increase in CA-induced cough in compound 48/80-pretreated animals. In addition, CA inhalation caused significant increase in plasma histamine concentration, which was blocked by compound 48/80 pretreatment. These results suggest that mast cells play an important role in CA aerosol inhalation-induced cough via perhaps mediators LTs and histamine.

  20. Fragmentation of amino acids induced by collisions with low-energy highly charged ions

    NASA Astrophysics Data System (ADS)

    Piekarski, D. G.; Maclot, S.; Domaracka, A.; Adoui, L.; Alcamí, M.; Rousseau, P.; Díaz-Tendero, S.; Huber, B. A.; Martín, F.

    2014-04-01

    Fragmentation of amino acids NH2-(CH2)n-COOH (n=1 glycine; n=2 β-alanine and n=3 γ-aminobutyric acid GABA) following collisions with slow highly charged ions has been studied in the gas phase by a combined experimental and theoretical approach. In the experiments, a multi-coincidence detection method was used to deduce the charge state of the molecules before fragmentation. Quantum chemistry calculations have been carried out in the basis of the density functional theory and ab initio molecular dynamics. The combination of both methodologies is essential to unambiguously unravel the different fragmentation pathways.

  1. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

    SciTech Connect

    Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm J.

    2006-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.

  2. Early molecular events during retinoic acid induced differentiation of neuromesodermal progenitors

    PubMed Central

    Cunningham, Thomas J.; Colas, Alexandre

    2016-01-01

    ABSTRACT Bipotent neuromesodermal progenitors (NMPs) residing in the caudal epiblast drive coordinated body axis extension by generating both posterior neuroectoderm and presomitic mesoderm. Retinoic acid (RA) is required for body axis extension, however the early molecular response to RA signaling is poorly defined, as is its relationship to NMP biology. As endogenous RA is first seen near the time when NMPs appear, we used WNT/FGF agonists to differentiate embryonic stem cells to NMPs which were then treated with a short 2-h pulse of 25 nM RA or 1 µM RA followed by RNA-seq transcriptome analysis. Differential expression analysis of this dataset indicated that treatment with 25 nM RA, but not 1 µM RA, provided physiologically relevant findings. The 25 nM RA dataset yielded a cohort of previously known caudal RA target genes including Fgf8 (repressed) and Sox2 (activated), plus novel early RA signaling targets with nearby conserved RA response elements. Importantly, validation of top-ranked genes in vivo using RA-deficient Raldh2−/− embryos identified novel examples of RA activation (Nkx1-2, Zfp503, Zfp703, Gbx2, Fgf15, Nt5e) or RA repression (Id1) of genes expressed in the NMP niche or progeny. These findings provide evidence for early instructive and permissive roles of RA in controlling differentiation of NMPs to neural and mesodermal lineages. PMID:27793834

  3. Lack of kainic acid-induced gamma oscillations predicts subsequent CA1 excitotoxic cell death

    PubMed Central

    Jinde, Seiichiro; Belforte, Juan E.; Yamamoto, Jun; Wilson, Matthew A.; Tonegawa, Susumu; Nakazawa, Kazu

    2009-01-01

    Gamma oscillations are a prominent feature of hippocampal network activity, but their functional role remains debated, ranging from mere epiphenomenon to crucial for information processing. Similarly, persistent gamma oscillations sometimes appear prior to epileptic discharges in patients with mesial temporal sclerosis. However, the significance of this activity in hippocampal excitotoxicity is unclear. We assessed the relationship between kainic acid (KA)-induced gamma oscillations and excitotoxicity in genetically-engineered mice in which N-methyl-D-aspartic acid (NMDA) receptor deletion was confined to CA3 pyramidal cells. Mutants showed reduced CA3 pyramidal cell firing and augmented sharp wave-ripple activity, resulting in higher susceptibility to KA-induced seizures, and leading to strikingly selective neurodegeneration in the CA1 subfield. Interestingly, the KA-induced gamma-aminobutyric acid (GABA) level increases and persistent 30-50 Hz gamma oscillations observed in control mice prior to the first seizure discharge was abolished in the mutants. Consequently, on subsequent days, mutants manifested prolonged epileptiform activity and massive neurodegeneration of CA1 cells, including local GABAergic neurons. Remarkably, pretreatment with the potassium channel blocker α-dendrotoxin (DTX) increased GABA levels, restored gamma oscillations, and prevented CA1 degeneration in the mutants. These results demonstrate that emergence of low frequency gamma oscillations predicts increased resistance to KA-induced excitotoxicity, raising the possibility that gamma oscillations may have potential prognostic value for the treatment of epilepsy. PMID:19735292

  4. Uric Acid Induces Endothelial Dysfunction by Activating the HMGB1/RAGE Signaling Pathway

    PubMed Central

    Cai, Wei; Duan, Xi-Mei; Liu, Ying; Yu, Jiao; Tang, Yun-Liang; Liu, Ze-Lin; Jiang, Shan; Zhang, Chun-Ping; Liu, Jian-Ying

    2017-01-01

    Uric acid (UA) is a risk factor for endothelial dysfunction, a process in which inflammation may play an important role. UA increases high mobility group box chromosomal protein 1 (HMGB1) expression and extracellular release in endothelial cells. HMGB1 is an inflammatory cytokine that interacts with the receptor for advanced glycation end products (RAGE), inducing an oxidative stress and inflammatory response, which leads to endothelial dysfunction. In this study, human umbilical vein endothelial cells (HUVECs) were incubated with a high concentration of UA (20 mg/dL) after which endothelial function and the expression of HMGB1, RAGE, nuclear factor kappa B (NF-κB), inflammatory cytokines, and adhesion molecules were evaluated. UA inhibited endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production in HUVECs, increased intracellular HMGB1 expression and extracellular HMGB1 secretion, and upregulated RAGE expression. UA also activated NF-κB and increased the level of inflammatory cytokines. Blocking RAGE significantly suppressed the upregulation of RAGE and HMGB1 and prevented the increase in DNA binding activity of NF-κB and the levels of inflammatory cytokines. It also blocked the decrease in eNOS expression and NO production induced by UA. Our results suggest that high concentrations of UA cause endothelial dysfunction via the HMGB1/RAGE signaling pathway. PMID:28116308

  5. Gibberellic Acid-Induced Synthesis of Protease by Isolated Aleurone Layers of Barley 1

    PubMed Central

    Jacobsen, John V.; Varner, J. E.

    1967-01-01

    The production of protease by isolated aleurone layers of barley in response to gibberellic acid has been examined. The protease arises in the aleurone layer and is mostly released from the aleurone cells. The courses of release of amylase and protease from aleurone layers, the dose responses to gibberellic acid and the effects of inhibitors on the production of both enzymes are parallel. As is the case for amylase, protease is made de novo in response to the hormone. These data give some credence to the hypothesis that the effect of gibberellic acid is to promote the simultaneous synthesis and secretion of a group of hydrolases. PMID:16656695

  6. All-trans retinoic acid induces oxidative phosphorylation and mitochondria biogenesis in adipocytes.

    PubMed

    Tourniaire, Franck; Musinovic, Hana; Gouranton, Erwan; Astier, Julien; Marcotorchino, Julie; Arreguin, Andrea; Bernot, Denis; Palou, Andreu; Bonet, M Luisa; Ribot, Joan; Landrier, Jean-François

    2015-06-01

    A positive effect of all-trans retinoic acid (ATRA) on white adipose tissue (WAT) oxidative and thermogenic capacity has been described and linked to an in vivo fat-lowering effect of ATRA in mice. However, little is known about the effects of ATRA on mitochondria in white fat. Our objective has been to characterize the effect of ATRA on mitochondria biogenesis and oxidative phosphorylation (OXPHOS) capacity in mature white adipocytes. Transcriptome analysis, oxygraphy, analysis of mitochondrial DNA (mtDNA), and flow cytometry-based analysis of mitochondria density were performed in mature 3T3-L1 adipocytes after 24 h incubation with ATRA (2 µM) or vehicle. Selected genes linked to mitochondria biogenesis and function and mitochondria immunostaining were analyzed in WAT tissues of ATRA-treated as compared with vehicle-treated mice. ATRA upregulated the expression of a large set of genes linked to mtDNA replication and transcription, mitochondrial biogenesis, and OXPHOS in adipocytes, as indicated by transcriptome analysis. Oxygen consumption rate, mtDNA content, and staining of mitochondria were increased in the ATRA-treated adipocytes. Similar results were obtained in WAT depots of ATRA-treated mice. We conclude that ATRA impacts mitochondria in adipocytes, leading to increased OXPHOS capacity and mitochondrial content in these cells.

  7. Agmatine rescues autistic behaviors in the valproic acid-induced animal model of autism.

    PubMed

    Kim, Ji-Woon; Seung, Hana; Kim, Ki Chan; Gonzales, Edson Luck T; Oh, Hyun Ah; Yang, Sung Min; Ko, Mee Jung; Han, Seol-Heui; Banerjee, Sourav; Shin, Chan Young

    2017-02-01

    Autism spectrum disorder (ASD) is an immensely challenging developmental disorder characterized primarily by two core behavioral symptoms of social communication deficits and restricted/repetitive behaviors. Investigating the etiological process and identifying an appropriate therapeutic target remain as formidable challenges to overcome ASD due to numerous risk factors and complex symptoms associated with the disorder. Among the various mechanisms that contribute to ASD, the maintenance of excitation and inhibition balance emerged as a key factor to regulate proper functioning of neuronal circuitry. Interestingly, our previous study involving the valproic acid animal model of autism (VPA animal model) has demonstrated excitatory-inhibitory imbalance (E/I imbalance) due to enhanced differentiation of glutamatergic neurons and reduced GABAergic neurons. Here, we investigated the potential of agmatine, an endogenous NMDA receptor antagonist, as a novel therapeutic candidate in ameliorating ASD symptoms by modulating E/I imbalance using the VPA animal model. We observed that a single treatment of agmatine rescued the impaired social behaviors as well as hyperactive and repetitive behaviors in the VPA animal model. We also observed that agmatine treatment rescued the overly activated ERK1/2 signaling in the prefrontal cortex and hippocampus of VPA animal models, possibly, by modulating over-excitability due to enhanced excitatory neural circuit. Taken together, our results have provided experimental evidence suggesting a possible therapeutic role of agmatine in ameliorating ASD-like symptoms in the VPA animal model of ASD.

  8. Valproic acid induces the glutamate transporter excitatory amino acid transporter-3 in human oligodendroglioma cells.

    PubMed

    Bianchi, M G; Franchi-Gazzola, R; Reia, L; Allegri, M; Uggeri, J; Chiu, M; Sala, R; Bussolati, O

    2012-12-27

    Glutamate transport in early, undifferentiated oligodendrocytic precursors has not been characterized thus far. Here we show that human oligodendroglioma Hs683 cells are not endowed with EAAT-dependent anionic amino acid transport. However, in these cells, but not in U373 human glioblastoma cells, valproic acid (VPA), an inhibitor of histone deacetylases, markedly induces SLC1A1 mRNA, which encodes for the glutamate transporter EAAT3. The effect is detectable after 8h and persists up to 120h of treatment. EAAT3 protein increase becomes detectable after 24h of treatment and reaches its maximum after 72-96h, when it is eightfold more abundant than control. The initial influx of d-aspartate increases in parallel, exhibiting the typical features of an EAAT3-mediated process. SLC1A1 mRNA induction is associated with the increased expression of PDGFRA mRNA (+150%), a marker of early oligodendrocyte precursor cells, while the expression of GFAP, CNP and TUBB3 remains unchanged. Short term experiments have indicated that the VPA effect is shared by trichostatin A, another inhibitor of histone deacetylases. On the contrary, EAAT3 induction is neither prevented by inhibitors of mitogen-activated protein kinases nor triggered by a prolonged incubation with lithium, thus excluding a role for the GSK3β/β-catenin pathway. Thus, the VPA-dependent induction of the glutamate transporter EAAT3 in human oligodendroglioma cells likely occurs through an epigenetic mechanism and may represent an early indicator of commitment to oligodendrocytic differentiation.

  9. Insulin Protects Pancreatic Acinar Cells from Palmitoleic Acid-induced Cellular Injury*

    PubMed Central

    Samad, Aysha; James, Andrew; Wong, James; Mankad, Parini; Whitehouse, John; Patel, Waseema; Alves-Simoes, Marta; Siriwardena, Ajith K.; Bruce, Jason I. E.

    2014-01-01

    Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca2+ ([Ca2+]i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca2+]i are linked critically by the ATP-driven plasma membrane Ca2+-ATPase (PMCA) important for maintaining low resting [Ca2+]i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca2+]i was measured by fura-2 imaging. An in situ [Ca2+]i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mm) and POAEE (100 μm) induced a small but irreversible Ca2+ overload response but had no significant effect on PMCA activity. POA (50–100 μm) induced a robust Ca2+ overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca2+ overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca2+ overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002. These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis. PMID:24993827

  10. Prometryn and humic acid induce Cytochrome P450 1A expression in Danio rerio (zebrafish).

    PubMed

    Zhao, Qian; Shi, Feng; Zhu, Lin

    2017-01-01

    Humic acid (HA) is a major component of dissolved organic matter, is ubiquitous in the aquatic environment and influences the biological toxicity of organic pollutants. In this study, we investigated the cytochrome P450 1A (CYP 1A) mRNA expression and ethoxyresorufin-O-deethylase (EROD) activity in the gills and liver of zebrafish following exposure to the s-triazine herbicide prometryn with or without HA present. Prometryn induced both CYP 1A mRNA expression and EROD activity. The CYP 1A mRNA expression of zebrafish that were exposed to a combination of prometryn and HA was increased compared to those exposed to prometryn alone. A likely cause for CYP 1A induction is the impact of special components of HA, functioning as aryl hydrocarbon receptor (AHR) agonists. In combination with HA, these increase prometryn levels in tissues. Similar results for EROD activity were evident. In our time course study, CYP 1A mRNA expression reached maximum values during 24h. This revealed CYP 1A mRNA transcription as a comparatively sensitive toxicity index. In a recovery experiment, we found a faster decrease of CYP 1A mRNA expression to control levels (CK) in gills compared to liver tissue. Following exposure to HA, CYP 1A mRNA expression in liver tissue displayed a faster decrease to CK levels. HA induced enhanced metabolic rates for prometryn. In contrast, recovery regularity of CYP 1A expression in gills was independent of the presence of HA. This result indicates different detoxification mechanisms for HA in liver and gills.

  11. A mechanistic study of cellular photodestruction with 5-aminolaevulinic acid-induced porphyrin.

    PubMed Central

    Iinuma, S.; Farshi, S. S.; Ortel, B.; Hasan, T.

    1994-01-01

    5-Aminolaevulinic acid (ALA)-induced porphyrin biosynthesis and phototoxicity in vitro was investigated in five malignant and two normal cell lines. Intracellular protoporphyrin IX (PpIX) content was quantified by extraction and fluorescence spectroscopy. Cellular PpIX content did not always correlate with cell proliferation rate as measured by the doubling times of cell lines. Cellular efflux of PpIX was also investigated. In a bladder carcinoma cell line, the observed rapid efflux was not blocked by verapamil, an inhibitor of the P-glycoprotein efflux pump. These data support the view that cellular PpIX accumulation is a dynamic process that is determined by both the efflux of PpIX from the cells and enzyme activities in the haem biosynthesis pathway. Desferrioxamine (desferal), a modulator of PpIX biosynthesis, enhanced ALA-induced cellular PpIX content significantly in all carcinoma cell lines but not in non-malignant cell lines. The enhanced PpIX cellular accumulation is attributed to inhibition of ferrochelatase activity, the enzyme responsible for the conversion of PpIX to haem. PpIX-mediated cellular photodestruction following irradiation with an argon ion laser at 514.5 nm was determined by the 'MTT assay'. There appeared to be a 'threshold' effect of cellular PpIX content; cells that synthesised less than 140 ng/mg-1 protein exhibited very little phototoxic damage, while cell lines having greater than 140 ng PpIX/mg-1 protein [corrected] exhibited a consistent phototoxic response. Among the cell lines which did undergo phototoxic damage, there was not a strict correlation between PpIX cellular content and ALA-induced phototoxicity. Desferal enhanced the PpIX content and phototoxic effect in the responsive cells. Fluorescence microscopy of the ALA-treated cells revealed marked accumulation of PpIX in mitochondria (rhodamine 123 co-staining). That the primary site of phototoxic damage is also the mitochondria was confirmed by electron micrographs of cells photosensitised with ALA-induced PpIX, which showed swelling of mitochondria within minutes after irradiation while other suborganelles appeared to be unaffected. The repair or further destruction of the mitochondria was fluence and cell-type dependent. The data from this study suggest that the basis of increased ALA-induced PpIX accumulation in tumours is a combination of various aspects of the metabolic process and pharmacokinetics and that the efficacy of photodestruction of malignancy will be determined not only by the rate of PpIX synthesis but also by specific cellular and tissue characteristics. Images Figure 4 Figure 5 Figure 7 PMID:8018536

  12. Regulation of c-myb expression in human neuroblastoma cells during retinoic acid-induced differentiation.

    PubMed Central

    Thiele, C J; Cohen, P S; Israel, M A

    1988-01-01

    We detected expression of the c-myb proto-oncogene, which was initially thought to be expressed in a tissue-specific manner in cells of hematopoietic lineage, in human tissues of neuronal origin. Since the level of c-myb expression declined during fetal development, we studied the regulation of its expression in human neuroblastoma cell lines induced to differentiate by retinoic acid. The expression of c-myb declined during the maturation of neuroblastoma cells, and this change was mediated by a decrease in c-myb transcription. Images PMID:3380093

  13. Ursodeoxycholic Acid Induces Death Receptor-mediated Apoptosis in Prostate Cancer Cells

    PubMed Central

    Lee, Won Sup; Jung, Ji Hyun; Panchanathan, Radha; Yun, Jeong Won; Kim, Dong Hoon; Kim, Hye Jung; Kim, Gon Sup; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun; Jung, Jin-Myung

    2017-01-01

    Background Bile acids have anti-cancer properties in a certain types of cancers. We determined anticancer activity and its underlying molecular mechanism of ursodeoxycholic acid (UDCA) in human DU145 prostate cancer cells. Methods Cell viability was measured with an MTT assay. UDCA-induced apoptosis was determined with flow cytometric analysis. The expression levels of apoptosis-related signaling proteins were examined with Western blotting. Results UDCA treatment significantly inhibited cell growth of DU145 in a dose-dependent manner. It induced cellular shrinkage and cytoplasmic blebs and accumulated the cells with sub-G1 DNA contents. Moreover, UDCA activated caspase 8, suggesting that UDCA-induced apoptosis is associated with extrinsic pathway. Consistent to this finding, UDCA increased the expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and death receptor 5 (DR5), and TRAIL augmented the UDCA-induced cell death in DU145 cells. In addition, UDCA also increased the expressions of Bax and cytochrome c and decreased the expression of Bcl-xL in DU145 cells. This finding suggests that UDCA-induced apoptosis may be involved in intrinsic pathway. Conclusions UDCA induces apoptosis via extrinsic pathway as well as intrinsic pathway in DU145 prostate cancer cells. UDCA may be a promising anti-cancer agent against prostate cancer. PMID:28382282

  14. Phosphatidic Acid Induces Ligand-independent Epidermal Growth Factor Receptor Endocytic Traffic through PDE4 Activation

    PubMed Central

    Norambuena, Andrés; Metz, Claudia; Jung, Juan E.; Silva, Antonia; Otero, Carolina; Cancino, Jorge; Retamal, Claudio; Valenzuela, Juan C.; Soza, Andrea

    2010-01-01

    Endocytosis modulates EGFR function by compartmentalizing and attenuating or enhancing its ligand-induced signaling. Here we show that it can also control the cell surface versus intracellular distribution of empty/inactive EGFR. Our previous observation that PKA inhibitors induce EGFR internalization prompted us to test phosphatidic acid (PA) generated by phospholipase D (PLD) as an endogenous down-regulator of PKA activity, which activates rolipram-sensitive type 4 phosphodiesterases (PDE4) that degrade cAMP. We found that inhibition of PA hydrolysis by propranolol, in the absence of ligand, provokes internalization of inactive (neither tyrosine-phosphorylated nor ubiquitinated) EGFR, accompanied by a transient increase in PA levels and PDE4s activity. This EGFR internalization is mimicked by PA micelles and is strongly counteracted by PLD2 silencing, rolipram or forskolin treatment, and PKA overexpression. Accelerated EGFR endocytosis seems to be mediated by clathrin-dependent and -independent pathways, leading to receptor accumulation in juxtanuclear recycling endosomes, also due to a decreased recycling. Internalized EGFR can remain intracellular without degradation for several hours or return rapidly to the cell surface upon discontinuation of the stimulus. This novel regulatory mechanism of EGFR, also novel function of signaling PA, can transmodulate receptor accessibility in response to heterologous stimuli. PMID:20554760

  15. Saturated fatty acids induce c-Src clustering within membrane subdomains leading to JNK activation

    PubMed Central

    Holzer, Ryan G.; Park, Eek-Joong; Li, Ning; Tran, Helen; Chen, Monica; Choi, Crystal; Solinas, Giovanni; Karin, Michael

    2011-01-01

    Saturated fatty acids (FA) exert adverse health effects and are more likely to cause insulin resistance and type 2 diabetes than unsaturated FA, some of which exert protective and beneficial effects. Saturated FA, but not unsaturated FA, activate Jun N terminal kinase (JNK), which has been linked to obesity and insulin resistance in mice and men. However, it is unknown how saturated and unsaturated FA are discriminated. We now demonstrate that saturated FA activate JNK and induce insulin resistance by altering the membrane distribution of c-Src, causing it to partition into intracellular membrane subdomains where it may become activated. Conversely, unsaturated FA with known beneficial effects on glucose metabolism prevent c-Src membrane partitioning and activation, which are dependent on its myristoylation, and block JNK activation. Consumption of a diabetogenic high fat diet causes the partitioning and activation of c-Src within detergent insoluble membrane subdomains of adipocytes. PMID:21962514

  16. Butoxyacetic acid-induced hemolysis of rat red blood cells: effect of external osmolarity and cations.

    PubMed

    Udden, M M; Patton, C S

    2005-03-28

    Hemolysis is the principal toxicity of acute exposure to ethylene glycol monobutyl ether (EGBE) in rats. EGBE itself is not an active hemolytic agent, but its metabolite, butoxyacetic acid (BAA) formed as a result of dehydrogenase activity is a potent hemolysin. Here we address the role of osmolarity and cation composition of the suspending buffers in the mechanism of BAA-induced hemolysis of rat red blood cells in vitro. Rat erythrocytes were protected from BAA-induced cell swelling and hemolysis by the addition of sucrose to the suspending media. Hemolysis and cell swelling were also reduced by replacing external sodium with potassium. When calcium was not present in the suspending medium or when chelated by EGTA, hemolysis was increased after 2 h incubation with 1 mM or 2 mM BAA. Addition of as little as 0.05 mM CaCl(2) reduced hemolysis significantly while the addition of MgCl(2) had no effect. The dose-response relationship between BAA concentration and hemolysis determined in the presence or absence of calcium showed an increased effect of BAA in the absence of calcium. BAA-induced spherocytosis and cell fragmentation were more pronounced in the absence of calcium. The time course of BAA-induced hemolysis in the presence and absence of calcium demonstrated that the effect of calcium is to delay the onset of hemolysis. Increased intracellular calcium as a result of exposure to BAA was verified by atomic absorption spectroscopy. Charybdotoxin, an inhibitor of the calcium activated potassium channel, blocked the protective effect of calcium suggesting that the delay of onset of hemolysis in the presence of calcium is due to potassium loss caused by this channel. We conclude that the mode of action of BAA is to cause a colloid osmotic lysis of the rat red blood cell. Hemolysis requires external sodium and is associated with calcium uptake. Calcium appears to delay the onset of hemolysis. We speculate that: (1) BAA causes sodium and calcium to enter the cell; (2) calcium initially has a protective effect via the calcium activated potassium channel which facilitates the loss of potassium thereby, compensating for the osmotic effect of increased cell sodium; (3) calcium subsequently may have other deleterious effects through activation of proteases and externalization of phosphatidylserine in the exterior leaflet of the membrane.

  17. Protective Effect of Edaravone in Primary Cerebellar Granule Neurons against Iodoacetic Acid-Induced Cell Injury

    PubMed Central

    Zhou, Xinhua; Zhu, Longjun; Wang, Liang; Guo, Baojian; Zhang, Gaoxiao; Sun, Yewei; Zhang, Zaijun; Lee, Simon Ming-Yuen; Yu, Pei; Wang, Yuqiang

    2015-01-01

    Edaravone (EDA) is clinically used for treatment of acute ischemic stroke in Japan and China due to its potent free radical-scavenging effect. However, it has yet to be determined whether EDA can attenuate iodoacetic acid- (IAA-) induced neuronal death in vitro. In the present study, we investigated the effect of EDA on damage of IAA-induced primary cerebellar granule neurons (CGNs) and its possible underlying mechanisms. We found that EDA attenuated IAA-induced cell injury in CGNs. Moreover, EDA significantly reduced intracellular reactive oxidative stress production, loss of mitochondrial membrane potential, and caspase 3 activity induced by IAA. Taken together, EDA protected CGNs against IAA-induced neuronal damage, which may be attributed to its antiapoptotic and antioxidative activities. PMID:26557222

  18. Sulforaphane prevents quinolinic acid-induced mitochondrial dysfunction in rat striatum.

    PubMed

    Luis-García, Erika Rubí; Limón-Pacheco, Jorge Humberto; Serrano-García, Norma; Hernández-Pérez, Alma Delia; Pedraza-Chaverri, José; Orozco-Ibarra, Marisol

    2017-02-01

    Quinolinic acid (QA) triggers striatal neuronal death by an excitotoxic cascade that involves oxidative stress, which in turns is tightly linked to mitochondria. Mitochondrial dysfunction is a molecular feature described in several brain pathologies. In this work, we determined whether the sulforaphane-neuroprotective effect in the rodent experimental model of Huntington's disease induced by QA is associated with mitochondrial function preservation. We found that QA impaired mitochondrial function within 24 h post-lesion. Sulforaphane effectively disrupted the mitochondrial dysfunction by preventing the decrease in respiratory control ratio, transmembrane potential, ability to synthetize ATP, and the activity of mitochondrial complexes I, II, and IV.

  19. Ursolic acid improves domoic acid-induced cognitive deficits in mice

    SciTech Connect

    Wu, Dong-mei; Lu, Jun; Zhang, Yan-qiu; Zheng, Yuan-lin; Hu, Bin; Cheng, Wei; Zhang, Zi-feng; Li, Meng-qiu

    2013-09-01

    Our previous findings suggest that mitochondrial dysfunction is the mechanism underlying cognitive deficits induced by domoic acid (DA). Ursolic acid (UA), a natural triterpenoid compound, possesses many important biological functions. Evidence shows that UA can activate PI3K/Akt signaling and suppress Forkhead box protein O1 (FoxO1) activity. FoxO1 is an important regulator of mitochondrial function. Here we investigate whether FoxO1 is involved in the oxidative stress-induced mitochondrial dysfunction in DA-treated mice and whether UA inhibits DA-induced mitochondrial dysfunction and cognitive deficits through regulating the PI3K/Akt and FoxO1 signaling pathways. Our results showed that FoxO1 knockdown reversed the mitochondrial abnormalities and cognitive deficits induced by DA in mice through decreasing HO-1 expression. Mechanistically, FoxO1 activation was associated with oxidative stress-induced JNK activation and decrease of Akt phosphorylation. Moreover, UA attenuated the mitochondrial dysfunction and cognitive deficits through promoting Akt phosphorylation and FoxO1 nuclear exclusion in the hippocampus of DA-treated mice. LY294002, an inhibitor of PI3K/Akt signaling, significantly decreased Akt phosphorylation in the hippocampus of DA/UA mice, which weakened UA actions. These results suggest that UA could be recommended as a possible candidate for the prevention and therapy of cognitive deficits in excitotoxic brain disorders. - Highlights: • Ursolic acid (UA) is a naturally triterpenoid compound. • UA attenuated the mitochondrial dysfunction and cognitive deficits. • Mechanistically, UA activates PI3K/Akt signaling and suppresses FoxO1 activity. • UA could be recommended as a possible candidate for anti-excitotoxic brain disorders.

  20. Lithocholic acid induces endoplasmic reticulum stress, autophagy and mitochondrial dysfunction in human prostate cancer cells.

    PubMed

    Gafar, Ahmed A; Draz, Hossam M; Goldberg, Alexander A; Bashandy, Mohamed A; Bakry, Sayed; Khalifa, Mahmoud A; AbuShair, Walid; Titorenko, Vladimir I; Sanderson, J Thomas

    2016-01-01

    Lithocholic acid (LCA) is a secondary bile acid that is selectively toxic to human neuroblastoma, breast and prostate cancer cells, whilst sparing normal cells. We previously reported that LCA inhibited cell viability and proliferation and induced apoptosis and necrosis of androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cells. In the present study, we investigated the roles of endoplasmic reticulum (ER) stress, autophagy and mitochondrial dysfunction in the toxicity of LCA in PC-3 and autophagy deficient, androgen-independent DU-145 cells. LCA induced ER stress-related proteins, such as CCAAT-enhancer-binding protein homologous protein (CHOP), and the phosphorylation of eukaryotic initiation factor 2-alpha (p-eIF2α) and c-Jun N-terminal kinases (p-JNK) in both cancer cell-types. The p53 upregulated modulator of apoptosis (PUMA) and B cell lymphoma-like protein 11 (BIM) levels were decreased at overtly toxic LCA concentrations, although PUMA levels increased at lower LCA concentrations in both cell lines. LCA induced autophagy-related conversion of microtubule-associated proteins 1A/1B light chain 3B (LC3BI-LC3BII), and autophagy-related protein ATG5 in PC-3 cells, but not in autophagy-deficient DU-145 cells. LCA (>10 µM) increased levels of reactive oxygen species (ROS) concentration-dependently in PC-3 cells, whereas ROS levels were not affected in DU-145 cells. Salubrinal, an inhibitor of eIF2α dephosphorylation and ER stress, reduced LCA-induced CHOP levels slightly in PC-3, but not DU-145 cells. Salubrinal pre-treatment increased the cytotoxicity of LCA in PC-3 and DU-145 cells and resulted in a statistically significant loss of cell viability at normally non-toxic concentrations of LCA. The late-stage autophagy inhibitor bafilomycin A1 exacerbated LCA toxicity at subtoxic LCA concentrations in PC-3 cells. The antioxidant α-tocotrienol strongly inhibited the toxicity of LCA in PC-3 cells, but not in DU-145 cells. Collectively, although LCA induces autophagy and ER stress in PC-3 cells, these processes appear to be initially of protective nature and subsequently consequential to, but not critical for the ROS-mediated mitochondrial dysfunction and cytotoxicity of LCA. The full mechanism of LCA-induced mitochondrial dysfunction and cytotoxicity in the similarly sensitive DU-145 cells remains to be elucidated.

  1. Protective effect of glutathione on kainic acid-induced neuropathological changes in the rat brain.

    PubMed

    Saija, A; Princi, P; Pisani, A; Lanza, M; Scalese, M; Aramnejad, E; Ceserani, R; Costa, G

    1994-01-01

    1. Glutathione (GSH), injected by slow intravenous (i.v.) infusion (7.9 microliters/min, for 4 hr; total dose: 1.5 g/kg), starting 10 min after i.v. injection of kainic acid (KA; 12 mg/kg) in the rat reduced the decrease in local cerebral glucose utilization observed 48 hr following the administration of the neurotoxin. 2. Furthermore, it blocked the neuronal loss in hippocampal CA1 and CA3 regions, and prevented, in the hippocampus, the development of edema and the marked depletion in the endogenous brain GSH pool. 3. One can speculate that this protective effect of exogenous GSH is correlated to its capacity to scavenge free radicals, thus preventing the accumulation of oxidant chemical species and the consequent reduction of cellular antioxidant defense.