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Sample records for interleukin-12 p40 homodimer

  1. Diagnostic value of interleukin-12 p40 in tuberculous pleural effusions.

    PubMed

    Valdés, L; San José, E; Alvarez Dobaño, J M; Golpe, A; Valle, J M; Penela, P; González Barcala, F J

    2009-04-01

    The diagnosis of tuberculous pleural effusion (TBPE) is frequently problematic. Several markers of TBPE in pleural fluid have been evaluated, with different results. Pleural effusions from 96 patients were classified on the basis of definitive diagnosis as tuberculous (n = 39), neoplastic (n = 42) or parapneumonic (n = 15). Adenosine deaminase (ADA), ADA isoform ADA-2, interferon (IFN)-gamma, CD3(+)/DR(+) T-lymphocytes and interleukin (IL)-12 p40 were determined in all 96 effusions. The efficiency of IL-12 p40 for diagnosis of TBPEs was evaluated, in comparison with those of the other parameters, by comparing the areas under their receiver operating characteristics. With the threshold value of 550 pg.mL(-1), IL-12 p40 had a sensitivity of 92.3% (36 out of 39) and specificity of 70.2% (17 false positives). The misclassification rate of IL-12 p40 was significantly greater than those of ADA-2 and ADA. Among TBPEs, ADA correlated significantly with ADA-2, and IFN-gamma with ADA and IL-12 p40. Although tuberculous pleural effusions show values of interleukin-12 p40 that are significantly higher than neoplastic and parapneumonic fluids, this parameter is less efficient than adenosine deaminase, adenosine deaminase isoform 2 and interferon-gamma. Its routine determination is, accordingly, not justified.

  2. The Structure of Interleukin-23 Reveals in the Molecular Basis of P40 Subunit Sharing With Interleukin-12

    SciTech Connect

    Lupardus, P.J.; Garcia, K.C.

    2009-05-19

    Interleukin-23 is a recently identified member of the IL-12 family of heterodimeric cytokines that modulate subpopulations of T helper cells, and both IL-12 and IL-23 are attractive targets for therapy of autoimmune diseases. IL-23 is a binary complex of a four-helix bundle cytokine (p19) and a soluble class I cytokine receptor p40. IL-12 and IL-23 share p40 as an {alpha}-receptor subunit, yet show only 15% sequence homology between their four-helix cytokines p19 and p35, respectively, and signal through different combinations of shared receptors. In order to elucidate the structural basis of p40 sharing, we have determined a 2.3{angstrom} crystal structure of IL-23 for comparison to the previously determined structure of IL-12. The docking mode of p19 to p40 is altered compared to p35, deviating by a 'tilt' and 'roll' that results in an altered footprint of p40 on the A and D helices of the respective cytokines. Binding of p19 to p40 is mediated primarily by an Arginine residue on helix D of p19 that forms an extensive charge and hydrogen-bonding network with residues at the base of the pocket on p40. This 'Arginine pocket' is lined with an inner shell of hydrophobic interactions that are ringed by an outer shell of polar interactions. Comparative analysis indicates that the IL-23 and IL-12 complexes 'mimic' the network of interactions constituting the central Arginine pocket despite p19 and p35 having limited sequence homology. The majority of the structural epitopes in the two complexes are composed of unique p19 and p35 pair-wise contacts with common residues on p40. Thus, while the critical hotspot is maintained in the two complexes, the majority of the interfaces are structurally distinct and, therefore, provide a basis for the therapeutic targeting of IL-12 versus IL-23 heterodimer formation despite their use of a common receptor subunit.

  3. Cloning, promoter analysis and expression in response to bacterial exposure of sea bass (Dicentrarchus labrax L.) interleukin-12 p40 and p35 subunits.

    PubMed

    Nascimento, Diana S; do Vale, Ana; Tomás, Ana M; Zou, Jun; Secombes, Christopher J; dos Santos, Nuno M S

    2007-03-01

    Interleukin-12 (IL-12) is a heterodimeric cytokine pivotal in resistance to microbial and viral infections. In the search for immunoregulatory genes in sea bass the genes for the two IL-12 subunits p40 and p35 were cloned and sequenced. Molecular characterization of these two genes was performed at both the cDNA and genomic levels. Sea bass IL-12 p40 and p35 conserve most cysteines involved in the intra-chain disulfide bonds of human IL-12 subunits as well as the important structural residues for human IL-12 heterodimerization. The gene organization of sea bass IL-12 p40 is similar to the human orthologue, whilst the sea bass IL-12 p35 gene structure, as reported for pufferfish, differs from the human one in containing an additional exon and lacking a second copy of a duplicated exon present in the mammalian genes. The promoter analysis of both sea bass and pufferfish IL-12 genes showed the presence of the main cis-acting elements involved in the transcriptional regulation of human and mouse orthologues. The involvement of IL-12 in sea bass anti-bacterial immune responses was demonstrated by investigating the expression profiles of IL-1beta, IL-12 p40 and p35 in the head-kidney and spleen following intraperitoneal injection of UV-killed and live Photobacterium damselae ssp. piscicida (Phdp). Finally, the importance of nuclear factor (NF)-kappaB on UV-killed Phdp-induced IL-12 p40 and p35 gene transcription was shown by the use of pyrrolidine dithiocarbamate (PDTC).

  4. Ciclosporin A inhibits production of interleukin-12/23p40 and interleukin-23 by the human monocyte cell line, THP-1.

    PubMed

    Kamata, M; Tada, Y; Tatsuta, A; Kawashima, T; Shibata, S; Mitsui, H; Asano, Y; Sugaya, M; Kadono, T; Kanda, N; Watanabe, S; Sato, S

    2013-07-01

    Ciclosporin (Cs)A is an effective treatment for psoriasis. However, to date, the effect of CsA on the production of interleukins (ILs) is unknown. We investigated how CsA affects production of IL-12/23p40 and IL-23 production by the human monocyte cell line, THP-1, which is able to differentiate into macrophage-like cells or normal human keratinocytes (NHKs). THP-1 cells were preincubated with CsA, then stimulated with lipopolysaccharide (LPS), polyinosinic:polycytidylic acid or adenosine triphosphate. The levels of IL-12/23p40 and IL-23 released into the supernatant were assayed by ELISA. CsA significantly reduced both IL-12/23p40 and IL-23 production by LPS-stimulated THP-1 cells, but not in LPS-stimulated macrophage-like differentiated THP-1 cells. None of the stimuli used significantly induced either IL-12/23p40 or IL-23 production in NHKs. CsA inhibits not only IL-12/23p40 and IL-12p70, but also heterodimeric IL-23 production by human monocytes, which may be one possible mechanism for the therapeutic efficacy of CsA in psoriasis.

  5. Scavenger receptor for lipoteichoic acid is involved in the potent ability of Lactobacillus plantarum strain L-137 to stimulate production of interleukin-12p40.

    PubMed

    Hatano, Shinya; Hirose, Yoshitaka; Yamamoto, Yoshihiro; Murosaki, Shinji; Yoshikai, Yasunobu

    2015-04-01

    Heat-killed Lactobacillus plantarum strain L-137 (HK L-137) is a more potent inducer of interleukin (IL)-12 than other heat-killed Lactobacillus strains. To elucidate the mechanism involved in this IL-12p40 induction, we compared HK L-137 with heat-killed L. plantarum strain JCM1149 (HK JCM1149) by nuclear magnetic resonance and mass spectrometry. Results showed that HK L-137 contained lipoteichoic acid (LTA) with a chemical structure similar to that of JCM1149, except for a lower degree of glucosyl substitution in the poly(glycerol phosphate) backbone. Lysozyme sensitivity and electrophoretic moiety analysis revealed that HK L-137 exposed more LTA on its cell surface than HK JCM1149. Phagocytosis of HK L-137 by splenic adherent cells was significantly greater than that of HK JCM1149. Anti-LTA antibody and anti-scavenger receptor-A (SR-A) antibody selectively inhibited phagocytosis of HK L-137, as well as IL-12p40 production, by splenic adherent cells. Thus, a higher efficiency of phagocytosis of HK L-137 via SR-A for LTA is responsible for the potent IL-12p40 induction.

  6. Whole blood stimulation with Toll-like receptor (TLR)-7/8 and TLR-9 agonists induces interleukin-12p40 expression in plasmacytoid dendritic cells in rhesus macaques but not in humans.

    PubMed

    Koopman, G; Beenhakker, N; Burm, S; Bouwhuis, O; Bajramovic, J; Sommandas, V; Mudde, G; Mooij, P; 't Hart, B A; Bogers, W M J M

    2013-10-01

    Macaques provide important animal models in biomedical research into infectious and chronic inflammatory disease. Therefore, a proper understanding of the similarities and differences in immune function between macaques and humans is needed for adequate interpretation of the data and translation to the human situation. Dendritic cells are important as key regulators of innate and adaptive immune responses. Using a new whole blood assay we investigated functional characteristics of blood plasmacytoid dendritic cells (pDC), myeloid dendritic cells (mDC) and monocytes in rhesus macaques by studying induction of activation markers and cytokine expression upon Toll-like receptor (TLR) stimulation. In a head-to-head comparison we observed that rhesus macaque venous blood contained relatively lower numbers of pDC than human venous blood, while mDC and monocytes were present at similar percentages. In contrast to humans, pDC in rhesus macaques expressed the interleukin (IL)-12p40 subunit in response to TLR-7/8 as well as TLR-9 stimulation. Expression of IL-12p40 was confirmed by using different monoclonal antibodies and by reverse transcription-polymerase chain reaction (RT-PCR). Both in humans and rhesus macaques, TLR-4 stimulation induced IL-12p40 expression in mDC and monocytes, but not in pDC. The data show that, in contrast to humans, pDC in macaques are able to express IL-12p40, which could have consequences for evaluation of human vaccine candidates and viral infection.

  7. A protease-activated receptor 2 agonist (AC-264613) suppresses interferon regulatory factor 5 and decreases interleukin-12p40 production by lipopolysaccharide-stimulated macrophages: Role of p53.

    PubMed

    Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Yamaguchi, Yasuo

    2016-06-01

    The transcription factor interferon regulatory factor 5 (IRF5) has a key role in the production of interleukin (IL)-12 by macrophages. IRF5 is also a central mediator of toll-like receptor signaling and is a direct target of p53. Activation of protease-activated receptor 2 (PAR-2) upregulates p53 and suppresses apoptosis. This study investigated the influence of human neutrophil elastase (HNE) and PAR-2 agonists on expression of IRF5 and IL-12p40 by macrophages stimulated with lipopolysaccharide. Granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent macrophages showed upregulation of IRF5 expression, while HNE reduced expression of p53 and IRF5 in a concentration-dependent manner. HNE also caused a concentration-dependent decrease of IRF5 in macrophages transfected with small interfering RNA to silence p53, while silencing of β-arrestin 2 blunted the reduction of p53 or IRF5 by HNE. Incubation of macrophages with a PAR-2 agonist, AC-264613, caused a decrease of IRF5 expression and also significantly reduced p53 protein expression. HNE upregulated the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) and caused transactivation of TLR4, while AC-264613 did not promote TLR4 transactivation. In conclusion, the PAR-2 agonist AC-264613 attenuated IRF5-associated IL-12p40 production by macrophages.

  8. Association of interferon γ T+874A and interleukin 12 p40 promoter CTCTAA/GC polymorphism with the need for respiratory support and perinatal complications in low birthweight neonates

    PubMed Central

    Bokodi, G; Derzbach, L; Bányász, I; Tulassay, T; Vásárhelyi, B

    2007-01-01

    Background Data support the role of interferon (IFN)γ and interleukin (IL)12 in perinatal complications. IFNγ T+874A and IL12 p40 promoter CTCTAA/GC polymorphisms may have an effect on cytokine production. Methods DNA was extracted from dried blood samples of 153 low birthweight (LBW) infants and 172 healthy term infants. IFNγ and IL12 genetic polymorphisms were determined to investigate the association between polymorphisms and ventilation characteristics, bronchopulmonary dysplasia (BPD) and other perinatal disorders. Results The IFNγ+874A allele was over‐represented in LBW infants. Carriers of the IFNγ+874T allele required mechanical ventilation and oxygen supplementation for time periods 41% and 35%, respectively, shorter than those required by those not carrying the IFNγ+874T allele. Stepwise logistic regression analysis showed that carriers of the IFNγ+874T allele were protected against BPD (odds ratio (OR) 0.35 (95% confidence interval (CI) (0.12 to 0.99))) and patent ductus arteriosus (OR 0.43 (95% CI 0.19 to 0.97)), whereas carriers of the IFNγ+874A allele were at higher risk of severe hypotension (OR 3.40 (95% CI 1.01 to 11.52)) and respiratory distress syndrome (OR 4.03 (95% CI 1.30 to 12.50)). Carriers of the IL12 GC allele were protected against pneumonia (OR 0.32 (95% CI 0.14 to 0.75)). Carriers of the IL12 CTCTAA allele were at higher risk of developing necrotising enterocolitis (NEC; OR 2.37 (95% CI 1.01 to 5.53)). Conclusions Carrier state of the IFNγ+874A allele presents an increased risk for premature birth and lung damage, as well as other perinatal complications. The risks of pneumonia and NEC are higher in heterozygotic carriers of the IL12 CTCTAA/GC polymorphism. Further studies are needed to determine whether these associations are the result of altered cytokine‐producing capacity in infants carrying the tested alleles. PMID:16754651

  9. Interleukin-12 in infectious diseases.

    PubMed Central

    Romani, L; Puccetti, P; Bistoni, F

    1997-01-01

    Interleukin-12 (IL-12) is a potent immunoregulatory cytokine that is crucially involved in a wide range of infectious diseases. In several experimental models of bacterial, parasitic, viral, and fungal infection, endogenous IL-12 is required for early control of infection and for generation and perhaps maintenance of acquired protective immunity, directed by T helper type 1 (Th1) cells and mediated by phagocytes. Although the relative roles of IL-12 and gamma interferon in Th1-cell priming may be to a significant extent pathogen dependent, common to most infections is that IL-12 regulates the magnitude of the gamma interferon response at the initiation of infection, thus potentiating natural resistance, favoring Th1-cell development; and inhibiting Th2 responses. Treatment of animals with IL-12, either alone or as a vaccine adjuvant, has been shown to prevent disease by many of the same infectious agents, by stimulating innate resistance or promoting specific reactivity. Although IL-12 may enhance protective memory responses in vaccination or in combination with antimicrobial chemotherapy, it is yet unclear whether exogenous IL-12 can alter established responses in humans. Continued investigation into the possible application of IL-12 therapy to human infections is warranted by the role of the cytokine in inflammation, immunopathology, and autoimmunity. PMID:9336665

  10. Interleukin-12 induction of Th1 cytokines is important for viral clearance in chronic hepatitis B.

    PubMed Central

    Rossol, S; Marinos, G; Carucci, P; Singer, M V; Williams, R; Naoumov, N V

    1997-01-01

    Interleukin-12, a cytokine with an important role against intracellular pathogens, promotes Th1 cell development, cellmediated cytotoxicity, and interferon-gamma production. We investigated the immunoregulatory role of IL-12 in 72 chronic hepatitis B virus (HBV) carriers, 33 of whom were monitored longitudinally during interferon-alpha treatment. Serum levels of IL-12 heterodimer, IL-12 p40 subunit, IL-4, and Th1 cytokines were determined by specific ELISAs, and hepatitis B core antigen-specific T cell response by a proliferation assay. Chronic HBV carriers had higher serum levels of IL-12 and IL-12 p40 in comparison with controls (P < 0.01), suggesting that IL-12 production is not impaired. The longitudinal analysis revealed a further substantial increase (> 2.5x baseline level) of bioactive IL-12 and Th1 cytokines in patients who cleared HBV and seroconverted to anti- hepatitis B e, unlike the 23 nonresponders with persistent HBV replication (P < 0.01). The IL-12 peak followed the peak of hepatocytolysis by 9.8+/-2.8 wk and occurred either before or simultaneously with hepatitis B e seroconversion. Hepatitis B core antigen-specific T cell proliferation closely correlated with hepatocytolysis and increased significantly in all patients (8 responders and 15 nonresponders) who developed hepatitis flare, irrespective of the virological outcome. These results provide in vivo evidence that IL-12 may have an important role for viral clearance in chronic HBV infection. PMID:9185527

  11. Interleukin-12 (IL-12), but not IL-23, deficiency ameliorates viral encephalitis without affecting viral control.

    PubMed

    Kapil, Parul; Atkinson, Roscoe; Ramakrishna, Chandran; Cua, Daniel J; Bergmann, Cornelia C; Stohlman, Stephen A

    2009-06-01

    The relative contributions of interleukin-12 (IL-12) and IL-23 to viral pathogenesis have not been extensively studied. IL-12p40 mRNA rapidly increases after neurotropic coronavirus infection. Infection of mice defective in both IL-12 and IL-23 (p40(-/-)), in IL-12 alone (p35(-/-)), and in IL-23 alone (p19(-/-)) revealed that the symptoms of coronavirus-induced encephalitis are regulated by IL-12. IL-17-producing cells never exceeded background levels, supporting a redundant role of IL-23 in pathogenesis. Viral control, tropism, and demyelination were all similar in p35(-/-), p19(-/-), and wild-type mice. Reduced morbidity in infected IL-12 deficient mice was also not associated with altered recruitment or composition of inflammatory cells. However, gamma interferon (IFN-gamma) levels and virus-specific IFN-gamma-secreting CD4 and CD8 T cells were all reduced in the central nervous systems (CNS) of infected p35(-/-) mice. Transcription of the proinflammatory cytokines IL-1beta and IL-6, but not tumor necrosis factor, were initially reduced in infected p35(-/-) mice but increased to wild-type levels during peak inflammation. Furthermore, although transforming growth factor beta mRNA was not affected, IL-10 was increased in the CNS in the absence of IL-12. These data suggest that IL-12 does not contribute to antiviral function within the CNS but enhances morbidity associated with viral encephalitis by increasing the ratio of IFN-gamma to protective IL-10.

  12. Efficient plant-based production of chicken interleukin-12 yields a strong immunostimulatory cytokine.

    PubMed

    Medrano, Giuliana; Dolan, Maureen C; Stephens, Nathan T; McMickle, Anthony; Erf, Gisela; Radin, David; Cramer, Carole L

    2010-03-01

    Interleukin-12 (IL-12), an important immunomodulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anticancer therapeutic in mammals. Therapeutic strategies to develop mammalian IL-12 as a vaccine adjuvant/immunomodulator for promoting cellular immunity and establishing a Th1-biased immune response further support the potential value of ChIL-12. Transgenic plants show promise as scalable bioproduction platforms for challenging biopharmaceutical proteins. We have expressed, characterized, and purified biologically active ChIL-12 in plants using a rapid Agrobacterium-mediated tobacco plant-based transient expression system. To ensure the stoichiometric expression and assembly of p35 and p40, we expressed a single-chain version of chicken IL-12 (ChIL-12). A histidine 6x tag was used for identity and purification of ChIL-12(His) protein. Our results demonstrated precise cleavage of the endogenous chicken p40 signal peptide in plants as well as addition of N-linked glycans. Biological activity was confirmed in vitro by interferon-gamma secretion of ChIL-12-treated chicken splenocytes. In addition, splenocytes treated with ChIL-12 expressed with or without the His tag demonstrated comparable ChIFN-gamma induction. These studies indicate that plant-based platforms for bioproduction of complex pharmaceutical proteins produce functional ChIL-12 and provide key advantages in safety, scale, and cost-effective platform for veterinary vaccine and therapeutic applications.

  13. An engineered antibody-interleukin-12 fusion protein with enhanced tumor vascular targeting properties.

    PubMed

    Gafner, Verena; Trachsel, Eveline; Neri, Dario

    2006-11-01

    The antibody-mediated targeted delivery of interleukin-12 (IL12) to the EDB domain of fibronectin, a marker of angiogenesis, is a promising avenue for enhancing the therapeutic index of this anti-cancer cytokine. Previous experiments, based on sequential fusion of a single-chain IL12 derivative to the anti-EDB antibody fragment scFv(L19) had yielded a therapeutic fusion protein [IL12-scFv(L19)-FLAG], which displayed an impressive therapeutic activity in murine models of cancer, in spite of a tumor uptake, which was less efficient compared to the parental unmodified scFv(L19). In this article, we describe the comparative analysis of 3 recombinant fusion proteins comprising the scFv(L19) and IL12 moieties. One of them, in which the p40 and p35 form a covalent heterodimer and in which each subunit is fused to a molecule of scFv(L19), displays an excellent tumor targeting performance in vivo, as assessed by quantitative biodistribution analysis, and a potent anti-tumor effect, superior to the one of IL12-scFv(L19)-FLAG. These results may have a clinical impact, considering the fact that the tumor targeting ability of scFv(L19) in patients with cancer has been demonstrated using scintigraphic methods, and that 2 scFv(L19)-based antibody-cytokine fusion are currently entering clinical trials.

  14. Electrogene therapy with interleukin-12 in canine mast cell tumors

    PubMed Central

    Pavlin, Darja; Cemazar, Maja; Cör, Andrej; Sersa, Gregor; Pogacnik, Azra; Tozon, Natasa

    2011-01-01

    Background Mast cell tumors (MCT) are the most common malignant cutaneous tumors in dogs with extremely variable biological behaviour. Different treatment approaches can be used in canine cutaneous MCT, with surgical excision being the treatment of choice. In this study, electrogene therapy (EGT) as a new therapeutic approach to canine MCTs, was established. Materials and methods. Eight dogs with a total of eleven cutaneous MCTs were treated with intratumoral EGT using DNA plasmid encoding human interleukin-12 (IL-12). The local response to the therapy was evaluated by repeated measurements of tumor size and histological examination of treated tumors. A possible systemic response was assessed by determination of IL-12 and interferon- γ (IFN-γ) in patients’ sera. The occurence of side effects was monitored with weekly clinical examinations of treated animals and by performing basic bloodwork, consisting of the complete bloodcount and determination of selected biochemistry parameters. Results Intratumoral EGT with IL-12 elicits significant reduction of treated tumors’ size, ranging from 13% to 83% (median 50%) of the initial tumor volume. Additionally, a change in the histological structure of treated nodules was seen. There was a reduction in number of malignant mast cells and inflammatory cell infiltration of treated tumors. Systemic release of IL-12 in four patients was detected, without any noticeable local or systemic side effects. Conclusions These data suggest that intratumoral EGT with plasmid encoding IL-12 may be useful in the treatment of canine MCTs, exerting a local antitumor effect. PMID:22933932

  15. Selective suppression of interleukin-12 induction after macrophage receptor ligation.

    PubMed

    Sutterwala, F S; Noel, G J; Clynes, R; Mosser, D M

    1997-06-02

    Interleukin (IL)-12 is a monocyte- and macrophage-derived cytokine that plays a crucial role in both the innate and the acquired immune response. In this study, we examined the effects that ligating specific macrophage receptors had on the induction of IL-12 by lipopolysaccharide (LPS). We report that ligation of the macrophage Fcgamma, complement, or scavenger receptors inhibited the induction of IL-12 by LPS. Both mRNA synthesis and protein secretion were diminished to near-undetectable levels following receptor ligation. Suppression was specific to IL-12 since IL-10 and tumor necrosis factor-alpha (TNF-alpha) production were not inhibited by ligating macrophage receptors. The results of several different experimental approaches suggest that IL-12 downregulation was due to extracellular calcium influxes that resulted from receptor ligation. First, preventing extracellular calcium influxes, by performing the assays in EGTA, abrogated FcgammaR-mediated IL-12(p40) mRNA suppression. Second, exposure of macrophages to the calcium ionophores, ionomycin or A23187, mimicked receptor ligation and inhibited IL-12(p40) mRNA induction by LPS. Finally, bone marrow-derived macrophages from FcR gamma chain-deficient mice, which fail to flux calcium after receptor ligation, failed to inhibit IL-12(p40) mRNA induction. These results indicate that the calcium influxes that occur as a result of receptor ligation are responsible for inhibiting the induction of IL-12 by LPS. Hence, the ligation of phagocytic receptors on macrophages can lead to a dramatic decrease in IL-12 induction. This downregulation may be a way of limiting proinflammatory responses of macrophages to extracellular pathogens, or suppressing the development of cell-mediated immunity to intracellular pathogens.

  16. Interleukin-12 synthesis is a required step in trehalose dimycolate-induced activation of mouse peritoneal macrophages.

    PubMed Central

    Oswald, I P; Dozois, C M; Petit, J F; Lemaire, G

    1997-01-01

    Trehalose dimycolate (TDM), a glycolipid present in the cell wall of Mycobacterium spp., is a powerful immunostimulant. TDM primes murine macrophages (Mphi) to produce nitric oxide (NO) and to develop antitumoral activity upon activation with low doses of lipopolysaccharide (LPS). In this study, we investigated the ability of TDM to induce interleukin 12 (IL-12) and the role of this cytokine in TDM-induced activation of murine Mphi. RNA isolated from peritoneal exudate cells (PEC) collected at different times after TDM injection was used to determine IL-12 (p35 and p40 subunits) and gamma interferon (IFN-gamma) mRNA levels by semiquantitative reverse transcriptase-PCR. Constitutive expression of IL-12p35 was observed in PEC from untreated as well as from TDM-injected mice. In contrast, expression of the IL-12p40 subunit was almost undetectable in control PEC but was dramatically upregulated in PEC from TDM-injected mice. IL-12p40 expression peaked at 8 h and subsided to baseline levels at 39 h postinjection. TDM was also able to induce IFN-gamma expression; however, kinetics of induction of IFN-gamma was different from that of IL-12p40. Maximal levels of IFN-gamma mRNA were reached by 24 h and did not return to baseline by 4 days. In addition, pretreatment of mice with neutralizing monoclonal antibodies directed against IL-12 (C15.6.7 and C15.1.2) blocked IFN-gamma mRNA induction in PEC from TDM-treated mice. We further determined if the induction of IL-12 and/or IFN-gamma contributes to the in vivo priming effect of TDM on peritoneal Mphi. TDM-injected mice were treated in vivo with anti-IL-12 or anti-IFN-gamma (XMG.1.6) monoclonal antibodies. TDM-primed Mphi were then activated in vitro with LPS and tested for their ability to produce NO and to develop cytostatic activity toward cocultivated L1210 tumor cells. Priming of Mphi by TDM was completely blocked by in vivo neutralization of either IL-12 or IFN-gamma as demonstrated by an absence of tumoricidal activity

  17. A case of interleukin-12 receptor beta-1 deficiency with recurrent leishmaniasis.

    PubMed

    Sanal, Ozden; Turkkani, Gulten; Gumruk, Fatma; Yel, Leman; Secmeer, Gulten; Tezcan, Ilhan; Kara, Ates; Ersoy, Fugen

    2007-04-01

    Interleukin-12 receptor beta-1 (IL-12Rbeta1) defect is generally associated with selective susceptibility to weakly pathogenic mycobacteria and Salmonella species. Patients rarely experience infections caused by other organisms. We report a 5-year-old patient with IL-12Rbeta1 deficiency who developed recurrent visceral leishmaniasis 6 months apart. The patient responded to lyposomal amphotericin B treatment reasonably well.

  18. Successful therapy of chronic, nonhealing murine cutaneous leishmaniasis with sodium stibogluconate and gamma interferon depends on continued interleukin-12 production.

    PubMed Central

    Li, J; Sutterwala, S; Farrell, J P

    1997-01-01

    Treatment of nonhealing forms of human leishmaniasis with antimonial drugs in combination with gamma interferon (IFN-gamma) may promote healing more effectively than conventional drug therapy. Although the natures of immune responses in patients prior to treatment are often unclear, it is generally assumed that such therapy also promotes a switch from a Th2-type response to a dominant Th1-type response. We have examined the efficacy of IFN-gamma therapy, in combination with drug therapy, to promote healing and a Th2-to-Th1 switch in highly susceptible BALB/c mice infected with Leishmania major. Short-term treatment with the antileishmanial drug sodium stibogluconate failed to significantly alter the course of disease or the immune response when it was given during the third and fourth weeks of infection. IFN-gamma therapy, administered over the same time period, also failed to induce cure or a Th1 dominant response. In contrast, mice treated with a combination of drug and IFN-gamma therapy resolved their infections and developed Th1-type responses. However, administration of an antibody to interleukin 12 (IL-12) reversed the therapeutic effects of therapy with drug plus IFN-gamma, suggesting that IFN-gamma promotes cure through an IL-12-dependent mechanism. Analysis of mRNA levels within parasitized lesions suggests that drug treatment plus IFN-gamma treatment, in addition to reducing parasite numbers, results in reduced levels of IL-4, IL-10, and transforming growth factor beta transcripts but increased levels of transcripts of the p40 chain of IL-12 and inducible nitric oxide synthase, which catalyzes the production of nitric oxide. Together, these results suggest that such immunotherapy may promote the development of a protective Th1-type response in susceptible mice by a mechanism which involves both suppression of regulatory cytokines and enhancement of IL-12 and nitric oxide production. PMID:9234779

  19. Immunological mechanisms of intravesical chitosan/interleukin-12 immunotherapy against murine bladder cancer

    PubMed Central

    2017-01-01

    ABSTRACT There is a critical unmet clinical need for bladder cancer immunotherapies capable of inducing durable antitumor immunity. We have shown that four intravesical treatments with a simple co-formulation of interleukin-12 and the biopolymer chitosan not only destroy orthotopic bladder tumors, but also promote a potent long-lasting systemic immune response as evidenced through tumor-specific in vitro killing assays, complete protection from rechallenge, and abscopal antitumor responses at distant non-treated tumors. This study investigates the immunological kinetics underlying these results. We show through depletion studies that CD8+ T cells are required for initial tumor rejection, but CD4+ T cells protect against rechallenge. We also show that even a single intravesical treatment can eliminate tumors in 50% of mice with 6/9 and 7/8 mice eliminating tumors after three or four treatments respectively. We then performed immunophenotyping studies to analyze shifts in immune cell populations after each treatment within the tumor itself as well as in secondary lymphoid organs. These studies demonstrated an initial infiltration of macrophages and granulocytes followed by increased CD4+ and CD8+ effector-memory cells. This was coupled with a decreased level of regulatory T cells in peripheral lymph nodes as well as decreased myeloid-derived suppressor cell infiltration in the bladder. Taken together, these data demonstrate the ability of properly delivered interleukin-12-based therapies to engage adaptive immunity within the tumor itself as well as throughout the body and strengthen the case for clinical translation of chitosan/interleukin-12 as an intravesical treatment for bladder cancer. PMID:28197381

  20. Immunological mechanisms of intravesical chitosan/interleukin-12 immunotherapy against murine bladder cancer.

    PubMed

    Smith, Sean G; Baltz, John L; Koppolu, Bhanu Prasanth; Ravindranathan, Sruthi; Nguyen, Khue; Zaharoff, David A

    2017-01-01

    There is a critical unmet clinical need for bladder cancer immunotherapies capable of inducing durable antitumor immunity. We have shown that four intravesical treatments with a simple co-formulation of interleukin-12 and the biopolymer chitosan not only destroy orthotopic bladder tumors, but also promote a potent long-lasting systemic immune response as evidenced through tumor-specific in vitro killing assays, complete protection from rechallenge, and abscopal antitumor responses at distant non-treated tumors. This study investigates the immunological kinetics underlying these results. We show through depletion studies that CD8(+) T cells are required for initial tumor rejection, but CD4(+) T cells protect against rechallenge. We also show that even a single intravesical treatment can eliminate tumors in 50% of mice with 6/9 and 7/8 mice eliminating tumors after three or four treatments respectively. We then performed immunophenotyping studies to analyze shifts in immune cell populations after each treatment within the tumor itself as well as in secondary lymphoid organs. These studies demonstrated an initial infiltration of macrophages and granulocytes followed by increased CD4(+) and CD8(+) effector-memory cells. This was coupled with a decreased level of regulatory T cells in peripheral lymph nodes as well as decreased myeloid-derived suppressor cell infiltration in the bladder. Taken together, these data demonstrate the ability of properly delivered interleukin-12-based therapies to engage adaptive immunity within the tumor itself as well as throughout the body and strengthen the case for clinical translation of chitosan/interleukin-12 as an intravesical treatment for bladder cancer.

  1. Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag

    PubMed Central

    Jayanthi, Srinivas; Koppolu, Bhanu prasanth; Smith, Sean G.; Jalah, Rashmi; Bear, Jenifer; Rosati, Margherita; Pavlakis, George N.; Felber, Barbara K.; Zaharoff, David A.; Kumar, Thallapuranam Krishnaswamy Suresh

    2014-01-01

    Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immunity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration. In this context, we report a simple and effective heparin-affinity based purification of recombinant human IL-12 (hIL-12) from the serum-free supernatants of stable IL-12-transduced HEK293 cells. Fractionation of culture supernatants on heparin Sepharose columns revealed that hIL-12 elutes as a single peak in 500 mM NaCl. Coomassie staining and Western blot analysis showed that hIL-12 eluted in 500 mM NaCl is homogeneous.Purity of hIL-12 was ascertained by RP-HPLC and ESI-MS analysis, and found to be ~98%. Western blot analysis, using monoclonal antibodies, demonstrated that the crucial inter-subunit disulfide bond linking the p35 and p40 subunits is intact in the purified hIL-12. Results of far UV circular dichrosim, steady-state tryptophan fluorescence, and differential scanning calorimetry experiments suggest that purified hIL-12 is in its stable native conformation. Enzyme linked immunosorbent assays (ELISAs) and bioactivity studies demonstrate that hIL-12 is obtained in high yields (0.31 ± 0.05 mg/ mL of the culture medium) and is also fully bioactive. Isothermal titration calorimetry data show that IL-12 exhibits a moderate binding affinity (Kd(app) = 69 ± 1 μM) to heparin. The purification method described in this study is expected to provide greater impetus for research on the role of heparin in the regulation of the function of IL-12. In addition, the results of this study provide an avenue to obtain high amounts of IL-12 required for structural studies which are aimed at the development of novel IL-12-based therapeutics. PMID:25123642

  2. The SNARE VAMP7 Regulates Exocytic Trafficking of Interleukin-12 in Dendritic Cells

    PubMed Central

    Chiaruttini, Giulia; Piperno, Giulia M.; Jouve, Mabel; De Nardi, Francesca; Larghi, Paola; Peden, Andrew A.; Baj, Gabriele; Müller, Sabina; Valitutti, Salvatore; Galli, Thierry; Benvenuti, Federica

    2016-01-01

    Summary Interleukin-12 (IL-12), produced by dendritic cells in response to activation, is central to pathogen eradication and tumor rejection. The trafficking pathways controlling spatial distribution and intracellular transport of IL-12 vesicles to the cell surface are still unknown. Here, we show that intracellular IL-12 localizes in late endocytic vesicles marked by the SNARE VAMP7. Dendritic cells (DCs) from VAMP7-deficient mice are partially impaired in the multidirectional release of IL-12. Upon encounter with antigen-specific T cells, IL-12-containing vesicles rapidly redistribute at the immune synapse and release IL-12 in a process entirely dependent on VAMP7 expression. Consistently, acquisition of effector functions is reduced in T cells stimulated by VAMP7-null DCs. These results provide insights into IL-12 intracellular trafficking pathways and show that VAMP7-mediated release of IL-12 at the immune synapse is a mechanism to transmit innate signals to T cells. PMID:26972013

  3. Clinical Features of Candidiasis in Patients With Inherited Interleukin 12 Receptor β1 Deficiency

    PubMed Central

    Ouederni, Monia; Sanal, Ozden; Ikincioğullari, Aydan; Tezcan, Ilhan; Dogu, Figen; Sologuren, Ithaisa; Pedraza-Sánchez, Sigifredo; Keser, Melike; Tanir, Gonul; Nieuwhof, Chris; Colino, Elena; Kumararatne, Dinakantha; Levy, Jacov; Kutukculer, Necil; Aytekin, Caner; Herrera-Ramos, Estefanía; Bhatti, Micah; Karaca, Neslihan; Barbouche, Ridha; Broides, Arnon; Goudouris, Ekaterini; Franco, José Luis; Parvaneh, Nima; Reisli, Ismail; Strickler, Alexis; Shcherbina, Anna; Somer, Ayper; Segal, Anthony; Angel-Moreno, Alfonso; Lezana-Fernandez, José Luis; Bejaoui, Mohamed; Bobadilla-Del Valle, Miriam; Kachboura, Salem; Sentongo, Timothy; Ben-Mustapha, Imen; Bustamante, Jacinta; Picard, Capucine; Puel, Anne; Boisson-Dupuis, Stéphanie; Abel, Laurent; Casanova, Jean-Laurent; Rodríguez-Gallego, Carlos

    2014-01-01

    Background. Interleukin 12Rβ1 (IL-12Rβ1)–deficient patients are prone to clinical disease caused by mycobacteria, Salmonella, and other intramacrophagic pathogens, probably because of impaired interleukin 12–dependent interferon γ production. About 25% of patients also display mucocutaneous candidiasis, probably owing to impaired interleukin 23–dependent interleukin 17 immunity. The clinical features and outcome of candidiasis in these patients have not been described before, to our knowledge. We report here the clinical signs of candidiasis in 35 patients with IL-12Rβ1 deficiency. Results. Most (n = 71) of the 76 episodes of candidiasis were mucocutaneous. Isolated oropharyngeal candidiasis (OPC) was the most common presentation (59 episodes, 34 patients) and was recurrent or persistent in 26 patients. Esophageal candidiasis (n = 7) was associated with proven OPC in 2 episodes, and cutaneous candidiasis (n = 2) with OPC in 1 patient, whereas isolated vulvovaginal candidiasis (VVC; n = 3) was not. Five episodes of proven invasive candidiasis were documented in 4 patients; 1 of these episodes was community acquired in the absence of any other comorbid condition. The first episode of candidiasis occurred earlier in life (median age±standard deviation, 1.5 ± 7.87 years) than infections with environmental mycobacteria (4.29 ± 11.9 years), Mycobacterium tuberculosis (4 ± 3.12 years), or Salmonella species (4.58 ± 4.17 years) or other rare infections (3 ± 11.67 years). Candidiasis was the first documented infection in 19 of the 35 patients, despite the vaccination of 10 of these 19 patients with live bacille Calmette-Guérin. Conclusions. Patients who are deficient in IL-12Rβ1 may have candidiasis, usually mucocutaneous, which is frequently recurrent or persistent. Candidiasis may be the first clinical manifestation in these patients. PMID:24186907

  4. Regulation of IL-12p40 by HIF controls Th1/Th17 responses to prevent mucosal inflammation.

    PubMed

    Marks, E; Naudin, C; Nolan, G; Goggins, B J; Burns, G; Mateer, S W; Latimore, J K; Minahan, K; Plank, M; Foster, P S; Callister, R; Veysey, M; Walker, M M; Talley, N J; Radford-Smith, G; Keely, S

    2017-01-25

    Intestinal inflammatory lesions are inherently hypoxic, due to increased metabolic demands created by cellular infiltration and proliferation, and reduced oxygen supply due to vascular damage. Hypoxia stabilizes the transcription factor hypoxia-inducible factor-1α (HIF) leading to a coordinated induction of endogenously protective pathways. We identified IL12B as a HIF-regulated gene and aimed to define how the HIF-IL-12p40 axis influenced intestinal inflammation. Intestinal lamina propria lymphocytes (LPL) were characterized in wild-type and IL-12p40(-/-) murine colitis treated with vehicle or HIF-stabilizing prolyl-hydroxylase inhibitors (PHDi). IL12B promoter analysis was performed to examine hypoxia-responsive elements. Immunoblot analysis of murine and human LPL supernatants was performed to characterize the HIF/IL-12p40 signaling axis. We observed selective induction of IL-12p40 following PHDi-treatment, concurrent with suppression of Th1 and Th17 responses in murine colitis models. In the absence of IL-12p40, PHDi-treatment was ineffective. Analysis of the IL12B promoter identified canonical HIF-binding sites. HIF stabilization in LPLs resulted in production of IL-12p40 homodimer which was protective against colitis. The selective induction of IL-12p40 by HIF-1α leads to a suppression of mucosal Th1 and Th17 responses. This HIF-IL12p40 axis may represent an endogenously protective mechanism to limit the progression of chronic inflammation, shifting from pro-inflammatory IL-12p70 to an antagonistic IL-12p40 homodimer.Mucosal Immunology advance online publication, 25 January 2017; doi:10.1038/mi.2016.135.

  5. Combination electro-gene therapy using herpes virus thymidine kinase and interleukin-12 expression plasmids is highly efficient against murine carcinomas in vivo.

    PubMed

    Goto, Tomoaki; Nishi, Toru; Kobayashi, Osamu; Tamura, Takahiko; Dev, Sukhendu B; Takeshima, Hideo; Kochi, Masato; Kuratsu, Jun-ichi; Sakata, Tsuneaki; Ushio, Yukitaka

    2004-11-01

    We report the use of plasmid DNA-mediated combination gene therapy for tumor-bearing mice using in vivo electroporation, also called electro-gene therapy (EGT), that resulted in uncomplicated and complete cures in more than 90% of the mice. Subcutaneously inoculated CT26 tumors in syngeneic BALB/c mice were subjected to repeated EGT treatments consisting of intratumoral co-injection of naked plasmids encoding the cytokine interleukin-12 (IL-12) (p35 and p40 subunits) and the suicide gene herpes simplex virus thymidine kinase (HSV-tk), followed by in vivo electroporation. The early anti-tumor effect was always stronger, and the rate of cure, as seen in the long-term follow-up, was always greater in the groups treated with combination EGT than in those treated with IL-12 or HSV-tk EGT alone. Systemic levels of IL-12 and IFN-gamma increased in both combination and IL-12-alone EGT-treated groups. Moreover, combination EGT for established subcutaneous tumors strongly reduced hematogenous lung metastases and increased survival time when live CT26 tumor cells were injected through the tail vein. Limited experiments on C57/B16 mice with murine melanoma also showed very similar trends. These results suggest that this simple and safe method of plasmid-mediated combination EGT may provide a potentially effective gene therapy for cancer.

  6. Definition of polymorphisms and haplotypes in the interleukin-12B gene: association with IL-12 production but not with Crohn's disease.

    PubMed

    Zwiers, A; Seegers, D; Heijmans, R; Koch, A; Hampe, J; Nikolaus, S; Peña, A S; Schreiber, S; Bouma, G

    2004-12-01

    Interleukin-12 (IL-12) is a key cytokine for the induction of Th1 immune responses. Recently, functional polymorphisms in IL-12p40 (IL12B) were found to be associated with susceptibility to several autoimmune diseases. Similarly, variation in IL12B might be involved in susceptibility to Crohn's disease (CD), a chronic inflammatory bowel disorder associated with high IL-12 expression. We searched for additional polymorphism in IL12B and genotyped a large cohort of CD patients. Differential in vitro secretors of IL-12 were tested for polymorphism. Polymorphisms were analyzed using the intrafamilial transmission disequilibrium test (TDT) and by case-control analysis. A novel polymorphism was strongly associated with differential expression of IL-12. However, no association with susceptibility to CD was seen for this and other polymorphisms. The high level of conservation is consistent with the key regulatory role of IL-12. The lack of association with IL12B makes it unlikely that this gene is directly involved in the susceptibility to CD.

  7. Interleukin-12 Induces a Th1-Like Response to Burkholderia mallei and Limited Protection in BALB/c Mice

    DTIC Science & Technology

    2006-01-01

    dependent on the concentration of IL-12. Mahon et al. [21] demonstrated that IL-12 increased the efficacy of a Bordetella pertussis acellular vaccine...Interleukin-12 is pro- duced by macrophages in response to live or killed Bordetella per- tussis and enhances the efficacy of an acellular pertussis

  8. Interleukin-12 Induces a Th1-like Response to Burkholderia mallei and Limited Protection in BALB/c Mice

    DTIC Science & Technology

    2005-09-02

    which was dependent on the concentration of IL-12. Mahon et al. [21] demonstrated that IL-12 increased the efficacy of a Bordetella pertussis...Mills KHG. Interleukin-12 is pro- duced by macrophages in response to live or killed Bordetella per- tussis and enhances the efficacy of an acellular

  9. Cancer Therapeutic Based on T Cell Receptors Designed to Regiospecifically Release Interleukin-12 | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute's Surgery Branch is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize a potential cancer therapeutic based on T cells genetically engineered to express the human interleukin 12 (IL-12) cytokine only in the tumor environment.

  10. Interleukin-12 Preserves the Cutaneous Physical and Immunological Barrier after Radiation Exposure

    PubMed Central

    Gerber, Scott A.; Cummings, Ryan J.; Judge, Jennifer L.; Barlow, Margaret L.; Nanduri, Julee; Milano Johnson, Doug E.; Palis, James; Pentland, Alice P.; Lord, Edith M.; Ryan, Julie L.

    2015-01-01

    The United States continues to be a prime target for attack by terrorist organizations in which nuclear detonation and dispersal of radiological material are legitimate threats. Such attacks could have devastating consequences to large populations, in the form of radiation injury to various human organ systems. One of these at risk organs is the cutaneous system, which forms both a physical and immunological barrier to the surrounding environment and is particularly sensitive to ionizing radiation. Therefore, increased efforts to develop medical countermeasures for treatment of the deleterious effects of cutaneous radiation exposure are essential. Interleukin-12 (IL-12) was shown to elicit protective effects against radiation injury on radiosensitive systems such as the bone marrow and gastrointestinal tract. In this article, we examined if IL-12 could protect the cutaneous system from a combined radiation injury in the form of sublethal total body irradiation and beta-radiation burn (β-burn) directly to the skin. Combined radiation injury resulted in a breakdown in skin integrity as measured by transepidermal water loss, size of β-burn lesion and an exacerbated loss of surveillant cutaneous dendritic cells. Interestingly, intradermal administration of IL-12 48 h postirradiation reduced transepidermal water loss and burn size, as well as retention of cutaneous dendritic cells. Our data identify IL-12 as a potential mitigator of radiation-induced skin injury and argue for the further development of this cytokine as a radiation countermeasure. PMID:25564716

  11. Systemic interleukin 12 displays anti-tumour activity in the mouse central nervous system.

    PubMed Central

    Kishima, H.; Shimizu, K.; Miyao, Y.; Mabuchi, E.; Tamura, K.; Tamura, M.; Sasaki, M.; Hakakawa, T.

    1998-01-01

    In various systemic cancers, interleukin 12 (IL-12) induces anti-tumour immunity mediated by T lymphocytes and natural killer cells. To determine whether IL-12 has anti-tumour activity against malignant gliomas in the central nervous system (CNS), which is considered to be an immunologically privileged site, we treated mice with meningeal gliomatosis by intraperitoneal (i.p.) or intrathecal (i.t.) administration of recombinant murine IL-12. Although untreated mice revealed symptoms, such as body weight loss or paraplegia as a result of the meningeal gliomatosis within 8 days after tumour inoculation, 80% of the mice treated with IL-12 at 0.5 microg i.p. were cured. Many lymphocytes, mostly CD4+ and CD8+ cells, infiltrated to the tumours of IL-12-treated mice. The numbers of these cells increased in the cervical lymph nodes, into which the cerebrospinal fluid drains, and there they secreted a considerable amount of interferon-gamma. Mice cured by IL-12 rejected subcutaneous or i.t. rechallenge with their original glioma cells, but the same mice were not able to reject other syngeneic tumour cells. These results indicate that the immune system recognizes malignant glioma cells in the subarachnoid space of the CNS and that systemic IL-12 may produce effective anti-tumour activity and long-lasting tumour-specific immunity. Images Figure 1 Figure 4 PMID:9716025

  12. Interleukin-12- and interferon-gamma-mediated natural killer cell activation by Agaricus blazei Murill.

    PubMed

    Yuminamochi, Eri; Koike, Taisuke; Takeda, Kazuyoshi; Horiuchi, Isao; Okumura, Ko

    2007-06-01

    Dried fruiting bodies of Agaricus blazei Murill (A. blazei) and its extracts have generally used as complementary and alternative medicines (CAMs). Here, we report that the oral administration of A. blazei augmented cytotoxicity of natural killer (NK) cells in wild-type (WT) C57BL/6, C3H/HeJ, and BALB/c mice. Augmented cytotoxicity was demonstrated by purified NK cells from treated wild-type (WT) and RAG-2-deficient mice, but not from interferon-gamma (IFN-gamma) deficient mice. NK cell activation and IFN-gamma production was also observed in vitro when dendritic cell (DC)-rich splenocytes of WT mice were coincubation with an extract of A. blazei. Both parameters were largely inhibited by neutralizing anti-interleukin-12 (IL-12) monoclonal antibody (mAb) and completely inhibited when anti-IL-12 mAb and anti-IL-18 mAb were used in combination. An aqueous extract of the hemicellulase-digested compound of A. blazei particle; (ABPC) induced IFN-gamma production more effectively, and this was completely inhibited by anti-IL-12 mAb alone. NK cell cytotoxicty was augmented with the same extracts, again in an IL-12 and IFN-gamma-dependent manner. These results clearly demonstrated that A. blazei and ABPC augmented NK cell activation through IL-12-mediated IFN-gamma production.

  13. Bleomycin/interleukin-12 electrochemogene therapy for treating naturally occurring spontaneous neoplasms in dogs.

    PubMed

    Reed, S D; Fulmer, A; Buckholz, J; Zhang, B; Cutrera, J; Shiomitsu, K; Li, S

    2010-07-01

    On the basis of superior outcomes from electrochemogene therapy (ECGT) compared with electrochemotherapy in mice, we determined the efficacy of ECGT applied to spontaneous canine neoplasms. Intralesional bleomycin and feline interleukin-12 DNA (fIL-12 DNA) injection combined with translesional electroporation resulted in complete cure of two recurrent World Health Organization stage T(2b)N(0)M(0) oral squamous cell carcinomas (SCCs) and one T(2)N(0)M(0) acanthomatous ameloblastoma. Three remaining dogs, which had no other treatment options, had partial responses to ECGT; one had mandibular T(3b)N(2b)M(1) melanoma with pulmonary and lymph node metastases; one had cubital T(3)N(0)M(1) histiocytic sarcoma with spleen metastases; and one had soft palate T(3)N(0)M(0) fibrosarcoma. The melanoma dog had decrease in size of the primary tumor before recrudescence and euthanasia. The histiocytic sarcoma dog had resolution of the primary tumor, but was euthanized because of metastases 4 months after the only treatment. The dog with T(3)N(0)M(0) fibrosarcoma had tumor regression with recrudescence. Treatment was associated with minimal side effects and was easy to perform. It was associated with repair of bone lysis in cured dogs, it improved quality of life of dogs with partial responses and extended overall survival time. ECGT seems to be a safe and resulted in complete responses in SCC and acanthomatous ameloblastoma.

  14. Bleomycin/interleukin-12 electrochemogenetherapy for treating naturally occurring spontaneous neoplasms in dogs.

    PubMed

    Reed, S D; Fulmer, A; Buckholz, J; Zhang, B; Cutrera, J; Shiomitsu, K; Li, S

    2010-08-01

    On the basis of superior outcomes from electrochemogenetherapy (ECGT) compared with electrochemotherapy in mice, we determined the efficacy of ECGT applied to spontaneous canine neoplasms. Intralesional bleomycin (BLM) and feline interleukin-12 DNA injection combined with translesional electroporation resulted in complete cure of two recurrent World Health Organization stage T(2b)N(0)M(0) oral squamous cell carcinomas (SCCs) and one T(2)N(0)M(0) acanthomatous ameloblastoma. Three remaining dogs, which had no other treatment options, had partial responses to ECGT; one had mandibular T(3b)N(2b)M(1) melanoma with pulmonary and lymph node metastases; one had cubital T(3)N(0)M(1) histiocytic sarcoma with spleen metastases; and one had soft palate T(3)N(0)M(0) fibrosarcoma. The melanoma dog had decrease in the size of the primary tumor before recrudescence and euthanasia. The histiocytic sarcoma dog had resolution of the primary tumor, but was euthanized because of metastases 4 months after the only treatment. The dog with T(3)N(0)M(0) fibrosarcoma had tumor regression with recrudescence. Treatment was associated with minimal side effects and was easy to perform, was associated with repair of bone lysis in cured dogs, improved quality of life for dogs with partial responses and extended overall survival time. ECGT seems to be a safe and resulted in complete responses in SCC and acanthomatous ameloblastoma.

  15. Interleukin-12 reverses the inhibitory impact of photodynamic therapy (PDT) on the murine contact hypersensitivity response

    NASA Astrophysics Data System (ADS)

    Simkin, Guillermo O.; Levy, Julia G.; Hunt, David W. C.

    1998-05-01

    Treatment of mice with certain photosensitizers combined with exposure to visible light limits the development of the immunologically-mediated contact hypersensitivity (CHS) response against topically-applied chemical haptens. Understanding of the inhibitory action of photosensitizers upon the CHS response is incomplete. Benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin), a photosensitizer with immunomodulatory activity, strongly depressed CHS responses to the hapten dinitrofluorobenzene (DNFB). However, if mice were administered 1 (mu) g of a recombinant preparation of the pro- inflammatory cytokine interleukin-12 (rIL-12), full-fledged CHS responses to DNFB ensued in animals treated with BPD-MA and light. In contrast, when rIL-12 was given in combination with an anti-IL-12 antibody the restorative effect of rIL-12 on the CHS response of PDT-treated mice was blocked. Evaluation of the cytokine status of spleen and draining lymph node cells showed for DNFB painted animals, that the release of the immunosuppressive cytokine IL-10 was increased by PDT and rIL-12 counter-acted the increase in IL-10 liberation associated with PDT. These studies indicate that IL-10 formation is upregulated and the availability of IL-12 may be limited in mice treated with PDT. These features may contribute to deficient CHS responses observed with PDT.

  16. Neoadjuvant immunotherapy with chitosan and interleukin-12 to control breast cancer metastasis

    PubMed Central

    Vo, Jimmy LN; Yang, Lirong; Kurtz, Samantha L; Smith, Sean G; Koppolu, Bhanu prasanth; Ravindranathan, Sruthi; Zaharoff, David A

    2015-01-01

    Metastasis accounts for approximately 90% of breast cancer-related deaths. Therefore, novel approaches which prevent or control breast cancer metastases are of significant clinical interest. Interleukin-12 (IL-12)-based immunotherapies have shown promise in controlling metastatic disease, yet modest responses and severe toxicities due to systemic administration of IL-12 in early trials have hindered clinical application. We hypothesized that localized delivery of IL-12 co-formulated with chitosan (chitosan/IL-12) could elicit tumor-specific immunity and provide systemic protection against metastatic breast cancer while minimizing systemic toxicity. Chitosan is a biocompatible polysaccharide derived primarily from the exoskeletons of crustaceans. In a clinically relevant resection model, mice bearing spontaneously metastatic 4T1 mammary adenocarcinomas received intratumoral injections of chitosan/IL-12, or appropriate controls, prior to tumor resection. Neoadjuvant chitosan/IL-12 immunotherapy resulted in long-term tumor-free survival in 67% of mice compared to only 24% or 0% of mice treated with IL-12 alone or chitosan alone, respectively. Antitumor responses following chitosan/IL-12 treatment were durable and provided complete protection against rechallenge with 4T1, but not RENCA renal adenocarcinoma, cells. Lymphocytes from chitosan/IL-12-treated mice demonstrated robust tumor-specific lytic activity and interferon-γ production. Cell-mediated immune memory was confirmed in vivo via clinically relevant delayed-type hypersensitivity (DTH) assays. Comprehensive hematology and toxicology analyses revealed that chitosan/IL-12 induced transient, reversible leukopenia with no changes in critical organ function. Results of this study suggest that neoadjuvant chitosan/IL-12 immunotherapy prior to breast tumor resection is a promising translatable strategy capable of safely inducing to tumor-specific immunity and, in the long term, reducing breast cancer mortality due to

  17. Gene therapy based on interleukin-12 loaded chitosan nanoparticles in a mouse model of fibrosarcoma

    PubMed Central

    Soofiyani, Saiedeh Razi; Hallaj-Nezhadi, Somayeh; Lotfipour, Farzaneh; Hosseini, Akbar Mohammad; Baradaran, Behzad

    2016-01-01

    Objective(s): Interleukin-12 (IL-12) as a cytokine has been proved to have a critical role in stimulating the immune system and has been used as immunotherapeutic agents in cancer gene therapy. Chitosan as a polymer, with high ability of binding to nucleic acids is a good candidate for gene delivery since it is biodegradable, biocompatible and non-allergenic polysaccharide. The objective of the present study was to investigate the effects of cells transfected with IL-12 loaded chitosan nanoparticles on the regression of fibrosarcoma tumor cells (WEHI-164) in vivo. Materials and Methods: WEHI-164 tumor cells were transfected with IL-12 loaded chitosan nanoparticles and then were injected subcutaneously to inoculate tumor in BALB/c mice. Tumor volumes were determined and subsequently extracted after mice sacrifice. The immunohistochemistry staining was performed for analysis of Ki-67 expression (a tumor proliferation marker) in tumor masses. The expression of IL-12 and IFN-γ were studied using real-time polymerase chain reaction and immunoblotting. Results: The group treated with IL-12 loaded chitosan nanoparticles indicated decreasing of tumor mass[r1] volume (P<0.001). The results of western blotting and real-time PCR showed that the IL-12 expression was increased in the group. Immunohistochemistry staining indicated that the Ki-67expression was reduced in the group treated with IL-12 loaded chitosan nanoparticles. Conclusion: IL-12 gene therapy using chitosan nanoparticles has therapeutic effects on the regression of tumor masses in fibrosarcoma mouse model. PMID:27917281

  18. Anti-inflammatory cytokines in asthma and allergy: interleukin-10, interleukin-12, interferon-gamma.

    PubMed Central

    Chung, F

    2001-01-01

    Interleukin-10 (IL-10) is a cytokine derived from CD4+ T-helper type 2 (T(H2)) cells identified as a suppressor of cytokines from T-helper type 1(T(H1)) cells. Interleukin-12 (IL-12) is produced by B cells, macrophages and dendritic cells, and primarily regulates T(H1) cell differentiation, while suppressing the expansion of T(H2) cell clones. Interferon-gamma (IFN-gamma) is a product of T(H1) cells and exerts inhibitory effects on T(H2) cell differentiation. These cytokines have been implicated in the pathogenesis of asthma and allergies. In this context, IL-12 and IFN-gamma production in asthma have been found to be decreased, and this may reduce their capacity to inhibit IgE synthesis and allergic inflammation. IL-10 is a potent inhibitor of monocyte/macrophage function, suppressing the production of many pro-inflammatory cytokines. A relative underproduction of IL-10 from alveolar macrophages of atopic asthmatics has been reported. Therapeutic modulation of T(H1)/T(H2) imbalance in asthma and allergy by mycobacterial vaccine, specific immunotherapy and cytoline-guanosine dinucleotide motif may lead to increases in IL-12 and IFN-gamma production. Stimulation of IL-10 production by antigen-specific T-cells during immunotherapy may lead to anergy through inhibition of CD28-costimulatory molecule signalling by IL-10s anti-inflammatory effect on basophils, mast cells and eosinophils. PMID:11405550

  19. Mechanisms by Which Interleukin-12 Corrects Defective NK Cell Anticryptococcal Activity in HIV-Infected Patients

    PubMed Central

    Kyei, Stephen K.; Ogbomo, Henry; Li, ShuShun; Timm-McCann, Martina; Xiang, Richard F.; Huston, Shaunna M.; Ganguly, Anutosh; Colarusso, Pina; Gill, M. John

    2016-01-01

    ABSTRACT Cryptococcus neoformans is a pathogenic yeast and a leading cause of life-threatening meningitis in AIDS patients. Natural killer (NK) cells are important immune effector cells that directly recognize and kill C. neoformans via a perforin-dependent cytotoxic mechanism. We previously showed that NK cells from HIV-infected patients have aberrant anticryptococcal killing and that interleukin-12 (IL-12) restores the activity at least partially through restoration of NKp30. However, the mechanisms causing this defect or how IL-12 restores the function was unknown. By examining the sequential steps in NK cell killing of Cryptococcus, we found that NK cells from HIV-infected patients had defective binding of NK cells to C. neoformans. Moreover, those NK cells that bound to C. neoformans failed to polarize perforin-containing granules to the microbial synapse compared to healthy controls, suggesting that binding was insufficient to restore a defect in perforin polarization. We also identified lower expression of intracellular perforin and defective perforin release from NK cells of HIV-infected patients in response to C. neoformans. Importantly, treatment of NK cells from HIV-infected patients with IL-12 reversed the multiple defects in binding, granule polarization, perforin content, and perforin release and restored anticryptococcal activity. Thus, there are multiple defects in the cytolytic machinery of NK cells from HIV-infected patients, which cumulatively result in defective NK cell anticryptococcal activity, and each of these defects can be reversed with IL-12. PMID:27555306

  20. Logical Analysis of Regulation of Interleukin-12 Expression Pathway Regulation During HCV Infection.

    PubMed

    Farooqi, Zia-Ur-Rehman; Tareen, Samar H K; Ahmed, Jamil; Zaidi, Najam-Us-Sahar S

    2016-01-01

    Hepatitis C virus (HCV) triggers coordinated innate and adaptive response in host cell. HCV genome and proteins of the replicating virus are recognized as non-self-antigens by host cell to activate Toll Like Receptors (TLRs). Activated TLRs ultimately express cytokines, which can clear virus either by activating interferon (IFN), protein kinase C (PKC) and RNA Lase system or through activation of cytotoxic T-lymphocytes. Interleukin-12 (IL-12) is a potent antiviral cytokine, capable of clearing HCV by bridging both innate and adaptive antiviral immune response. Activation of TLR-4 on macrophages surface induces expression of IL-12 via NF-κB and AP-1 transcriptional pathway. After expression, IL- 12 releases IFN-γ, which activates anti-HCV cytotoxic lymphocytes. Conversely, in chronic HCV infection downregulation of IL-12 has been reported instead of by number of studies. Keeping in view of the above mentioned facts, this study was designed to evaluate HCV-core mediated down-regulation of IL-12 transcriptional pathway by employing a logical modeling approach based on the Ren´e Thomas formalism. The logical parameters of entities were estimated by using SMBioNet. The Logical model represents all possible dynamics of protein expression involved during course of HCV pathology. Results demonstrated that at chronic stage of infection, though TLR-4 was constantly active but yet it failed to express the NF-κB, AP-1, IL-12 and IFN-γ. This mechanism was indicative of incorporation of core mediated changes in IL-12 regulatory pathway. Moreover, results also indicate that HCV adopts different trajectories to accomplish the persistence of chronic phase of infection. It also implicated that human immune system tries to clear HCV but core is capable of inducing system oscillations to evade the immunity.

  1. Role of interferon-gamma in interleukin 12-induced pathology in mice.

    PubMed Central

    Car, B. D.; Eng, V. M.; Schnyder, B.; LeHir, M.; Shakhov, A. N.; Woerly, G.; Huang, S.; Aguet, M.; Anderson, T. D.; Ryffel, B.

    1995-01-01

    Interleukin 12 (IL-12) activates natural killer (NK) and T cells with the secondary synthesis and release of interferon-gamma (IFN-gamma) and other cytokines. IL-12-induced organ alterations are reported for mice and the pathogenetic role of IFN-gamma is investigated by the use of mice deficient in the IFN-gamma receptor (IFN-gamma R-/-). IL-12 caused a rapid infiltration of liver and splenic red pulp with activated macrophages; this and increased NK cells resulted in a fivefold increase of splenic weight in wild-type mice. Splenomegaly was associated with myelosuppression and decreasing peripheral leukocyte counts. IL-12-induced changes in wild-type mice were associated with markedly increased IFN-gamma serum levels and up-regulation of major histocompatibility complex (MHC) class I and II expression in various epithelia. IL-12 induced a qualitatively similar macrophage infiltration in IFN-gamma R-/- mice, less marked splenomegaly (to 2 x normal), and no MHC upregulation. Strikingly increased vascular endothelial intercellular adhesion molecule-1 expression was apparent in both IFN-gamma R-/- and IFN-gamma R+/+ mice. Restricted to mutant mice was a severe, invariably lethal, interstitial, and perivascular pulmonary macrophage infiltration with diffuse pulmonary edema. Extensive quantitative reverse transcriptase polymerase chain reaction analysis revealed an increase of only IL-6 and IL-10 pulmonary gene transcripts in IFN-gamma R-/- mice compared with wild-type mice. IL-12-induced myelosuppression is due to IFN-gamma-release from NK cells and T cells, and is associated with macrophage activation and distinct MHC class I and II antigen upregulation. The pulmonary pathology in IFN-gamma R-/- mice, however, reveals a toxic potential for IL-12 and suggests that endogenous IFN-gamma plays a protective role in preventing fatal pulmonary disease in these mice. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 Figure 8 Figure 9 PMID:7495294

  2. Interleukin-12 plasmid DNA delivery using l-thyroxine-conjugated polyethylenimine nanocarriers

    NASA Astrophysics Data System (ADS)

    Dehshahri, Ali; Sadeghpour, Hossein; Kazemi Oskuee, Reza; Fadaei, Mahin; Sabahi, Zahra; Alhashemi, Samira Hossaini; Mohazabieh, Erfaneh

    2014-05-01

    In this study, l-thyroxine was covalently grafted on 25 kDa branched polyethylenimine (PEI), and the ability of the nano-sized polyplexes for transferring plasmid encoding interleukin-12 (IL-12) gene was evaluated. As there are several problems in systemic administration of recombinant IL-12 protein, local expression of the plasmid encoding IL-12 gene inside the tumor tissue has been considered as an effective alternative approach. The l-thyroxine-conjugated PEI polyplexes were prepared using pUMVC3-hIL12 plasmid, and their transfection activity was determined in HepG2 human liver carcinoma and Neuro2A neuroblastoma cell lines. The polyplexes characterized in terms of DNA condensation ability, particle size, zeta potential, and buffering capacity as well as cytotoxicity and resistance to enzyme digestion. The results revealed that l-thyroxine conjugation of PEI increased gene transfer ability by up to two fold relative to unmodified 25 kDa PEI, the gold standard for non-viral gene delivery, with the highest increase occurring at degrees of conjugation around 10 %. pDNA condensation tests and dynamic light scattering measurements exhibited the ability of PEI conjugates to optimally condense the plasmid DNA into polyplexes in the size range around 200 nm. The modified polymers showed remarkable buffering capacity and protection against enzymatic degradation comparable to that of unmodified PEI. These results suggest that l-thyroxine conjugation of PEI is a simple modification strategy for future investigations aimed at developing a targeting gene vehicle.

  3. Leishmania promastigotes evade interleukin 12 (IL-12) induction by macrophages and stimulate a broad range of cytokines from CD4+ T cells during initiation of infection

    PubMed Central

    1994-01-01

    Leishmania major are intramacrophage parasites whose eradication requires the induction of T helper 1 (Th1) effector cells capable of activating macrophages to a microbicidal state. Interleukin 12 (IL-12) has been recently identified as a macrophage-derived cytokine capable of mediating Th1 effector cell development, and of markedly enhancing interferon gamma (IFN-gamma) production by T cells and natural killer cells. Infection of macrophages in vitro by promastigotes of L. major caused no induction of IL-12 p40 transcripts, whereas stimulation using heat-killed Listeria or bacterial lipopolysaccharide induced readily detectable IL-12 mRNA. Using a competitor construct to quantitate a number of transcripts, a kinetic analysis of cytokine induction during the first few days of infection by L. major was performed. All strains of mice examined, including susceptible BALB/c and resistant C57BL/6, B10.D2, and C3H/HeN, had the appearance of a CD4+ population in the draining lymph nodes that contained transcripts for IL-2, IL-4, and IFN- gamma (and in some cases, IL-10) that peaked 4 d after infection. In resistant mice, the transcripts for IL-2, IL-4, and IL-10 were subsequently downregulated, whereas in susceptible BALB/c mice, these transcripts were only slightly decreased, and IL-4 continued to be reexpressed at high levels. IL-12 transcripts were first detected in vivo by 7 d after infection, consistent with induction by intracellular amastigotes. Challenge of macrophages in vitro confirmed that amastigotes, in contrast to promastigotes, induced IL-12 p40 mRNA. Reexamination of the cytokine mRNA at 4 d revealed expression of IL-13 in all strains analyzed, suggesting that IL-2 and IL-13 may mediate the IL-12-independent production of IFN-gamma during the first days after infection. Leishmania have evolved to avoid inducing IL-12 from host macrophages during transmission from the insect vector, and cause a striking induction of mRNAs for IL-2, IL-4, IL-10, and IL-13 in

  4. Interleukin-12 inhibits pathological neovascularization in mouse model of oxygen-induced retinopathy

    PubMed Central

    Zhou, Yedi; Yoshida, Shigeo; Kubo, Yuki; Kobayashi, Yoshiyuki; Nakama, Takahito; Yamaguchi, Muneo; Ishikawa, Keijiro; Nakao, Shintaro; Ikeda, Yasuhiro; Ishibashi, Tatsuro; Sonoda, Koh-Hei

    2016-01-01

    Hypoxia-induced retinal neovascularization is a major pathological condition in many vision-threatening diseases. In the present study, we determined whether interleukin (IL)-12, a cytokine that regulates angiogenesis, plays a role in the neovascularization in a mouse model of oxygen-induced retinopathy (OIR). We found that the expressions of the mRNAs of both IL-12p35 and IL-12p40 were significantly reduced in the OIR retinas compared to that of the room air-raised control. The sizes of the avascular areas and neovascular tufts were larger in IL-12p40 knock-out (KO) mice than that in wild type (WT) mice. In addition, an intravitreal injection of recombinant IL-12 reduced both avascular areas and neovascular tufts. IL-12 injection enhanced the expressions of interferon-gamma (IFN-γ) and other downstream chemokines. In an in vitro system, IL-12 had no significant effect on tube formation of human retinal microvascular endothelial cells (HRECs). Moreover, a blockade of IFN-γ suppressed the inhibitory effect of IL-12 on pathological neovascularization. These results suggest that IL-12 plays important roles in inhibiting pathological retinal neovascularization. PMID:27312090

  5. Effect of recombinant interleukin-12 on murine skin regeneration and cell dynamics using in vivo multimodal microscopy

    PubMed Central

    Li, Joanne; Bower, Andrew J.; Vainstein, Vladimir; Gluzman-Poltorak, Zoya; Chaney, Eric J.; Marjanovic, Marina; Basile, Lena A.; Boppart, Stephen A.

    2015-01-01

    Interleukin-12 (IL-12) is a pro-inflammatory cytokine known for its role in immunity, and previous studies have shown that IL-12 provides mitigation of radiation injury. In this study, we utilize a multimodal microscopy system equipped with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM) to examine the effect of IL-12 on collagen structure and cellular metabolic activity in vivo during skin wound healing. This preliminary study illustrates the highly dynamic and heterogeneous in vivo microenvironment of the wounded skin. In addition, results suggest that IL-12 triggers a significantly more rapid and greater cellular metabolic response in the wounded animals. These results can elucidate insights into the response mechanism of IL-12 in both wound healing and acute radiation syndrome. PMID:26600994

  6. A Model to Explain How the Bacille Calmette Guérin (BCG) Vaccine Drives Interleukin-12 Production in Neonates.

    PubMed

    Kativhu, Chido Loveness; Libraty, Daniel H

    2016-01-01

    The Bacille Calmette Guérin (BCG) vaccine is the only routine vaccination at birth that effectively induces neonatal T-helper 1 (Th1)-polarized immune responses. The primary cytokine that drives CD4+ T-cell Th1 differentiation is interleukin (IL)-12 p70, a heterodimeric cytokine composed of the IL-12 p35 and IL-12 p40 subunits. We therefore examined the mechanisms involved in BCG vaccine stimulation of IL-12 p35 and p40 production from human umbilical cord (neonatal) cells. We found that BCG bacilli did not upregulate IL-12 p35 mRNA production, but upregulated IL-12 p40 mRNA production in a Toll-like receptor (TLR)2-dependent manner, in human neonatal monocyte-derived dendritic cells (mdDCs). The combination of TLR2 signaling, Type I interferon (IFN), and Type II IFN induced maximal levels of IL-12 p35 and p40 mRNA production in human neonatal mdDCs. The cell-free supernatants of reconstituted BCG vaccine vials contained extracellular mycobacterial (BCG) DNA which could induce IFN-α (Type I IFN) production in human neonatal plasmacytoid dendritic cells (pDCs). BCG bacilli also stimulated human neonatal CD16lo natural killer (NK) cells to produce IFN-γ (Type II IFN) in a TLR2-dependent manner. We have therefore proposed a model where BCG vaccine could stimulate the combination of neonatal conventional DCs (cDCs), pDCs, and CD16lo NK cells to produce optimal neonatal IL-12 p35 and p40 (IL-12 p70) production and subsequent CD4+ T-cell Th1 polarization. An adjuvant that emulates the mechanism by which the BCG vaccine stimulates neonatal IL-12 p35 and p40 production could improve vaccine strategies at birth for protection against intracellular pathogens and toxins.

  7. A Model to Explain How the Bacille Calmette Guérin (BCG) Vaccine Drives Interleukin-12 Production in Neonates

    PubMed Central

    Kativhu, Chido Loveness; Libraty, Daniel H.

    2016-01-01

    The Bacille Calmette Guérin (BCG) vaccine is the only routine vaccination at birth that effectively induces neonatal T-helper 1 (Th1)-polarized immune responses. The primary cytokine that drives CD4+ T-cell Th1 differentiation is interleukin (IL)-12 p70, a heterodimeric cytokine composed of the IL-12 p35 and IL-12 p40 subunits. We therefore examined the mechanisms involved in BCG vaccine stimulation of IL-12 p35 and p40 production from human umbilical cord (neonatal) cells. We found that BCG bacilli did not upregulate IL-12 p35 mRNA production, but upregulated IL-12 p40 mRNA production in a Toll-like receptor (TLR)2-dependent manner, in human neonatal monocyte-derived dendritic cells (mdDCs). The combination of TLR2 signaling, Type I interferon (IFN), and Type II IFN induced maximal levels of IL-12 p35 and p40 mRNA production in human neonatal mdDCs. The cell-free supernatants of reconstituted BCG vaccine vials contained extracellular mycobacterial (BCG) DNA which could induce IFN-α (Type I IFN) production in human neonatal plasmacytoid dendritic cells (pDCs). BCG bacilli also stimulated human neonatal CD16lo natural killer (NK) cells to produce IFN-γ (Type II IFN) in a TLR2-dependent manner. We have therefore proposed a model where BCG vaccine could stimulate the combination of neonatal conventional DCs (cDCs), pDCs, and CD16lo NK cells to produce optimal neonatal IL-12 p35 and p40 (IL-12 p70) production and subsequent CD4+ T-cell Th1 polarization. An adjuvant that emulates the mechanism by which the BCG vaccine stimulates neonatal IL-12 p35 and p40 production could improve vaccine strategies at birth for protection against intracellular pathogens and toxins. PMID:27571272

  8. Intranasal Administration of an Inactivated Yersinia pestis Vaccine with Interleukin-12 Generates Protective Immunity against Pneumonic Plague ▿ #

    PubMed Central

    Kumar, Devender; Kirimanjeswara, Girish; Metzger, Dennis W.

    2011-01-01

    Inhalation of Yersinia pestis causes pneumonic plague, which rapidly progresses to death. A previously licensed killed whole-cell vaccine is presently unavailable due to its reactogenicity and inconclusive evidence of efficacy. The present study now shows that vaccination intranasally (i.n.) with inactivated Y. pestis CO92 (iYp) adjuvanted with interleukin-12 (IL-12) followed by an i.n. challenge with a lethal dose of Y. pestis CO92 prevented bacterial colonization and protected 100% of mice from pneumonic plague. Survival of the vaccinated mice correlated with levels of systemic and lung antibodies, reduced pulmonary pathology and proinflammatory cytokines, and the presence of lung lymphoid cell aggregates. Protection against pneumonic plague was partially dependent upon Fc receptors and could be transferred to naïve mice with immune mouse serum. On the other hand, protection was not dependent upon complement, and following vaccination, depletion of CD4 and/or CD8 T cells before challenge did not affect survival. In summary, the results demonstrate the safety, immunogenicity, and protective efficacy of i.n. administered iYp plus IL-12 in a mouse model of pneumonic plague. PMID:21880856

  9. Tailor-made fibroblast-specific and antibiotic-free interleukin 12 plasmid for gene electrotransfer-mediated cancer immunotherapy.

    PubMed

    Kamensek, Urska; Tesic, Natasa; Sersa, Gregor; Kos, Spela; Cemazar, Maja

    2017-01-01

    Electrotransfer mediated delivery of interleukin-12 (IL-12) gene, encoded on a plasmid vector, has already been demonstrated to have a potent antitumor efficacy and great potential for clinical application. In the present study, our aim was to construct an optimized IL-12-encoding plasmid that is safe from the regulatory point of view. In light of previous studies demonstrating that IL-12 should be released in a tumor localized manner for optimal efficacy, the strong ubiquitous promoter was replaced with a weak endogenous promoter of the collagen 2 gene, which is specific for fibroblasts. Next, to comply with increasing regulatory demands for clinically used plasmids, the expression cassette was cloned in a plasmid lacking the antibiotic resistance gene. The constructed fibroblast-specific and antibiotic-free IL-12 plasmid was demonstrated to support low IL-12 expression after gene electrotransfer in selected cell lines. Furthermore, the removal of antibiotic resistance did not affect the plasmid expression profile and lowered its cytotoxicity. With optimal IL-12 expression and minimal transgene non-specific effects, i.e., low cytotoxicity, the constructed plasmid could be especially valuable for different modern immunological approaches to achieve localized boosting of the host's immune system.

  10. In vivo activity of plant-based interleukin-12 in the lung of Balb/c mouse

    PubMed Central

    2010-01-01

    Background In the last years, plants are being used for the production of a wide variety of biopharmaceuticals, including cytokines, and have the potential to serve as vehicles for mucosal administration of these molecules. We had previously reported the expression of a cytokine, interleukin-12 (IL-12), in transgenic tomato plants and had demonstrated that it retained its biologic activity in vitro. Findings In this work, we administered crude extracts of IL-12-containing tomato fruits to mice through the intratracheal route, measuring endogenous IL-12 and determining biologic activity by quantification of interferon-gamma (IFN-γ) in lungs and by histological analysis. IFN-γ expression in lungs, as well as histological analysis, indicate that tomato-expressed IL-12 retains its biologic activity and, most importantly, its effects are restricted to the site of administration. Conclusion Our results indicate that the functional activity of tomato-expressed IL-12 is comparable to that of commercial recombinant IL-12 when given via the mucosal route. This opens the possibility of using crude extracts prepared from tomatoes expressing IL-12 for certain immunotherapies. PMID:20507618

  11. Interleukin-12 and interleukin-2 alone or in combination against the infection in invasive pulmonary aspergillosis mouse model.

    PubMed

    Zhang, Chang-Ran; Lin, Jian-Cong; Xu, Wen-Ming; Li, Ming; Ye, Hui-Shao; Cui, Wei-Ling; Lin, Qing

    2013-03-01

    Aspergillus fumigatus is an intracellular opportunistic fungus causing invasive pulmonary mycosis, characterised by hyphal invasion and destruction of pulmonary tissue. Th1 cytokines could enhance fungicidal activity. The effects from the combination of interleukin-12 (IL-12) and IL-2 are rarely known in invasive pulmonary aspergillosis infection. To assess the cleaning of A. fumigatus infection in the pulmonary tissues by IL-12 and IL-2, interferon-γ (IFN-γ) was detected in the sera using ELISA, quantification of IFN-γ mRNA using real-time RT-PCR and lung Colony-forming unit was assayed by cultivation. Morphology was analysed by histopathological examination. Our results showed that IL-12 and/or IL-2 could enhance the IFN-γ expression in the pulmonary tissue, reduce the colony load in the pulmonary tissue and increase the survival rate of mouse. The combination of IL-12 and IL-2 could assist in increasing the IFN-γ expression in the pulmonary tissue, but neither reduce colony load in the pulmonary tissue nor increase the survival rate of mouse significantly. It was demonstrated that IL-12 and IL-2 were strong immunomodulatory cytokines as a prerequisite for protecting the host from infectious agents.

  12. Interleukin-12 and photocarcinogenesis

    SciTech Connect

    Katiyar, Santosh K.

    2007-11-01

    UV radiation induces immunosuppression and inflammatory responses, as well as oxidative stress and DNA damage, in skin cells and these various effects have been implicated in melanoma and nonmelanoma skin cancers, i.e., photocarcinogenesis. The cytokine interleukin (IL)-12 has been shown to possess potent antitumor activity in a wide variety of murine tumor models. In this review, we summarize the evidence that IL-12 plays a role in preventing photocarcinogenesis, and present a model of its possible mechanisms of action. Treatment of mice with IL-12 prevents UV-induced immunosuppression in a process mediated by repair of UV-induced damaged DNA. After exposure to the photocarcinogenesis protocol, the development of UV-induced tumors is more rapid and the tumor multiplicity and tumor size are significantly greater in IL-12-deficient or knockout (KO) mice than their wild-type counterparts. IL-12-deficiency in mice enhances the proliferation potential of tumor cells, and this may be one of the reasons for the rapid growth of the tumors and their greater size. The rate of malignant transformation of UV-induced papillomas to carcinomas also is higher in the IL-12 KO mice than in their wild-type counterparts in terms of carcinoma incidence and carcinoma multiplicity. UV-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) and sunburn cells is lower, or repaired more rapidly, in wild-type mice than IL-12 KO mice. The IL-12-associated reduction in UV-specific CPDs is due to induction of DNA repair, and particularly enhancement of nucleotide-excision repair. We suggest that endogenous stimulation of IL-12 may protect the skin from UV-induced immunosuppression, DNA damage, and, ultimately, the risk of photocarcinogenesis. Taken together, this information suggests that augmentation of IL-12 should be considered as a strategy for the prevention and treatment of photocarcinogenesis.

  13. Vector description of electric and hydrophobic interactions in protein homodimers.

    PubMed

    Mozo-Villarías, Angel; Cedano, Juan; Querol, Enrique

    2016-05-01

    This article describes the formation of homodimers from their constituting monomers, based on the rules set by a simple model of electric and hydrophobic interactions. These interactions are described in terms of the electric dipole moment (D) and hydrophobic moment vectors (H) of proteins. The distribution of angles formed by the two dipole moments of monomers constituting dimers were analysed, as well as the distribution of angles formed by the two hydrophobic moments. When these distributions were fitted to Gaussian curves, it was found that for biological dimers, the D vectors tend mostly to adopt a perpendicular arrangement with respect to each other, in which the constituting dipoles have the least interaction. A minor population tends towards an antiparallel arrangement implying maximum electric attraction. Also in biological dimers, the H vectors of most monomers tend to interact in such a way that the total hydrophobic moment of the dimer increases with respect to those of the monomers. This shows that hydrophobic moments have a tendency to align. In dimers originating in the crystallisation process, the distribution of angles formed by both hydrophobic and electric dipole moments appeared rather featureless, probably because of unspecific interactions in the crystallisation processes. The model does not describe direct interactions between H and D vectors although the distribution of angles formed by both vectors in dimers was analysed. It was found that in most cases these angles tended to be either small (both moments aligned parallel to each other) or large (antiparallel disposition).

  14. Interleukin-12 is critical for induction of nitric oxide-mediated immunosuppression following vaccination of mice with attenuated Salmonella typhimurium.

    PubMed

    Schwacha, M G; Eisenstein, T K

    1997-12-01

    Studies from our laboratory have shown that infection of mice with an attenuated strain of Salmonella typhimurium causes a marked suppression in the capacity of splenocytes to generate an in vitro plaque-forming cell (PFC) response to sheep erythrocytes. The suppression has been shown to be mediated by mature, adherent macrophages (Mphis) and nonadherent, precursor Mphis. Nitric oxide has been identified as the suppressor factor. The present study investigated the role of interleukin-12 (IL-12) in the generation of nitric oxide-mediated immunosuppression in this model. Salmonella inoculation resulted in marked suppression of PFC responses and high levels of nitrite production. When mice were treated with anti-IL-12 prior to inoculation, nitrite levels in splenocyte cultures were reduced by 75% and the suppression of PFC responses was prevented. The nonadherent splenocyte fraction from Salmonella-inoculated mice, which contains precursor Mphis and is weakly immunosuppressive, was treated with IL-12 in vitro. IL-12 augmented the capacity of this fraction to suppress PFC responses by normal splenocytes in a coculture system. Additionally, IL-12 induced nitrite and gamma interferon (IFN-gamma) production in a dose-dependent manner. Treatment with anti-IFN-gamma blocked nitrite production and suppression, indicating that IFN-gamma is an important intermediary in the pathway of IL-12-induced immunosuppression. These results indicate that IL-12 is critical for the induction of nitric oxide-mediated immunosuppression following S. typhimurium inoculation and, through its ability to stimulate IFN-gamma production, can induce nitric oxide-producing suppressor Mphis.

  15. Borna disease virus accelerates inflammation and disease associated with transgenic expression of interleukin-12 in the central nervous system.

    PubMed

    Freude, Susanna; Hausmann, Jürgen; Hofer, Markus; Pham-Mitchell, Ngan; Campbell, Iain L; Staeheli, Peter; Pagenstecher, Axel

    2002-12-01

    Targeted expression of biologically active interleukin-12 (IL-12) in astrocytes of the central nervous system (CNS) results in spontaneous neuroimmunological disease of aged mice. Borna disease virus (BDV) can readily multiply in the mouse CNS but does not trigger disease in most strains. Here we show that a large percentage of IL-12 transgenic mice developed severe ataxia within 5 to 10 weeks after infection with BDV. By contrast, no disease developed in mock-infected IL-12 transgenic and wild-type mice until 4 months of age. Neurological symptoms were rare in infected wild-type animals, and if they occurred, these were milder and appeared later. Histological analyses showed that the cerebellum of infected IL-12 transgenic mice, which is the brain region with strongest transgene expression, contained large numbers of CD4(+) and CD8(+) T cells as well as lower numbers of B cells, whereas other parts of the CNS showed only mild infiltration by lymphocytes. The cerebellum of diseased mice further showed severe astrogliosis, calcifications and signs of neurodegeneration. BDV antigen and nucleic acids were present in lower amounts in the inflamed cerebellum of infected transgenic mice than in the noninflamed cerebellum of infected wild-type littermates, suggesting that IL-12 or IL-12-induced cytokines exhibited antiviral activity. We propose that BDV infection accelerates the frequency by which immune cells such as lymphocytes and NK cells enter the CNS and then respond to IL-12 present in the local milieu causing disease. Our results illustrate that infection of the CNS with a virus that is benign in certain hosts can be harmful in such normally disease-resistant hosts if the tissue is unfavorably preconditioned by proinflammatory cytokines.

  16. Chemical shift imprint of intersubunit communication in a symmetric homodimer

    PubMed Central

    Falk, Bradley T.; Sapienza, Paul J.; Lee, Andrew L.

    2016-01-01

    Allosteric communication is critical for protein function and cellular homeostasis, and it can be exploited as a strategy for drug design. However, unlike many protein–ligand interactions, the structural basis for the long-range communication that underlies allostery is not well understood. This lack of understanding is most evident in the case of classical allostery, in which a binding event in one protomer is sensed by a second symmetric protomer. A primary reason why study of interdomain signaling is challenging in oligomeric proteins is the difficulty in characterizing intermediate, singly bound species. Here, we use an NMR approach to isolate and characterize a singly ligated state (“lig1”) of a homodimeric enzyme that is otherwise obscured by rapid exchange with apo and saturated forms. Mixed labeled dimers were prepared that simultaneously permit full population of the lig1 state and isotopic labeling of either protomer. Direct visualization of peaks from lig1 yielded site-specific ligand-state multiplets that provide a convenient format for assessing mechanisms of intersubunit communication from a variety of NMR measurements. We demonstrate this approach on thymidylate synthase from Escherichia coli, a homodimeric enzyme known to be half-the-sites reactive. Resolving the dUMP1 state shows that active site communication occurs not upon the first dUMP binding, but upon the second. Surprisingly, for many sites, dUMP1 peaks are found beyond the limits set by apo and dUMP2 peaks, indicating that binding the first dUMP pushes the enzyme ensemble to further conformational extremes than the apo or saturated forms. The approach used here should be generally applicable to homodimers. PMID:27466406

  17. Deprotonated Dicarboxylic Acid Homodimers: Hydrogen Bonds and Atmospheric Implications

    SciTech Connect

    Hou, Gao-Lei; Valiev, Marat; Wang, Xue-Bin

    2016-03-31

    Dicarboxylic acids represent an important class of water-soluble organic compounds found in the atmosphere. In this work we are studying properties of dicarboxylic acid homodimer complexes (HO2(CH2)nCO2-[HO2(CH2)nCO2H], n = 0-12), as potentially important intermediates in aerosol formation processes. Our approach is based on experimental data from negative ion photoelectron spectra of the dimer complexes combined with updated measurements of the corresponding monomer species. These results are analyzed with quantum-mechanical calculations, which provide further information about equilibrium structures, thermochemical parameters associated with the complex formation, and evaporation rates. We find that upon formation of the dimer complexes the electron binding energies increase by 1.3–1.7 eV (30.0–39.2 kcal/mol), indicating increased stability of the dimerized complexes. Calculations indicate that these dimer complexes are characterized by the presence of strong intermolecular hydrogen bonds with high binding energies and are thermodynamically favorable to form with low evaporation rates. Comparison with previously studied HSO4-[HO2(CH2)2CO2H] complex (J. Phys. Chem. Lett. 2013, 4, 779-785) shows that HO2(CH2)2CO2-[HO2(CH2)2CO2H] has very similar thermochemical properties. These results imply that dicarboxylic acids not only can contribute to the heterogeneous complexes formation involving sulfuric acid and dicarboxylic acids, but also can promote the formation of homogenous complexes by involving dicarboxylic acids themselves.

  18. Presentation of interleukin-12/-23 receptor beta1 deficiency with various clinical symptoms of Salmonella infections.

    PubMed

    Sanal, Ozden; Turul, Tuba; De Boer, Tijtske; Van de Vosse, Esther; Yalcin, Işik; Tezcan, Ilhan; Sun, Cağman; Memis, L; Ottenhoff, Tom H M; Ersoy, Fugen

    2006-01-01

    Clinical disease caused by weakly pathogenic mycobacterial species, Mycobacterium bovis Bacille Calmette-Guérin (BCG) and non-tuberculous environmental mycobacteria (EM), which is known as Mendelian susceptibility to mycobacterial disease (MSMD), is a rare entity defined recently. Infections with the more virulent Mycobacterium species, M. tuberculosis, may have largely gone unnoticed in these patients due to early death. Mutations in five proteins (IFNgammaR1, IFNgammaR2, IL-12/IL-23Rbeta1, IL-12/IL-23p40 and STAT1) have been found in MSMD. These patients are prone to surprisingly few other infectious diseases mainly to salmonellosis. Here we present three IL-12/IL-23Rbeta1 deficient patients from three different families and with different genetic mutations, who presented exclusively with Salmonella infections. Bacteremia and lymph node involvement were common clinical expressions. Leukocytoclastic vasculitis developed in one of these patients. Two patients were not inoculated with BCG, the third patient did not develop BCG infection although BCG vaccine had been given twice at ages of 1 and 7 years. All three patients responded well to antibiotic treatment. In conclusion, patients with chronic, recurrent or complicated Salmonella infections should be screened for MSMD, particularly for IL-12/IL-23p40/IL-12R/-23Rbeta1 deficiency. Conversely, in patients with genetic IL-12/-23Rbeta1 deficiency a full evaluation for Salmonella infection is required. IL-12/IL-23p40/IL-12R/IL-23Rbeta1 deficiency seem to be underdiagnosed in patients with salmonellosis, and since such patients need prolonged therapy, diagnosis is important.

  19. Intranasal Coadministration of Live Lactococci Producing Interleukin-12 and a Major Cow's Milk Allergen Inhibits Allergic Reaction in Mice▿

    PubMed Central

    Cortes-Perez, Naima G.; Ah-Leung, Sandrine; Bermúdez-Humarán, Luis G.; Corthier, Gérard; Wal, Jean-Michel; Langella, Philippe; Adel-Patient, Karine

    2007-01-01

    The Th1/Th2 balance deregulation toward a Th2 immune response plays a central role in allergy. We previously demonstrated that administration of recombinant Lactococcus lactis strains expressing bovine β-lactoglobulin (BLG), a major cow's milk allergen, partially prevents mice from sensitization. In the present study, we aimed to improve this preventive effect by coadministration of L. lactis BLG and a second recombinant L. lactis strain producing biologically active interleukin-12 (IL-12). This L. lactis strain producing IL-12 was previously used to enhance the Th1 immune response in a tumoral murine model (L. G. Bermúdez-Humarán et al., J. Immunol. 175:7297-7302, 2005). A comparison of the administration of either BLG alone or BLG in the presence of IL-12 was conducted. A BLG-specific primary Th1 immune response was observed only after intranasal coadministration of both L. lactis BLG and IL-12-producing L. lactis, as demonstrated by the induction of serum-specific immunoglobulin G2a (IgG2a) concomitant with gamma interferon secretion by splenocytes, confirming the adjuvanticity of IL-12-producing L. lactis. Immunized mice were further sensitized by intraperitoneal administration of purified BLG, and the allergic reaction was elicited by intranasal challenge with purified BLG. Mice pretreated with BLG in either the presence or the absence of IL-12 were rendered completely tolerant to further allergic sensitization and elicitation. Pretreatment with either L. lactis BLG or L. lactis BLG and IL-12-producing L. lactis induces specific anti-BLG IgG2a production in serum and bronchoalveolar lavage (BAL) fluid. Although specific serum IgE was not affected by these pretreatments, the levels of eosinophilia and IL-5 secretion in BAL fluid were significantly reduced after BLG challenge in the groups pretreated with L. lactis BLG and L. lactis BLG-IL-12-producing L. lactis, demonstrating a decreased allergic reaction. Our data demonstrate for the first time (i) the

  20. T cell-, interleukin-12-, and gamma interferon-driven viral clearance in measles virus-infected brain tissue.

    PubMed

    Stubblefield Park, Samantha R; Widness, Mi; Levine, Alan D; Patterson, Catherine E

    2011-04-01

    Genetic studies with immunocompetent mice show the importance of both T cells and gamma interferon (IFN-γ) for survival of a measles virus (MV) challenge; however, the direct role of T cells and IFN-γ within the MV-infected brain has not been addressed. Organotypic brain explants represent a successful ex vivo system to define central nervous system (CNS)-specific mechanisms of leukocyte migration, activation, and MV clearance. Within the heterogeneous, brain-derived, primed leukocyte population which reduced MV RNA levels in brain explants by 60%, CD3 T cells are the active antiviral cells, as purified CD3-positive cells are highly antiviral and CD3-negative leukocytes are unable to reduce the viral load. Neutralization of CCL5 and CXCL10 decreases leukocyte migration to areas of infection by 70%. However, despite chemokines directing the migration of T cells to infected neurons, chemokine neutralization revealed that migration is not required for viral clearance, suggesting a cytokine-mediated antiviral mechanism. In accordance with our hypothesis, the ability of leukocytes to clear the virus is abrogated when explants are treated with anti-IFN-γ neutralizing antibodies. IFN-γ applied to infected slices in the absence of primed leukocytes reduces the viral load by more than 80%; therefore, in brain tissue, IFN-γ is both necessary and sufficient to clear MV. Secretion of IFN-γ is stimulated by interleukin-12 (IL-12) in the brain, as neutralization of IL-12 results in loss of antiviral activity and stimulation of leukocytes with IL-12/IL-18 enhances their immune effector function of viral clearance. MV-primed leukocytes can reduce both West Nile and mouse hepatitis viral RNAs, indicating that cytokine-mediated viral clearance occurs in an antigen-independent manner. The IFN-γ signal is transduced within the brain explant by the Jak/STAT signaling pathway, as inhibition of Jak kinases results in a loss of antiviral activity driven by either brain

  1. Intranasal coadministration of live lactococci producing interleukin-12 and a major cow's milk allergen inhibits allergic reaction in mice.

    PubMed

    Cortes-Perez, Naima G; Ah-Leung, Sandrine; Bermúdez-Humarán, Luis G; Corthier, Gérard; Wal, Jean-Michel; Langella, Philippe; Adel-Patient, Karine

    2007-03-01

    The Th1/Th2 balance deregulation toward a Th2 immune response plays a central role in allergy. We previously demonstrated that administration of recombinant Lactococcus lactis strains expressing bovine beta-lactoglobulin (BLG), a major cow's milk allergen, partially prevents mice from sensitization. In the present study, we aimed to improve this preventive effect by coadministration of L. lactis BLG and a second recombinant L. lactis strain producing biologically active interleukin-12 (IL-12). This L. lactis strain producing IL-12 was previously used to enhance the Th1 immune response in a tumoral murine model (L. G. Bermúdez-Humarán et al., J. Immunol. 175:7297-7302, 2005). A comparison of the administration of either BLG alone or BLG in the presence of IL-12 was conducted. A BLG-specific primary Th1 immune response was observed only after intranasal coadministration of both L. lactis BLG and IL-12-producing L. lactis, as demonstrated by the induction of serum-specific immunoglobulin G2a (IgG2a) concomitant with gamma interferon secretion by splenocytes, confirming the adjuvanticity of IL-12-producing L. lactis. Immunized mice were further sensitized by intraperitoneal administration of purified BLG, and the allergic reaction was elicited by intranasal challenge with purified BLG. Mice pretreated with BLG in either the presence or the absence of IL-12 were rendered completely tolerant to further allergic sensitization and elicitation. Pretreatment with either L. lactis BLG or L. lactis BLG and IL-12-producing L. lactis induces specific anti-BLG IgG2a production in serum and bronchoalveolar lavage (BAL) fluid. Although specific serum IgE was not affected by these pretreatments, the levels of eosinophilia and IL-5 secretion in BAL fluid were significantly reduced after BLG challenge in the groups pretreated with L. lactis BLG and L. lactis BLG-IL-12-producing L. lactis, demonstrating a decreased allergic reaction. Our data demonstrate for the first time (i) the

  2. RNA of Enterococcus faecalis Strain EC-12 Is a Major Component Inducing Interleukin-12 Production from Human Monocytic Cells

    PubMed Central

    Nishibayashi, Ryoichiro; Inoue, Ryo; Harada, Yuri; Watanabe, Takumi; Makioka, Yuko; Ushida, Kazunari

    2015-01-01

    Interleukin-12 (IL-12) is an important cytokine for the immunomodulatory effects of lactic acid bacteria (LAB). Using murine immune cells, we previously reported that the RNA of Enterococcus faecalis EC-12, a LAB strain exerting probiotic-like beneficial effects, is the major IL-12-inducing immunogenic component. However, it was recently revealed that bacterial RNA can be a ligand for Toll-like receptor (TLR) 13, which is only expressed in mice. Because TLR13 is not expressed in humans, the immuno-stimulatory and -modulatory effects of LAB RNA in human cells should be augmented excluding TLR13 contribution. In experiment 1 of this study, the role of LAB RNA in IL-12 induction in human immune cells was studied using three LAB strains, E.faecalis EC-12, Lactobacillus gasseri JCM5344, and Bifidobacterium breve JCM1192. RNase A treatment of heat-killed LAB significantly decreased the IL-12 production of human peripheral blood mononuclear cells on stimulation, while RNase III treatment revealed virtually no effects. Further, IL-12 production against heat-killed E. faecalis EC-12 was abolished by depleting monocytes. These results demonstrated that single stranded RNA (ssRNA) of LAB is a strong inducer of IL-12 production from human monocytes. In experiment 2, major receptor for ssRNA of E. faecalis EC-12 was identified using THP-1 cells, a human monocytic cell line. The type of RNA molecules of E. faecalis EC-12 responsible for IL-12 induction was also identified. IL-12 production induced by the total RNA of E. faecalis EC-12 was significantly reduced by the treatment of siRNA for TLR8 but not for TLR7. Furthermore, both 23S and 16S rRNA, but not mRNA, of E. faecalis EC-12 markedly induced IL-12 production from THP-1 cells. These results suggested that the recognition of ssRNA of E. faecalis EC-12 was mediated by TLR8 and that rRNA was the RNA molecule that exhibited IL-12-inducing ability in human cells. PMID:26083838

  3. Non-heat pipe/P-40 Stirling engine

    NASA Technical Reports Server (NTRS)

    Haglund, R. A.

    1980-01-01

    The non-heat-pipe receiver/P-40 Stirling engine system design is described. A 25 kW direct-driven induction-type alternator will be mounted directly to the P-40 engine to produce to a 60 Hz, 115/230 volt output.

  4. Interleukin-12B gene polymorphism frequencies in Egyptians and sex-related susceptibility to hepatitis C infection.

    PubMed

    Youssef, Samar Samir; Abd El Aal, Asmaa Mostafa; Nasr, Amal Soliman; el Zanaty, Taher; Seif, Sameh Mohamed

    2013-08-01

    Hepatitis C virus (HCV) infection is a major health problem worldwide. Egypt is the country with the highest HCV infection epidemic in the world. Interleukin (IL)-12 is a cytokine that has been shown to have a potent role as an antiviral cytokine. IL-12 is a heterodimer of the polypeptides p35 and p40. IL-12 B, the gene encoding IL-12 p40, is polymorphic, and a functional single-nucleotide polymorphism (SNP) of the 3'-untranslated region at position rs3212227 was associated with apparent resistance to HCV. The genotype distribution of this polymorphism differs by race. This study is sought to identify the genotype distribution of the IL-12 SNP rs3212227 polymorphism in Egyptians and to assess its role in susceptibility to chronic HCV infection alone or in a sex-dependent way. The study included 238 subjects: 100 healthy controls and 138 patients with HCV infection. The IL-12 SNP rs3212227 was genotyped by the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). Results showed a genotype frequency of 46%, 39%, and 15% for AA, AC, and CC IL-12 genotypes, respectively. No significant result (P=0.5) was shown in the differential distribution of the IL-12 SNP genotypes between controls and patients with HCV infection. Nonetheless, this difference in the IL-12 genotype distribution was significant (0.005) when it was stratified according to sex; moreover, the C allele distribution in men and women differed with a statistically high significance (P=0.0001) in controls versus HCV patients. In conclusion, the IL-12 SNP rs3212227 polymorphism confers a susceptibility to HCV infection in a sex-dependent way in Egyptians.

  5. Relish2 mediates bursicon homodimer-induced prophylactic immunity in the mosquito Aedes aegypti.

    PubMed

    Zhang, Hongwei; Dong, Shengzhang; Chen, Xi; Stanley, David; Beerntsen, Brenda; Feng, Qili; Song, Qisheng

    2017-02-22

    Bursicon is a neuropeptide hormone consisting of two cystine-knot proteins (burs α and burs β), responsible for cuticle tanning and other developmental processes in insects. Recent studies show that each bursicon subunit forms homodimers that induce prophylactic immunity in Drosophila melanogaster. Here, we investigated the hypothesis that bursicon homodimers act in prophylactic immunity in insects, and possibly arthropods, generally, using the mosquito, Aedes aegypti. We found that burs α and burs β are expressed in larvae, pupae and newly emerged adults. Treating newly emerged Ae. aegypti and D. melanogaster adults with recombinant bursicon (r-bursicon) heterodimer led to cuticle tanning in both species. Treating larvae and adults with r-bursicon homodimers led to up-regulation of five anti-microbial peptide (AMP) genes, noting the possibility that bursicon heterodimers also lead to up-regulation of these genes can not been excluded. The induced AMPs effectively suppressed the growth of bacteria in vitro. RNAi knock-down of the transcriptional factor Relish2 abolished the influence of r-bursicon homodimers on AMP production. We infer the bursicon homodimers induce expression of AMP genes via Relish2 in Ae. aegypti, as prophylactic immunity to protect mosquitoes during the vulnerable stages of each molt.

  6. Relish2 mediates bursicon homodimer-induced prophylactic immunity in the mosquito Aedes aegypti

    PubMed Central

    Zhang, Hongwei; Dong, Shengzhang; Chen, Xi; Stanley, David; Beerntsen, Brenda; Feng, Qili; Song, Qisheng

    2017-01-01

    Bursicon is a neuropeptide hormone consisting of two cystine-knot proteins (burs α and burs β), responsible for cuticle tanning and other developmental processes in insects. Recent studies show that each bursicon subunit forms homodimers that induce prophylactic immunity in Drosophila melanogaster. Here, we investigated the hypothesis that bursicon homodimers act in prophylactic immunity in insects, and possibly arthropods, generally, using the mosquito, Aedes aegypti. We found that burs α and burs β are expressed in larvae, pupae and newly emerged adults. Treating newly emerged Ae. aegypti and D. melanogaster adults with recombinant bursicon (r-bursicon) heterodimer led to cuticle tanning in both species. Treating larvae and adults with r-bursicon homodimers led to up-regulation of five anti-microbial peptide (AMP) genes, noting the possibility that bursicon heterodimers also lead to up-regulation of these genes can not been excluded. The induced AMPs effectively suppressed the growth of bacteria in vitro. RNAi knock-down of the transcriptional factor Relish2 abolished the influence of r-bursicon homodimers on AMP production. We infer the bursicon homodimers induce expression of AMP genes via Relish2 in Ae. aegypti, as prophylactic immunity to protect mosquitoes during the vulnerable stages of each molt. PMID:28225068

  7. Brucella abortus as a potential vaccine candidate: induction of interleukin-12 secretion and enhanced B7.1 and B7.2 and intercellular adhesion molecule 1 surface expression in elutriated human monocytes stimulated by heat-inactivated B. abortus.

    PubMed Central

    Zaitseva, M; Golding, H; Manischewitz, J; Webb, D; Golding, B

    1996-01-01

    Development of a vaccine which is capable of generating a strong cellular immune response associated with gamma interferon (IFN-gamma) production and cytotoxic T-cell development requires that the immunogen be capable of inducing the secretion of interleukin-12 (IL-12), which is a pivotal factor for the differentiation of Th1 or Tc1 cells. We have previously shown that the heat-inactivated gram-negative bacterium Brucella abortus can induce IFN-gamma secretion by T cells. In the present study, we demonstrate that B. abortus and lipopolysaccharide (LPS) from B. abortus can induce IL-12 p40 mRNA expression and protein secretion by human elutriated monocytes (99% pure). p40 mRNA was detected within 4 h, and p40 protein could be measured at 24 h. This induction was abrogated by anti-CD14 monoclonal antibody, suggesting that monocytes recognize B. abortus via their receptor for LPS. The biological activity of IL-12 secreted by B. abortus-stimulated monocytes was demonstrated by its ability to upregulate IFN-gamma mRNA expression in T cells separated from monocytes and B. abortus by a transwell membrane. The B. abortus-induced IL-12 also enhanced NK cytolytic activity against K562 target cells. B. abortus was shown to rapidly increase the expression of the costimulatory molecules B7.1 and B7.2 and intercellular adhesion molecule 1 on human monocytes. Together, these data indicate that B. abortus can directly activate human monocytes and provide the cytokine milieu which would direct the immune response towards Th1-Tc1 differentiation. PMID:8757841

  8. Nicotinamidase/pyrazinamidase of Mycobacterium tuberculosis forms homo-dimers stabilized by disulfide bonds

    PubMed Central

    Rueda, Daniel; Sheen, Patricia; Gilman, Robert H.; Bueno, Carlos; Santos, Marco; Pando-Robles, Victoria; Batista, Cesar V.; Zimic, Mirko

    2014-01-01

    Recombinant wild-pyrazinamidase from H37Rv M. tuberculosis was analyzed by gel electrophoresis under differential reducing conditions to evaluate its quaternary structure. PZAse was fractionated by size exclusion chromatography under non-reducing conditions. PZAse activity was measured and mass spectrometry analysis was performed to determine the identity of proteins by de novo sequencing and to determine the presence of disulfide bonds. This study confirmed that M. tuberculosis wild type PZAse was able to form homo-dimers in vitro. Homo-dimers showed a slightly lower specific PZAse activity compared to monomeric PZAse. PZAse dimers were dissociated into monomers in response to reducing conditions. Mass spectrometry analysis confirmed the existence of disulfide bonds (C72-C138 and C138-C138) stabilizing the quaternary structure of the PZAse homo-dimer. PMID:25199451

  9. Effects of the Japanese Herbal Medicine “Sho-saiko-to” (TJ-9) on Interleukin-12 Production in Patients with HCV-Positive Liver Cirrhosis

    PubMed Central

    Nishimura, Akira; Huang, Xian-Xi; Nobori, Tsutomu; Sakaguchi, Seigo; Suzuki, Hiroyuki

    1999-01-01

    Interleukin-12 (IL-12) is an important cytokine for maintainence of normal systemic defense and bioregulation. The Japanese herbal medicine Sho-saiko-to (TJ-9) has been administered to 1.5 million Japanese patients with chronic liver diseases. TJ-9 is known to significantly suppress cancer development in the liver and has macrobiotic effects. In the present study, we examined the in vitro production of IL-12 by circulating mononuclear cells from liver cirrhosis patients and the effects of TJ-9 on IL-12 production. The monocyte/macrophage fraction and the lymphocyte fraction of peripheral blood were obtained from 11 HCV-positive liver cirrhosis patients and 12 healthy subjects. Interleukin-12 levels in the supernatants were measured using ELISA kits. The levels of IL-12 produced by the patients fractions were significantly lower than those produced by healthy subjects (p < 0.01, p < 0.05). However, when TJ-9 was added to the cultures, the IL-12 production levels in both cell fractions increased approximately three fold, and the levels from the monocyte/macrophage fraction were almost the same as those from healthy subjects. This effect of TJ-9 was attributable to two of its seven herb components, that is, scutellaria root and glycyrrhiza root. One possible mechanism for the macrobiotic effects of TJ-9 on liver cirrhosis patients may be the improvement in IL-12 production. PMID:10636475

  10. Phosphorylated nuclear receptor CAR forms a homodimer to repress its constitutive activity for ligand activation.

    PubMed

    Shizu, Ryota; Osabe, Makoto; Perera, Lalith; Moore, Rick; Sueyoshi, Tatsuya; Negishi, Masahiko

    2017-03-06

    Nuclear receptor CAR (NR1I3) regulates hepatic drug and energy metabolism as well as cell fate. Its activation can be a critical factor in drug-induced toxicity and disease development such as diabetes and tumors. CAR inactivates its constitutive activity by phosphorylation at threonine 38. Utilizing receptor for protein kinase 1 (RACK1) as the regulatory subunit, protein phosphatase PP2A dephosphorylates threonine 38 to activate CAR. Here we have demonstrated that CAR undergoes its homodimer-monomer conversion to regulate this dephosphorylation. By co-expressing two differently-tagged CAR proteins in Huh-7 cells, mouse primary hepatocytes and mouse livers, co-immunoprecipitation and two-dimensional gel electrophoresis revealed that CAR can form a homodimer in a configuration in which the PP2A/RACK1 binding site is buried within its dimer interface. Epidermal growth factor (EGF) was found to stimulate CAR homo-dimerization, thus constraining CAR in its inactive form. The agonistic ligand CITCO binds directly to the CAR homodimer and dissociates phosphorylated CAR into its monomer, exposing the PP2A/RACK1 binding site for dephosphorylation. Phenobarbital, which is not a CAR ligand, binds the EGF receptor, reversing the EGF signal to monomerize CAR for its indirect activation. Thus, the homodimer-monomer conversion is the underlying molecular mechanism that regulates CAR activation, by placing phosphorylated threonine 38 as the common target for both direct and in direct activation of CAR.

  11. A Set of Computationally Designed Orthogonal Antiparallel Homodimers that Expands the Synthetic Coiled-Coil Toolkit

    PubMed Central

    2015-01-01

    Molecular engineering of protein assemblies, including the fabrication of nanostructures and synthetic signaling pathways, relies on the availability of modular parts that can be combined to give different structures and functions. Currently, a limited number of well-characterized protein interaction components are available. Coiled-coil interaction modules have been demonstrated to be useful for biomolecular design, and many parallel homodimers and heterodimers are available in the coiled-coil toolkit. In this work, we sought to design a set of orthogonal antiparallel homodimeric coiled coils using a computational approach. There are very few antiparallel homodimers described in the literature, and none have been measured for cross-reactivity. We tested the ability of the distance-dependent statistical potential DFIRE to predict orientation preferences for coiled-coil dimers of known structure. The DFIRE model was then combined with the CLASSY multistate protein design framework to engineer sets of three orthogonal antiparallel homodimeric coiled coils. Experimental measurements confirmed the successful design of three peptides that preferentially formed antiparallel homodimers that, furthermore, did not interact with one additional previously reported antiparallel homodimer. Two designed peptides that formed higher-order structures suggest how future design protocols could be improved. The successful designs represent a significant expansion of the existing protein-interaction toolbox for molecular engineers. PMID:25337788

  12. Structures of the Yeast Ribonucleotide Reductase Rnr2 and Rnr4 Homodimers

    SciTech Connect

    Sommerhalter, M.; Voegtli, W.C.; Perlstein, D.L.; Ge, J.; Stubbe, J.; Rosenzweig, A.C.

    2010-03-08

    Class I ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides. Eukaryotic RNRs comprise two subunits, the R1 subunit, which contains substrate and allosteric effector binding sites, and the R2 subunit, which houses a catalytically essential diiron-tyrosyl radical cofactor. In Saccharomyces cerevisiae, there are two variants of the R2 subunit, called Rnr2 and Rnr4. Rnr4 is unique in that it lacks three iron-binding residues conserved in all other R2s. Nevertheless, Rnr4 is required to activate Rnr2, and the functional species in vivo is believed to be a heterodimeric complex between the two proteins. The crystal structures of the Rnr2 and Rnr4 homodimers have been determined and are compared to that of the heterodimer. The homodimers are very similar to the heterodimer and to mouse R2 in overall fold, but there are several key differences. In the Rnr2 homodimer, one of the iron-binding helices, helix {alpha}B, is not well-ordered. In the heterodimer, interactions with a loop region connecting Rnr4 helices {alpha}A and {alpha}3 stabilize this Rnr2 helix, which donates iron ligand Asp 145. Sequence differences between Rnr2 and Rnr4 prevent the same interactions from occurring in the Rnr2 homodimer. These findings provide a structural rationale for why the heterodimer is the preferred complex in vivo. The active-site region in the Rnr4 homodimer reveals interactions not apparent in the heterodimer, supporting previous conclusions that this subunit does not bind iron. When taken together, these results support a model in which Rnr4 stabilizes Rnr2 for cofactor assembly and activity.

  13. From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle

    PubMed Central

    Gresock, Michael G.; Kastead, Kyle A.

    2015-01-01

    ABSTRACT The TonB system actively transports large, scarce, and important nutrients through outer membrane (OM) transporters of Gram-negative bacteria using the proton gradient of the cytoplasmic membrane (CM). In Escherichia coli, the CM proteins ExbB and ExbD harness and transfer proton motive force energy to the CM protein TonB, which spans the periplasmic space and cyclically binds OM transporters. TonB has two activity domains: the amino-terminal transmembrane domain with residue H20 and the periplasmic carboxy terminus, through which it binds to OM transporters. TonB is inactivated by all substitutions at residue H20 except H20N. Here, we show that while TonB trapped as a homodimer through its amino-terminal domain retained full activity, trapping TonB through its carboxy terminus inactivated it by preventing conformational changes needed for interaction with OM transporters. Surprisingly, inactive TonB H20A had little effect on homodimerization through the amino terminus and instead decreased TonB carboxy-terminal homodimer formation prior to reinitiation of an energy transduction cycle. That result suggested that the TonB carboxy terminus ultimately interacts with OM transporters as a monomer. Our findings also suggested the existence of a separate equimolar pool of ExbD homodimers that are not in contact with TonB. A model is proposed where interaction of TonB homodimers with ExbD homodimers initiates the energy transduction cycle, and, ultimately, the ExbD carboxy terminus modulates interactions of a monomeric TonB carboxy terminus with OM transporters. After TonB exchanges its interaction with ExbD for interaction with a transporter, ExbD homodimers undergo a separate cycle needed to re-energize them. IMPORTANCE Canonical mechanisms of active transport across cytoplasmic membranes employ ion gradients or hydrolysis of ATP for energy. Gram-negative bacterial outer membranes lack these resources. The TonB system embodies a novel means of active transport

  14. Encapsulation and Characterization of Proton-Bound Amine Homodimers in a Water Soluble, Self-Assembled Supramolecular Host

    SciTech Connect

    Pluth, Michael; Fiedler, Dorothea; Mugridge, Jeffrey; Bergman, Robert; Raymond, Kenneth

    2008-10-01

    Cyclic amines can be encapsulated in a water-soluble self-assembled supramolecular host upon protonation. The hydrogen bonding ability of the cyclic amines, as well as the reduced degrees of rotational freedom, allows for the formation of proton-bound homodimers inside of the assembly which are otherwise not observable in aqueous solution. The generality of homodimer formation was explored with small N-alkyl aziridines, azetidines, pyrrolidines and piperidines. Proton-bound homodimer formation is observed for N-alkylaziridines (R = methyl, isopropyl, tert-butyl), N-alkylazetidines (R = isopropyl, tertbutyl), and N-methylpyrrolidine. At high concentration, formation of a proton-bound homotrimer is observed in the case of N-methylaziridine. The homodimers stay intact inside the assembly over a large concentration range, thereby suggesting cooperative encapsulation. Both G3(MP2)B3 and G3B3 calculations of the proton-bound homodimers were used to investigate the enthalpy of the hydrogen bond in the proton-bound homodimers and suggest that the enthalpic gain upon formation of the proton-bound homodimers may drive guest encapsulation.

  15. HorC, a hop-resistance related protein, presumably functions in homodimer form.

    PubMed

    Iijima, Kazumaru; Suzuki, Koji; Asano, Shizuka; Ogata, Tomoo; Kitagawa, Yasushi

    2009-08-01

    To determine whether two HorC molecules coordinately form a single unit, the functional properties of covalently linked dimers of HorC encoded by tandemly fused horC genes were studied. Lactobacillus brevis introduced with the fused horC genes and a single horC gene exhibited same degree of resistance to hop compounds and cetyltrimethylammonium bromide. This suggests that HorC functions as a homodimer.

  16. Modulation of the FGF14:FGF14 homodimer interaction through short peptide fragments

    PubMed Central

    Ali, Syed; Shavkunov, Alexander; Panova, Neli; Stoilova-McPhie, Svetla; Laezza, Fernanda

    2016-01-01

    Fibroblast growth factor 14 (FGF14) is a member of the intracellular FGF (iFGFs) family and a functionally relevant component of the neuronal voltage-gated Na+ (Nav) channel complex. Through a monomeric interaction with the intracellular C-terminus of neuronal Nav channels, FGF14 modulates Na+ currents in an Nav isoform-specific manner serving as a fine-tuning regulator of excitability. Previous studies based on the highly homologous FGF13 homodimer crystal structure have proposed a conserved protein:protein interaction (PPI) interface common to both Nav channel binding and iFGF homodimer formation. This interface could provide a novel target for drug design against neuronal Nav channels. Here, we provide the first in-cell reconstitution of the FGF14:FGF14 protein complex and measure the dimer interaction using the split-luciferase complementation assay (LCA). Based on the FGF14 dimer structure generated in silico, we designed short peptide fragments against the FGF14 dimer interface. One of these fragments, FLPK aligns with the pocket defined by the β12-strand and β8-β9 loop, reducing the FGF14:FGF14 dimer interaction by 25% as measured by LCA. We further compared the relative interaction strength of FGF14 wild type homodimers with FGF14 hetero- and homodimers carrying double N mutations at the Y153 and V155 residues, located at the β8-β9 loop. The Y153N/V155N double mutation counteracts the FLPK effect by increasing the strength of the dimer interaction. These data suggest that the β12 strand of FGF14 might serve as scaffold for drug design against neuronal FGF14 dimers and Nav channels. PMID:25426956

  17. Assembly of Bak homodimers into higher order homooligomers in the mitochondrial apoptotic pore

    PubMed Central

    Mandal, Tirtha; Shin, Seungjin; Aluvila, Sreevidya; Chen, Hui-Chen; Grieve, Carter; Choe, Jun-Yong; Cheng, Emily H.; Hustedt, Eric J.; Oh, Kyoung Joon

    2016-01-01

    In mitochondrial apoptosis, Bak is activated by death signals to form pores of unknown structure on the mitochondrial outer membrane via homooligomerization. Cytochrome c and other apoptotic factors are released from the intermembrane space through these pores, initiating downstream apoptosis events. Using chemical crosslinking and double electron electron resonance (DEER)-derived distance measurements between specific structural elements in Bak, here we clarify how the Bak pore is assembled. We propose that previously described BH3-in-groove homodimers (BGH) are juxtaposed via the ‘α3/α5’ interface, in which the C-termini of helices α3 and α5 are in close proximity between two neighboring Bak homodimers. This interface is observed concomitantly with the well-known ‘α6:α6’ interface. We also mapped the contacts between Bak homodimers and the lipid bilayer based on EPR spectroscopy topology studies. Our results suggest a model for the lipidic Bak pore, whereby the mitochondrial targeting C-terminal helix does not change topology to accommodate the lining of the pore lumen by BGH. PMID:27488021

  18. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    SciTech Connect

    Xu, Yu; Liu, Zhengchun; Kong, Haiyan; Sun, Wenjie; Liao, Zhengkai; Zhou, Fuxiang; Xie, Conghua; and others

    2011-09-09

    Highlights: {yields} A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. {yields} The promoter was characterized with radiation-inducibility and tumor-specificity. {yields} Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. {yields} Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  19. Endotoxin free hyaluronan and hyaluronan fragments do not stimulate TNF-α, interleukin-12 or upregulate co-stimulatory molecules in dendritic cells or macrophages

    PubMed Central

    Dong, Yifei; Arif , Arif; Olsson, Mia; Cali, Valbona; Hardman, Blair; Dosanjh, Manisha; Lauer, Mark; Midura, Ronald J.; Hascall, Vincent C.; Brown, Kelly L.; Johnson, Pauline

    2016-01-01

    The extracellular matrix glycosaminoglycan, hyaluronan, has been described as a regulator of tissue inflammation, with hyaluronan fragments reported to stimulate innate immune cells. High molecular mass hyaluronan is normally present in tissues, but upon inflammation lower molecular mass fragments are generated. It is unclear if these hyaluronan fragments induce an inflammatory response or are a consequence of inflammation. In this study, mouse bone marrow derived macrophages and dendritic cells (DCs) were stimulated with various sizes of hyaluronan from different sources, fragmented hyaluronan, hyaluronidases and heavy chain modified-hyaluronan (HA-HC). Key pro-inflammatory molecules, tumour necrosis factor alpha, interleukin-1 beta, interleukin-12, CCL3, and the co-stimulatory molecules, CD40 and CD86 were measured. Only human umbilical cord hyaluronan, bovine testes and Streptomyces hyaluronlyticus hyaluronidase stimulated macrophages and DCs, however, these reagents were found to be contaminated with endotoxin, which was not fully removed by polymyxin B treatment. In contrast, pharmaceutical grade hyaluronan and hyaluronan fragments failed to stimulate in vitro-derived or ex vivo macrophages and DCs, and did not induce leukocyte recruitment after intratracheal instillation into mouse lungs. Hence, endotoxin-free pharmaceutical grade hyaluronan does not stimulate macrophages and DCs in our inflammatory models. These results emphasize the importance of ensuring hyaluronan preparations are endotoxin free. PMID:27869206

  20. Evaluation of a technetium-99m labeled bombesin homodimer for GRPR imaging in prostate cancer.

    PubMed

    Yu, Zilin; Carlucci, Giuseppe; Ananias, Hildo J K; Dierckx, Rudi A J O; Liu, Shuang; Helfrich, Wijnand; Wang, Fan; de Jong, Igle J; Elsinga, Philip H

    2013-02-01

    Multimerization of peptides can improve the binding characteristics of the tracer by increasing local ligand concentration and decreasing dissociation kinetics. In this study, a new bombesin homodimer was developed based on an ε-aminocaproic acid-bombesin(7-14) (Aca-bombesin(7-14)) fragment, which has been studied for targeting the gastrin-releasing peptide receptor (GRPR) in prostate cancer. The bombesin homodimer was conjugated to 6-hydrazinopyridine-3-carboxylic acid (HYNIC) and labeled with (99m)Tc for SPECT imaging. The in vitro binding affinity to GRPR, cell uptake, internalization and efflux kinetics of the radiolabeled bombesin dimer were investigated in the GRPR-expressing human prostate cancer cell line PC-3. Biodistribution and the GRPR-targeting potential were evaluated in PC-3 tumor-bearing athymic nude mice. When compared with the bombesin monomer, the binding affinity of the bombesin dimer is about ten times lower. However, the (99m)Tc labeled bombesin dimer showed a three times higher cellular uptake at 4 h after incubation, but similar internalization and efflux characters in vitro. Tumor uptake and in vivo pharmacokinetics in PC-3 tumor-bearing mice were comparable. The tumor was visible on the dynamic images in the first hour and could be clearly distinguished from non-targeted tissues on the static images after 4 h. The GRPR-targeting ability of the (99m)Tc labeled bombesin dimer was proven in vitro and in vivo. This bombesin homodimer provides a good starting point for further studies on enhancing the tumor targeting activity of bombesin multimers.

  1. Rock bream (Oplegnathus fasciatus) IL-12p40: identification, expression, and effect on bacterial infection.

    PubMed

    Zhang, Lu; Zhang, Bao-Cun; Hu, Yong-Hua

    2014-08-01

    IL-12p40, also called IL-12β, is a subunit of the proinflammatory cytokines interleukin (IL)-12 and IL-23. In teleost, IL-12p40 homologues have been identified in several species, however, the biological function of fish IL-12p40 is essentially unknown. In this work, we reported the identification and analysis of an IL-12p40, OfIL-12p40, from rock bream (Oplegnathus fasciatus). OfIL-12p40 is composed of 361 amino acids and possesses a conserved IL-12p40 domain and a WSxWS signature motif characteristic of known IL-12p40. Constitutive expression of OfIL-12p40 occurred in multiple tissues and was highest in kidney. Experimental infection with bacterial pathogen upregulated the expression of OfIL-12p40 in kidney and spleen in a time-dependent manner. Purified recombinant OfIL-12p40 (rOfIL-12p40) stimulated the respiratory burst activity of peripheral blood leukocytes in a dose-dependent manner. rOfIL-12p40 also enhanced the resistance of rock bream against bacterial infection and upregulated the expression of innate immune genes in kidney. Taken together, these results indicate that OfIL-12p40 possesses cytokine-like property and plays a role in immune defense against bacterial infection.

  2. Silibinin inhibits ultraviolet B radiation-induced DNA-damage and apoptosis by enhancing interleukin-12 expression in JB6 cells and SKH-1 hairless mouse skin.

    PubMed

    Narayanapillai, Sreekanth; Agarwal, Chapla; Deep, Gagan; Agarwal, Rajesh

    2014-06-01

    Recent studies have demonstrated silibinin efficacy against ultraviolet B (UVB)-induced skin carcinogenesis via different mechanisms in cell lines and animal models; however, its role in regulating interleukin-12 (IL-12), an immunomodulatory cytokine that reduces UVB-induced DNA damage and apoptosis, is not known. Here, we report that UVB irradiation causes caspase 3 and PARP cleavage and apoptosis, and addition of recombinant IL-12 or silibinin immediately after UVB significantly protects UVB-induced apoptosis in JB6 cells. IL-12 antibody-mediated blocking of IL-12 activity compromised the protective effects of both IL-12 and silibinin. Both silibinin and IL-12 also accelerated the repair of UVB-caused cyclobutane-pyrimidine dimers (CPDs) in JB6 cells. Additional studies confirmed that indeed silibinin causes a significant increase in IL-12 levels in UVB-irradiated JB6 cells as well as in mouse skin epidermis, and that similar to cell-culture findings, silibinin topical application immediately after UVB exposure causes a strong protection against UVB-induced TUNEL positive cells in epidermis possibly through a significantly accelerated repair of UVB-caused CPDs. Together, these findings for the first time provide an important insight regarding the pharmacological mechanism wherein silibinin induces endogenous IL-12 in its efficacy against UVB-caused skin damages. In view of the fact that an enhanced endogenous IL-12 level could effectively remove UVB-caused DNA damage and associated skin cancer, our findings suggest that the use of silibinin in UVB-damaged human skin would also be a practical and translational strategy to manage solar radiation-caused skin damages as well as skin cancer.

  3. Combination of interleukin-12 gene therapy, metronomic cyclophosphamide and DNA cancer vaccination directs all arms of the immune system towards tumor eradication.

    PubMed

    Denies, Sofie; Cicchelero, Laetitia; Van Audenhove, Isabel; Sanders, Niek N

    2014-08-10

    In this work a combination therapy that acts upon the immune suppressive, the innate and specific arms of the immune system is proposed. This combination therapy, which consists of intratumoral interleukin-12 (IL-12) gene therapy, human tyrosinase (hTyr) DNA vaccination and metronomic cyclophosphamide (CPX), was evaluated in a B16-F10 mouse model. The following groups were compared: (1) no treatment, (2) control vector, (3) intratumoral IL-12 gene therapy, (4) intratumoral IL-12 gene therapy+metronomic CPX, (5) intratumoral IL-12 gene therapy+metronomic CPX+hTyr DNA vaccination. Next to clinical efficacy and safety, we characterized acute effects of IL-12 and anti-tumor immune response after a second tumor challenge. All treatment groups showed increased survival and higher cure rates than control groups. Survival of non-cured mice was increased when metronomic CPX was combined with IL-12 gene therapy. Furthermore, mice that received metronomic CPX had significantly lower percentages of regulatory T cells. Addition of the hTyr DNA vaccine increased cure rate and resulted in increased survival compared to other treatment groups. We also demonstrated that the manifest necrosis within days after IL-12 gene therapy is at least partly due to IL-12 mediated activation of NK cells. All cured mice were resistant to a second challenge. A humoral memory response against the tumor cells was observed in all groups that received IL-12 gene therapy, while a cellular memory response was observed only in the vaccinated mice. In conclusion, every component of this combination treatment contributed a unique immunologic trait with associated clinical benefits.

  4. Therapeutic effect of interleukin 12 on mouse haemangiosarcomas is not associated with an increased anti-tumour cytotoxic T-lymphocyte activity.

    PubMed Central

    Vizler, C.; Rosato, A.; Calderazzo, F.; Quintieri, L.; Fruscella, P.; Wainstok de Calmanovici, R.; Mantovani, A.; Vecchi, A.; Zanovello, P.; Collavo, D.

    1998-01-01

    In syngeneic mice, the H5V polyoma middle-T oncogene-transformed endothelioma cell line induces Kaposi's sarcoma-like cavernous haemangiomas that regress transiently, probably because of an anti-tumour immune response, but eventually grow progressively and kill the host. To evaluate the generation of tumour-specific cytotoxic T lymphocytes (CTLs), spleen cells of tumour-bearing mice were restimulated with irradiated H5V cells in mixed leucocyte-tumour cell cultures. Tumour-specific CTLs were demonstrable only when low numbers of H5V stimulator cells were used (<1 H5V cell per 50 splenocytes). We found that H5V cells secrete immunosuppressive mediators because CTL generation was blocked when H5V cells culture supernatants were added to allogeneic mixed leucocyte cultures. As numerous tumour-derived immunosuppressive mediators may interfere with interleukin 12 (IL-12) production, we tested whether IL-12 treatment of the tumour-bearing mice would augment their immune response and thus suppress tumour growth. Indeed, IL-12 inhibited tumour growth and prevented mortality, but did not increase anti-H5V CTL generation either in vitro or in vivo. Moreover, the anti-tumour activity in IL-12-treated mice was abrogated by anti-interferon (IFN)-gamma monoclonal antibody (MAb) co-administration. These results strongly suggest that the anti-tumour effect of IL-12 is principally mediated by IFN-gamma release that in turn blocks H5V cell proliferation and induces the release of factors that suppress angiogenesis. PMID:9484826

  5. A safety and efficacy study of local delivery of interleukin-12 transgene by PPC polymer in a model of experimental glioma.

    PubMed

    Sonabend, Adam M; Velicu, Simona; Ulasov, Ilya V; Han, Yu; Tyler, Betty; Brem, Henry; Matar, Majed M; Fewell, Jason G; Anwer, Khursheed; Lesniak, Maciej S

    2008-02-01

    Interleukin-12 (IL-12) triggers an antitumoral immune response and an antiangiogenic effect against cancer. In this study, we tested a novel polymeric vehicle for IL-12 gene therapy along with adjuvant local biodegradable carmustine (BCNU) chemotherapy for the treatment of malignant glioma. Highly concentrated DNA/PPC (polyethylenimine covalently modified with methoxypolyethyleneglycol and cholesterol) complexes were used to deliver a murine plasmid encoding IL-12 (pmIL-12). For toxicity assessment, mice received intracranial injections with different volumes of pmIL-12/PPC. For efficacy, mice with intracranial GL261 glioma were treated with local delivery of pmIL-12/PPC and/or BCNU-containing polymers. Intracranial injections of 5-10 microl of pmIL-12/PPC were well tolerated and led to IL-12 expression in the brains of treated animals. Treatment with pmIL-12/PPC led to a significant increase in survival compared with untreated mice (median survival 57 days; 25% long-term survival >95 vs. 45 days for control; P<0.05). Treatment with BCNU led to a significant increase in survival compared with untreated mice, with 75% of treated mice having a long-term survival >95 days, (P<0.05). Most importantly, the combination of BCNU and pmIL-12/PPC led to a survival of 100% of the mice for 95 days after treatment (P<0.0001). This novel strategy is safe and effective for the treatment of malignant glioma. The synergy resultant from the combination of locally administered pmIL-12/PPC and BCNU suggests a role for this approach in the treatment of malignant brain tumors.

  6. Interferon-α and interleukin-12 are induced, respectively, by double-stranded DNA and single-stranded RNA in human myeloid dendritic cells.

    PubMed

    Katashiba, Yuichi; Miyamoto, Rie; Hyo, Akira; Shimamoto, Keiko; Murakami, Naoko; Ogata, Makoto; Amakawa, Ryuichi; Inaba, Muneo; Nomura, Shosaku; Fukuhara, Shirou; Ito, Tomoki

    2011-02-01

    Dendritic cells (DCs) are initiators of innate immunity and acquired immunity as cells linking these two bio-defence systems through the production of cytokines such as interferon-α (IFN-α) and interleukin-12 (IL-12). Nucleic acids such as DNA from damaged cells or pathogens are important activators not only for anti-microbial innate immune responses but also in the pathogenesis of IFN-related autoimmune diseases. Plasmacytoid DCs are regarded as the main effectors for the DNA-mediated innate immunity by possessing DNA-sensing toll-like receptor 9 (TLR9). We here found that double-stranded DNA (dsDNA) complexed with lipotransfectants triggered activation of human monocyte-derived DCs (moDCs), leading to the preferential production of IFN-α but not IL-12. This indicates that myeloid DCs also function as supportive effectors against the invasion of pathogenic microbes through the DNA-mediated activation in innate immunity. The dsDNA with lipotransfectants can be taken up by moDCs without co-localization of endosomal LAMP1 staining, and the dsDNA-mediated IFN-α production was not impaired by chloroquine. These findings indicate that moDC activation by dsDNA does not involve the endosomal TLR pathway. In contrast, single-stranded RNA (ssRNA) stimulated moDCs to secrete IL-12 but not IFN-α. This process was inhibited by chloroquine, suggesting an involvement of the TLR pathway in ssRNA-mediated moDC activation. As might be inferred from our findings, myeloid DCs may function as a traffic control between innate immunity via IFN-α production and acquired immunity via IL-12 production, depending on the type of nucleic acids. Our results provide a new insight into the biological action of myeloid DCs underlying the DNA-mediated activation of protective or pathogenic immunity.

  7. High Interleukin-12 Levels May Prevent an Increase in the Amount of Fungi in the Gastrointestinal Tract during the First Years of Diabetes Mellitus Type 1

    PubMed Central

    Kowalewska, Beata; Szmigiero-Kawko, Małgorzata; Wąż, Piotr; Myśliwiec, Małgorzata

    2016-01-01

    The objective of the research was to investigate serum levels of interleukin-12 (IL12) in relation to percentage of yeast-like fungi colonies residing in the gastrointestinal tract in children and adolescents with type 1 diabetes mellitus (T1DM). The study involved 83 children and adolescents, including 53 T1DM patients and 30 healthy control subjects. In the studied population biochemical tests were performed and yeast-like fungi were identified in the faeces. Moreover, IL12 absorbance was measured and measurements of Candida albicans IgG and IgM antibodies were performed with microplate reader ChroMate 4300 (Awareness Technology, Inc., USA) at wavelength λ = 450 nm. In the group of T1DM children and adolescents with disease duration ≤ 2 years, high levels of IL12 were found with lower percentage of yeast-like fungal colonies versus T1DM patients with disease duration > 2 years and ≤5 years, as well as versus T1DM patients with disease duration > 5 years. Additionally, serum levels of IL12 were found to be decreasing by 18.1 pg/ml with each year of diabetes duration. IL12 serum levels were also found to be decreasing by 52.9 pg/ml with each 1% increase in HbA1c. We suggest that high IL12 levels can inhibit infection with yeast-like fungi colonizing the gastrointestinal tract in children and adolescents with T1DM. Further studies are needed to confirm the antifungal activity of IL12. PMID:28127111

  8. The Safety and Immunogenicity of an Interleukin-12-Enhanced Multiantigen DNA Vaccine Delivered by Electroporation for the Treatment of HIV-1 Infection

    PubMed Central

    Jacobson, Jeffrey M.; Zheng, Lu; Wilson, Cara C.; Tebas, Pablo; Matining, Roy M.; Egan, Michael A.; Eldridge, John; Landay, Alan L.; Clifford, David B.; Luetkemeyer, Anne F.; Tiu, Jennifer; Martinez, Ana; Janik, Jennifer; Spitz, Teresa A.; Hural, John; McElrath, Juliana; Frahm, Nicole

    2015-01-01

    Background Therapeutic vaccination is being studied in eradication and “functional cure” strategies for HIV-1. The Profectus Biosciences (Tarrytown, NY) multiantigen (MAG) HIV-1 DNA vaccine encodes HIV-1 Gag/Pol, Nef/Tat/Vif, and Envelope, and interleukin-12 (IL-12) and is delivered by electroporation (EP) combined with intramuscular injection (IM-EP). Methods Sixty-two HIV-1-infected patients on antiretroviral therapy (plasma HIV-1 RNA levels ≤200 copies/mL; CD4+ T-cell counts ≥500 cells/mm3) were randomly allocated 5:1 to receive vaccine or placebo. At weeks 0, 4 and 12, four consecutive cohorts received 3000 μg HIV MAG pDNA with 0, 50, 250, or 1000 μg of IL-12 pDNA by IM-EP. A 5th cohort received HIV MAG pDNA plus 1000 μg of IL-12 pDNA by standard IM injection. Results CD4+ T cells expressing IL-2 in response to Gag and Pol and interferon-γ responses to Gag, Pol, and Env increased from baseline to week 14 in the low-dose (50-μg) IL-12 arm vs. placebo (P < 0.05; intracellular cytokine staining). The total increase in the IL-2 expressing CD4+ T-cell responses to any antigen was also higher in the low-dose IL-12 arm vs. placebo (P = 0.04). Cytokine responses by CD8 T cells to HIV antigens were not increased in any vaccine arm relative to placebo. Conclusions HIV-1 MAG/low-dose IL-12 DNA vaccine delivered by IM-EP augmented CD4+ but not CD8+ T-cell responses to multiple HIV-1 antigens. PMID:26761518

  9. The HhH domain of the human DNA repair protein XPF forms stable homodimers.

    PubMed

    Das, Devashish; Tripsianes, Konstantinos; Jaspers, Nicolaas G J; Hoeijmakers, Jan H J; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E

    2008-03-01

    The human XPF-ERCC1 protein complex plays an essential role in nucleotide excision repair by catalysing positioned nicking of a DNA strand at the 5' side of the damage. We have recently solved the structure of the heterodimeric complex of the C-terminal domains of XPF and ERCC1 (Tripsianes et al., Structure 2005;13:1849-1858). We found that this complex comprises a pseudo twofold symmetry axis and that the helix-hairpin-helix motif of ERCC1 is required for DNA binding, whereas the corresponding domain of XPF is functioning as a scaffold for complex formation with ERCC1. Despite the functional importance of heterodimerization, the C-terminal domain of XPF can also form homodimers in vitro. We here compare the stabilities of homodimeric and heterodimeric complexes of the C-terminal domains of XPF and ERCC1. The higher stability of the XPF HhH complexes under various experimental conditions, determined using CD and NMR spectroscopy and mass spectrometry, is well explained by the structural differences that exist between the HhH domains of the two complexes. The XPF HhH homodimer has a larger interaction interface, aromatic stacking interactions, and additional hydrogen bond contacts as compared to the XPF/ERCC1 HhH complex, which accounts for its higher stability.

  10. Requirement for natural killer cell-produced interferon gamma in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration

    PubMed Central

    1995-01-01

    killing at levels comparable to those observed in control-treated mice. The consequences of interleukin 12 (IL-12) administration, a known potent inducer of IFN- gamma production by NK cells, were evaluated in MCMV-infected mice. Low IL-12 doses, i.e., 1 ng/d, increased NK cell cytotoxicity and IFN-gamma production up to twofold and resulted in improved antiviral status; virus-induced hepatitis was decreased as much as fivefold, and viral burdens were decreased to levels below detection.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7561678

  11. HemaMax™, a Recombinant Human Interleukin-12, Is a Potent Mitigator of Acute Radiation Injury in Mice and Non-Human Primates

    PubMed Central

    Basile, Lena A.; Ellefson, Dolph; Gluzman-Poltorak, Zoya; Junes-Gill, Katiana; Mar, Vernon; Mendonca, Sarita; Miller, Joseph D.; Tom, Jamie; Trinh, Alice; Gallaher, Timothy K.

    2012-01-01

    HemaMax, a recombinant human interleukin-12 (IL-12), is under development to address an unmet medical need for effective treatments against acute radiation syndrome due to radiological terrorism or accident when administered at least 24 hours after radiation exposure. This study investigated pharmacokinetics, pharmacodynamics, and efficacy of m-HemaMax (recombinant murine IL-12), and HemaMax to increase survival after total body irradiation (TBI) in mice and rhesus monkeys, respectively, with no supportive care. In mice, m-HemaMax at an optimal 20 ng/mouse dose significantly increased percent survival and survival time when administered 24 hours after TBI between 8–9 Gy (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by increases in plasma interferon-γ (IFN-γ) and erythropoietin levels, recovery of femoral bone hematopoiesis characterized with the presence of IL-12 receptor β2 subunit–expressing myeloid progenitors, megakaryocytes, and osteoblasts. Mitigation of jejunal radiation damage was also examined. At allometrically equivalent doses, HemaMax showed similar pharmacokinetics in rhesus monkeys compared to m-HemaMax in mice, but more robustly increased plasma IFN-γ levels. HemaMax also increased plasma erythropoietin, IL-15, IL-18, and neopterin levels. At non-human primate doses pharmacologically equivalent to murine doses, HemaMax (100 ng/Kg and 250 ng/Kg) administered at 24 hours after TBI (6.7 Gy/LD50/30) significantly increased percent survival of HemaMax groups compared to vehicle (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by a significantly higher leukocyte (neutrophils and lymphocytes), thrombocyte, and reticulocyte counts during nadir (days 12–14) and significantly less weight loss at day 12 compared to vehicle. These findings indicate successful interspecies dose conversion and provide proof of concept that HemaMax increases survival in irradiated rhesus monkeys by promoting

  12. Spectral-luminescent and photochemical characteristics of homodimers of the styrylcyanine dye Sbt in solutions.

    PubMed

    Kurtaliev, Eldar N

    2011-10-15

    The influence of concentration, solvent nature on the intermolecular interaction and its spectroscopic manifestations in solutions of styrylcyanine dye Sbt ((E)-2-(4-(dimethylamino)styryl)-3-methylbenzo[d]thiazol-3-ium iodide) and its homodimers, i.e. dyes with two interconnected chromophores, was studied. It was found out that, depending on the concentration, the structure of dye molecules, the nature of the solvent various forms of associated molecules are forms; each of these forms is manifested in the spectra of absorption and fluorescence in its own way. It is shown that the intensity of the main absorption band of the studied dyes in aqueous solutions and in a mixture of water+ethanol decreases in the process of light irradiation and a new band is observed in the region of shorter wavelengths; the intensity of the new band increases with increase of radiation exposure of solutions.

  13. COUP-TF II homodimers are formed in preference to heterodimers with RXR alpha or TR beta in intact cells.

    PubMed Central

    Butler, A J; Parker, M G

    1995-01-01

    Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) represses the transcriptional activity of a number of nuclear receptors, including that of retinoid receptors (RAR and RXR) and thyroid hormone receptors (TR). Since COUP-TF is capable of binding to DNA in vitro either as a homodimer or as a heterodimer with RXR or TR, it has not been possible to distinguish between competitive DNA binding and heterodimer formation as a mechanism to account for the repression. Using a two-hybrid system we have investigated the dimerisation properties of COUP-TF II in intact cells. In conditions where COUP-TF II homodimers and RXR alpha-RAR alpha heterodimers were formed we were unable to detect the formation of heterodimers between COUP-TF II and RXR alpha. Moreover, we were unable to detect an interaction between COUP-TF II and RXR alpha on DNA. Similarly COUP-TF II homodimers and RXR alpha-TR beta heterodimers are favoured over COUP-TF II-TR beta heterodimers. We conclude that the formation of functionally inactive heterodimers is unlikely to represent a general mechanism by which COUP-TF represses the transcriptional activity of nuclear receptors and favour a model in which repression is mediated by COUP-TF homodimers competing for binding to DNA. Images PMID:7479078

  14. Heterodimers and homodimers of inhibin subunits have different paracrine action in the modulation of luteinizing hormone-stimulated androgen biosynthesis

    SciTech Connect

    Hsueh, A.J.W.; Dahl, K.D.; Vaughan, J.; Tucker, E.; Rivier, J.; Bardin, C.W.; Vale, W.

    1987-07-01

    Inhibin, a gonadal hormone capable of preferential suppression of pituitary follicle-stimulating hormone (FSH) secretion, has recently been purified. The major form of this protein is an ..cap alpha beta.. heterodimer encoded by two separate genes. In contrast to the FSH-suppressing action of the ..cap alpha beta.. heterodimer, the ..beta beta.. homodimer stimulates FSH secretion. Luteinizing hormone (LH)-secreting pituitary cells and gonadal androgen-producing cells have long been shown to form a closed-loop feedback axis. Based on recent studies demonstrated the FSH stimulation of inhibin biosynthesis by ovarian granulosa and testis Sertoli cells, an additional closed-loop feedback axis exists between pituitary FSH- and gonadal inhibin-producing cells. Because uncharacterized Sertoli cell factors have been suggested to either stimulate or inhibit androgen production by testicular Leydig cells, the authors have tested the intragonadal paracrine actions of heterodimers and homodimers of inhibin subunits. In primary cultures of testis cells, the ..cap alpha beta.. heterodimer of inhibin enhances Leydig cell androgen biosynthesis stimulated by LH, whereas the ..beta beta.. homodimer suppresses androgen production. The data indicate that the inhibin-related gene products synthesized by Sertoli and granulosa cells may form heterodimers or homodimers to serve as intragonadal paracrine signals in the modulation of LH-stimulated androgen biosynthesis and allow cross-communication between the two feedback loops.

  15. A stable double-stranded DNA-ethidium homodimer complex: application to picogram fluorescence detection of DNA in agarose gels.

    PubMed Central

    Glazer, A N; Peck, K; Mathies, R A

    1990-01-01

    The complex between double-stranded DNA and ethidium homodimer (5,5'-diazadecamethylene)bis(3,8-diamino-6-phenylphenanthridini um) cation, formed at a ratio of 1 homodimer per 4 or 5 base pairs, is stable in agarose gels under the usual conditions for electrophoresis. This unusual stability allows formation of the complex before electrophoresis and then separation and detection in the absence of background stain. Competition experiments between the preformed DNA-ethidium homodimer complex and a 50-fold molar excess of unlabeled DNA show that approximately one-third of the dye is retained within the original complex independent of the duration of the competition. However, dye-extraction experiments show that these are not covalent complexes. After electrophoretic separation, detection of bands containing 25 pg of DNA was readily achieved in 1-mm thick agarose gels with laser excitation at 488 nm and a scanning confocal fluorescence imaging system. The band intensity was linear with the amount of DNA applied from 0.2 to 1.0 ng per lane and with the number of kilobase pairs (kbp) per band within a lane. Analysis of an aliquot of a polymerase-chain-reaction mixture permitted ready detection of 80 pg of a 1.6-kbp amplified fragment. The use of the ethidium homodimer complex together with laser excitation for DNA detection on gels is at least two orders of magnitude more sensitive than conventional fluorescence-based procedures. The homodimer-DNA complex exemplifies a class of fluorescent probes where the intercalation of dye chromophores in DNA forms a stable, highly fluorescent ensemble. Images PMID:2339125

  16. A stable double-stranded DNA-ethidium homodimer complex: Application to picogram fluorescence detection of DNA in agarose gels

    SciTech Connect

    Glazer, A.N.; Mathies, R.A. Lawrence Berkeley Laboratory, CA ); Peck, K. )

    1990-05-01

    The complex between double-stranded DNA and ethidium homodimer (5,5{prime}-diazadecamethylene)bis(3,8-diamino-6-phenylphenanthridinium) cation, formed at a ratio of 1 homodimer per 4 or 5 base pairs, is stable in agarose gels under the usual conditions for electrophoresis. This unusual stability allows formation of the complex before electrophoresis and then separation and detection in the absence of background stain. Competition experiments between the performed DNA-ethidium homodimer complex and a 50-fold molar excess of unlabeled DNA show that approximately one-third of the dye is retained within the original complex independent of the duration of the competition. However, dye-extraction experiments show that these are not covalent complexes. After electrophoretic separation, detection of bands containing 25 pg of DNA was readily achieved in 1-mm thick agarose gels with laser excitation at 488 nm and a scanning confocal fluorescence imaging system. The band intensity was linear with the amount of DNA applied from 0.2 to 1.0 ng per lane and with the number of kilobase pairs (kbp) per band within a lane. Analysis of an aliquot of a polymerase-chain-reaction mixture permitted ready detection of 80 pg of a 1.6-kbp amplified fragment. The use of the ethidium homodimer complex together with laser excitation for DNA detection on gels is at least two orders of magnitude more sensitive than conventional fluorescence-based procedures. The homodimer-DNA complex exemplifies a class of fluorescent probes where the intercalation of dye chromophores in DNA forms a stable, highly fluorescent ensemble.

  17. Insect Neuropeptide Bursicon Homodimers Induce Innate Immune and Stress Genes during Molting by Activating the NF-κB Transcription Factor Relish

    PubMed Central

    Li, Sheng; Gilbert, Lawrence I.; Stanley, David; Song, Qisheng

    2012-01-01

    Background Bursicon is a heterodimer neuropeptide composed of two cystine knot proteins, bursicon α (burs α) and bursicon β (burs β), that elicits cuticle tanning (melanization and sclerotization) through the Drosophila leucine-rich repeats-containing G protein-coupled receptor 2 (DLGR2). Recent studies show that both bursicon subunits also form homodimers. However, biological functions of the homodimers have remained unknown until now. Methodology/Principal Findings In this report, we show in Drosophila melanogaster that both bursicon homodimers induced expression of genes encoding antimicrobial peptides (AMPs) in neck-ligated adults following recombinant homodimer injection and in larvae fat body after incubation with recombinant homodimers. These AMP genes were also up-regulated in 24 h old unligated flies (when the endogenous bursicon level is low) after injection of recombinant homodimers. Up-regulation of AMP genes by the homodimers was accompanied by reduced bacterial populations in fly assay preparations. The induction of AMP expression is via activation of the NF-κB transcription factor Relish in the immune deficiency (Imd) pathway. The influence of bursicon homodimers on immune function does not appear to act through the heterodimer receptor DLGR2, i.e. novel receptors exist for the homodimers. Conclusions/Significance Our results reveal a mechanism of CNS-regulated prophylactic innate immunity during molting via induced expression of genes encoding AMPs and genes of the Turandot family. Turandot genes are also up-regulated by a broader range of extreme insults. From these data we infer that CNS-generated bursicon homodimers mediate innate prophylactic immunity to both stress and infection during the vulnerable molting cycle. PMID:22470576

  18. Metal-Mediated Affinity and Orientation Specificity in a Computationally Designed Protein Homodimer

    SciTech Connect

    Der, Bryan S.; Machius, Mischa; Miley, Michael J.; Mills, Jeffrey L.; Szyperski, Thomas; Kuhlman, Brian

    2015-10-15

    Computationally designing protein-protein interactions with high affinity and desired orientation is a challenging task. Incorporating metal-binding sites at the target interface may be one approach for increasing affinity and specifying the binding mode, thereby improving robustness of designed interactions for use as tools in basic research as well as in applications from biotechnology to medicine. Here we describe a Rosetta-based approach for the rational design of a protein monomer to form a zinc-mediated, symmetric homodimer. Our metal interface design, named MID1 (NESG target ID OR37), forms a tight dimer in the presence of zinc (MID1-zinc) with a dissociation constant <30 nM. Without zinc the dissociation constant is 4 {micro}M. The crystal structure of MID1-zinc shows good overall agreement with the computational model, but only three out of four designed histidines coordinate zinc. However, a histidine-to-glutamate point mutation resulted in four-coordination of zinc, and the resulting metal binding site and dimer orientation closely matches the computational model (C{alpha} rmsd = 1.4 {angstrom}).

  19. Regulation of the PI3K pathway through a p85α monomer–homodimer equilibrium

    PubMed Central

    Cheung, Lydia WT; Walkiewicz, Katarzyna W; Besong, Tabot MD; Guo, Huifang; Hawke, David H; Arold, Stefan T; Mills, Gordon B

    2015-01-01

    The canonical action of the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is to associate with the p110α catalytic subunit to allow stimuli-dependent activation of the PI3K pathway. We elucidate a p110α-independent role of homodimerized p85α in the positive regulation of PTEN stability and activity. p110α-free p85α homodimerizes via two intermolecular interactions (SH3:proline-rich region and BH:BH) to selectively bind unphosphorylated activated PTEN. As a consequence, homodimeric but not monomeric p85α suppresses the PI3K pathway by protecting PTEN from E3 ligase WWP2-mediated proteasomal degradation. Further, the p85α homodimer enhances the lipid phosphatase activity and membrane association of PTEN. Strikingly, we identified cancer patient-derived oncogenic p85α mutations that target the homodimerization or PTEN interaction surface. Collectively, our data suggest the equilibrium of p85α monomer–dimers regulates the PI3K pathway and disrupting this equilibrium could lead to disease development. DOI: http://dx.doi.org/10.7554/eLife.06866.001 PMID:26222500

  20. Apelin receptor homodimer-oligomers revealed by single-molecule imaging and novel G protein-dependent signaling

    PubMed Central

    Cai, Xin; Bai, Bo; Zhang, Rumin; Wang, Chunmei; Chen, Jing

    2017-01-01

    The apelin receptor (APJ) belongs to family A of the G protein-coupled receptors (GPCRs) and is a potential pharmacotherapeutic target for heart failure, hypertension, and other cardiovascular diseases. There is evidence APJ heterodimerizes with other GPCRs; however, the existence of APJ homodimers and oligomers remains to be investigated. Here, we measured APJ monomer-homodimer-oligomer interconversion by monitoring APJ dynamically on cells and compared their proportions, spatial arrangement, and mobility using total internal reflection fluorescence microscopy, resonance energy transfer, and proximity biotinylation. In cells with <0.3 receptor particles/μm2, approximately 60% of APJ molecules were present as dimers or oligomers. APJ dimers were present on the cell surface in a dynamic equilibrium with constant formation and dissociation of receptor complexes. Furthermore, we applied interference peptides and MALDI-TOF mass spectrometry to confirm APJ homo-dimer and explore the dimer-interfaces. Peptides corresponding to transmembrane domain (TMD)1, 2, 3, and 4, but not TMD5, 6, and 7, disrupted APJ dimerization. APJ mutants in TMD1 and TMD2 also decreased bioluminescence resonance energy transfer of APJ dimer. APJ dimerization resulted in novel functional characteristics, such as a distinct G-protein binding profile and cell responses after agonist stimulation. Thus, dimerization may serve as a unique mechanism for fine-tuning APJ-mediated functions. PMID:28091541

  1. Structure of a Thyroid Hormone Receptor DNA-Binding Domain Homodimer Bound to an Inverted Palindrome DNA Response Element

    SciTech Connect

    Chen, Yi; Young, Matthew A.

    2010-10-22

    Thyroid hormone receptor (TR), as a member of the nuclear hormone receptor family, can recognize and bind different classes of DNA response element targets as either a monomer, a homooligomer, or a heterooligomer. We report here the first crystal structure of a homodimer TR DNA-binding domain (DBD) in complex with an inverted repeat class of thyroid response element (TRE). The structure shows a nearly symmetric structure of the TR DBD assembled on the F2 TRE where the base recognition contacts in the homodimer DNA complex are conserved relative to the previously published structure of a TR-9-cis-retinoic acid receptor heterodimer DNA complex. The new structure also reveals that the T-box region of the DBD can function as a structural hinge that enables a large degree of flexibility in the position of the C-terminal extension helix that connects the DBD to the ligand-binding domain. Although the isolated TR DBDs exist as monomers in solution, we have measured highly cooperative binding of the two TR DBD subunits onto the inverted repeat DNA sequence. This suggests that elements of the DBD can influence the specific TR oligomerization at target genes, and it is not just interactions between the ligand-binding domains that are responsible for TR oligomerization at target genes. Mutational analysis shows that intersubunit contacts at the DBD C terminus account for some, but not all, of the cooperative homodimer TR binding to the inverted repeat class TRE.

  2. Cholate-solubilized erythrocyte glucose transporters exist as a mixture of homodimers and homotetramers.

    PubMed

    Hebert, D N; Carruthers, A

    1991-05-14

    The molecular size of purified, human erythrocyte glucose transport protein (GLUT1) solubilized in cholic acid was determined by size-exclusion chromatography (SEC) and sucrose gradient ultracentrifugation. GLUT1 purified in the presence of dithiothreitol (GLUT1 + DTT) is resolved as a complex of average Stokes' radius 5.74 nm by SEC. This complex displays D-glucose-inhibitable cytochalasin B binding and, upon reconstitution into proteoliposomes, catalyzes cytochalasin B inhibitable D-glucose transport. GLUT1 purified in the absence of dithiothreitol (GLUT1-DTT) is resolved by SEC as at least two particles of average Stokes' radii 5.74 (minor component) and 7.48 nm (major component). Solubilization of GLUT1-DTT in the presence of dithiothreitol reduces the amount of 7.48-nm complex and increases the amount of 5.74-nm complex resolved by SEC. GLUT1-DTT displays D-glucose-inhibitable cytochalasin B binding and, upon reconstitution into proteoliposomes, catalyzes cytochalasin B inhibitable D-glucose transport. Sucrose gradient ultracentrifugation of GLUT1 + DTT in cholate resolves GLUT1 into two components of 4.8 and 7.6 S. The 4.8S complex is the major component of GLUT1 + DTT. The reverse profile is observed upon sucrose gradient ultracentrifugation of GLUT1-DTT. SEC of human erythrocyte membrane proteins resolves GLUT1 as a major broad peak of average Stokes' radius 7.48 nm and a minor component of 5.74 nm. Both components are characterized by D-glucose-inhibitable cytochalasin B binding. Purified GLUT1 is associated with approximately 26 tightly bound lipid molecules per monomer of transport protein. These data suggest that purified GLUT1 exists as a mixture of homodimers and homotetramers in cholate-lipid micelles and that the presence of reductant during solubilization favors dimer formation.

  3. Comparative analysis of three-dimensional structures of homodimers of uridine phosphorylase from Salmonella typhimurium in the unligated state and in a complex with potassium ion

    SciTech Connect

    Lashkov, A. A.; Zhukhlistova, N. E.; Gabdulkhakov, A. G.; Mikhailov, A. M.

    2009-03-15

    The spatial organization of the homodimer of unligated uridine phosphorylase from Salmonella typhimurium (St UPh) was determined with high accuracy. The structure was refined at 1.80 A resolution to R{sub work} = 16.1% and R{sub free} = 20.0%. The rms deviations for the bond lengths, bond angles, and chiral angles are 0.006 A, 1.042{sup o}, and 0.071{sup o}, respectively. The coordinate error estimated by the Luzzati plot is 0.166 A. The coordinate error based on the maximum likelihood is 0.199 A. A comparative analysis of the spatial organization of the homodimer in two independently refined structures and the structure of the homodimer St UPh in the complex with a K{sup +} ion was performed. The substrate-binding sites in the homodimers StUPhs in the unligated state were found to act asynchronously. In the presence of a potassium ion, the three-dimensional structures of the subunits in the homodimer are virtually identical, which is apparently of importance for the synchronous action of both substrate-binding sites. The atomic coordinates of the refined structure of the homodimer and structure factors have been deposited in the Protein Data Bank (PDB ID code 3DPS).

  4. Comparative analysis of three-dimensional structures of homodimers of uridine phosphorylase from Salmonella typhimurium in the unligated state and in a complex with potassium ion

    NASA Astrophysics Data System (ADS)

    Lashkov, A. A.; Zhukhlistova, N. E.; Gabdulkhakov, A. G.; Mikhailov, A. M.

    2009-03-01

    The spatial organization of the homodimer of unligated uridine phosphorylase from Salmonella typhimurium ( St UPh) was determined with high accuracy. The structure was refined at 1.80 Å resolution to R work = 16.1% and R free = 20.0%. The rms deviations for the bond lengths, bond angles, and chiral angles are 0.006 Å, 1.042°, and 0.071°, respectively. The coordinate error estimated by the Luzzati plot is 0.166 Å. The coordinate error based on the maximum likelihood is 0.199 Å. A comparative analysis of the spatial organization of the homodimer in two independently refined structures and the structure of the homodimer St UPh in the complex with a K+ ion was performed. The substrate-binding sites in the homodimers StUPhs in the unligated state were found to act asynchronously. In the presence of a potassium ion, the three-dimensional structures of the subunits in the homodimer are virtually identical, which is apparently of importance for the synchronous action of both substrate-binding sites. The atomic coordinates of the refined structure of the homodimer and structure factors have been deposited in the Protein Data Bank (PDB ID code 3DPS).

  5. Aryl hydrocarbon receptor modulates NADPH oxidase activity via direct transcriptional regulation of p40phox expression.

    PubMed

    Wada, Taira; Sunaga, Hiroshi; Ohkawara, Reiko; Shimba, Shigeki

    2013-05-01

    A member of the NADPH oxidase subunits, p40(phox) plays an important role in the regulation of NADPH oxidase activity and the subsequent production of reactive oxygen species (ROS). In this study, we show that mouse p40(phox) is a novel transcriptional target of the aryl hydrocarbon receptor (AhR), known as a dioxin receptor or xenobiotic receptor, in the liver. Treatment of mice with 3-methylcholanthrene (3MC) increased p40(phox) gene expression in the liver, but this induction of p40(phox) gene expression was diminished by the deletion of the AhR gene in the liver. Consistent with the in vivo results, the expression of the p40(phox) gene was increased in 3MC-treated Hepa1c1c7 cells in an AhR-dependent manner. In addition, promoter analysis established p40(phox) as a transcriptional target of AhR. Studies using the RNA-interference technique revealed that p40(phox) is involved in the increase of NADPH oxidase activity and the subsequent ROS production in AhR-activated Hepa1c1c7 cells. Consequently, the results obtained here may provide a novel molecular mechanism for ROS production after exposure to dioxins.

  6. RNA and a cell wall component of Enterococcus faecalis IC-1 are required for phagocytosis and interleukin 12 production by the mouse macrophage cell line J774.1.

    PubMed

    Nakase, Junpei; Ukawa, Yuuichi; Takemoto, Syoji; Kubo, Takayoshi; Sagesaka, Yuko M; Aoki-Yoshida, Ayako; Totsuka, Mamoru

    2017-04-13

    Enterococcus faecalis is a resident lactic acid bacterium in the human intestine. Its immunostimulatory action was reported to be enhanced by heat sterilization. To investigate its beneficial actions, we evaluated the ability of 10 E. faecalis strains to induce interleukin-12 (IL-12) production in a mouse macrophage cell line, J774.1 and found that the strain, E. faecalis IC-1, had a potent IL-12-inducing ability. Furthermore, we investigated the underlying mechanism by treating IC-1 cells with RNase or lysozyme. Its activity almost disappeared and an antagonist of Toll-like receptor (TLR) 7 inhibited this activity. Moreover, lysozyme-treated IC-1 bacteria were not phagocytized by J774.1 cells, and did not induce IL-12 production. Based on our results, we propose that macrophages recognize the cell wall components of IC-1, leading to phagocytosis. The IC-1 RNA is then recognized by TLR7, which induces the production of IL-12.

  7. Cooperative Signaling between Homodimers of Metabotropic Glutamate Receptors 1 and 5

    PubMed Central

    Sevastyanova, Tatyana N.

    2014-01-01

    Metabotropic glutamate receptors (mGluRs) function as dimers. Recent work suggests that mGluR1 and mGluR5 may physically interact, but the nature and functional consequences of this relationship have not been addressed. In this study, the functional and pharmacological consequences of this interaction were investigated. Using heterologous expression of mGluR cDNA in rat sympathetic neurons from the superior cervical ganglion and inhibition of the native calcium currents as an assay for receptor activation, a functional interdependence between mGluR1 and mGluR5 was demonstrated. In neurons coexpressing these receptors, combining a selective mGluR1 competitive antagonist with either an mGluR1- or mGluR5-selective negative allosteric modulator (NAM) BAY36-7620 [(3aS,6aS)-hexahydro-5-methylene-6a-(2-naphthalenylmethyl)-1H-cyclopenta[c]furan-1-one] or MPEP [2-methyl-6-(phenylethynyl)pyridine hydrochloride], respectively, strongly occluded signaling by both receptors to an approximately equal degree. By contrast, in cells coexpressing mGluR1 and mGluR2, combining the same mGluR1 competitive inhibitor with an mGluR1 or mGluR2 NAM yielded partial and full inhibition of the response, respectively, as expected for independently acting receptors. In neurons expressing mGluR1 and mGluR5, the selective NAMs each strongly inhibited the response to glutamate, suggesting that these receptors do not interact as heterodimers, which would not be inhibited by selective NAMs. Finally, evidence for a similar mGluR1/mGluR5 functional dependence is shown in medium spiny striatal neurons. Together, these data demonstrate cooperative signaling between mGluR1 and mGluR5 in a manner inconsistent with heterodimerization, and thus suggest an interaction between homodimers. PMID:25113912

  8. Modulation of the Immune Response to DNA Vaccine Encoding Gene of 8-kDa Subunit of Echinococcus granulosus Antigen B Using Murine Interleukin-12 Plasmid in BALB/c Mice

    PubMed Central

    AZIZI, Hakim; KAZEMI, Bahram; BANDEHPOUR, Mojgan; MOHEBALI, Mehdi; KHAMESIPOUR, Ali; ARYAEIPOUR, Mojgan; YAGHOOBI, Hajar; ROKNI, Mohammad Bagher

    2016-01-01

    Background: The current study was designed to evaluate immune responses induced by DNA vaccines encoding 8-kDa subunit of antigen B (HydI) of Echinococcus granulosus and murine interleukin 12 (IL-12) as genetic adjuvants in BALB/c mice. Methods: Expression plasmid pcDNA3.1 containing HydI (pcHyd1) as vaccine along with the murine interleukin 12 (pcMIL12) as adjuvant were used. Thirty-five mice in the five experimental groups received PBS, empty pcDNA3.1, pcHydІ, pcMIL-12, and pcHydІ+ pcMIL-12 in days zero, 14th and 28th. Two weeks after the last immunization, evaluation of the immune response was performed by evaluating the proliferation of splenic lymphocytes, IFN-γ and IL-4, determination of IgG isotyping titer. Results: Mice that received the pcHydI+pcMIL12 exhibited higher levels of lymphocyte proliferation compared to mice that received the pcHydI alone (P<0.001), and produced significantly more IFN-γ in comparison to other groups (P< 0.001). In addition, they produced significantly less IL-4 than mice receiving the PBS and the empty plasmid (P<0.023). The IgG2a levels were clearly higher in pcHydI+pcMIL12 group in comparison with the groups of pcHydI alone, empty plasmid, and PBS. In contrast, IgG1 was elevated in the group of pcHydI. Conclusion: Co-delivery of IL-12 with DNA encoding 8-kDa subunit of antigen B was effective significantly in inducing the immune response in mice. PMID:28127359

  9. The Ability to Form Homodimers Is Essential for RDM1 to Function in RNA-Directed DNA Methylation

    PubMed Central

    Sasaki, Taku; Lorković, Zdravko J.; Liang, Shih-Chieh; Matzke, Antonius J. M.; Matzke, Marjori

    2014-01-01

    RDM1 (RNA-DIRECTED DNA METHYLATION1) is a small plant-specific protein required for RNA-directed DNA methylation (RdDM). RDM1 interacts with RNA polymerase II (Pol II), ARGONAUTE4 (AGO4), and the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) and binds to methylated single stranded DNA. As the only protein identified so far that interacts directly with DRM2, RDM1 plays a pivotal role in the RdDM mechanism by linking the de novo DNA methyltransferase activity to AGO4, which binds short interfering RNAs (siRNAs) that presumably base-pair with Pol II or Pol V scaffold transcripts synthesized at target loci. RDM1 also acts together with the chromatin remodeler DEFECTIVE IN RNA-DIRECTED DNA METHYLATION1 (DRD1) and the structural-maintenance-of-chromosomes solo hinge protein DEFECTIVE IN MERISTEM SILENCING3 (DMS3) to form the DDR complex, which facilitates synthesis of Pol V scaffold transcripts. The manner in which RDM1 acts in both the DDR complex and as a factor bridging DRM2 and AGO4 remains unclear. RDM1 contains no known protein domains but a prior structural analysis suggested distinct regions that create a hydrophobic pocket and promote homodimer formation, respectively. We have tested several mutated forms of RDM1 altered in the predicted pocket and dimerization regions for their ability to complement defects in RdDM and transcriptional gene silencing, support synthesis of Pol V transcripts, form homodimers, and interact with DMS3. Our results indicate that the ability to form homodimers is essential for RDM1 to function fully in the RdDM pathway and may be particularly important during the de novo methylation step. PMID:24498436

  10. The active form of the norovirus RNA-dependent RNA polymerase is a homodimer with cooperative activity.

    PubMed

    Högbom, Martin; Jäger, Katrin; Robel, Ivonne; Unge, Torsten; Rohayem, Jacques

    2009-02-01

    Norovirus (NV) is a leading cause of gastroenteritis worldwide and a major public health concern. So far, the replication strategy of NV remains poorly understood, mainly because of the lack of a cell system to cultivate the virus. In this study, the function and the structure of a key viral enzyme of replication, the RNA-dependent RNA polymerase (RdRp, NS7), was examined. The overall structure of the NV NS7 RdRp was determined by X-ray crystallography to a 2.3 A (0.23 nm) resolution (PDB ID 2B43), displaying a right-hand fold typical of the template-dependent polynucleotide polymerases. Biochemical analysis evidenced that NV NS7 RdRp is active as a homodimer, with an apparent K(d) of 0.649 microM and a positive cooperativity (Hill coefficient n(H)=1.86). Crystals of the NV NS7 homodimer displayed lattices containing dimeric arrangements with high shape complementarity statistics. This experimental data on the structure and function of the NV RdRp may set the cornerstone for the development of polymerase inhibitors to control the infection with NV, a medically relevant pathogen.

  11. ThWRKY4 from Tamarix hispida Can Form Homodimers and Heterodimers and Is Involved in Abiotic Stress Responses.

    PubMed

    Wang, Liuqiang; Zheng, Lei; Zhang, Chunrui; Wang, Yucheng; Lu, Mengzhu; Gao, Caiqiu

    2015-11-13

    WRKY proteins are a large family of transcription factors that are involved in diverse developmental processes and abiotic stress responses in plants. However, our knowledge of the regulatory mechanisms of WRKYs participation in protein-protein interactions is still fragmentary, and such protein-protein interactions are fundamental in understanding biological networks and the functions of proteins. In this study, we report that a WRKY protein from Tamarix hispida, ThWRKY4, can form both homodimers and heterodimers with ThWRKY2 and ThWRKY3. In addition, ThWRKY2 and ThWRKY3 can both bind to W-box motif with binding affinities similar to that of ThWRKY4. Further, the expression patterns of ThWRKY2 and ThWRKY3 are similar to that of ThWRKY4 when plants are exposed to abscisic acid (ABA). Subcellular localization shows that these three ThWRKY proteins are nuclear proteins. Taken together, these results demonstrate that ThWRKY4 is a dimeric protein that can form functional homodimers or heterodimers that are involved in abiotic stress responses.

  12. Crystal structure of endonuclease G in complex with DNA reveals how it nonspecifically degrades DNA as a homodimer

    PubMed Central

    Lin, Jason L. J.; Wu, Chyuan-Chuan; Yang, Wei-Zen; Yuan, Hanna S.

    2016-01-01

    Endonuclease G (EndoG) is an evolutionarily conserved mitochondrial protein in eukaryotes that digests nucleus chromosomal DNA during apoptosis and paternal mitochondrial DNA during embryogenesis. Under oxidative stress, homodimeric EndoG becomes oxidized and converts to monomers with diminished nuclease activity. However, it remains unclear why EndoG has to function as a homodimer in DNA degradation. Here, we report the crystal structure of the Caenorhabditis elegans EndoG homologue, CPS-6, in complex with single-stranded DNA at a resolution of 2.3 Å. Two separate DNA strands are bound at the ββα-metal motifs in the homodimer with their nucleobases pointing away from the enzyme, explaining why CPS-6 degrades DNA without sequence specificity. Two obligatory monomeric CPS-6 mutants (P207E and K131D/F132N) were constructed, and they degrade DNA with diminished activity due to poorer DNA-binding affinity as compared to wild-type CPS-6. Moreover, the P207E mutant exhibits predominantly 3′-to-5′ exonuclease activity, indicating a possible endonuclease to exonuclease activity change. Thus, the dimer conformation of CPS-6 is essential for maintaining its optimal DNA-binding and endonuclease activity. Compared to other non-specific endonucleases, which are usually monomeric enzymes, EndoG is a unique dimeric endonuclease, whose activity hence can be modulated by oxidation to induce conformational changes. PMID:27738134

  13. Functional analysis of the p40 and p75 proteins from Lactobacillus casei BL23.

    PubMed

    Bäuerl, Christine; Pérez-Martínez, Gaspar; Yan, Fang; Polk, D Brent; Monedero, Vicente

    2010-01-01

    The genomes of Lactobacillus casei/paracasei and Lactobacillus rhamnosus strains carry two genes encoding homologues of p40 and p75 from L. rhamnosus GG, two secreted proteins which display anti-apoptotic and cell protective effects on human intestinal epithelial cells. p40 and p75 carry cysteine, histidine-dependent aminohydrolase/peptidase (CHAP) and NLPC/P60 domains, respectively, which are characteristic of proteins with cell-wall hydrolase activity. In L. casei BL23 both proteins were secreted to the growth medium and were also located at the bacterial cell surface. The genes coding for both proteins were inactivated in this strain. Inactivation of LCABL_00230 (encoding p40) did not result in a significant difference in phenotype, whereas a mutation in LCABL_02770 (encoding p75) produced cells that formed very long chains. Purified glutathione-S-transferase (GST)-p40 and -p75 fusion proteins were able to hydrolyze the muropeptides from L. casei cell walls. Both fusions bound to mucin, collagen and to intestinal epithelial cells and, similar to L. rhamnosus GG p40, stimulated epidermal growth factor receptor phosphorylation in mouse intestine ex vivo. These results indicate that extracellular proteins belonging to the machinery of cell-wall metabolism in the closely related L. casei/paracasei-L. rhamnosus group are most likely involved in the probiotic effects described for these bacteria.

  14. Functional Analysis of the p40 and p75 Proteins from Lactobacillus casei BL23

    PubMed Central

    Bäuerl, Christine; Pérez-Martínez, Gaspar; Yan, Fang; Polk, D. Brent; Monedero, Vicente

    2011-01-01

    The genomes of Lactobacillus casei/paracasei and Lactobacillus rhamnosus strains carry two genes encoding homologues of p40 and p75 from L. rhamnosus GG, two secreted proteins which display anti-apoptotic and cell protective effects on human intestinal epithelial cells. p40 and p75 carry cysteine, histidine-dependent aminohydrolase/peptidase (CHAP) and NLPC/P60 domains, respectively, which are characteristic of proteins with cell-wall hydrolase activity. In L. casei BL23 both proteins were secreted to the growth medium and were also located at the bacterial cell surface. The genes coding for both proteins were inactivated in this strain. Inactivation of LCABL_00230 (encoding p40) did not result in a significant difference in phenotype, whereas a mutation in LCABL_02770 (encoding p75) produced cells that formed very long chains. Purified glutathione-S-transferase (GST)-p40 and -p75 fusion proteins were able to hydrolyze the muropeptides from L. casei cell walls. Both fusions bound to mucin, collagen and to intestinal epithelial cells and, similar to L. rhamnosus GG p40, stimulated epidermal growth factor receptor phosphorylation in mouse intestine ex vivo. These results indicate that extracellular proteins belonging to the machinery of cell-wall metabolism in the closely related L. casei/paracasei-L. rhamnosus group are most likely involved in the probiotic effects described for these bacteria PMID:21178363

  15. Formation of retinoid X receptor homodimers leads to repression of T3 response: hormonal cross talk by ligand-induced squelching.

    PubMed Central

    Lehmann, J M; Zhang, X K; Graupner, G; Lee, M O; Hermann, T; Hoffmann, B; Pfahl, M

    1993-01-01

    Thyroid hormone receptors (TRs) form heterodimers with retinoid X receptors (RXRs). Heterodimerization is required for efficient TR DNA binding to most response elements and transcriptional activation by thyroid hormone. RXRs also function as auxiliary proteins for several other receptors. In addition, RXR alpha can be induced by specific ligands to form homodimers. Here we report that RXR-specific retinoids that induce RXR homodimers are effective repressors of the T3 response. We provide evidence that this repression by RXR-specific ligands occurs by sequestering of RXR from TR-RXR heterodimers into RXR homodimers. This ligand-induced squelching may represent an important mechanism by which RXR-specific retinoids and 9-cis retinoic acid mediate hormonal cross talk among a subfamily of nuclear receptors activated by structurally unrelated ligands. Images PMID:8246986

  16. Homodimer of p50 (NF kappa B1) does not introduce a substantial directed bend into DNA according to three different experimental assays.

    PubMed Central

    Kuprash, D V; Rice, N R; Nedospasov, S A

    1995-01-01

    Transcription factors can distort the conformation of the DNA double helix upon binding to their target sites. Previously, studies utilizing circular permutation--electrophoretic mobility shift assay suggested that the homodimer of p50 (NF kappa B1), canonical NF-kappa B (p65-p50), as well as several non-canonical NF-kappa B/Rel complexes, may induce substantial DNA bending at the binding site. Here we have applied three additional experimental approaches, helical phasing analysis, minicircle binding and cyclization kinetics, and conclude that the homodimer of p50 introduces virtually no directed bend into the consensus kappa B sequences GGGACTTTCC or GGGAATTCCC. Images PMID:7885838

  17. The effects of Alcea rosea L., Malva sylvestris L. and Salvia libanotica L. water extracts on the production of anti-egg albumin antibodies, interleukin-4, gamma interferon and interleukin-12 in BALB/c mice.

    PubMed

    El Ghaoui, Walid Bou Jaber; Ghanem, Elsa Bou; Chedid, Lara Abou; Abdelnoor, Alexander M

    2008-12-01

    Polysaccharides obtained from certain plants have been reported to have immunomodulatory properties. As a consequence of these reports the aim of this study was to investigate some immunomodulatory properties of water extracts of Alcea rosea L. (ARE), Malva sylvestris L. (MSE) and Salvia libanotica L. (SLE).Groups of egg albumin (EA)-immunized and -non-immunized Balb/c mice were treated with the carbohydrate-rich water extracts. Mice from each group were bled and their spleens removed at 3, 6 and 10 days post-immunization/treatment. Anti-egg albumin antibody levels in the processed sera were determined by an enzyme linked immunosorbent assay (ELISA). RNA was extracted from spleen cells and interleukin-4 (IL-4), interleukin-12 (IL-12) and gamma-interferon transcripts were determined by the reverse transcription polymerase chain reaction (RT-PCR).ARE appeared to boost the antibody response to EA, but had no effect on IL-4 and gamma-interferon gene transcription. MSE and SLE appeared to have no effect on anti-EA antibody production, but enhanced IL-12 and gamma-interferon gene transcription. MSE appeared to switch off, and SLE had no effect on, IL-4 transcription.In conclusion, it appears that ARE is a B-lymphocyte polyclonal activator, and MSE and SLE are macrophage and T helper-1 (Th-1) activators.

  18. A single intratumoral injection of a fiber-mutant adenoviral vector encoding interleukin 12 induces remarkable anti-tumor and anti-metastatic activity in mice with Meth-A fibrosarcoma.

    PubMed

    Gao, Jian-Qing; Sugita, Toshiki; Kanagawa, Naoko; Iida, Keisuke; Eto, Yusuke; Motomura, Yoshiaki; Mizuguchi, Hiroyuki; Tsutsumi, Yasuo; Hayakawa, Takao; Mayumi, Tadanori; Nakagawa, Shinsaku

    2005-03-25

    Cytokine-encoding viral vectors are considered to be promising in cancer gene immunotherapy. Interleukin 12 (IL-12) has been used widely for anti-tumor treatment, but the administration route and tumor characteristics strongly influence therapeutic efficiency. Meth-A fibrosarcoma has been demonstrated to be insensitive to IL-12 treatment via systemic administration. In the present study, we developed an IL-12-encoding fiber-mutant adenoviral vector (AdRGD-IL-12) that showed enhanced gene transfection efficiency in Meth-A tumor cells, and the production of IL-12 p70 in the culture supernatant from transfected cells was confirmed by ELISA. In therapeutic experiments, a single low-dose (2 x 10(7) plaque-forming units) intratumoral injection of AdRGD-IL-12 elicited pronounced anti-tumor activity and notably prolonged the survival of Meth-A fibrosarcoma-bearing mice. Immunohistochemical staining revealed that the IL-12 vector induced the accumulation of T cells in tumor tissue. Furthermore, intratumoral administration of the vector induced an anti-metastasis effect as well as long-term specific immunity against syngeneic tumor challenge.

  19. Thermal and chemical unfolding pathways of PaSdsA1 sulfatase, a homo-dimer with topologically interlinked chains.

    PubMed

    Aguirre, César; Goto, Yuji; Costas, Miguel

    2016-01-01

    Understanding the mechanisms as to how interlinked proteins entangle and fold is a challenge. PaSdsA1 sulfatase is a homo-dimer containing two zinc atoms per monomer. The monomer chains are interlinked in a dimerization domain. To study the unfolding pathways denaturation experiments were performed. In the native protein three forms coexist in chemical equilibrium, each with a different number of zinc atoms. In the chemical unfolding of the holo-dimers the entanglement of the chains is preserved and acts as a 'folding seed', allowing the unfolding process to be reversible. Thermal irreversible unfolding of the holo-dimers favours dissociation, producing monomers that are SDS-stabilized. The thermal unfolding of these monomers is reversible. However, it is not possible to form dimers from unfolded monomers.

  20. Structure of the homodimer of uridine phosphorylase from Salmonella typhimurium in the native state at 1.9 Å resolution

    NASA Astrophysics Data System (ADS)

    Timofeev, V. I.; Pavlyuk, B. F.; Lashkov, A. A.; Seregina, T. A.; Gabdulkhakov, A. G.; Vaĭnshteĭn, B. K.; Mikhaĭlov, A. M.

    2007-11-01

    Uridine phosphorylase ( UPh) belongs to pyrimidine nucleoside phosphorylases. This enzyme catalyzes cleavage of the C-N glycoside bond in uridine to form uracil and ribose-1’-phosphate. Uridine phosphorylase supplies cells with nucleotide precursors by catalyzing the phosphorolysis of purine and pyrimidine nucleosides. This is an alternative to de novo nucleotide synthesis. The three-dimensional structure of native uridine phosphorylase from Salmonella typhimurium ( StUPh) in a new crystal form was solved and refined at 1.90 Å resolution ( R st = 20.37%; R free = 24.69%; the rmsd of bond lengths and bond angles are 0.009 Å and 1.223°, respectively). A homodimer containing two asynchronously functioning active sites was demonstrated to be the minimum structural unit necessary for function of the hexameric StUPh molecule ( L 33 L 2). Each active site is formed by amino acid residues of both subunits.

  1. A novel EID family member, EID-3, inhibits differentiation and forms a homodimer or heterodimer with EID-2

    SciTech Connect

    Sasajima, Yuka; Tanaka, Hiroyuki; Miyake, Satoshi; Yuasa, Yasuhito . E-mail: yuasa.monc@tmd.ac.jp

    2005-08-05

    The EID family members, i.e., E1A-like inhibitor of differentiation-1 (EID-1) and EID-1-like inhibitor of differentiation-2 (EID-2), were identified as negative regulators of cellular differentiation. EID-1 seems to inhibit differentiation by blocking histone acetyltransferase activity and EID-2 possibly inhibits differentiation through binding to class I histone deacetylases (HDACs). Here, we report a novel inhibitor of differentiation exhibiting homology with EID-2 termed EID-3 (EID-2-like inhibitor of differentiation-3). Like EID-2, EID-3 inhibited MyoD- and GR{alpha}-dependent transcription and blocked muscle differentiation in cultured cells by binding to class I HDACs. Unlike that of EID-2, the C-terminus, but not the N-terminus, of EID-3 was required for nuclear localization. EID-3 formed a homodimer or heterodimer with EID-2. These results suggest that EID-3 inhibits differentiation by blocking transcription as a complex in cells.

  2. DNA binding of Jun and Fos bZip domains: homodimers and heterodimers induce a DNA conformational change in solution.

    PubMed Central

    John, M; Leppik, R; Busch, S J; Granger-Schnarr, M; Schnarr, M

    1996-01-01

    We constructed plasmids encoding the sequences for the bZip modules of c-Jun and c-Fos which could then be expressed as soluble proteins in Escherichia coli. The purified bZip modules were tested for their binding capacities of synthetic oligonucleotides containing either TRE or CRE recognition sites in electrophoretic mobility shift assays and circular dichroism (CD). Electrophoretic mobility shift assays showed that bZip Jun homodimers and bZip Jun/Fos heterodimers bind a collagenase-like TRE (CTGACTCAT) with dissociation constants of respectively 1.4 x 10(-7) M and 5 x 10(-8) M. As reported earlier [Patel et al. (1990) Nature 347, 572-575], DNA binding induces a marked change of the protein structure. However, we found that the DNA also undergoes a conformational change. This is most clearly seen with small oligonucleotides of 13 or 14 bp harboring respectively a TRE (TGACTCA) or a CRE (TGACGTCA) sequence. In this case, the positive DNA CD signal at 280 nm increases almost two-fold with a concomitant blue-shift of 3-4 nm. Within experimental error the same spectral changes are observed for TRE and CRE containing DNA fragments. The spectral changes observed with a non-specific DNA fragment are weaker and the signal of free DNA is recovered upon addition of much smaller salt concentrations than required for a specific DNA fragment. Surprisingly the spectral changes induced by Jun/Jun homodimers are not identical to those induced by Jun/Fos heterodimers. However, in both cases the increase of the positive CD band and the concomitant blue shift would be compatible with a B to A-transition of part of the binding site or a DNA conformation intermediate between the canonical A and B structures. PMID:8948639

  3. Communication between the ERRalpha homodimer interface and the PGC-1alpha binding surface via the helix 8-9 loop.

    PubMed

    Greschik, Holger; Althage, Magnus; Flaig, Ralf; Sato, Yoshiteru; Chavant, Virginie; Peluso-Iltis, Carole; Choulier, Laurence; Cronet, Philippe; Rochel, Natacha; Schüle, Roland; Strömstedt, Per-Erik; Moras, Dino

    2008-07-18

    Although structural studies on the ligand-binding domain (LBD) have established the general mode of nuclear receptor (NR)/coactivator interaction, determinants of binding specificity are only partially understood. The LBD of estrogen receptor-alpha (ERalpha), for example, interacts only with a region of peroxisome proliferator-activated receptor coactivator (PGC)-1alpha, which contains the canonical LXXLL motif (NR box2), whereas the LBD of estrogen-related receptor-alpha (ERRalpha) also binds efficiently an untypical, LXXYL-containing region (NR box3) of PGC-1alpha. Surprisingly, in a previous structural study, the ERalpha LBD has been observed to bind NR box3 of transcriptional intermediary factor (TIF)-2 untypically via LXXYL, whereas the ERRalpha LBD binds this region of TIF-2 only poorly. Here we present a new crystal structure of the ERRalpha LBD in complex with a PGC-1alpha box3 peptide. In this structure, residues N-terminal of the PGC-1alpha LXXYL motif formed contacts with helix 4, the loop connecting helices 8 and 9, and with the C terminus of the ERRalpha LBD. Interaction studies using wild-type and mutant PGC-1alpha and ERRalpha showed that these contacts are functionally relevant and are required for efficient ERRalpha/PGC-1alpha interaction. Furthermore, a structure comparison between ERRalpha and ERalpha and mutation analyses provided evidence that the helix 8-9 loop, which differs significantly in both nuclear receptors, is a major determinant of coactivator binding specificity. Finally, our results revealed that in ERRalpha the helix 8-9 loop allosterically links the LBD homodimer interface with the coactivator cleft, thus providing a plausible explanation for distinct PGC-1alpha binding to ERRalpha monomers and homodimers.

  4. Characterisation and stability of anthocyanins in purple-fleshed sweet potato P40.

    PubMed

    Xu, Jianteng; Su, Xiaoyu; Lim, Soyoung; Griffin, Jason; Carey, Edward; Katz, Benjamin; Tomich, John; Smith, J Scott; Wang, Weiqun

    2015-11-01

    Purple-fleshed sweet potato P40 has been shown to prevent colorectal cancer in a murine model. This study is to identify anthocyanins by using HPLC/MS-MS and assess the stability during various cooking conditions. P40 possesses a high content of anthocyanins up to 14 mg/g dry matter. Total 12 acylated anthocyanins are identified. Top three anthocyanins, e.g., cyanidin 3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside, peonidin 3-caffeoyl sophoroside-5-glucoside, and cyanidin 3-(6"-caffeoyl-6"-feruloylsophoroside)-5-glucoside, account for half of the anthocyanin contents. Over 80% of anthocyanins measured by acid hydrolysis were cyanidin derivatives, indicating P40 is unique when compared with other purple-fleshed sweet potatoes that usually contain more peonidin than cyanidin. Steaming, pressure cooking, microwaving, and frying but not baking significantly reduced 8-16% of total anthocyanin contents. Mono-acylated anthocyanins showed a higher resistance against heat than di- and non-acylated. Among of which, cyanidin 3-p-hydroxybenzoylsophoroside-5-glucoside exhibited the best thermal stability. The stable acylated and cyanidin-predominated anthocyanins in P40 may provide extra benefits for cancer prevention.

  5. A new quadruple hydrogen-bonding module with a DDAA array: formation of a stable homodimer without competition from undesired hydrogen-bonded dimers.

    PubMed

    Hisamatsu, Yosuke; Shirai, Naohiro; Ikeda, Shin-ichi; Odashima, Kazunori

    2009-10-01

    A new DDAA hydrogen-bonding module (UImp-2), based on a ureidoimidazo[1,2-a]pyrimidine structure, forms a highly stable homodimer (K(dim) > 1.1 x 10(5) M(-1) in CDCl(3)) without competition from undesired hydrogen-bonded dimers.

  6. Interleukin-12- and Gamma Interferon-Dependent Innate Immunity Are Essential and Sufficient for Long-Term Survival of Passively Immunized Mice Infected with Herpes Simplex Virus Type 1

    PubMed Central

    Vollstedt, Sabine; Franchini, Marco; Alber, Gottfried; Ackermann, Mathias; Suter, Mark

    2001-01-01

    Interferon (IFN) type I (alpha/beta IFN [IFN-α/β]) is very important in directly controlling herpes simplex virus type I (HSV-1) replication as well as in guiding and upregulating specific immunity against this virus. By contrast, the roles of IFN type II (IFN-γ) and antibodies in the defense against HSV-1 are not clear. Mice without a functional IFN system and no mature B and T cells (AGR mice) did not survive HSV-1 infection in the presence or absence of neutralizing antibodies to the virus. Mice without a functional IFN type I system and with no mature B and T cells (AR129 mice) were unable to control infection with as little as 10 PFU of HSV-1 strain F. By contrast, in the presence of passively administered neutralizing murine antibodies to HSV-1, some AR129 mice survived infection with up to104 PFU of HSV-1. This acute immune response was dependent on the presence of interleukin-12 (IL-12) p75. Interestingly, some virus-infected mice stayed healthy for several months, at which time antibody to HSV-1 was no longer detectable. Treatment of these virus-exposed mice with dexamethasone led to death in approximately 40% of the mice. HSV-1 was found in brains of mice that did not survive dexamethasone treatment, whereas HSV-1 was absent in those that survived the treatment. We conclude that in the presence of passively administered HSV-1-specific antibodies, the IL-12-induced IFN-γ-dependent innate immune response is able to control low doses of virus infection. Surprisingly, in a significant proportion of these mice, HSV-1 appears to persist in the absence of antibodies and specific immunity. PMID:11559791

  7. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-06-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

  8. An Optimized Hepatitis C Virus E2 Glycoprotein Core Adopts a Functional Homodimer That Efficiently Blocks Virus Entry.

    PubMed

    McCaffrey, Kathleen; Boo, Irene; Owczarek, Catherine M; Hardy, Matthew P; Perugini, Matthew A; Fabri, Louis; Scotney, Pierre; Poumbourios, Pantelis; Drummer, Heidi E

    2017-03-01

    The hepatitis C virus (HCV) envelope glycoprotein E2 is the major target of broadly neutralizing antibodies in vivo and is the focus of efforts in the rational design of a universal B cell vaccine against HCV. The E2 glycoprotein exhibits a high degree of amino acid variability which localizes to three discrete regions: hypervariable region 1 (HVR1), hypervariable region 2 (HVR2), and the intergenotypic variable region (igVR). All three variable regions contribute to immune evasion and/or isolate-specific structural variations, both important considerations for vaccine design. A high-resolution structural definition of the intact HCV envelope glycoprotein complex containing E1 and E2 remains to be elucidated, while crystallographic structures of a recombinant E2 ectodomain failed to resolve HVR1, HVR2, and a major neutralization determinant adjacent to HVR1. To obtain further information on E2, we characterized the role of all three variable regions in E2 ectodomain folding and function in the context of a recombinant ectodomain fragment (rE2). We report that removal of the variable regions accelerates binding to the major host cell receptor CD81 and that simultaneous deletion of HVR2 and the igVR is required to maintain wild-type CD81-binding characteristics. The removal of the variable regions also rescued the ability of rE2 to form a functional homodimer. We propose that the rE2 core provides novel insights into the role of the variable motifs in the higher-order assembly of the E2 ectodomain and may have implications for E1E2 structure on the virion surface. IMPORTANCE Hepatitis C virus (HCV) infection affects ∼2% of the population globally, and no vaccine is available. HCV is a highly variable virus, and understanding the presentation of key antigenic sites at the virion surface is important for the design of a universal vaccine. This study investigates the role of three surface-exposed variable regions in E2 glycoprotein folding and function in the context

  9. The effects of nonidet P40 on the function of rat peritoneal mast cells in vitro.

    PubMed

    Batchelor, K W; Stanworth, D R

    1981-01-01

    1 Treatment of purified rat peritoneal mast cells at 37 degrees C with concentrations of the non-ionic detergent nonidet P40 (NP40) up to 0.005% (v/v) failed to reduce their viability. 2 There was a marked reduction in the histamine releasing capacity of NP40-treated mast cells upon challenge with a variety of selective (adrenocorticotrophic hormone 1-24 (Synacthen), rabbit anti-rat IgE antiserum, adenosine triphosphate (ATP) and the calcium ionophore, A 23187) and non-selective (rabbit anti-rat mast cell antiserum plus complement) histamine liberators. 3 Nonidet P40 (0.005%) was found to reduce the activity of a mast cell membrane 'ecto-enzyme', calcium-activated ATPase, by about 45% when presented at the time of its assay.

  10. Nonidet P40 and the retrograde transport of horseradish peroxidase in undamaged visceral nerves.

    PubMed

    Rogers, R C; Liholtz, L A; Ebly, E M

    1982-06-01

    The cell bodies of origin of peripheral nerves, in particular visceral nerves, are often difficult to identify using standard horseradish peroxidase (HRP) methods. The non-ionic surfactant Nonidet-P40, when applied to intact peripheral nerve along with HRP, allows the investigator to examine the neurons of origin of the nerve without cutting the fibers or injecting label into its peripheral terminal field.

  11. Prismatic-cased Li/(CF(x))n P-40 Cell Performance under Resistive Loads

    NASA Technical Reports Server (NTRS)

    King, L. M.; Margalit, N.

    1996-01-01

    It was concluded that the P-40 cell performed well in the -23.5 deg. C to 71 deg. range with resistive loads from 1.35 ohms to 675 ohms. Storage with subsequent discharge: tests performed to date show little self-discharge up to 2 years. And good pulse capability with 3, 2, and 1 ohm loads at 0, 24, and 49 deg. C.

  12. Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue.

    PubMed

    Davies, A A; Wigglesworth, N M; Allan, D; Owens, R J; Crumpton, M J

    1984-04-01

    Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5'-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.

  13. Impaired Endothelial Nitric Oxide Synthase Homodimer Formation Triggers Development of Transplant Vasculopathy - Insights from a Murine Aortic Transplantation Model

    PubMed Central

    Oberhuber, Rupert; Riede, Gregor; Cardini, Benno; Bernhard, David; Messner, Barbara; Watschinger, Katrin; Steger, Christina; Brandacher, Gerald; Pratschke, Johann; Golderer, Georg; Werner, Ernst R.; Maglione, Manuel

    2016-01-01

    Transplant vasculopathy (TV) represents a major obstacle to long-term graft survival and correlates with severity of ischemia reperfusion injury (IRI). Donor administration of the nitric oxide synthases (NOS) co-factor tetrahydrobiopterin has been shown to prevent IRI. Herein, we analysed whether tetrahydrobiopterin is also involved in TV development. Using a fully allogeneic mismatched (BALB/c to C57BL/6) murine aortic transplantation model grafts subjected to long cold ischemia time developed severe TV with intimal hyperplasia (α-smooth muscle actin positive cells in the neointima) and endothelial activation (increased P-selectin expression). Donor pretreatment with tetrahydrobiopterin significantly minimised these changes resulting in only marginal TV development. Severe TV observed in the non-treated group was associated with increased protein oxidation and increased occurrence of endothelial NOS monomers in the aortic grafts already during graft procurement. Tetrahydrobiopterin supplementation of the donor prevented all these early oxidative changes in the graft. Non-treated allogeneic grafts without cold ischemia time and syngeneic grafts did not develop any TV. We identified early protein oxidation and impaired endothelial NOS homodimer formation as plausible mechanistic explanation for the crucial role of IRI in triggering TV in transplanted aortic grafts. Therefore, targeting endothelial NOS in the donor represents a promising strategy to minimise TV. PMID:27883078

  14. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo

    PubMed Central

    Tropak, Michael B; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J; Walia, Jagdeep S; Mark, Brian L; Mahuran, Don

    2016-01-01

    Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels. PMID:26966698

  15. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo.

    PubMed

    Tropak, Michael B; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J; Walia, Jagdeep S; Mark, Brian L; Mahuran, Don

    2016-01-01

    Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels.

  16. Albumin Homodimers in Patients with Cirrhosis: Clinical and Prognostic Relevance of a Novel Identified Structural Alteration of the Molecule

    PubMed Central

    Baldassarre, Maurizio; Domenicali, Marco; Naldi, Marina; Laggetta, Maristella; Giannone, Ferdinando A.; Biselli, Maurizio; Patrono, Daniela; Bertucci, Carlo; Bernardi, Mauro; Caraceni, Paolo

    2016-01-01

    Decompensated cirrhosis is associated to extensive post-transcriptional changes of human albumin (HA). This study aims to characterize the occurrence of HA homodimerization in a large cohort of patients with decompensated cirrhosis and to evaluate its association with clinical features and prognosis. HA monomeric and dimeric isoforms were identified in peripheral blood by using a HPLC-ESI-MS technique in 123 cirrhotic patients hospitalized for acute decompensation and 50 age- and sex-comparable healthy controls. Clinical and biochemical parameters were recorded and patients followed up to one year. Among the monomeric isoforms identified, the N- and C-terminal truncated and the native HA underwent homodimerization. All three homodimers were significantly more abundant in patients with cirrhosis, acute-on-chronic liver failure and correlate with the prognostic scores. The homodimeric N-terminal truncated isoform was independently associated to disease complications and was able to stratify 1-year survival. As a result of all these changes, the monomeric native HA was significantly decreased in patients with cirrhosis, being also associated with a poorer prognosis. In conclusion homodimerization is a novel described structural alteration of the HA molecule in decompensated cirrhosis and contributes to the progressive reduction of the monomeric native HA, the only isoform provided of structural and functional integrity. PMID:27782157

  17. Structure of the human NF-kappaB p52 homodimer-DNA complex at 2.1 A resolution.

    PubMed Central

    Cramer, P; Larson, C J; Verdine, G L; Müller, C W

    1997-01-01

    The crystal structure of human NF-kappaB p52 in its specific complex with the natural kappaB DNA binding site MHC H-2 has been solved at 2.1 A resolution. Whereas the overall structure resembles that of the NF-kappaB p50-DNA complex, pronounced differences are observed within the 'insert region'. This sequence segment differs in length between different Rel proteins. Compared with NF-kappaB p50, the compact alpha-helical insert region element is rotated away from the core of the N-terminal domain, opening up a mainly polar cleft. The insert region presents potential interaction surfaces to other proteins. The high resolution of the structure reveals many water molecules which mediate interactions in the protein-DNA interface. Additional complexity in Rel protein-DNA interaction comes from an extended interfacial water cavity that connects residues at the edge of the dimer interface to the central DNA bases. The observed water network might acount for differences in binding specificity between NF-kappaB p52 and NF-kappaB p50 homodimers. PMID:9384586

  18. PDGF A chain homodimers drive proliferation of bipotential (O-2A) glial progenitor cells in the developing rat optic nerve.

    PubMed Central

    Pringle, N; Collarini, E J; Mosley, M J; Heldin, C H; Westermark, B; Richardson, W D

    1989-01-01

    The bipotential glial progenitor cells (O-2A progenitors), which during development of the rat optic nerve give rise to oligodendrocytes and type 2 astrocytes, are stimulated to divide in culture by platelet-derived growth factor (PDGF), and there is evidence that PDGF is important for development of the O-2A cell lineage in vivo. We have visualized PDGF mRNA in the rat optic nerve by in situ hybridization, and its spatial distribution is compatible with the idea that type 1 astrocytes are the major source of PDGF in the nerve. We can detect mRNA encoding the A chain, but not the B chain of PDGF in the brain and optic nerve, suggesting that the major form of PDGF in the central nervous system is a homodimer of A chains (PDGF-AA). PDGF-AA is a more potent mitogen for O-2A progenitor cells than is PDGF-BB, while the reverse is true for human or rat fibroblasts. Fibroblasts display two types of PDGF receptors, type A receptors which bind to all three dimeric isoforms of PDGF, and type B receptors which bind PDGF-BB and PDGF-AB, but have low affinity for PDGF-AA. Our results suggest that O-2A progenitor cells possess predominantly type A receptors, and proliferate during development in response to PDGF-AA secreted by type 1 astrocytes. Images PMID:2545439

  19. PDGF A chain homodimers drive proliferation of bipotential (O-2A) glial progenitor cells in the developing rat optic nerve.

    PubMed

    Pringle, N; Collarini, E J; Mosley, M J; Heldin, C H; Westermark, B; Richardson, W D

    1989-04-01

    The bipotential glial progenitor cells (O-2A progenitors), which during development of the rat optic nerve give rise to oligodendrocytes and type 2 astrocytes, are stimulated to divide in culture by platelet-derived growth factor (PDGF), and there is evidence that PDGF is important for development of the O-2A cell lineage in vivo. We have visualized PDGF mRNA in the rat optic nerve by in situ hybridization, and its spatial distribution is compatible with the idea that type 1 astrocytes are the major source of PDGF in the nerve. We can detect mRNA encoding the A chain, but not the B chain of PDGF in the brain and optic nerve, suggesting that the major form of PDGF in the central nervous system is a homodimer of A chains (PDGF-AA). PDGF-AA is a more potent mitogen for O-2A progenitor cells than is PDGF-BB, while the reverse is true for human or rat fibroblasts. Fibroblasts display two types of PDGF receptors, type A receptors which bind to all three dimeric isoforms of PDGF, and type B receptors which bind PDGF-BB and PDGF-AB, but have low affinity for PDGF-AA. Our results suggest that O-2A progenitor cells possess predominantly type A receptors, and proliferate during development in response to PDGF-AA secreted by type 1 astrocytes.

  20. Definition of the surface in the thyroid hormone receptor ligand binding domain for association as homodimers and heterodimers with retinoid X receptor.

    PubMed

    Ribeiro, R C; Feng, W; Wagner, R L; Costa, C H; Pereira, A C; Apriletti, J W; Fletterick, R J; Baxter, J D

    2001-05-04

    Thyroid hormone receptors (TRs) bind as homodimers or heterodimers with retinoid X receptors (RXRs) to DNA elements with diverse orientations of AGGTCA half-sites. We performed a comprehensive x-ray crystal structure-guided mutation analysis of the TR ligand binding domain (TR LBD) surface to map the functional interface for TR homodimers and heterodimers with RXR in the absence and/or in the presence of DNA. We also identified the molecular contacts in TR LBDs crystallized as dimers. The results show that crystal dimer contacts differ from those found in the functional studies. We found that identical TR LBD residues found in helices 10 and 11 are involved in TR homodimerization and heterodimerization with RXR. Moreover, the same TR LBD surface is operative for dimerization with direct repeats spaced by 4 base pairs (DR-4) and with the inverted palindrome spaced by 6 base pairs (F2), but not with TREpal (unspaced palindrome), where homodimers appear to be simply two monomers binding independently to DNA. We also demonstrate that interactions between the TR and RXR DNA binding domains stabilize TR-RXR heterodimers on DR-4. The dimer interface can be functional in the cell, because disruption of key residues impairs transcriptional activity of TRs mediated through association with RXR LBD linked to GAL4 DNA-binding domain.

  1. BMP2/7 heterodimer is a stronger inducer of bone regeneration in peri-implant bone defects model than BMP2 or BMP7 homodimer.

    PubMed

    Sun, Ping; Wang, Jingxiao; Zheng, Yuanna; Fan, Yi; Gu, Zhiyuan

    2012-01-01

    This study aimed to compare the effects of bone morphogenetic protein BMP2/7 heterodimer and BMP homodimers on bone regeneration in bone defects model. Identical peri-implant bone defects model were created using proper controls on the frontal skull in 18 minipigs. Collagen sponges with low-dose (30 ng/mL) BMP2/7 heterodimer, BMP2 or BMP7 homodimer were filled in the defects. New bone formation and the expression of type I collagen (Col1), alkaline phosphatase (ALP) and osteocalcin (OCN) were evaluated after 2, 3, and 6 weeks of implantation. BMP2/7 resulted in significantly higher new bone areas percentage in the defect region than BMP2 and BMP7 (p<0.05). Immunohistochemical staining of Col1, ALP and OCN was stronger in BMP2/7 group than BMP2, BMP7 and control group (p<0.05). These results demonstrate that BMP2/7 heterodimer is a stronger inducer of osteoblastogenesis and could be applied at low dose to reduce the cost and side effects of BMP homodimers.

  2. Structural Analysis of Guanylyl Cyclase-Activating Protein-2 (GCAP-2) Homodimer by Stable Isotope-Labeling, Chemical Cross-Linking, and Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Pettelkau, Jens; Thondorf, Iris; Theisgen, Stephan; Lilie, Hauke; Schröder, Thomas; Arlt, Christian; Ihling, Christian H.; Sinz, Andrea

    2013-12-01

    The topology of the GCAP-2 homodimer was investigated by chemical cross-linking and high resolution mass spectrometry. Complementary conducted size-exclusion chromatography and analytical ultracentrifugation studies indicated that GCAP-2 forms a homodimer both in the absence and in the presence of Ca2+. In-depth MS and MS/MS analysis of the cross-linked products was aided by 15 N-labeled GCAP-2. The use of isotope-labeled protein delivered reliable structural information on the GCAP-2 homodimer, enabling an unambiguous discrimination between cross-links within one monomer (intramolecular) or between two subunits (intermolecular). The limited number of cross-links obtained in the Ca2+-bound state allowed us to deduce a defined homodimeric GCAP-2 structure by a docking and molecular dynamics approach. In the Ca2+-free state, GCAP-2 is more flexible as indicated by the higher number of cross-links. We consider stable isotope-labeling to be indispensable for deriving reliable structural information from chemical cross-linking data of multi-subunit protein assemblies.

  3. Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant

    SciTech Connect

    Atwell, Shane; Brouillette, Christie G.; Conners, Kris; Emtage, Spencer; Gheyi, Tarun; Guggino, William B.; Hendle, Jorg; Hunt, John F.; Lewis, Hal A.; Lu, Frances; Protasevich, Irina I.; Rodgers, Logan A.; Romero, Rich; Wasserman, Stephen R.; Weber, Patricia C.; Wetmore, Diana; Zhang, Feiyu F.; Zhao, Xun

    2010-04-26

    Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646({Delta}405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-{angstrom} resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The {Delta}F508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

  4. A conserved proline residue in the leucine zipper region of AtbZIP34 and AtbZIP61 in Arabidopsis thaliana interferes with the formation of homodimer

    SciTech Connect

    Shen Huaishun; Cao Kaiming; Wang Xiping

    2007-10-19

    Two putative Arabidopsis E group bZIP transcript factors, AtbZIP34 and AtbZIP61, are nuclear-localized and their transcriptional activation domain is in their N-terminal region. By searching GenBank, we found other eight plant homologues of AtbZIP34 and AtbZIP61. All of them have a proline residue in the third heptad of zipper region. Yeast two-hybrid assay and EMSA showed that AtbZIP34 and AtbZIP61 could not form homodimer while their mutant forms, AtbZIP34m and AtbZIP61m, which the proline residue was replaced by an alanine residue in the zipper region, could form homodimer and bind G-box element. These results suggest that the conserved proline residue interferes with the homodimer formation. However, both AtbZIP34 and AtbZIP61 could form heterodimers with members of I group and S group transcription factors in which some members involved in vascular development. So we speculate that AtbZIP34 and AtbZIP61 may participate in plant development via interacting with other group bZIP transcription factors.

  5. Two new crystal forms of the choline-binding domain of the major pneumococcal autolysin: insights into the dynamics of the active homodimer.

    PubMed

    Fernández-Tornero, Carlos; García, Ernesto; López, Rubens; Giménez-Gallego, Guillermo; Romero, Antonio

    2002-08-02

    Very little is known about the in vivo regulation of the catalytic activity of the major pneumococcal autolysin (LytA), a surface-exposed enzyme that rules the self-destruction of pneumococcal cells through degradation of their peptidoglycan backbone. Two new crystal forms of the cell wall anchoring domain of LytA were obtained, and their structures were solved and refined to 2.4A and 2.8A resolution. The domain is a homodimer with a boomerang-like shape in which the tertiary structure of each monomer is comprised by six independent beta hairpins arranged in a superhelical fashion. Choline molecules at the hydrophobic interface of consecutive hairpins maintain this unique structure. The C-terminal hairpin (last 13 residues of LytA) in the solenoid is responsible for the formation of the catalytically active homodimer. Although the general fold in the structures derived from both crystal forms is essentially the same, two different conformations of the basic homodimer are observed. Biochemical approaches have demonstrated the fundamental role of the 11 C-terminal residues in the catalytic activity of LytA. The studies reported here reveal the importance of some amino acid residues at the C terminus in the determination of the relative distance of the active dimeric form of the autolysin, which appears to be essential for the catalytic activity of this enzyme.

  6. Electrophoretic separation of A gamma and G gamma human globin chains in Nonidet P-40.

    PubMed

    Guerrasio, A; Saglio, G; Mazza, U; Pich, P; Camaschella, C; Ricco, G; Gianazza, E; Righetti, P G

    1979-11-15

    Electrophoresis in cellulose acetate in the presence of 3% Nonidet P-40 can resolve two neutral genetic variants, A gamma and G gamma human fetal globin chains. The ratio of these two chains, determined by densitometry of the electrophoretic strips, is in excellent agreement with the Gly-Ala ratio obtained by chemical analysis of the cyanogen bromide fragment gamma CB3. It is suggested that the detergent binds preferentially to the hydrophobic amino acid segment 133-141 in the A gamma chain, thus masking either a Lys or an Arg residue at the two extremes.

  7. Phosphorylation of phosphatase inhibitor-2 (i-2) by a bovine thymus tyrosine protein kinase, p40

    SciTech Connect

    DePaoli-Roach, A.A.; Votaw, P.; Zioncheck, T.F.; Harrison, M.L.; Geahlen, R.L.

    1987-05-01

    Phosphatase inhibitor-2, a heat stable protein of Mr 22,800, is a regulatory component of the ATP-Mg-dependent phosphatase. It has been shown that in the cell tyrosine kinase activation can result in altered phosphorylation at serine and/or threonine residues, but the mechanism involved is unknown. The authors have found that i-2 is a substrate for a tyrosine specific protein kinase, p40, purified from bovine thymus. The purified enzyme is a monomer of Mr 40,000 that is autophosphorylated at tyrosine residue(s). The stoichiometry of phosphorylation of i-2 by this tyrosine protein kinase is up to 1 mol of phosphate per mol of i-2. Phosphoaminoacid analysis revealed that all the phosphate introduced was associated with tyrosine residues. Mapping of TSP-tryptic peptides by TLE and isoelectric focusing showed one major labeled fragment. Using the ATP-Mg-dependent phosphatase, a lesser extent of phosphorylation of i-2 by p40 was obtained although partial activation of the phosphatase was observed. The effect on the activity was not due to FA/GSK-3 contamination. These results could provide an important link between tyrosine protein kinase activity and modulation of phosphorylation at serine and/or threonine residues.

  8. p40 (ΔNp63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma.

    PubMed

    Bishop, Justin A; Teruya-Feldstein, Julie; Westra, William H; Pelosi, Giuseppe; Travis, William D; Rekhtman, Natasha

    2012-03-01

    Immunohistochemistry has recently emerged as a powerful ancillary tool for differentiating lung adenocarcinoma and squamous cell carcinoma-a distinction with important therapeutic implications. Although the most frequently recommended squamous marker p63 is extremely sensitive, it suffers from low specificity due to its reactivity in a substantial proportion of lung adenocarcinomas and other tumor types, particularly lymphomas. p40 is a relatively unknown antibody that recognizes ΔNp63-a p63 isoform suggested to be highly specific for squamous/basal cells. Here we compared the standard p63 antibody (4A4) and p40 in a series of 470 tumors from the archives of Memorial Sloan-Kettering Cancer Center and The Johns Hopkins Hospital, which included lung squamous cell carcinomas (n=81), adenocarcinomas (n=237), and large cell lymphomas (n=152). The p63 was positive in 100% of squamous cell carcinomas, 31% of adenocarcinomas, and 54% of large cell lymphomas (sensitivity 100%, specificity 60%). In contrast, although p40 was also positive in 100% of squamous cell carcinomas, only 3% of adenocarcinomas, and none of large cell lymphomas had p40 labeling (sensitivity 100%, specificity 98%). The mean percentage of p63 versus p40-immunoreactive cells in squamous cell carcinomas was equivalent (97 vs 96%, respectively, P=0.73). Rare adenocarcinomas with p40 labeling had reactivity in no more than 5% of tumor cells, whereas the mean (range) of p63-positive cells in adenocarcinomas and lymphomas was 26% (1-90%) and 48% (2-100%), respectively. In summary, p40 is equivalent to p63 in sensitivity for squamous cell carcinoma, but it is markedly superior to p63 in specificity, which eliminates a potential pitfall of misinterpreting a p63-positive adenocarcinoma or unsuspected lymphoma as squamous cell carcinoma. These findings strongly support the routine use of p40 in place of p63 for the diagnosis of pulmonary squamous cell carcinoma.

  9. Activation of Epidermal Growth Factor Receptor Mediates Mucin Production Stimulated by p40, a Lactobacillus rhamnosus GG-derived Protein*

    PubMed Central

    Wang, Lihong; Cao, Hailong; Liu, Liping; Wang, Bangmao; Walker, W. Allan; Acra, Sari A.; Yan, Fang

    2014-01-01

    The mucus layer coating the gastrointestinal tract serves as the first line of intestinal defense against infection and injury. Probiotics promote mucin production by goblet cells in the intestine. p40, a Lactobacillus rhamnosus GG-derived soluble protein, has been shown to transactivate the EGF receptor (EGFR) in intestinal epithelial cells, which is required for inhibition of apoptosis and preservation of barrier function in the colon, thereby ameliorating intestinal injury and colitis. Because activation of EGFR has been shown to up-regulate mucin production in goblet cells, the purpose of this study was to investigate the effects and mechanisms of p40 regulation of mucin production. p40 activated EGFR and its downstream target, Akt, in a concentration-dependent manner in LS174T cells. p40 stimulated Muc2 gene expression and mucin production in LS174T cells, which were abolished by inhibition of EGFR kinase activity, down-regulation of EGFR expression by EGFR siRNA transfection, or suppression of Akt activation. Treatment with p40 increased mucin production in the colonic epithelium, thus thickening the mucus layer in the colon of wild type, but not of Egfrwa5 mice, which have a dominant negative mutation in the EGFR kinase domain. Furthermore, inhibition of mucin-type O-linked glycosylation suppressed the effect of p40 on increasing mucin production and protecting intestinal epithelial cells from TNF-induced apoptosis in colon organ culture. Thus, these results suggest that p40-stimulated activation of EGFR mediates up-regulation of mucin production, which may contribute to the mechanisms by which p40 protects the intestinal epithelium from injury. PMID:24895124

  10. Molecular size and amino acid composition of H-2d antigen solubilized in Nonidet P-40.

    PubMed

    Rossowski, W; Kloczewiak, M; Radzikowski, C; Strzadala, L

    1976-01-01

    H-2d antigenic material solubilized by the detergent Nonidet P-40 from L-1210 mouse leukemia cells was isolated by gel filtration on Bio-Gel P-100. A single peak eluted in the void volume consisted of about 90% protein, 8% hexose and traces of sialic acids. In sedimentation velocity runs, the antigen sedimented as a single peak of 3-1 S. Molecular weight determined by sedimentation equilibrium as well as calculated from amino acid composition was found to be in the range of 53,000 daltons and approx. 45,000-51,000 when calculated from sodium dodecyl sulfate polyacrylamide gel electrophoresis. Secondary structure of H-2d glycoprotein was predicted from the amino acid composition. For NP-40-solubilized H-2d antigen, about 34% of helix, 13% beta sheet and 41% turns was found.

  11. Base-pairing energies of proton-bound homodimers determined by guided ion beam tandem mass spectrometry: application to cytosine and 5-substituted cytosines.

    PubMed

    Yang, Bo; Wu, R R; Rodgers, M T

    2013-11-19

    Base-pairing interactions in proton-bound dimers of cytosine (C(+)·C) are the major forces responsible for stabilization of DNA i-motif conformations. Permethylation of cytosine in extended (CCG)·(CGG)n trinucleotide repeats has been shown to cause fragile-X syndrome, the most widespread inherited cause of mental retardation in humans. Oligonucleotides containing 5-bromo- or 5-fluorocytosine can bind to proteins that selectively bind methylated DNA, suggesting that halogenated cytosine damage products can potentially mimic methylation signals. However, the influence of methylation or halogenation on the base-pairing energies (BPEs) of proton-bound dimers of cytosine and their impact on the stability of DNA i-motif conformations is presently unknown. To address this, proton-bound homodimers of cytosine and 5-methyl-, 5-fluoro-, 5-bromo-, and 5-iodocytosine are investigated in detail both experimentally and theoretically. The BPEs of proton-bound homodimers of cytosine and the modified cytosines are measured by threshold collision-induced dissociation (TCID) techniques. 5-Methylation of cytosine is found to increase the BPE and would therefore tend to stabilize DNA i-motif conformations. In contrast, 5-halogenation lowers the BPE. However, the BPEs of the proton-bound 5-halocytosine homodimers examined here still significantly exceed that of Watson-Crick G·C base pairs, such that DNA i-motif conformations should be preserved in the presence of these modifications. Excellent agreement between TCID measured and B3LYP calculated BPEs is found, suggesting that B3LYP calculations can be used to provide reliable energetic predictions for related systems.

  12. Effect of p40tax trans-activator of human T cell lymphotropic virus type I on expression of autoantigens.

    PubMed

    Banki, K; Ablonczy, E; Nakamura, M; Perl, A

    1994-03-01

    The possibility of a retroviral etiology has long been raised in a number of autoimmune disorders. More recently, Sjögren's syndrome and rheumatoid arthritis were noted in transgenic mice carrying the tax gene of human T cell leukemia virus type I (HTLV-I). To evaluate the involvement of HTLV-I Tax in autoimmunity, its effect on expression of autoantigens was investigated. A metallothionein promoter-driven p40tax expression plasmid, pMAXRHneo-1, was stably transfected into Molt4 and Jurkat cells and the p40tax protein was induced with CdCl2. trans-Activation or trans-repression of autoantigens by HTLV-I Tax was studied by Western blot analysis utilizing autoantigen-specific murine monoclonal and rabbit polyvalent antibodies as well as sera from 161 autoimmune patients. Induction of p40tax of HTLV-I had no significant effect on levels of expression of common autoantigens U1 snRNP, Sm, Ro, La, HSP-70, topoisomerase I/Scl70, PCNA, and HRES-1. Expression of two potentially novel autoantigens, 44 and 46 kDa, was induced by p40tax as detected by sera of progressive systemic sclerosis patients, BAK and VAR. By contrast, expression of 24- and 34-kDa proteins was suppressed in response to induction of p40tax as detected by sera of systemic lupus erythematosus patients PUS and HOR. Because none of these patients were infected by HTLV-I, a protein functionally similar to p40tax may be involved in eliciting autoantigen expression and a subsequent autoantibody response in a minority of patients with PSS and SLE. Sera of autoimmune patients may also be utilized to detect novel proteins trans-activated or trans-repressed by p40tax of HTLV-I.

  13. Homodimers of cytosine and 1-methylcytosine. A DFT study of geometry, relative stability and H-NMR shifts in gas-phase and selected solvents.

    PubMed

    Paytakov, Guvanchmyrat; Gorb, Leonid; Stepanyugin, Andriy; Samiylenko, Svitlana; Hovorun, Dmytro; Leszczynski, Jerzy

    2014-03-01

    Dimers of cytosine and its N¹-methylated counterpart were investigated in gas-phase and in various solvents including chloroform, dimethylsulfoxide, and water. The studies were performed at DFT/M06-2X/6-31+G(d,p) level of theory. Relative stabilities of tautomers of cytosine solvated explicitly by a small number of solvent molecules were evaluated. Further solvation effect calculations for homodimers were carried out with conductor-like polarizable continuum model (CPCM). H-NMR shifts and IR frequencies for optimized structures were calculated and compared with available experimental data.

  14. Use of substitute Nonidet P-40 nonionic detergents in intracellular tubulin polymerization assays for screening of microtubule targeting agents.

    PubMed

    Sinha, Saptarshi; Field, Jessica J; Miller, John H

    2017-01-10

    Shell Chemical Company Nonidet P-40 has been used for decades in many biochemical assays as a nonionic, nondenaturing detergent; however, Shell no longer produces this product. Four commercially available substitutes were investigated and their activities titrated in an intracellular tubulin polymerization assay. Although claimed by the supply companies to be identical to the Shell Nonidet P-40, all four substitutes were about 10-fold more potent and needed to be diluted accordingly. As microtubule targeting drugs are a major class of anticancer agent, and many researchers use the intracellular tubulin polymerization assay, this information is important to help troubleshoot assay development with the new substitutes. As the Shell Nonidet P-40 has been used in many biochemical buffers, these results will be of general interest to the biochemical, cell, and molecular research community.

  15. CD8+ immunoregulatory cells in the graft-versus-host reaction: CD8 T cells activate dendritic cells to secrete interleukin-12/interleukin-18 and induce T helper 1 autoantibody

    PubMed Central

    Noble, Alistair; Leggat, Jamie A; Inderberg, Else M

    2003-01-01

    Initiation of cell-mediated immunity or autoimmunity requires secretion of interleukin (IL)-12 from dendritic cells (DC), which drives the generation of T helper 1 (Th1) effector cells in synergy with IL-18. Induction of IL-12 can be triggered by microbial stimuli but also requires signals from activated T cells. We investigated interactions between alloreactive CD4 and CD8 T cells in mixed lymphocyte reactions (MLR) in vitro and in the graft-versus-host reaction (GVHR) in vivo. In a parent-into-F1 model of GVHR, donor CD8 cells were found to suppress the hyper-immunoglobulin E (IgE) syndrome, anti-DNA immunoglobulin G1 (IgG1) autoantibodies and donor CD4-cell expansion, but were essential for Th1-dependent immunoglobulin G2a (IgG2a) autoantibody production and release of serum IL-12 p40. In vitro, addition of alloreactive CD8 cells to CD4 cells and mature DC enhanced Th1 development. CD4 and CD8 T cells induced IL-18 from DC and primed for IL-12 p70 secretion via interferon-γ (IFN-γ) or tumour necrosis factor-α (TNF-α). However CD8 T cells, but not CD4 cells, released IFN-γ/TNF-α after primary stimulation. The data suggest that rapid release of inflammatory cytokines from central memory-type CD8 cells early in immunity is critical for induction of Th1 cells via DC activation and IL-12 production. This pathway could provide a means for amplification of cell-mediated autoimmunity in the absence of microbial stimuli. PMID:12871213

  16. On a fully closed state of native human type-1 VDAC enriched in Nonidet P40.

    PubMed

    Thinnes, Friedrich P; Burckhardt, Gerhard

    2012-11-01

    There is indication that human type-1 VDAC/Porin31HL complexes, when purified from highly enriched cell membrane preparations of human B-lymphocytes by classical ion-exchange chromatography in the detergent Nonidet P40, rest in fully closed state, its N-terminus being accessible for mAbs. Cholesterol appears to be involved as a channel modulator. The channel switches to anion-selective or "open state" while being incorporated into black membranes at zero transmembrane potential. In this case, its N-terminus is hidden in the channel lumen. The cation-selective or "closed state" can be induced by transmembrane potentials beyond 30 mV, the N-terminus putatively now being positioned outside the channel lumen. The latter situation might allow one to decide if type-1 VDAC, preincubated with adequate antibodies against its N-terminal part, would enter black membranes in fully closed state or stay in the application medium, respectively, may be complexed to dimers.

  17. Properties of a ribonucleoprotein particle isolated from Nonidet P-40-treated Rous sarcoma virus.

    PubMed

    Davis, N L; Rueckert, R R

    1972-11-01

    A ribonucleoprotein particle containing about 20% ribonucleic acid (RNA), and containing little if any phospholipid or glucosamine, was recovered in high yield after treatment of Schmidt-Ruppin strain of Rous sarcoma virus and B77 virus with the nonionic detergent Nonidet P-40. This structure, which probably derives from the internal ribonucleoprotein filament described in electron microscopy studies, contained 80 to 90% of the viral 60 to 70S RNA and only about 10% of the protein present in intact virions. It sedimented in glycerol density gradients at approximately 130S and had a buoyant density in sucrose of about 1.34 g/ml. Studies with (32)P-labeled virus indicated that the ribonucleoprotein particle contained approximately 30 4S RNA molecules per 10(7) daltons of high-molecular-weight viral RNA. Intact virions contained about 70 4S RNA molecules per 10(7) daltons of high-molecular-weight RNA. Electrophoretic studies in dodecyl sulfate-containing polyacrylamide gels showed that the ribonucleoprotein particle contained only 5 of the 11 polypeptides found in the virion; of these the major component was a polypeptide weighing 14,000 daltons.

  18. Polymorphous low grade adenocarcinoma has a consistent p63+/p40- immunophenotype that helps distinguish it from adenoid cystic carcinoma and cellular pleomorphic adenoma.

    PubMed

    Rooper, Lisa; Sharma, Rajni; Bishop, Justin A

    2015-03-01

    Polymorphous low grade adenocarcinoma (PLGA) is a tumor of minor salivary glands that exhibits considerable morphologic overlap with adenoid cystic carcinoma and cellular pleomorphic adenoma, especially in small biopsy specimens. Unlike these other tumor types. PLGAs do not harbor a myoepithelial component, yet their frequent positivity for p63 diminishes the usefulness of this particular myoepithelial marker as a discriminating immunostain. p40 is an antibody that recognizes ΔNp63, a p63 isoform that is more specific for true myoepithelial differentiation. As such, p40 immunostaining could help distinguish PLGAs from adenoid cystic carcinomas and pleomorphic adenomas. In this study, p63 and p40 immunohistochemistry was performed on paraffin embedded, formalin fixed tissue from 11 PLGAs, 101 adenoid cystic carcinomas, and 31 pleomorphic adenomas. All 11 PLGAs (100 %) were positive for p63 but completely negative for p40. Among adenoid cystic carcinomas, 91 of 101 (90 %) were positive for p63 and 90/101 (89 %) were positive for p40. The single discordant p63+/p40- adenoid cystic carcinoma exhibited solid architecture and high grade features not typically seen in PLGA. Among pleomorphic adenomas, 21/31 (68 %) were positive for p63 and 13/31 (42 %) were positive for p40. For the pleomorphic adenomas, the discordant p63+/p40- staining pattern was seen only in the overtly mesenchymal chondromyxoid stroma. The cellular epithelial component of the pleomorphic adenomas demonstrated concordant p63+/p40+ or p63-/p40- immunophenotypes. PLGA consistently exhibits a p63+/p40- immunophenotype that can help distinguish it from adenoid cystic carcinoma and cellular pleomorphic adenoma, tumors that characteristically demonstrate concordant p63 and p40 immunostaining patterns. A p63/p40 immunohistochemical panel can provide a valuable tool for making the distinction between these morphologically similar but clinically divergent entities.

  19. [Cloning and functional research of Arp2/3-P40/ARPC1 subunit of Sf9 cells].

    PubMed

    Han, Shi-Li; Mu, Jing-Fang; Zhang, Yong-Li; Chen, Xin-Wen; Wang, Yun; Li, Lu-Lin

    2012-11-01

    The baculovirus-induced actin polymerization is mainly associated with the virus nucleocapsid protein P78/83, which is homologous with WASP proteins that can activate Arp2/3 complex and induce the actin polymerization. In order to explore the role of Arp2/3 complex in the baculovirus replication, the P40 subunit of Arp2/3 complex from Sf9 (Spodoptera frugiperda 9) cell line was cloned and characterized. Immunofluorescent microscopy assay indicated that P40 was recruited to the inner-side of nuclear membrane during virus infection, which was in accordance with nuclear F-actin distribution in virus-infected cells as documented in our previous research, suggesting P40 could be used to track Arp2/3 complex subcellular distribution changes during virus infection. In addition, co-immunoprecipitation assay demonstrated that P40 interacted with P78/83 only in virus-infected cells, suggesting that actin polymerization induced by P78/83-Arp2/3 complex during baculovirus infection was regulated by some unidentified virus factors.

  20. An in Vitro and in Vivo Investigation of Bivalent Ligands That Display Preferential Binding and Functional Activity for Different Melanocortin Receptor Homodimers.

    PubMed

    Lensing, Cody J; Freeman, Katie T; Schnell, Sathya M; Adank, Danielle N; Speth, Robert C; Haskell-Luevano, Carrie

    2016-04-14

    Pharmacological probes for the melanocortin receptors have been utilized for studying various disease states including cancer, sexual function disorders, Alzheimer's disease, social disorders, cachexia, and obesity. This study focused on the design and synthesis of bivalent ligands to target melanocortin receptor homodimers. Lead ligands increased binding affinity by 14- to 25-fold and increased cAMP signaling potency by 3- to 5-fold compared to their monovalent counterparts. Unexpectedly, different bivalent ligands showed preferences for particular melanocortin receptor subtypes depending on the linker that connected the binding scaffolds, suggesting structural differences between the various dimer subtypes. Homobivalent compound 12 possessed a functional profile that was unique from its monovalent counterpart providing evidence of the discrete effects of bivalent ligands. Lead compound 7 significantly decreased feeding in mice after intracerebroventricular administration. To the best of our knowledge, this is the first report of a melanocortin bivalent ligand's in vivo physiological effects.

  1. Carcinogenic heavy metals replace Ca{sup 2+} for DNA binding and annealing activities of mono-ubiquitinated annexin A1 homodimer

    SciTech Connect

    Hirata, Aiko; Corcoran, George B.; Hirata, Fusao

    2010-10-01

    Mono-ubiquitinated annexin A1 was purified from rat liver nuclei. The homodimer form of mono-ubiquitinated annexin A1 was able to unwind dsDNA in a Mg{sup 2+}- and ATP-dependent manner, and to anneal ssDNA in a Ca{sup 2+}-dependent manner. Phospholipids decreased the concentration of Ca{sup 2+} required for maximal annealing activity. Heavy metals such as As{sup 3+}, Cr{sup 6+}, Pb{sup 2+} and Cd{sup 2+} substituted for Ca{sup 2+} in the ssDNA binding and annealing activities of annexin A1. While these metals inhibited the unwinding of dsDNA by nuclear annexin A1 in the presence of Mg{sup 2+} and ATP, they enhanced dsDNA-dependent ATPase activity of annexin A1. Heavy metals may have produced dsDNA, a substrate for the DNA unwinding reaction, via the DNA annealing reaction. DNA synthesomes were isolated from L5178Y tk(+/-) mouse lymphoma cells in exponential growth, and were found to contain helicase activities. The As{sup 3+}- or Cr{sup 6+}-induced increases in ssDNA binding activity of DNA synthesomes were reduced by a mono-specific anti-annexin A1 antibody, but not by anti-Ig antibody. Anti-annexin A1 antibody also blocked the inhibitory and stimulatory effects of As{sup 3+} or Cr{sup 6+} towards DNA unwinding and annealing activities of DNA synthesomes. Based on these observations, it can be concluded that the effects of heavy metals on DNA annealing and unwinding activities are mediated, at least in substantial part, through actions of the mono-ubiquitinated annexin A1 homodimer.

  2. Latent membrane protein 1 of Epstein-Barr virus sensitizes cancer cells to cisplatin by enhancing NF-κB p50 homodimer formation and downregulating NAPA expression.

    PubMed

    Wu, Zchong-Zcho; Chow, Kai-Ping N; Kuo, Tzu-Ching; Chang, Yu-Sun; Chao, Chuck C-K

    2011-12-15

    Expression of the oncogenic latent membrane protein 1 (LMP1) of Epstein-Barr virus is involved in the pathogenesis of nasopharyngeal carcinoma (NPC) and lymphoma. In previous studies, we found that expression of LMP1 was sufficient to transform BALB/c-3T3 cells. In contrast, other studies have shown that LMP1 induces apoptosis in a NF-κB-dependent manner and also inhibits the growth of tumors in mice, thereby indicating that LMP1 may produce various biological effects depending on the biological and cellular context. Still, the mechanism underlying the pro-apoptotic activity of LMP1 remains unclear. In the present study, we found that LMP1 inhibits the expression of NAPA, an endoplasmic reticulum SNARE protein that possesses anti-apoptotic properties against the DNA-damaging drug cisplatin. Accordingly, LMP1-transformed BALB/c-3T3 cells were sensitized to cisplatin-induced apoptosis, whereas no sensitization effect was noted following treatment with the mitotic spindle-damaging drugs vincristine and taxol. Knockdown of LMP1 with antisense oligonucleotides restored NAPA protein level and rendered the cells resistant to cisplatin. Similarly, overexpression of NAPA reduced the effect of LMP1 and induced resistance to cisplatin. LMP1 was shown to upregulate the NF-κB subunit p50, leading to formation of p50 homodimers on the NAPA promoter. These findings suggest that the viral protein LMP1 may sensitize cancer cells to cisplatin chemotherapy by downregulating NAPA and by enhancing the formation of p50 homodimers which in turn inhibit the expression of NF-κB regulated anti-apoptotic genes. These findings provide an explanatory mechanism for the pro-apoptotic activity of LMP1 as well as new therapeutic targets to control tumor growth.

  3. Both JrWRKY2 and JrWRKY7 of Juglans regia mediate responses to abiotic stresses and abscisic acid through formation of homodimers and interaction.

    PubMed

    Yang, G; Zhang, W; Liu, Z; Yi-Maer, A-Y; Zhai, M; Xu, Z

    2017-03-01

    WRKY transcription factors belong to a large protein family that is involved in diverse developmental processes and abiotic stress responses. Currently, there is little understanding of the role of WRKY transcription factors in regulatory mechanisms in plants, especially in the protein-protein interactions that are essential for biological regulatory functions and networks. In the present study, yeast one-hybrid, yeast two-hybrid, transient expression and quantitative RT-PCR were applied to investigate the potential characteristics of two WRKY proteins from Juglans regia, JrWRKY2 (GenBank Accession No. KU057089) and JrWRKY7 (GenBank Accession No. KP784651). JrWRKY2 and JrWRKY7 can form homodimers and interact with each other. JrWRKY2 and JrWRKY7 can bind to W-box motifs. Similarly high levels of transcription were found for JrWRKY2 and JrWRKY7 under NaCl and polyethylene glycol (PEG) stresses, as well as at different developmental stages, e.g., the pistil or terminal leaf. JrWRKY2 and JrWRKY7 were transiently overexpressed in an independent manner in the terminal leaf. Analyses of superoxide dismutase (SOD) and peroxidase (POD) activities, proline and malondialdehyde (MDA) contents, and electrolyte leakage rate showed that JrWRKY2 and JrWRKY7 overexpression improved plant tolerance to NaCl, PEG, abscisic acid, and cold stress. Additionally, JrWRKY2 and JrWRKY7 overexpression elevated transcription of SOD, POD, glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX), and MYB genes, but downregulated the expression of NAC. Overall, the results demonstrate that JrWRKY2 and JrWRKY7 are dimeric proteins that can form functional homodimers and interact with each other and that they are involved in abiotic stress responses.

  4. Immunoblotting technique for the detection of allergens of Aspergillus fumigatus: influence of Nonidet P-40 on the sensitivity.

    PubMed

    Wijnands, L M; Deisz, W D; van Leusden, F M

    1999-01-01

    Immunoblotting provides a useful technique for the study of antigens, antibodies and allergens. To overcome problems regarding the loss of antigenic properties during the blotting and developing procedures, several solutions have been described. The inclusion of Nonidet P-40, recommended to increase the sensitivity of developing procedures for immunoblots, in an existing procedure for the detection of allergens of Aspergillus fumigatus, however, led to decreased sensitivity of the method.

  5. A lactobacillus rhamnosus GG-derived soluble protein, p40, stimulates ligand release from intestinal epithelial cells to transactivate epidermal growth factor receptor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis and preserves barrier function by activation of EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study was to determine the mechanisms by which p40...

  6. Mast cell growth-enhancing activity (MEA) is structurally related and functionally identical to the novel mouse T cell growth factor P40/TCGFIII (interleukin 9).

    PubMed

    Hültner, L; Druez, C; Moeller, J; Uyttenhove, C; Schmitt, E; Rüde, E; Dörmer, P; Van Snick, J

    1990-06-01

    We have previously shown that certain bone marrow-derived mast cell (BMMC) lines proliferate in response to a mast cell growth-enhancing activity (MEA) that is distinct from interleukin (IL) 3 and IL 4. Here we provide evidence that MEA is identical with the recently cloned mouse T cell growth factor P40. The evidence is as follows: (a) recombinant P40 displayed all the biological activities ascribed to MEA: it supported the growth of MEA-sensitive BMMC lines, it induced IL 6 secretion by these cells, and it enhanced survival of primary mast cell cultures; (b) highly purified MEA stimulated the growth of P40-dependent cell lines; (c) a rabbit monospecific antiserum directed against P40 specifically inhibited the action of MEA on BMMC; (d) specific binding sites for P40 were detected on BMMC and (e) MEA competed with P40 for binding to P40-dependent T cells, indicating that the two molecules interact with the same receptor. These observations further extend the range of biological activities ascribed to P40 and warrant its proposed designation as IL9.

  7. Homo-Dimers of Vanillin and Apocynin Decrease Metastatic Potential of Human Cancer Cells by Inhibiting the FAK/PI3K/Akt Signaling Pathway.

    PubMed

    Jantaree, Phatcharida; Lirdprapamongkol, Kriengsak; Kaewsri, Wilailak; Thongsornkleeb, Charnsak; Choowongkomon, Kiattawee; Atjanasuppat, Korakot; Ruchirawat, Somsak; Svasti, Jisnuson

    2017-03-01

    The spread of cancer cells to distant organs, in a process called metastasis, is the main factor that contributes to most death in cancer patients. Vanillin, the vanilla flavoring agent, has been shown to suppress metastasis in a mouse model. Here, we evaluated the anti-metastatic potential of the food additive divanillin, the homo-dimer of vanillin, and their structurally related compounds, apocynin and diapocynin, in hepatocellular carcinoma cells. The Transwell invasion assay showed that the dimeric forms exhibited higher potency than vanillin and apocynin in inhibiting invasion, with IC50 values of 23.3±7.4 to 41.3±4.2 μM for the dimers, which are 26-34 fold lower than IC50 values of vanillin and apocynin (p<0.05). Both monomeric and dimeric forms target regulation of the invasion process, by inhibiting phosphorylation of FAK and Akt. Molecular docking studies suggested that the dimers should bind more tightly than vanillin and apocynin to the Y397 pocket of FAK FERM domain. Thus, the food additive divanillin has greater anti-metastatic potential than the flavoring agent vanillin.

  8. The APC/C subunit Cdc16/Cut9 is a contiguous tetratricopeptide repeat superhelix with a homo-dimer interface similar to Cdc27

    PubMed Central

    Zhang, Ziguo; Kulkarni, Kiran; Hanrahan, Sarah J; Thompson, Andrew J; Barford, David

    2010-01-01

    The anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase responsible for controlling cell cycle transitions, is a multisubunit complex assembled from 13 different proteins. Numerous APC/C subunits incorporate multiple copies of the tetratricopeptide repeat (TPR). Here, we report the crystal structure of Schizosaccharomyces pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26), showing that Cdc16/Cut9 is a contiguous TPR superhelix of 14 TPR units. A C-terminal block of TPR motifs interacts with Hcn1, whereas an N-terminal TPR block mediates Cdc16/Cut9 self-association through a homotypic interface. This dimer interface is structurally related to the N-terminal dimerization domain of Cdc27, demonstrating that both Cdc16/Cut9 and Cdc27 form homo-dimers through a conserved mechanism. The acetylated N-terminal Met residue of Hcn1 is enclosed within a chamber created from the Cut9 TPR superhelix. Thus, in complex with Cdc16/Cut9, the N-acetyl-Met residue of Hcn1, a putative degron for the Doa10 E3 ubiquitin ligase, is inaccessible for Doa10 recognition, protecting Hcn1/Cdc26 from ubiquitin-dependent degradation. This finding may provide a structural explanation for a mechanism to control the stoichiometry of proteins participating in multisubunit complexes. PMID:20924356

  9. Replication-specific conversion of the Staphylococcus aureus pT181 initiator protein from an active homodimer to an inactive heterodimer.

    PubMed Central

    Rasooly, A; Wang, P Z; Novick, R P

    1994-01-01

    The Staphylococcus aureus rolling circle plasmid pT181 regulates its replication by controlling the synthesis of its initiator protein RepC. RepC is inactivated during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. We analyzed RepC and RepC* in four classes of mutants: plasmid copy number mutants, two classes of RepC mutants affecting different portions of the protein and oriC (origin) mutants. We have found that in the cell with wild-type RepC there are similar relative amounts of RepC and RepC*, regardless of copy number, and that the conversion of RepC to RepC* is replication dependent. Genetic and biochemical evidence is presented that RepC functions as a dimer and that during replication the RepC homodimer is converted to the RepC/RepC* heterodimer. Images PMID:7957090

  10. Cerebrospinal Fluid IL-12p40, CXCL13 and IL-8 as a Combinatorial Biomarker of Active Intrathecal Inflammation

    PubMed Central

    Bielekova, Bibiana; Komori, Mika; Xu, Quangang; Reich, Daniel S.; Wu, Tianxia

    2012-01-01

    Diagnosis and management of the neuroinflammatory diseases of the central nervous system (CNS) are hindered by the lack of reliable biomarkers of active intrathecal inflammation. We hypothesized that measuring several putative inflammatory biomarkers simultaneously will augment specificity and sensitivity of the biomarker to the clinically useful range. Based on our pilot experiment in which we measured 18 inflammatory biomarkers in 10-fold concentrated cerebrospinal fluid (CSF) derived from 16 untreated patients with highly active multiple sclerosis (MS) we selected a combination of three CSF biomarkers, IL-12p40, CXCL13 and IL-8, for further validation. Concentrations of IL-12p40, CXCL13 and IL-8 were determined in a blinded fashion in CSF samples from an initial cohort (n = 72) and a confirmatory cohort (n = 167) of prospectively collected, untreated subjects presenting for a diagnostic work-up of possible neuroimmunological disorder. Diagnostic conclusion was based on a thorough clinical workup, which included laboratory assessment of the blood and CSF, neuroimaging and longitudinal follow-up. Receiver operating characteristic (ROC) curve analysis in conjunction with principal component analysis (PCA), which was used to combine information from all three biomarkers, assessed the diagnostic value of measured biomarkers. Each of the three biomarkers was significantly increased in MS and other inflammatory neurological disease (OIND) in comparison to non-inflammatory neurological disorder patients (NIND) at least in one cohort. However, considering all three biomarkers together improved accuracy of predicting the presence of intrathecal inflammation to the consistently good to excellent range (area under the ROC curve = 0.868–0.924). Future clinical studies will determine if a combinatorial biomarker consisting of CSF IL-12p40, CXCL13 and IL-8 provides utility in determining the presence of active intrathecal inflammation in diagnostically uncertain

  11. P40 and P90 from Mpn142 are Targets of Multiple Processing Events on the Surface of Mycoplasma pneumoniae

    PubMed Central

    Widjaja, Michael; Berry, Iain J.; Pont, Elsa J.; Padula, Matthew P.; Djordjevic, Steven P.

    2015-01-01

    Mycoplasma pneumoniae is a significant cause of community acquired pneumonia globally. Despite having a genome less than 1 Mb in size, M. pneumoniae presents a structurally sophisticated attachment organelle that (i) provides cell polarity, (ii) directs adherence to receptors presented on respiratory epithelium, and (iii) plays a major role in cell motility. The major adhesins, P1 (Mpn141) and P30 (Mpn453), are localised to the tip of the attachment organelle by the surface accessible cleavage fragments P90 and P40 derived from Mpn142. Two events play a defining role in the formation of P90 and P40; removal of a leader peptide at position 26 (23SLA↓NTY28) during secretion to the cell surface and cleavage at amino acid 455 (452GPL↓RAG457) generating P40 and P90. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) analysis of tryptic peptides generated by digesting size-fractionated cell lysates of M. pneumoniae identified 15 cleavage fragments of Mpn142 ranging in mass from 9–84 kDa. Further evidence for the existence of cleavage fragments of Mpn142 was generated by mapping tryptic peptides to proteins recovered from size fractionated eluents from affinity columns loaded with heparin, fibronectin, fetuin, actin, plasminogen and A549 surface proteins as bait. To define the sites of cleavage in Mpn142, neo-N-termini in cell lysates of M. pneumoniae were dimethyl-labelled and characterised by LC-MS/MS. Our data suggests that Mpn142 is cleaved to generate adhesins that are auxiliary to P1 and P30.

  12. PtdIns(3)P-DEPENDENT AND INDEPENDENT FUNCTIONS OF p40PHOX IN ACTIVATION OF THE NEUTROPHIL NADPH OXIDASE

    PubMed Central

    Bissonnette, Sarah A.; Glazier, Christina M.; Stewart, Mary Q.; Brown, Glenn E.; Ellson, Chris D.; Yaffe, Michael B.

    2009-01-01

    In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40phox, p47phox and p67phox, and the small GTPase Rac2, and is regulated through a process involving Protein Kinase Cs, MAP kinases, and PI 3-kinases. The role of p40phox remains less well defined than those of p47phox and p67phox. We investigated the biological role of p40phox in differentiated PLB-985 neutrophils, and show that depletion of endogenous p40phox using lentiviral shRNA reduces ROS production and impairs bacterial killing under conditions where p67phox levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40phox reduces both the maximal rate and total amount of ROS produced without altering the KM of the oxidase for NADPH. Using a series of mutants, p47PX-p40phox chimeras, and deletion constructs, we found that the p40phox PX domain has PtdIns(3)P-dependent and independent functions. Translocation of p67phox requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40phox, however, requires both PtdIns(3)P binding and an SH3 domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40phox can increase oxidase activity ∼2.5-fold above that of wild-type p40phox, but maintain the requirement for PX and SH3 domain function. We present a model where p40phox translocates p67phox to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement. PMID:18029359

  13. Effector-repressor interactions, binding of a single effector molecule to the operator-bound TtgR homodimer mediates derepression.

    PubMed

    Terán, Wilson; Krell, Tino; Ramos, Juan Luis; Gallegos, María-Trinidad

    2006-03-17

    The RND family transporter TtgABC and its cognate repressor TtgR from Pseudomonas putida DOT-T1E were both shown to possess multidrug recognition properties. Structurally unrelated molecules such as chloramphenicol, butyl paraben, 1,3-dihydroxynaphthalene, and several flavonoids are substrates of TtgABC and activate pump expression by binding to the TtgR-operator complex. Isothermal titration calorimetry was employed to determine the thermodynamic parameters for the binding of these molecules to TtgR. Dissociation constants were in the range from 1 to 150 microm, the binding stoichiometry was one effector molecule per dimer of TtgR, and the process was driven by favorable enthalpy changes. Although TtgR exhibits a large multidrug binding profile, the plant-derived compounds phloretin and quercetin were shown to bind with the highest affinity (K(D) of around 1 microm), in contrast to other effectors (chloramphenicol and aromatic solvents) for which exhibited a more reduced affinity. Structure-function studies of effectors indicate that the presence of aromatic rings as well as hydroxyl groups are determinants for TtgR binding. The binding of TtgR to its operator DNA does not alter the protein effector profile nor the effector binding stoichiometry. Moreover, we demonstrate here for the first time that the binding of a single effector molecule to the DNA-bound TtgR homodimer induces the dissociation of the repressor-operator complex. This provides important insight into the molecular mechanism of effector-mediated derepression.

  14. Biophysical characterization of the b-HLH-LZ of ΔMax, an alternatively spliced isoform of Max found in tumor cells: Towards the validation of a tumor suppressor role for the Max homodimers.

    PubMed

    Maltais, Loïka; Montagne, Martin; Bédard, Mikaël; Tremblay, Cynthia; Soucek, Laura; Lavigne, Pierre

    2017-01-01

    It is classically recognized that the physiological and oncogenic functions of Myc proteins depend on specific DNA binding enabled by the dimerization of its C-terminal basic-region-Helix-Loop-Helix-Leucine Zipper (b-HLH-LZ) domain with that of Max. However, a new paradigm is emerging, where the binding of the c-Myc/Max heterodimer to non-specific sequences in enhancers and promoters drives the transcription of genes involved in diverse oncogenic programs. Importantly, Max can form a stable homodimer even in the presence of c-Myc and bind DNA (specific and non-specific) with comparable affinity to the c-Myc/Max heterodimer. Intriguingly, alterations in the Max gene by germline and somatic mutations or changes in the gene product by alternative splicing (e.g. ΔMax) were recently associated with pheochromocytoma and glioblastoma, respectively. This has led to the proposition that Max is, by itself, a tumor suppressor. However, the actual mechanism through which it exerts such an activity remains to be elucidated. Here, we show that contrary to the WT motif, the b-HLH-LZ of ΔMax does not homodimerize in the absence of DNA. In addition, although ΔMax can still bind the E-box sequence as a homodimer, it cannot bind non-specific DNA in that form, while it can heterodimerize with c-Myc and bind E-box and non-specific DNA as a heterodimer with high affinity. Taken together, our results suggest that the WT Max homodimer is important for attenuating the binding of c-Myc to specific and non-specific DNA, whereas ΔMax is unable to do so. Conversely, the splicing of Max into ΔMax could provoke an increase in overall chromatin bound c-Myc. According to the new emerging paradigm, the splicing event and the stark reduction in homodimer stability and DNA binding should promote tumorigenesis impairing the tumor suppressor activity of the WT homodimer of Max.

  15. Biophysical characterization of the b-HLH-LZ of ΔMax, an alternatively spliced isoform of Max found in tumor cells: Towards the validation of a tumor suppressor role for the Max homodimers

    PubMed Central

    Maltais, Loïka; Montagne, Martin; Bédard, Mikaël; Tremblay, Cynthia; Soucek, Laura

    2017-01-01

    It is classically recognized that the physiological and oncogenic functions of Myc proteins depend on specific DNA binding enabled by the dimerization of its C-terminal basic-region-Helix-Loop-Helix-Leucine Zipper (b-HLH-LZ) domain with that of Max. However, a new paradigm is emerging, where the binding of the c-Myc/Max heterodimer to non-specific sequences in enhancers and promoters drives the transcription of genes involved in diverse oncogenic programs. Importantly, Max can form a stable homodimer even in the presence of c-Myc and bind DNA (specific and non-specific) with comparable affinity to the c-Myc/Max heterodimer. Intriguingly, alterations in the Max gene by germline and somatic mutations or changes in the gene product by alternative splicing (e.g. ΔMax) were recently associated with pheochromocytoma and glioblastoma, respectively. This has led to the proposition that Max is, by itself, a tumor suppressor. However, the actual mechanism through which it exerts such an activity remains to be elucidated. Here, we show that contrary to the WT motif, the b-HLH-LZ of ΔMax does not homodimerize in the absence of DNA. In addition, although ΔMax can still bind the E-box sequence as a homodimer, it cannot bind non-specific DNA in that form, while it can heterodimerize with c-Myc and bind E-box and non-specific DNA as a heterodimer with high affinity. Taken together, our results suggest that the WT Max homodimer is important for attenuating the binding of c-Myc to specific and non-specific DNA, whereas ΔMax is unable to do so. Conversely, the splicing of Max into ΔMax could provoke an increase in overall chromatin bound c-Myc. According to the new emerging paradigm, the splicing event and the stark reduction in homodimer stability and DNA binding should promote tumorigenesis impairing the tumor suppressor activity of the WT homodimer of Max. PMID:28350847

  16. Induction of asymmetry into homodimers.

    PubMed

    Bardsley, B; Cho, Y R; Westwell, M S; Williams, D H

    1998-01-01

    The self-regulation of biological signalling receptors via homodimerization is discussed in relation to the symmetry changes occurring when these receptors bind their target ligand. The idea of positive and negative cooperativity between dimerization and ligand binding, mediated by changes in the symmetry of the system as a source of signalling control is considered; an analogy made with the homodimerization of a glycopeptide antibiotic, ristocetin A, which displays negative cooperativity. Finally, the regulation of the bacterial aspartate receptor and the human growth hormone receptor is discussed as a function of ligand-induced asymmetry.

  17. Identification of the major proteins of an immune modulating fraction from adult Fasciola hepatica released by Nonidet P40.

    PubMed

    Morphew, Russell M; Hamilton, Clare M; Wright, Hazel A; Dowling, David J; O'Neill, Sandra M; Brophy, Peter M

    2013-01-31

    Fasciola hepatica NP-40 released protein extract (FhNPE) exhibits potent Th1 immunosuppressive properties in vitro and in vivo. However, the protein composition of this active fraction, responsible for Th1 immune modulatory activity, has yet to be resolved. Therefore, FhNPE, a Nonidet P-40 extract, was subjected to a proteomic analysis in order to identify individual protein components. This was performed using an in house F. hepatica EST database following 2D electrophoresis combined with de novo sequencing based mass spectrometry. The identified proteins, a mixture of excretory/secretory and membrane-associated proteins, are associated with stress response and chaperoning, energy metabolism and cytoskeletal components. The immune modulatory properties of these identified protein(s) are discussed and HSP70 from F. hepatica is highlighted as a potential host immune modulator for future study.

  18. The Human Sodium-Glucose Cotransporter (hSGLT1) Is a Disulfide-Bridged Homodimer with a Re-Entrant C-Terminal Loop

    PubMed Central

    Sasseville, Louis J.; Morin, Michael; Coady, Michael J.; Blunck, Rikard; Lapointe, Jean-Yves

    2016-01-01

    Na-coupled cotransporters are proteins that use the trans-membrane electrochemical gradient of Na to activate the transport of a second solute. The sodium-glucose cotransporter 1 (SGLT1) constitutes a well-studied prototype of this transport mechanism but essential molecular characteristics, namely its quaternary structure and the exact arrangement of the C-terminal transmembrane segments, are still debated. After expression in Xenopus oocytes, human SGLT1 molecules (hSGLT1) were labelled on an externally accessible cysteine residue with a thiol-reactive fluorophore (tetramethylrhodamine-C5-maleimide, TMR). Addition of dipicrylamine (DPA, a negatively-charged amphiphatic fluorescence “quencher”) to the fluorescently-labelled oocytes is used to quench the fluorescence originating from hSGLT1 in a voltage-dependent manner. Using this arrangement with a cysteine residue introduced at position 624 in the loop between transmembrane segments 12 and 13, the voltage-dependent fluorescence signal clearly indicated that this portion of the 12–13 loop is located on the external side of the membrane. As the 12–13 loop begins on the intracellular side of the membrane, this suggests that the 12–13 loop is re-entrant. Using fluorescence resonance energy transfer (FRET), we observed that different hSGLT1 molecules are within molecular distances from each other suggesting a multimeric complex arrangement. In agreement with this conclusion, a western blot analysis showed that hSGLT1 migrates as either a monomer or a dimer in reducing and non-reducing conditions, respectively. A systematic mutational study of endogenous cysteine residues in hSGLT1 showed that a disulfide bridge is formed between the C355 residues of two neighbouring hSGLT1 molecules. It is concluded that, 1) hSGLT1 is expressed as a disulfide bridged homodimer via C355 and that 2) a portion of the intracellular 12–13 loop is re-entrant and readily accessible from the extracellular milieu. PMID:27137918

  19. The Human Sodium-Glucose Cotransporter (hSGLT1) Is a Disulfide-Bridged Homodimer with a Re-Entrant C-Terminal Loop.

    PubMed

    Sasseville, Louis J; Morin, Michael; Coady, Michael J; Blunck, Rikard; Lapointe, Jean-Yves

    2016-01-01

    Na-coupled cotransporters are proteins that use the trans-membrane electrochemical gradient of Na to activate the transport of a second solute. The sodium-glucose cotransporter 1 (SGLT1) constitutes a well-studied prototype of this transport mechanism but essential molecular characteristics, namely its quaternary structure and the exact arrangement of the C-terminal transmembrane segments, are still debated. After expression in Xenopus oocytes, human SGLT1 molecules (hSGLT1) were labelled on an externally accessible cysteine residue with a thiol-reactive fluorophore (tetramethylrhodamine-C5-maleimide, TMR). Addition of dipicrylamine (DPA, a negatively-charged amphiphatic fluorescence "quencher") to the fluorescently-labelled oocytes is used to quench the fluorescence originating from hSGLT1 in a voltage-dependent manner. Using this arrangement with a cysteine residue introduced at position 624 in the loop between transmembrane segments 12 and 13, the voltage-dependent fluorescence signal clearly indicated that this portion of the 12-13 loop is located on the external side of the membrane. As the 12-13 loop begins on the intracellular side of the membrane, this suggests that the 12-13 loop is re-entrant. Using fluorescence resonance energy transfer (FRET), we observed that different hSGLT1 molecules are within molecular distances from each other suggesting a multimeric complex arrangement. In agreement with this conclusion, a western blot analysis showed that hSGLT1 migrates as either a monomer or a dimer in reducing and non-reducing conditions, respectively. A systematic mutational study of endogenous cysteine residues in hSGLT1 showed that a disulfide bridge is formed between the C355 residues of two neighbouring hSGLT1 molecules. It is concluded that, 1) hSGLT1 is expressed as a disulfide bridged homodimer via C355 and that 2) a portion of the intracellular 12-13 loop is re-entrant and readily accessible from the extracellular milieu.

  20. Seventeen copies of the human 37 kDa laminin receptor precursor/p40 ribosome-associated protein gene are processed pseudogenes arisen from retropositional events.

    PubMed

    Jackers, P; Clausse, N; Fernandez, M; Berti, A; Princen, F; Wewer, U; Sobel, M E; Castronovo, V

    1996-02-07

    A cDNA coding for a 37 kDa polypeptide has been identified in several species as both the potential precursor of the 67 kDa laminin receptor (37LRP) and a putative ribosome-associated protein (p40). Interestingly, increased expression of this polypeptide (37LRP/p40) is consistently observed in invasive and metastatic cancer cells and is associated with poor prognosis. Southern-blot analysis of human genomic DNA predicted multiple copies of the 37LRP/p40 gene. In this study, we report that the number of copies of this sequence in the human genome is 26 +/- 2. We have sequenced and analyzed 19 genomic clones corresponding to the 37LRP/p40 gene and found that they were all processed pseudogenes. They all lack intronic sequences and show multiple genetic alterations leading in some cases to the appearance of stop codons. Moreover, they all bear characteristic features of retroposons as the presence of a poly(A)-tail at their 3' end and short direct repeated flanking DNA sequences. None of the pseudogenes analyzed present cis-elements in their 5' flanking region such as TATA or GC boxes. Our date reveal that over 50% of the 37LRP/p40 gene copies are pseudogenes most probably generated by retropositional events. The finding of multiple pseudogenes for the 37LRP/p40 suggests that the accumulation of several copies of this gene might have given a survival advantage to the cell in the course of evolution.

  1. Difference in extractability of estradiol- and tamoxifen-receptor complex in the nuclei from MCF-7 cells with Nonidet P-40.

    PubMed

    Ikeda, M; Omukai, Y; Hosokawa, K; Senoo, T

    1984-05-01

    The extraction of [3H]estradiol- and [3H]tamoxifen-receptor complex in the nuclei from MCF-7 cells with the nonionic detergent Nonidet P-40 has been studied. We found that there is a striking difference in the extractability of estradiol- and tamoxifen-receptor complex from nuclei with 0.5% Nonidet P-40. The nuclear bound estradiol-receptor complex is scarcely extractable with Nonidet P-40. In contrast, almost all of the nuclear bound tamoxifen-receptor complex is extractable. The nuclear [3H]tamoxifen-receptor complex extracted in the presence of Nonidet P-40 sediments in two peaks at 7 S and 5 S. The latter sedimentation rate is the same with that of the nuclear [3H]tamoxifen-receptor complex extracted with 0.4 M KCl. The nuclear [3H]estradiol-receptor complex extracted with 0.4 M KCl sediments at 4 S. The results suggest that interaction of tamoxifen-receptor complex with chromatin is different from that of estradiol-receptor complex.

  2. A transcriptional enhancer sequence of HTLV-I is responsible for trans-activation mediated by p40 chi HTLV-I.

    PubMed Central

    Fujisawa, J; Seiki, M; Sato, M; Yoshida, M

    1986-01-01

    Human T-cell leukemia virus type I (HTLV-I) contains a unique sequence pX that is located between env and the 3' long terminal repeat (LTR) and codes for three pX proteins, p40 chi, pp27 chi-III and pp21 chi-III. One of these proteins, p40 chi, was previously shown to activate transcription from the LTR in a trans-acting manner, which suggested that it activated some cellular genes involved in leukemogenesis. In this study, the sequences in the LTR responsible for this trans-activation were analyzed. Construction of deletion mutants of the LTR in pLTR-CAT and measurement of their activities in trans-activated expression of the CAT gene showed that sequences upstream of the TATA box were responsible for the trans-activation mediated by p40 chi. The active unit was identified as an enhancer sequence containing direct repeats by inserting it into an enhancer-minus SV40 promoter. Thus, it was concluded that an enhancer sequence in HTLV-I LTR is responsible, at least in part, for transcriptional trans-activation mediated by the viral product p40 chi. Images Fig.2. Fig.4. PMID:3011423

  3. Solubilisation effect of Nonidet P-40, triton X-100 and CHAPS in the detection of MHC-like glycoproteins.

    PubMed

    Labeta, M O; Fernandez, N; Festenstein, H

    1988-08-09

    We have analysed the differential solubilisation effect of three detergents on cell-membrane histocompatibility glycoproteins. Two nonionic detergents (Nonidet P-40 and Triton X-100) which are extensively used in the extraction of MHC proteins and a zwitterionic detergent (CHAPS) which is sulphobetaine derivative of cholic acid were used. An AKR (H-2k) derived spontaneous leukaemic cell line--424--was used as the experimental model. In this tumour cell line a class I-like antigen is expressed but not directly detected by cell-binding radioimmunoassay or immunoprecipitation from NP-40 or Triton X-100 solubilised glycoproteins. However, 46 kDa and 12 kDa bands consistent with the classical H-2 class I pattern were seen by SDS-PAGE after immunoprecipitation with the 34.5.8 anti-H-2Dd MoAb using CHAPS solubilised 424 glycoproteins. The H-2Dd-reactive molecule appears to be associated with at least one of the syngeneic class I specificities (H-2Kk, H-2Dk) and not accessible to react with the specific anti H-2Dd MoAb. The detergents NP-40 and Triton X-100 appear to be less efficient than CHAPS in breaking protein-protein interactions. This property of CHAPS permitted the adequate solubilisation of the novel antigen and its direct detection. The results of this study suggest that the alternative use of a non-denaturing zwitterionic detergent may contribute to the detection and characterisation of MHC-related, membrane-bound proteins of tumours and normal cells.

  4. Early infiltration of p40IL12+CCR7+CD11b+ cells is critical for fibrosis development

    PubMed Central

    Correa‐Costa, Matheus; Azevedo, Hatylas; Silva, Reinaldo Correia; Cruz, Mario Costa; Almeida, Maira Estanislau Soares; Hiyane, Meire Ioshie; Moreira‐Filho, Carlos Alberto; Santos, Marinilce Fagundes; Perez, Katia Regina; Cuccovia, Iolanda Midea; Camara, Niels Olsen Saraiva

    2016-01-01

    Abstract Introduction Macrophages are heterogeneous and thus can be correlated with distinct tissue outcomes after injury. Conflicting data have indicated that the M2‐related phenotype directly triggers fibrosis. Conversely, we hypothesize here that the inflammatory milieu provided by early infiltration of pro‐inflammatory macrophages dictates tissue scarring after injury. Methods and Results We first determined that tissue‐localized macrophages exhibit a pro‐inflammatory phenotype (p40IL12+CCR7+CD11b+) during the early phase of a chronic injury model, in contrast to a pro‐resolving phenotype (Arg1+IL10+CD206+CD11b+) at a later stage. Then, we evaluated the effects of injecting macrophages differentiated in vitro in the presence of IFNγ + LPS or IL4 + IL13 or non‐differentiated macrophages (hereafter, M0) on promoting inflammation and progression of chronic injury in macrophage‐depleted mice. In addition to enhancing the expression of pro‐inflammatory cytokines, the injection of M (IFNγ + LPS), but not M (IL4 + IL13) or M0, accentuated fibrosis while augmenting levels of anti‐inflammatory molecules, increasing collagen deposition and impairing organ function. We observed a similar profile after injection of sorted CCR7+CD11b+ cells and a more pronounced effect of M (IFNγ + LPS) cells originated from Stat6−/− mice. The injection of M (IFNγ + LPS) cells was associated with the up‐regulation of inflammation‐ and fibrosis‐related proteins (Thbs1, Mmp7, Mmp8, and Mmp13). Conclusions Our results suggest that pro‐inflammatory macrophages promote microenvironmental changes that may lead to fibrogenesis by inducing an inflammatory milieu that alters a network of extracellular‐related genes, culminating in tissue fibrosis. PMID:27621813

  5. Lamin A and lamin C form homodimers and coexist in higher complex forms both in the nucleoplasmic fraction and in the lamina of cultured human cells.

    PubMed

    Kolb, Thorsten; Maass, Kendra; Hergt, Michaela; Aebi, Ueli; Herrmann, Harald

    2011-01-01

    We have investigated and quantified the nuclear A-type lamin pool from human HeLa S3 suspension cells with respect to their distribution to detergent soluble and insoluble fractions. We devised a sequential extraction protocol and found that maximally 10% of A-type lamins are recovered in the soluble fraction. Notably, lamin C is enriched in low detergent fractions and only with 0.5% Nonidet P-40 lamin A and C are recovered in ratios nearly equivalent to those found in whole cell extracts and in the lamina fraction. Authentic nucleoplasmic proteins such as LAP2a, pRB and p53 are co-extracted to a large part together with the A-type lamins in these fractions. By sucrose density centrifugation we revealed that the majority of lamins co-sedimented with human IgG indicating they form rather small complexes in the range of dimers and slightly larger complexes. Some lamin A - but not lamin C - is obtained in addition in a much faster sedimenting fraction. Authentic nuclear proteins such as PCNA, p53 and LAP2a were found both in the light and the heavy sucrose fractions together with lamin A. Last but not least, immunoprecipitation experiments from both soluble fractions and from RIPA lysates of whole cells revealed that lamin A and lamin C do not form heterodimers but segregate practically completely. Correspondingly, immunofluorescence microscopy of formaldehyde-fixed cells clearly demonstrated that lamin A and C are localized at least in part to distinct patches within the lamina. Hence, the structural segregation of lamin A and C is indeed retained in the nuclear envelope to some extent too.

  6. Low concentrations of the non-ionic detergent Nonidet P-40 interfere with sterol biogenesis and viability of the yeast Saccharomyces cerevisiae.

    PubMed

    Hronská, Lucia; Mrózová, Zuzana; Valachovic, Martin; Hapala, Ivan

    2004-09-01

    Mild non-ionic detergents are used for solubilization of hydrophobic substrates in yeast growth media at concentrations 0.1-1%. Our data show that low concentrations of Nonidet P-40 may significantly affect lipid biogenesis in the yeast Saccharomyces cerevisiae. The uptake and esterification of external [4-14C]-cholesterol is strongly reduced in hem1 mutants treated with low concentrations of Nonidet P-40. Significant inhibitory effect of NP-40 on sterol uptake and esterification was evident both in non-growing and growing cells supplemented with external cholesterol. Increased levels of sterol precursors (squalene, lanosterol) in hem1 cells grown in complex medium with cholesterol indicated general interference of NP-40 with sterol biosynthesis. NP-40 in the growth medium affected also cell viability estimated as the colony forming ability. More attention should be therefore paid to possible effects of mild detergents at low concentrations generally considered to be harmless, especially in cells with disturbed lipid biogenesis.

  7. Increased Production of IL-4 and IL-12p40 from Bronchoalveolar Lavage Cells Are Biomarkers of Mycobacterium tuberculosis in the Sputum

    PubMed Central

    Nolan, Anna; Fajardo, Elaine; Huie, Maryann L.; Condos, Rany; Pooran, Anil; Dawson, Rodney; Dheda, Keertan; Bateman, Eric; Rom, William N.; Weiden, Michael D.

    2013-01-01

    Background Tuberculosis (TB) causes 1.45 million deaths annually world wide, the majority of which occur in the developing world. Active TB disease represents immune failure to control latent infection from airborne spread. Acid-fast bacillus (AFB) seen on sputum smear is a biomarker for contagiousness. Methods We enrolled 73 tuberculosis patients with extensive infiltrates into a research study using bronchoalveolar lavage (BAL) to sample lung immune cells and assay BAL cell cytokine production. All patients had sputum culture demonstrating Mycobacterium tuberculosis and 59/73 (81%) had AFB identified by microscopy of the sputum. Compared with smear negative patients, smear positive patients at presentation had a higher proportion with smoking history, a higher proportion with temperature >38.50 C, higher BAL cells/ml, lower percent lymphocytes in BAL, higher IL-4 and IL-12p40 in BAL cell supernatants. There was no correlation between AFB smear and other BAL or serum cytokines. Increasing IL-4 was associated with BAL PMN and negatively associated with BAL lymphocytes. Each 10-fold increase in BAL IL-4 and IL-12p40 increased the odds of AFB smear positivity by 7.4 and 2.2-fold, respectively, in a multi-variable logistic model. Conclusion Increasing IL-4 and IL-12p40 production by BAL cells are biomarkers for AFB in sputum of patients who present with radiographically advanced TB. They likely reflect less effective immune control of pathways for controlling TB, leading to patients with increased infectiousness. PMID:23527200

  8. In silico analysis of the three-dimensional structures of the homodimer of uridine phosphorylase from Yersinia Pseudotuberculosis in the ligand-free state and in a complex with 5-fluorouracil

    NASA Astrophysics Data System (ADS)

    Lashkov, A. A.; Sotnichenko, S. E.; Mikhailov, A. M.

    2013-03-01

    Pseudotuberculosis is an acute infectious disease characterized by a lesion of the gastrointestinal tract. A positive therapeutic effect can be achieved by selectively suppressing the activity of uridine phosphorylase from the causative agent of the disease Yersinia pseudotuberculosis. The synergistic effect of a combination of the chemotherapeutic agent 5-fluorouracil and antimicrobial drugs, which block the synthesis of pyrimidine bases, on the cells of pathogenic protozoa and bacteria is described in the literature. The three-dimensional structures of uridine phosphorylase from Yersinia pseudotuberculosis ( YptUPh) both in the ligand-free state and in complexes with pharmacological agents are unknown, which hinders the search for and design of selective inhibitors of YptUPh. The three-dimensional structure of the ligand-free homodimer of YptUPh was determined by homology-based molecular modeling. The three-dimensional structure of the subunit of the YptUPh molecule belongs to α/β proteins, and its topology is a three-layer α/β/α sandwich. The subunit monomer of the YptUPh molecule consists of 38% helices and 24% β strands. A model of the homodimer structure of YptUPh in a complex with 5-FU was obtained by the molecular docking. The position of 5-FU in the active site of the molecule is very consistent with the known data on the X-ray diffraction structures of other bacterial uridine phosphorylases (the complex of uridine phosphorylase from Salmonella typhimurium ( StUPh) with 5-FU, ID PDB: 4E1V and the complex of uridine phosphorylase from Escherichia coli ( EcUPh) with 5-FU and ribose 1-phosphate, ID PDB: 1RXC).

  9. In silico analysis of the three-dimensional structures of the homodimer of uridine phosphorylase from Yersinia Pseudotuberculosis in the ligand-free state and in a complex with 5-fluorouracil

    SciTech Connect

    Lashkov, A. A. Sotnichenko, S. E.; Mikhailov, A. M.

    2013-03-15

    Pseudotuberculosis is an acute infectious disease characterized by a lesion of the gastrointestinal tract. A positive therapeutic effect can be achieved by selectively suppressing the activity of uridine phosphorylase from the causative agent of the disease Yersinia pseudotuberculosis. The synergistic effect of a combination of the chemotherapeutic agent 5-fluorouracil and antimicrobial drugs, which block the synthesis of pyrimidine bases, on the cells of pathogenic protozoa and bacteria is described in the literature. The three-dimensional structures of uridine phosphorylase from Yersinia pseudotuberculosis (YptUPh) both in the ligand-free state and in complexes with pharmacological agents are unknown, which hinders the search for and design of selective inhibitors of YptUPh. The three-dimensional structure of the ligand-free homodimer of YptUPh was determined by homology-based molecular modeling. The three-dimensional structure of the subunit of the YptUPh molecule belongs to {alpha}/{beta} proteins, and its topology is a three-layer {alpha}/{beta}/{alpha} sandwich. The subunit monomer of the YptUPh molecule consists of 38% helices and 24% {beta} strands. A model of the homodimer structure of YptUPh in a complex with 5-FU was obtained by the molecular docking. The position of 5-FU in the active site of the molecule is very consistent with the known data on the X-ray diffraction structures of other bacterial uridine phosphorylases (the complex of uridine phosphorylase from Salmonella typhimurium (StUPh) with 5-FU, ID PDB: 4E1V and the complex of uridine phosphorylase from Escherichia coli (EcUPh) with 5-FU and ribose 1-phosphate, ID PDB: 1RXC).

  10. Unveiling the non-covalent interactions of molecular homodimers by dispersion-corrected DFT calculations and collision-induced broadening of ro-vibrational transitions: application to (CH2F2)2 and (SO2)2.

    PubMed

    Tasinato, Nicola; Grimme, Stefan

    2015-02-28

    Thermodynamic and spectroscopic properties of molecular complexes featuring non-covalent interactions, such as van der Waals forces and hydrogen bonds, are of fundamental interest in many fields, ranging from chemistry and biology to nanotechnology. In the present work the homodimers of difluoromethane (CH2F2) and sulfur dioxide (SO2) are investigated theoretically using dispersion-corrected density functional theory (DFT-D3) and experimentally by tunable diode laser (TDL) infrared (IR) spectroscopy. The dissociation energies of (CH2F2)2 and (SO2)2 are determined experimentally from the broadening of the ro-vibrational transitions of the corresponding monomers collisionally perturbed by a range of damping gases. The resulting dissociation energies are 2.79 ± 0.32 and 2.62 ± 0.16 kcal mol(-1) for the CH2F2 and SO2 dimers, respectively. Six to nine different stationary points on the PES of the two complexes are investigated theoretically at the DFT-D3 level, retrieving the corresponding dissociation energies, structures and rotational constants. Computations are carried out by employing six different density functionals (BLYP, TPSS, B3LYP, PBE0, TPSSh, and PW6B95) in conjunction with def2-TZVP and in a few cases def2-QZVP basis sets. DFT-D3 dissociation energies are benchmarked against reference values from CCSD(T)/CBS computations, and furthermore compared to experimental ones. A very good agreement between theory and experiment is attained, showing that DFT-D3 provides a significant improvement over standard DFT. This work shows that dissociation energies of homodimers can be consistently derived from collisional broadening cross sections and that interaction energies at various DFT-D3 levels (nearly) reach the accuracy of highly correlated wavefunction methods.

  11. The human parasite Leishmania amazonensis downregulates iNOS expression via NF-κB p50/p50 homodimer: role of the PI3K/Akt pathway

    PubMed Central

    Calegari-Silva, Teresa C.; Vivarini, Áislan C.; Miqueline, Marina; Dos Santos, Guilherme R. R. M.; Teixeira, Karina Luiza; Saliba, Alessandra Mattos; Nunes de Carvalho, Simone; de Carvalho, Laís; Lopes, Ulisses G.

    2015-01-01

    Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis-infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho-9 Ser GSK3β in infected macrophages, which is associated with GSK3β inhibition and, consequently, its inability to phosphorylate p105. Remarkably, we found that the levels of pPTEN 370 Ser, a negative regulator of PI3K, increased due to L. amazonensis infection. Our data support the notion that PI3K/Akt activity is sustained during the parasite infection, leading to NF-κB 105 phosphorylation and further processing to originate p50/p50 homodimers and the consequent downregulation of iNOS expression. PMID:26400473

  12. Activation of endogenous c-fos proto-oncogene expression by human T-cell leukemia virus type I-encoded p40 sup tax protein in the human T-cell line, Jurkat

    SciTech Connect

    Nagata, Kinya; Ohtani, Kiyoshi; Nakamura, Masataka; Sugamura, Kazuo )

    1989-08-01

    The authors examined the ability of the trans-acting factor p40{sup tax} of human T-cell leukemia virus type I (HTLV-I), which is thought to be a crucial molecule in T-cell transformation by HTLV-I, to activate expression of a set of endogenous cellular genes related to T-cell proliferation. For this purpose, they established a subclone (JPX-9) of Jurkat cells that was stably transfected with an expression plasmid containing the p40{sup tax} gene, whose expression is definitively dependent on heavy-metal ions. Expression of the interleukin-2 receptor {alpha} chain in JPX-9 cells was induced in response to the induction of p40{sup tax} expression, as has been demonstrated by others in transient transfection experiments with Jurkat cells. In addition, they found that significant enhancement of expression of the nuclear proto-oncogene c-fos was closely associated with expression of p40{sup tax}. Continuous enhancement in the level of c-fos mRNA was observed in the presence of p40{sup tax}. These results suggest that (i) in addition to the interleukin-2-interleukin-2 receptor system, cellular genes such as c-fos, which regulate normal T-cell growth, are also activated directly or indirectly by p40{sup tax} and (ii) p40{sup tax}-induced modulation of gene expression plays a crucial role in T-cell transformation by HTLV-I.

  13. Myeloid-Restricted AMPKα1 Promotes Host Immunity and Protects against IL-12/23p40-Dependent Lung Injury during Hookworm Infection.

    PubMed

    Nieves, Wildaliz; Hung, Li-Yin; Oniskey, Taylor K; Boon, Louis; Foretz, Marc; Viollet, Benoit; Herbert, De'Broski R

    2016-06-01

    How the metabolic demand of parasitism affects immune-mediated resistance is poorly understood. Immunity against parasitic helminths requires M2 cells and IL-13, secreted by CD4(+) Th2 and group 2 innate lymphoid cells (ILC2), but whether certain metabolic enzymes control disease outcome has not been addressed. This study demonstrates that AMP-activated protein kinase (AMPK), a key driver of cellular energy, regulates type 2 immunity and restricts lung injury following hookworm infection. Mice with a selective deficiency in the AMPK catalytic α1 subunit in alveolar macrophages and conventional dendritic cells produced less IL-13 and CCL17 and had impaired expansion of ILC2 in damaged lung tissue compared with wild-type controls. Defective type 2 responses were marked by increased intestinal worm burdens, exacerbated lung injury, and increased production of IL-12/23p40, which, when neutralized, restored IL-13 production and improved lung recovery. Taken together, these data indicate that defective AMPK activity in myeloid cells negatively impacts type 2 responses through increased IL-12/23p40 production. These data support an emerging concept that myeloid cells and ILC2 can coordinately regulate tissue damage at mucosal sites through mechanisms dependent on metabolic enzyme function.

  14. T cell receptor complexes containing Fc epsilon RI gamma homodimers in lieu of CD3 zeta and CD3 eta components: a novel isoform expressed on large granular lymphocytes

    PubMed Central

    1992-01-01

    CD3 zeta and CD3 eta form disulfide-linked homo- or heterodimers important in targeting partially assembled Ti alpha-beta/CD3 gamma delta epsilon T cell receptor (TCR) complexes to the cell surface and transducing stimulatory signals after antigen recognition. Here we identify a new TCR isoform expressed on splenic CD2+, CD3/Ti alpha- beta+, CD4-, CD8-, CD16+, NK1.1+ mouse large granular lymphocytes (LGL), which are devoid of CD3 zeta and CD3 eta proteins. The TCRs of this subset contain homodimers of the gamma subunit of the high affinity receptor for IgE (Fc epsilon RI gamma) in lieu of CD3 zeta and/or CD3 eta proteins. The LGL display natural killer-like activity and are cytotoxic for B cell hybridomas producing anti-CD3 epsilon and anti-CD16 monoclonal antibodies, demonstrating the signaling capacity of both TCR and CD16 in this cell type. These findings provide evidence for an additional level of complexity of TCR signal transduction isoforms in naturally occurring T cell subsets. PMID:1530959

  15. Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark®) for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens

    PubMed Central

    Larson, Jeffrey S.; Goodman, Laurie J.; Tan, Yuping; Defazio-Eli, Lisa; Paquet, Agnes C.; Cook, Jennifer W.; Rivera, Amber; Frankson, Kristi; Bose, Jolly; Chen, Lili; Cheung, Judy; Shi, Yining; Irwin, Sarah; Kiss, Linda D. B.; Huang, Weidong; Utter, Shannon; Sherwood, Thomas; Bates, Michael; Weidler, Jodi; Parry, Gordon; Winslow, John; Petropoulos, Christos J.; Whitcomb, Jeannette M.

    2010-01-01

    We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7–10 times greater than conventional immunohistochemistry (IHC) (HercepTest). The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC) or on indirect assessments of gene amplification (FISH). PMID:21151530

  16. Covalent homodimers of murine secretory component induced by epitope substitution unravel the capacity of the polymeric Ig receptor to dimerize noncovalently in the absence of IgA ligand.

    PubMed

    Crottet, P; Peitsch, M C; Servis, C; Corthésy, B

    1999-10-29

    Recombinant secretory immunoglobulin A containing a bacterial epitope in domain I of the secretory component (SC) moiety can serve as a mucosal delivery vehicle triggering both mucosal and systemic responses (Corthésy, B., Kaufmann, M., Phalipon, A., Peitsch, M., Neutra, M. R., and Kraehenbuhl, J.-P. (1996) J. Biol. Chem. 271, 33670-33677). To load recombinant secretory IgA with multiple B and T epitopes and extend its biological functions, we selected, based on molecular modeling, five surface-exposed sites in domains II and III of murine SC. Loops predicted to be exposed at the surface of SC domains were replaced with the DYKDDDDK octapeptide (FLAG). Another two mutants were obtained with the FLAG inserted in between domains II and III or at the carboxyl terminus of SC. As shown by mass spectrometry, internal substitution of the FLAG into four of the mutants induced the formation of disulfide-linked homodimers. Three of the dimers and two of the monomers from SC mutants could be affinity-purified using an antibody to the FLAG, mapping them as candidates for insertion. FLAG-induced dimerization also occurred with the polymeric immunoglobulin receptor (pIgR) and might reflect the so-far nondemonstrated capacity of the receptor to oligomerize. By co-expressing in COS-7 cells and epithelial Caco-2 cells two pIgR constructs tagged at the carboxyl terminus with hexahistidine or FLAG, we provide the strongest evidence reported to date that the pIgR dimerizes noncovalently in the plasma membrane in the absence of polymeric IgA ligand. The implication of this finding is discussed in terms of IgA transport and specific antibody response at mucosal surfaces.

  17. An Investigation of the 40Ar(n,p)40Cl Reaction Cross-Section below 50MeV at Crocker Nuclear Laboratory

    NASA Astrophysics Data System (ADS)

    Walsh, Nicholas Ian

    Large underground liquid argon detectors are poised to detect neutrinos from the next galactic supernova. Liquid argon detectors are uniquely sensitive to the electron neutrino, thus giving them the capability to detect neutronization neutrinos for the first time. One background that may mimic the signal of this low-energy neutrino interaction in argon is the beta-decay of Cl-40 which is produced in argon by a fast neutron reaction. Previous measurements of this 40Ar+n->40Cl+p reaction cross section exist only below 15 MeV and the measurements differ by a factor of two. Using the U.C. Davis Crocker Nuclear Laboratory neutron beam this cross-section is determined by fitting to a parametrized model for neutron energies up to 50 MeV. Neutrons at this facility are generated from mono-energetic protons impinging on a thick beryllium target. Then, the neutrons that pass through the collimator are measured by time-of-flight and a fast-neutron activation technique. Using the neutron fluxes generated from five different proton energies, including 50 MeV protons, the 40Ar(n,p)40Cl reaction cross section is measured by irradiating liquid argon in each beam and counting the subsequent gammas from the Cl-40 decay in a high-purity germanium detector.

  18. Demonstration of non-infectious hemagglutinating particles of rabies virus and isolation of the hemagglutinin by disruption of the virion with Nonidet P-40.

    PubMed

    Arai, Y T; Kondo, A; Suzuki, K

    1976-01-01

    Non-infectious hemagglutinating particles of rabies virus accumulated in the fluid phase of chick embryo cell cultures at 6 days post-infection, though they were undetectable at 4 days. They were characterized as looped filaments resembling viral envelope as revealed by electron microscopy. Another form of hemagglutinin (HAnin) was obtained by solubilization of partially purified virions with Nonidet P-40 (NP-40) followed by successive high speed and CsCl density gradient centrifugations. The density of the isolated HAnin averaged 1.28 g/cm3. SDS-polyacrylamide gel electrophoresis of the HAnin demonstrated that it was mainly composed of a glycoprotein (G) with a molecular weight of 83,000. Electron microscopically, it differed from the above non-infectious hemagglutinating particles, being much smaller in size and showing a star- or rosette-like appearance with a diameter of about 25 nm, composed of a central particle surrounded by particles resembling envelope-spikes. Virus-neutralizing (VN) and hemagglutination inhibiting (HI) antibodies were produced in rabbits immunized with the HAnin isolated from virions.

  19. Infection Rate and Tissue Localization of Murine IL-12p40-Producing Monocyte-Derived CD103+ Lung Dendritic Cells during Pulmonary Tuberculosis

    PubMed Central

    Leepiyasakulchai, Chaniya; Taher, Chato; Chuquimia, Olga D.; Mazurek, Jolanta; Söderberg-Naucler, Cecilia; Fernández, Carmen; Sköld, Markus

    2013-01-01

    Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103+ dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40+ cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype. PMID:23861965

  20. Various Antibody Clones of Napsin A, Thyroid Transcription Factor 1, and p40 and Comparisons With Cytokeratin 5 and p63 in Histopathologic Diagnostics of Non-Small Cell Lung Carcinoma.

    PubMed

    Tran, Lena; Mattsson, Johanna S M; Nodin, Björn; Jönsson, Per; Planck, Maria; Jirström, Karin; Botling, Johan; Micke, Patrick; Brunnström, Hans

    2016-10-01

    Histopathologic classification of cancer in the lung is important for choice of treatment. Cytokeratin 5 (CK5), p63, and p40 are commonly used immunohistochemical markers for squamous cell carcinoma, and napsin A (NAPA) and thyroid transcription factor 1 (TTF-1) are markers for adenocarcinoma of the lung. The aim of the present study was to evaluate these 5 markers and to compare different commercially available antibody clones in lung cancer. Tissue microarrays including 557 cases of surgically treated primary tumors and 73 matched metastases of non-small cell lung carcinoma were stained with CK5, p63, p40 (monoclonal and polyclonal), NAPA (5 different clones/protocols), and TTF-1 (2 different clones). The sensitivity and specificity to separate squamous cell carcinomas from non-small cell carcinomas of nonsquamous type were 95% and 97%, respectively, for CK5, 95% and 87% for p63, 94% and 96% for p40, 75% to 79% and 96% to 98% for the NAPA clones/protocols and 80% to 85% and 95% to 97% for the TTF-1 clones. A combination of NAPA and TTF-1 resulted in a higher sensitivity (85% to 88%), whereas combining CK5 and p40 did not increase the diagnostic performance. The sensitivity was generally lower in evaluation of lung cancer metastases. The κ-values for comparison of staining results between monoclonal and polyclonal p40 and between the 5 NAPA clones/protocols were 0.97 to 1.0, whereas the corresponding figure for the 2 TTF-1 clones was 0.91 to 0.93. Conclusively, CK5 and p40 are good diagnostic markers for squamous cell carcinoma and superior to p63. In addition, it may be useful to combine NAPA and TTF-1 for increased sensitivity in lung cancer diagnostics. There is no substantial difference between monoclonal and polyclonal p40 and between different NAPA clones, whereas there is a difference between the TTF-1 clones 8G7G3/1 and SPT24.

  1. A Regulatory Element Near the 3′ End of the Adeno-Associated Virus rep Gene Inhibits Adenovirus Replication in cis by Means of p40 Promoter-Associated Short Transcripts

    PubMed Central

    Hammer, Eva; Gonsior, Melanie; Stutika, Catrin; Heilbronn, Regine

    2016-01-01

    ABSTRACT Adeno-associated virus (AAV) has long been known to inhibit helper adenovirus (Ad) replication independently of AAV Rep protein expression. More recently, replication of Ad serotype 5 (Ad5)/AAV serotype 2 (AAV-2) hybrid vectors was shown to be inhibited in cis by a sequence near the 3′ end of AAV rep, termed the Rep inhibition sequence for adenoviral replication (RIS-Ad). RIS-Ad functions independently of Rep protein expression. Here we demonstrate that inhibition of adenoviral replication by RIS-Ad requires an active AAV p40 promoter and the 5′ half of the intron. In addition, Ad inhibition is critically dependent on the integrity of the p40 transcription start site (TSS) leading to short p40-associated transcripts. These do not give rise to effector molecules capable of inhibiting adenoviral replication in trans, like small polypeptides or microRNAs. Our data point to an inhibitory mechanism in which RNA polymerase II (Pol II) pauses directly downstream of the p40 promoter, leading to interference of the stalled Pol II transcription complex with the adenoviral replication machinery. Whereas inhibition by RIS-Ad is mediated exclusively in cis, it can be overcome by providing a replication-competent adenoviral genome in trans. Moreover, the inhibitory effect of RIS-Ad is not limited to AAV-2 but could also be shown for the corresponding regions of other AAV serotypes, including AAV-5. These findings have important implications for the future generation of Ad5/AAV hybrid vectors. IMPORTANCE Insertion of sequences from the 3′ part of the rep gene of adeno-associated virus (AAV) into the genome of its helper adenovirus strongly reduces adenoviral genome replication. We could show that this inhibition is mediated exclusively in cis without the involvement of trans-acting regulatory RNAs or polypeptides but nevertheless requires an active AAV-2 p40 promoter and p40-associated short transcripts. Our results suggest a novel inhibitory mechanism that has so

  2. Interleukin-12 and Interleukin-2 in Treating Patients With Mycosis Fungoides

    ClinicalTrials.gov

    2013-01-15

    Recurrent Cutaneous T-cell Non-Hodgkin Lymphoma; Recurrent Mycosis Fungoides/Sezary Syndrome; Stage I Cutaneous T-cell Non-Hodgkin Lymphoma; Stage I Mycosis Fungoides/Sezary Syndrome; Stage II Cutaneous T-cell Non-Hodgkin Lymphoma; Stage II Mycosis Fungoides/Sezary Syndrome; Stage III Cutaneous T-cell Non-Hodgkin Lymphoma; Stage III Mycosis Fungoides/Sezary Syndrome; Stage IV Cutaneous T-cell Non-Hodgkin Lymphoma; Stage IV Mycosis Fungoides/Sezary Syndrome

  3. Interleukin-12 Followed by Interferon Alfa in Treating Patients With Advanced Cancer

    ClinicalTrials.gov

    2013-01-31

    Chronic Myeloproliferative Disorders; Leukemia; Lymphoma; Multiple Myeloma and Plasma Cell Neoplasm; Myelodysplastic Syndromes; Precancerous Condition; Unspecified Adult Solid Tumor, Protocol Specific

  4. Interleukin-12 bypasses common gamma-chain signalling in emergency natural killer cell lymphopoiesis

    PubMed Central

    Ohs, Isabel; van den Broek, Maries; Nussbaum, Kathrin; Münz, Christian; Arnold, Sebastian J.; Quezada, Sergio A.; Tugues, Sonia; Becher, Burkhard

    2016-01-01

    Differentiation and homeostasis of natural killer (NK) cells relies on common gamma-chain (γc)-dependent cytokines, in particular IL-15. Consequently, NK cells do not develop in mice with targeted γc deletion. Herein we identify an alternative pathway of NK-cell development driven by the proinflammatory cytokine IL-12, which can occur independently of γc-signalling. In response to viral infection or upon exogenous administration, IL-12 is sufficient to elicit the emergence of a population of CD122+CD49b+ cells by targeting NK-cell precursors (NKPs) in the bone marrow (BM). We confirm the NK-cell identity of these cells by transcriptome-wide analyses and their ability to eliminate tumour cells. Rather than using the conventional pathway of NK-cell development, IL-12-driven CD122+CD49b+ cells remain confined to a NK1.1lowNKp46low stage, but differentiate into NK1.1+NKp46+ cells in the presence of γc-cytokines. Our data reveal an IL-12-driven hard-wired pathway of emergency NK-cell lymphopoiesis bypassing steady-state γc-signalling. PMID:27982126

  5. Role of interleukin-12 family cytokines in the cellular response to mycobacterial disease.

    PubMed

    Méndez-Samperio, Patricia

    2010-05-01

    Interleukin (IL)-12 is a multifunctional cytokine acting as a key regulator of cell-mediated immune responses through the differentiation of naïve CD4+ T cells into type 1 helper T cells (Th1) producing interferon-gamma. As our knowledge of IL-12 family members is rapidly growing, it will be important to specify their involvement in the regulation of mycobacterial infection. This article is a review of the current knowledge regarding the functions of the IL-12 family cytokines in the immune host defense system against mycobacteria. Specifically, this review aims to describe recent scientific evidence concerning the protective role of some members of the IL-12 family cytokines for the control of mycobacterial infection, as well as to summarize knowledge of the potential use of the IL-12 family members as potent adjuvants in the prevention and treatment of mycobacterial infectious diseases. In addition, recent data supporting the importance of the IL-12 family members in mycobacterial diseases in relation to Th17 function are discussed. This examination will help to improve our understanding of the immune response to mycobacterial infection and also improve vaccine design and immunotherapeutic intervention against tuberculosis.

  6. Phase I Trial of Interleukin-12 Plasmid Electroporation in Patients With Metastatic Melanoma

    PubMed Central

    Daud, Adil I.; DeConti, Ronald C.; Andrews, Stephanie; Urbas, Patricia; Riker, Adam I.; Sondak, Vernon K.; Munster, Pamela N.; Sullivan, Daniel M.; Ugen, Kenneth E.; Messina, Jane L.; Heller, Richard

    2008-01-01

    Purpose Gene-based immunotherapy for cancer is limited by the lack of safe, efficient, reproducible, and titratable delivery methods. Direct injection of DNA into tissue, although safer than viral vectors, suffers from low gene transfer efficiency. In vivo electroporation, in preclinical models, significantly enhances gene transfer efficiency while retaining the safety advantages of plasmid DNA. Patients and Methods A phase I dose escalation trial of plasmid interleukin (IL)-12 electroporation was carried out in patients with metastatic melanoma. Patients received electroporation on days 1, 5, and 8 during a single 39-day cycle, into metastatic melanoma lesions with six 100-μs pulses at a 1,300-V/cm electric field through a penetrating six-electrode array immediately after DNA injection. Pre- and post-treatment biopsies were obtained at defined time points for detailed histologic evaluation and determination of IL-12 protein levels. Results Twenty-four patients were treated at seven dose levels, with minimal systemic toxicity. Transient pain after electroporation was the major adverse effect. Post-treatment biopsies showed plasmid dose proportional increases in IL-12 protein levels as well as marked tumor necrosis and lymphocytic infiltrate. Two (10%) of 19 patients with nonelectroporated distant lesions and no other systemic therapy showed complete regression of all metastases, whereas eight additional patients (42%) showed disease stabilization or partial response. Conclusion This report describes the first human trial, to our knowledge, of gene transfer utilizing in vivo DNA electroporation. The results indicated this modality to be safe, effective, reproducible, and titratable. PMID:19029422

  7. Interleukin 12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in mice

    PubMed Central

    1995-01-01

    Allergic asthma is characterized by airway hyperresponsiveness and pulmonary eosinophilia, and may be mediated by T helper (Th) lymphocytes expressing a Th2 cytokine pattern. Interleukin (IL) 12 suppresses the expression of Th2 cytokines and their associated responses, including eosinophilia, serum immunoglobulin E, and mucosal mastocytosis. We have previously shown in a murine model that antigen- induced increases in airway hyperresponsiveness and pulmonary eosinophilia are CD4+ T cell dependent. We used this model to determine the ability of IL-12 to prevent antigen-induced increases in airway hyperresponsiveness, bronchoalveolar lavage (BAL) eosinophils, and lung Th2 cytokine expression. Sensitized A/J mice developed airway hyperresponsiveness and increased numbers of BAL eosinophils and other inflammatory cells after single or repeated intratracheal challenges with sheep red blood cell antigen. Pulmonary mRNA and protein levels of the Th2 cytokines IL-4 and IL-5 were increased after antigen challenge. Administration of IL-12 (1 microgram/d x 5 d) at the time of a single antigen challenge abolished the airway hyperresponsiveness and pulmonary eosinophilia and promoted an increase in interferon (IFN) gamma and decreases in IL-4 and IL-5 expression. The effects of IL-12 were partially dependent on IFN-gamma, because concurrent treatment with IL-12 and anti-IFN-gamma monoclonal antibody partially reversed the inhibition of airway hyperresponsiveness and eosinophilia by IL-12. Treatment of mice with IL-12 at the time of a second antigen challenge also prevented airway hyperresponsiveness and significantly reduced numbers of BAL inflammatory cells, reflecting the ability of IL-12 to inhibit responses associated with ongoing antigen-induced pulmonary inflammation. These data show that antigen-induced airway hyperresponsiveness and inflammation can be blocked by IL-12, which suppresses Th2 cytokine expression. Local administration of IL-12 may provide a novel immunotherapy for the treatment of pulmonary allergic disorders such as atopic asthma. PMID:7595222

  8. Rituximab and Interleukin-12 in Treating Patients With B-Cell Non-Hodgkin's Lymphoma

    ClinicalTrials.gov

    2013-08-23

    Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Nodal Marginal Zone B-cell Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Small Lymphocytic Lymphoma; Splenic Marginal Zone Lymphoma

  9. Interleukin-12, Paclitaxel, and Trastuzumab in Treating Patients With Solid Tumors

    ClinicalTrials.gov

    2013-06-03

    Male Breast Cancer; Recurrent Breast Cancer; Recurrent Endometrial Carcinoma; Recurrent Gastric Cancer; Recurrent Non-small Cell Lung Cancer; Recurrent Ovarian Epithelial Cancer; Recurrent Small Cell Lung Cancer

  10. Differential regulation of interleukin-12- and interleukin-15-induced natural killer cell activation by interleukin-4.

    PubMed

    Salvucci, O; Mami-Chouaib, F; Moreau, J L; Thèze, J; Chehimi, J; Chouaib, S

    1996-11-01

    The regulation of human natural killer (NK) cell activation is under the control of a network of regulatory signals provided by cytokines. In the present study, we investigated the functional interaction between interleukin (IL)-4 and two monocyte/macrophage-derived cytokines, IL-12 and IL-15, during the process of NK stimulation. Using freshly isolated human NK cells, we have demonstrated that IL-4 negatively regulates lymphokine-activated killer (LAK) activity induced by IL-15 against the NK-resistant Daudi target cells. In contrast, IL-4 had no effect on IL-12-stimulated LAK generation. The differential effect of IL-4 on NK cell activation by IL-12 and IL-15 correlates with its ability to increase or to down-regulate the level of tumor necrosis factor-alpha and interferon-gamma release by NK cells, respectively. In contrast, endogenous transforming growth factor-beta 1 does not appear to be involved in the IL-4 regulatory pathway. Furthermore, while IL-4 was found to decrease the basal expression of the IL-2 receptor beta subunit utilized by IL-15, it had no effect on the expression of the beta 1 chain of the IL-12 receptor compared to untreated cells. Northern blot analysis indicated that the IL-4 regulatory effect on NK lytic function was associated with its capacity to down-regulate granzyme B and perforin gene transcription in response to IL-15 and its failure to affect the expression of both gene's in response to IL-12. Together, these data suggest the existence of a distinct cross-talk between IL-4 and IL-15 or IL-12 signaling pathways during the regulation of human non-major histocompatibility complex-restricted cytotoxicity.

  11. Interleukin-12 in Treating Patients With Hematologic Cancers or Solid Tumors

    ClinicalTrials.gov

    2014-09-09

    Breast Cancer; Chronic Myeloproliferative Disorders; Gestational Trophoblastic Tumor; Kidney Cancer; Leukemia; Lymphoma; Multiple Myeloma and Plasma Cell Neoplasm; Myelodysplastic Syndromes; Neuroblastoma; Ovarian Cancer; Testicular Germ Cell Tumor

  12. Role of chitosan co-formulation in enhancing interleukin-12 delivery and antitumor activity

    PubMed Central

    Yang, Lirong; Zaharoff, David A.

    2013-01-01

    Local delivery systems that provide sustained, high concentrations of antitumor cytokines in the tumor microenvironment while minimizing systemic dissemination are needed to realize the potential of cytokine-based immunotherapies. Recently, co-formulations of cytokines with chitosan solutions have been shown to increase local cytokine retention and bioactivity. In particular, intratumoral (i.t.) injections of chitosan/IL-12 can eliminate established tumors and generate tumor-specific immune responses. In the present study, we explored the mechanisms by which chitosan potentiated IL-12’s antitumor activity. The location of chitosan/IL-12 injection was found to be critical for optimal cytokine delivery. I.t. injections eliminated 9 of 10 MC38 adenocarcinomas while contralateral and peritumoral injections delayed tumor growth but could not eliminate tumors. Microdosing studies demonstrated that IL-12 depots, simulated through daily i.t. injections with IL-12 alone, were not as effective as weekly i.t. chitosan/IL-12. 50–75% of mice receiving daily IL-12 microdoses and 87.5% of mice receiving weekly chitosan/IL-12 were cured of MC38 tumors. Chitosan was found to increase IL-12-mediated leukocytic expansion in tumors and tumor-draining lymph nodes (TDLNs) by 40% and 100%, respectively. Immunophenotyping studies demonstrated that chitosan co-formulation amplified IL-12-induced increases in important effector populations, such as CD8+IFN-γ+ and NKT cells, in tumors and dendritic cell populations in TDLNs. Remarkable increases in Gr-1+CD11b+ tumor infiltrates were also observed in mice receiving chitosan or chitosan/IL-12. This population does not appear be suppressive and may facilitate the local antitumor response. Presented data suggest that chitosan-mediated depot formation and enhanced local cytokine retention is significantly, but not entirely, responsible for increased cytokine bioactivity. PMID:23453060

  13. Interleukin-12 in Treating Patients With Previously Treated Non-Hodgkin's Lymphoma or Hodgkin's Disease

    ClinicalTrials.gov

    2015-04-14

    Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Nodal Marginal Zone B-cell Lymphoma; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Adult Diffuse Mixed Cell Lymphoma; Recurrent Adult Diffuse Small Cleaved Cell Lymphoma; Recurrent Adult Hodgkin Lymphoma; Recurrent Adult Immunoblastic Large Cell Lymphoma; Recurrent Cutaneous T-cell Non-Hodgkin Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Grade 3 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Mycosis Fungoides/Sezary Syndrome; Recurrent Small Lymphocytic Lymphoma; Splenic Marginal Zone Lymphoma; Waldenström Macroglobulinemia

  14. Cyclooxygenase 2-mediated suppression of macrophage interleukin-12 production after thermal injury.

    PubMed

    Schwacha, Martin G; Chung, Chun-Shiang; Ayala, Alfred; Bland, Kirby I; Chaudry, Irshad H

    2002-02-01

    Macrophage (Mphi) prostaglandin (PG)E(2) production has been implicated in immunosuppression and increased susceptibility to sepsis after thermal injury. Deficient interleukin (IL)-12 production has also been implicated in these postburn complications. The present study examined the relationship between Mphi cyclooxygenase (COX)-2 activity and IL-12 production after thermal injury. C57BL/6 female mice were subjected to a 25% total body surface area full-thickness burn. Mphi were isolated 7 days later, or the mice were subjected to sepsis by cecal ligation and puncture (CLP). IL-12 production by Mphi from injured mice was suppressed by >50%, whereas COX-2 expression and PGE(2) production were increased twofold. The COX-2 inhibitor NS-398 suppressed PGE(2) production and normalized IL-12 production in the injury group, whereas it had no effect on IL-10 production. Injured mice subjected to CLP had lower IL-12 plasma levels compared with sham-treated mice subjected to CLP. NS-398 treatment prevented the suppression in plasma IL-12 levels in the injury group. Thus elevated Mphi COX-2 activity, independent of IL-10, suppresses Mphi IL-12 production after thermal injury and may play an important role in the observed immunosuppression under such conditions.

  15. Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis.

    PubMed Central

    Coughlin, C M; Salhany, K E; Wysocka, M; Aruga, E; Kurzawa, H; Chang, A E; Hunter, C A; Fox, J C; Trinchieri, G; Lee, W M

    1998-01-01

    The antitumor effect and mechanisms activated by murine IL-12 and IL-18, cytokines that induce IFN-gamma production, were studied using engineered SCK murine mammary carcinoma cells. In syngeneic A/J mice, SCK cells expressing mIL-12 or mIL-18 were less tumorigenic and formed tumors more slowly than control cells. Neither SCK.12 nor SCK.18 cells protected significantly against tumorigenesis by distant SCK cells. However, inoculation of the two cell types together synergistically protected 70% of mice from concurrently injected distant SCK cells and 30% of mice from SCK cells established 3 d earlier. Antibody neutralization studies revealed that the antitumor effects of secreted mIL-12 and mIL-18 required IFN-gamma. Interestingly, half the survivors of SCK.12 and/or SCK.18 cells developed protective immunity suggesting that anti-SCK immunity is unlikely to be responsible for protection. Instead, angiogenesis inhibition, assayed by Matrigel implants, appeared to be a property of both SCK.12 and SCK.18 cells and the two cell types together produced significantly greater systemic inhibition of angiogenesis. This suggests that inhibition of tumor angiogenesis is an important part of the systemic antitumor effect produced by mIL-12 and mIL-18. PMID:9502787

  16. Improved transport of horseradish peroxidase after injection with a non-ionic detergent (Nonidet P-40) into mouse cortex and observations on the relationship between spread at the injection site and amount of transported label.

    PubMed

    Lipp, H P; Schwegler, H

    1980-10-20

    Addition of the non-ionic detergent Nonidet P-40 (NP-40) to horseradish peroxidase (HRP) injected in small quantities into barrelfields of mouse somatosensory cortex results in a significant increase of labeled neurons in the contralateral barrelfield cortex as compared to normal HRP. Comparisons with lysolethicin as an additive to HRP show that with NP-40 neurons are labeled more reliably and spread of label is less extensive at the injection site. Using NP-40, the region of dense label spread at the injection site as revealed by the diaminobenzidine/cobalt procedure coincides rather precisely with the contralateral cortical region containing labeled neurons as visualized by tetramethylbenzidine.

  17. Biological and biochemical properties of Nonidet P40-solubilized and partially purified tumor-specific antigens of the transplantation type from plasma membranes of a methylcholanthrene-induced sarcoma.

    PubMed

    Natori, T; Law, L W; Appella, E

    1977-09-01

    Tumor-specific transplantation antigen (TSTA) was solubilized from cell membranes of sarcoma Meth-A with non-ionic detergent Nonidet P40. Soluble TSTA was partially characterized by chromatographic separation and electrophoresis. The antigen responsible for tumor rejection activity had a molecular weight of approximately 70,000 daltons in the presence of detergent and an electrophoretic mobility of alpha-globulin. TSTA was well separated from mouse histocompatibility antigen H-2 by a sequence of procedures, including gel filtration, lectin affinity chromatography, column electrophoresis, and rechromatography on agarose, showed only three major bands on polyacrylamide gel electrophoresis. TSTA was specific for sarcoma Meth-A.

  18. The low-virulent African swine fever virus (ASFV/NH/P68) induces enhanced expression and production of relevant regulatory cytokines (IFNalpha, TNFalpha and IL12p40) on porcine macrophages in comparison to the highly virulent ASFV/L60.

    PubMed

    Gil, S; Sepúlveda, N; Albina, E; Leitão, A; Martins, C

    2008-01-01

    The impact of infection by the low-virulent ASFV/NH/P68 (NHV) and the highly virulent ASFV/L60 (L60) isolates on porcine macrophages was assessed through the quantification of IFNalpha, TNFalpha, IL12p40, TGFbeta and ASFV genes by real-time PCR at 2, 4 and 6 h post-infection. Increased IFNalpha, TNFalpha and IL12p40 expression was found in infection with NHV, in which expression of TGFbeta was lower than in infection with L60. Principal component analysis showed a positive interaction of cytokines involved in cellular immune mechanisms, namely IFNalpha and IL12p40 in the NHV infection. Quantification by ELISA confirmed higher production of IFNalpha, TNFalpha and IL12p40 in the NHV-infected macrophages. Overall, our studies reinforce and clarify the effect of the NHV infection by targeting cellular and cellular-based immune responses relevant for pig survival against ASFV infection.

  19. A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation

    PubMed Central

    Wang, Xin; Luo, Cheng; Yu, Dongmei; Wang, Yuheng; Chen, Yucong; Lei, Wen; Gao, Xiangdong; Yao, Wenbing

    2015-01-01

    Interleukin (IL)-12 and IL-23 respectively driving polarization of T helper (Th) 1 and Th17 cells has been strongly implicated in the pathogenesis of both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). In this study, we first constructed, expressed and purified a novel human truncated IL12rβ1-Fc fusion protein (tIL12rβ1/Fc) binding multiple forms of the p40 subunit of human IL-12 and IL-23. tIL12rβ1/Fc was found to effectively ameliorate MOG35–55-induced EAE through reducing the production of Th1- and Th17-polarized pro-inflammatory cytokines and suppressing inflammation and demyelination in the focused parts. Moreover, tIL12rβ1/Fc suppressed Th1 (IFN-γ+ alone) and IFN-γ+ IL-17+ as well as the population of classic Th17 (IL-17+ alone) cells in vivo. Furthermore, tIL12rβ1/Fc ameliorated EAE at the peak of disease via the inhibition of STAT pathway, thereby causing a prominent reduction of RORγt (Th17) and T-bet (Th1) expression. Notably, tIL12rβ1/Fc could increase the relative number of CD4+ Foxp3+ regulatory T cells. These findings indicates that tIL12rβ1/Fc is a novel fusion protein for specific binding multiple forms of p40 subunit to exert potent anti-inflammatory effects and provides a valuable approach for the treatment of MS and other autoimmune diseases. PMID:26384304

  20. Dendritic Cell-Based Genetic Immunotherapy for Ovarian Cancer

    DTIC Science & Technology

    2007-12-01

    a gamma-counter. Maximum and spontaneous release of 51 Cr was obtained from the supernatants of the target cells in 1% Nonidet P-40 and in...16: 1045-9. 7. Piazzolla, G., C. Tortorella, G. Fiore, M. Fanelli, A. Pisconti, and S. Antonaci, Interleukin-12 p40 /p70 ratio and in vivo

  1. A unique enhancer element for the trans activator (p40 sup tax ) of human T-cell leukemia virus type I that is distinct from cyclic AMP- and 12-O-tetradecanoylphobol-13-acetate-responsive elements

    SciTech Connect

    Fujisawa, Junichi; Toita, Masami; Yoshida, Mitsuaki )

    1989-08-01

    The trans activator (p40{sup tax}) of human T-cell leukemia virus type I (HTLV-I) is a transcriptional factor that activates the long terminal repeat (LTR) of HTLV-I and interleukin-2 receptor {alpha}. The authors examined the HTLV-I enhancer responsible for tax-mediated trans activation and identified (A/T)(G/C)(G/C)CNNTGACG(T/A) as a plausible tax-responsive element (TRE). The putative TRE in the LTR was found to be different from the elements required for activation by cyclic AMP and 12-O-tetradecanoylphorbol-13-acetate, although these elements overlapped each other. The TRE was also different from a binding site of N-{kappa}B-like factor that was identified was identified in the interleukin-2 receptor {alpha} promoter and human immunodeficiency virus LTR as a TRE. The latter result was further demonstrated by the failure of the NF-{kappa}B sequence to compete with the TRE of the LTR in a protein-binding assay. These findings indicate that tax function and its cascade can modulate activities of various enhancer sequences, which are probably regulated by distinct DNA-binding factors.

  2. A comparative evaluation of the in vitro penetration performance of the improved Crest complete toothbrush versus the Current Crest complete toothbrush, the Colgate Precision toothbrush and the Oral-B P40 toothbrush.

    PubMed

    Volpenhein, D W; Handel, S E; Hughes, T J; Wild, J

    1996-01-01

    Removal of plaque and debris from interproximal surfaces during toothbrushing has generally been difficult to achieve, in large part because traditional flat-bristled toothbrushes do not offer good interproximal penetration. As a result, a number of varying bristle designs have been developed, with the rippled-design brush shown to be particularly effective at removing interproximal plaque. Recently, an existing brush, the original Crest Complete, was modified to offer a more deeply rippled version. This study evaluated the interproximal penetration of four bristle designs: rippled pattern (original Crest Complete), deeper rippled pattern (improved Crest Complete), multi-level (Colgate Precision), and flat-tufted (Oral-B P40). The study used a previously reported in vitro model for determining interproximal penetration of manual toothbrushes (J Clin Dent 5:27-33, 1994). In order to effectively mimic the in-use characteristics of toothbrushing, this model is based on analysis of videotaped consumer brushing habits, tooth morphology, and in vivo plaque tenacity characteristics and uses the three most predominantly used brushing techniques (circular, up-and-down, and back-and-forth, with the brush held at both 45 and 90 degrees to the tooth surface). In addition, the model's brush stroke length, brush force, and brush speed are likewise based on analysis of consumer brushing patterns. The results of the study indicate that the new Crest Complete with deeper rippled bristles provided significantly superior (p < or = 0.05) interproximal penetration than the Colgate Precision and Oral-B brushes overall and for three of the four brush strokes tested. In addition, the new Crest Complete was found to provide significantly superior interproximal penetration to the original Crest Complete overall and in circular and up-and-down strokes, and the original Crest Complete provided superior overall interproximal penetration to the Colgate and Oral-B brushes.

  3. Sirtuin 1 is a key regulator of the interleukin-12 p70/interleukin-23 balance in human dendritic cells.

    PubMed

    Alvarez, Yolanda; Rodríguez, Mario; Municio, Cristina; Hugo, Etzel; Alonso, Sara; Ibarrola, Nieves; Fernández, Nieves; Crespo, Mariano Sánchez

    2012-10-12

    Stimulation of human dendritic cells with the fungal surrogate zymosan produces IL-23 and a low amount of IL-12 p70. Trans-repression of il12a transcription, which encodes IL-12 p35 chain, by proteins of the Notch family and lysine deacetylation reactions have been reported as the underlying mechanisms, but a number of questions remain to be addressed. Zymosan produced the location of sirtuin 1 (SIRT1) to the nucleus, enhanced its association with the il12a promoter, increased the nuclear concentration of the SIRT1 co-substrate NAD(+), and decreased chromatin accessibility in the nucleosome-1 of il12a, which contains a κB-site. The involvement of deacetylation reactions in the inhibition of il12a transcription was supported by the absence of Ac-Lys-14-histone H3 in dendritic cells treated with zymosan upon coimmunoprecipitation of transducin-like enhancer of split. In contrast, we did not obtain evidence of a possible effect of SIRT1 through the deacetylation of c-Rel, the central element of the NF-κB family involved in il12a regulation. These data indicate that an enhancement of SIRT1 activity in response to phagocytic stimuli may reduce the accessibility of c-Rel to the il12a promoter and its transcriptional activation, thus regulating the IL-12 p70/IL-23 balance and modulating the ongoing immune response.

  4. Sirtuin 1 Is a Key Regulator of the Interleukin-12 p70/Interleukin-23 Balance in Human Dendritic Cells*

    PubMed Central

    Alvarez, Yolanda; Rodríguez, Mario; Municio, Cristina; Hugo, Etzel; Alonso, Sara; Ibarrola, Nieves; Fernández, Nieves; Crespo, Mariano Sánchez

    2012-01-01

    Stimulation of human dendritic cells with the fungal surrogate zymosan produces IL-23 and a low amount of IL-12 p70. Trans-repression of il12a transcription, which encodes IL-12 p35 chain, by proteins of the Notch family and lysine deacetylation reactions have been reported as the underlying mechanisms, but a number of questions remain to be addressed. Zymosan produced the location of sirtuin 1 (SIRT1) to the nucleus, enhanced its association with the il12a promoter, increased the nuclear concentration of the SIRT1 co-substrate NAD+, and decreased chromatin accessibility in the nucleosome-1 of il12a, which contains a κB-site. The involvement of deacetylation reactions in the inhibition of il12a transcription was supported by the absence of Ac-Lys-14-histone H3 in dendritic cells treated with zymosan upon coimmunoprecipitation of transducin-like enhancer of split. In contrast, we did not obtain evidence of a possible effect of SIRT1 through the deacetylation of c-Rel, the central element of the NF-κB family involved in il12a regulation. These data indicate that an enhancement of SIRT1 activity in response to phagocytic stimuli may reduce the accessibility of c-Rel to the il12a promoter and its transcriptional activation, thus regulating the IL-12 p70/IL-23 balance and modulating the ongoing immune response. PMID:22893703

  5. Adjuvant activity of chicken interleukin-12 co-administered with infectious bursal disease virus recombinant VP2 antigen in chickens.

    PubMed

    Su, Bor Sheu; Chiu, Hua Hsien; Lin, Cheng Chung; Shien, Jui Hung; Yin, Hsien Sheng; Lee, Long Huw

    2011-02-15

    A recombinant fowlpox virus (rFPV/VP2) expressing infectious bursal diseases virus (IBDV) VP2 gene has been constructed. After purification and identification of rFPV/VP2, the adjuvant activity of the recombinant chicken IL-12 (rchIL-12), synthesized by our previous construct of rFPV/chIL-12, in rFPV/VP2-expressed rVP2 antigen was assessed in one-week-old specific-pathogen free chickens. The results indicated that rchIL-12 alone or rchIL-12 plus mineral oil (MO) co-administered with rVP2 antigen significantly enhanced the production of serum neutralization (SN) antibody against IBDV, compared to those with MO alone. The SN titers in groups receiving rVP2 antigen with MO alone were more inconsistent after vaccination. On the other hand, rchIL-12 significantly stimulated IFN-γ production in serum and in splenocyte cultured supernatant, suggesting that rchIL-12 alone or plus MO significantly induced a cell-mediated immune response. Finally, bursal lesion protection from very virulent IBDV (vvIBDV) challenge in chickens receiving rVP2 antigen with rchIL-12 alone or plus MO was much more effective than that with MO alone at two weeks after boosting. Taken together, rchIL-12 alone augmented in vivo the induction of a primary and also a secondary SN antibody production and a cell-mediated immunity against IBDV rVP2 antigen, which conferred the enhancement of bursal lesion protective efficacy from vvIBDV challenge. These data indicated that a potential for chIL-12 as immunoadjuvant for chicken vaccine development such as IBDV rVP2 antigen.

  6. Effects of interleukin 12 on immune responses and host protection in mice infected with intestinal nematode parasites

    PubMed Central

    1994-01-01

    The cytokine interleukin (IL) 12 stimulates T cell and natural killer cell production of interferon (IFN) gamma and inhibits T cell production of IL-4. We investigated the effects of IL-12 on cytokine gene expression, immunoglobulin (Ig)E, mucosal mast cell, and eosinophil responses, and the course of infection in mice inoculated with the nematode parasite Nippostrongylus brasiliensis, as well as the IFN-gamma dependence of these effects. IL-12 stimulated IFN-gamma and IL-10 gene expression during primary and secondary N. brasiliensis infections and inhibited IL-3, IL-4, IL-5, and IL-9 gene expression during primary infections but had little inhibitory effect during secondary infections. IL-12 inhibited IgE, mucosal mast cell, and blood and tissue eosinophil responses during primary infections, but only eosinophil responses during secondary infections. IL-12 enhanced adult worm survival and egg production during primary, but not secondary infections. IL-12 needed to be administered by day 4 of a primary infection to inhibit IgE and mucosal mast cell responses, and by day 6 to strongly inhibit eosinophil responses and to enhance worm survival and fecundity. Anti-IFN-gamma mAb inhibited the effects of IL-12 on IgE secretion, intestinal mucosal mastocytosis, and parasite survival and fecundity, but did not affect IL-12 inhibition of eosinophilia. These observations indicate that IL-12, if administered during the initiation of eosinophilia. These observations indicate that IL-12, if administered during the initiation of an immune response, can change the response from one that is characterized by the production of T helper (Th)2-associated cytokines to one characterized by the production of Th-1 associated cytokines. However, IL-12 treatment has less of an effect once the production of Th2-associated cytokines has become established. In addition, our results provide evidence that Th2- associated responses protect against, and/or Th1-associated responses exacerbate, nematode infections. PMID:7909327

  7. Interleukin-12 and Trastuzumab in Treating Patients With Cancer That Has High Levels of HER2/Neu

    ClinicalTrials.gov

    2013-02-27

    Advanced Adult Primary Liver Cancer; Anaplastic Thyroid Cancer; Bone Metastases; Carcinoma of the Appendix; Distal Urethral Cancer; Fallopian Tube Cancer; Gastrinoma; Glucagonoma; Inflammatory Breast Cancer; Insulinoma; Liver Metastases; Localized Unresectable Adult Primary Liver Cancer; Lung Metastases; Male Breast Cancer; Malignant Pericardial Effusion; Malignant Pleural Effusion; Metastatic Gastrointestinal Carcinoid Tumor; Metastatic Parathyroid Cancer; Metastatic Transitional Cell Cancer of the Renal Pelvis and Ureter; Newly Diagnosed Carcinoma of Unknown Primary; Occult Non-small Cell Lung Cancer; Pancreatic Polypeptide Tumor; Primary Peritoneal Cavity Cancer; Proximal Urethral Cancer; Pulmonary Carcinoid Tumor; Recurrent Adenoid Cystic Carcinoma of the Oral Cavity; Recurrent Adrenocortical Carcinoma; Recurrent Adult Primary Liver Cancer; Recurrent Anal Cancer; Recurrent Bladder Cancer; Recurrent Breast Cancer; Recurrent Carcinoma of Unknown Primary; Recurrent Cervical Cancer; Recurrent Colon Cancer; Recurrent Endometrial Carcinoma; Recurrent Esophageal Cancer; Recurrent Extrahepatic Bile Duct Cancer; Recurrent Gallbladder Cancer; Recurrent Gastric Cancer; Recurrent Gastrointestinal Carcinoid Tumor; Recurrent Islet Cell Carcinoma; Recurrent Malignant Testicular Germ Cell Tumor; Recurrent Mucoepidermoid Carcinoma of the Oral Cavity; Recurrent Non-small Cell Lung Cancer; Recurrent Ovarian Epithelial Cancer; Recurrent Pancreatic Cancer; Recurrent Parathyroid Cancer; Recurrent Prostate Cancer; Recurrent Rectal Cancer; Recurrent Renal Cell Cancer; Recurrent Salivary Gland Cancer; Recurrent Small Intestine Cancer; Recurrent Squamous Cell Carcinoma of the Larynx; Recurrent Squamous Cell Carcinoma of the Lip and Oral Cavity; Recurrent Squamous Cell Carcinoma of the Nasopharynx; Recurrent Squamous Cell Carcinoma of the Oropharynx; Recurrent Thyroid Cancer; Recurrent Transitional Cell Cancer of the Renal Pelvis and Ureter; Recurrent Urethral Cancer; Recurrent Vaginal Cancer; Recurrent Vulvar Cancer; Skin Metastases; Small Intestine Adenocarcinoma; Somatostatinoma; Stage III Adenoid Cystic Carcinoma of the Oral Cavity; Stage III Adrenocortical Carcinoma; Stage III Bladder Cancer; Stage III Cervical Cancer; Stage III Colon Cancer; Stage III Endometrial Carcinoma; Stage III Esophageal Cancer; Stage III Follicular Thyroid Cancer; Stage III Gastric Cancer; Stage III Malignant Testicular Germ Cell Tumor; Stage III Mucoepidermoid Carcinoma of the Oral Cavity; Stage III Ovarian Epithelial Cancer; Stage III Pancreatic Cancer; Stage III Papillary Thyroid Cancer; Stage III Prostate Cancer; Stage III Rectal Cancer; Stage III Renal Cell Cancer; Stage III Salivary Gland Cancer; Stage III Squamous Cell Carcinoma of the Larynx; Stage III Squamous Cell Carcinoma of the Lip and Oral Cavity; Stage III Squamous Cell Carcinoma of the Nasopharynx; Stage III Squamous Cell Carcinoma of the Oropharynx; Stage III Vaginal Cancer; Stage III Vulvar Cancer; Stage IIIA Anal Cancer; Stage IIIA Breast Cancer; Stage IIIA Non-small Cell Lung Cancer; Stage IIIB Anal Cancer; Stage IIIB Breast Cancer; Stage IIIB Non-small Cell Lung Cancer; Stage IV Adenoid Cystic Carcinoma of the Oral Cavity; Stage IV Adrenocortical Carcinoma; Stage IV Anal Cancer; Stage IV Bladder Cancer; Stage IV Breast Cancer; Stage IV Colon Cancer; Stage IV Endometrial Carcinoma; Stage IV Esophageal Cancer; Stage IV Follicular Thyroid Cancer; Stage IV Gastric Cancer; Stage IV Mucoepidermoid Carcinoma of the Oral Cavity; Stage IV Non-small Cell Lung Cancer; Stage IV Ovarian Epithelial Cancer; Stage IV Pancreatic Cancer; Stage IV Papillary Thyroid Cancer; Stage IV Prostate Cancer; Stage IV Rectal Cancer; Stage IV Renal Cell Cancer; Stage IV Salivary Gland Cancer; Stage IV Squamous Cell Carcinoma of the Larynx; Stage IV Squamous Cell Carcinoma of the Lip and Oral Cavity; Stage IV Squamous Cell Carcinoma of the Nasopharynx; Stage IV Squamous Cell Carcinoma of the Oropharynx; Stage IVA Cervical Cancer; Stage IVA Vaginal Cancer; Stage IVB Cervical Cancer; Stage IVB Vaginal Cancer; Stage IVB Vulvar Cancer; Thyroid Gland Medullary Carcinoma; Unresectable Extrahepatic Bile Duct Cancer; Unresectable Gallbladder Cancer; Urethral Cancer Associated With Invasive Bladder Cancer; WDHA Syndrome

  8. Induction of interleukin-8 and interleukin-12 in neonatal ovine lung following experimental inoculation of bovine respiratory syncytial virus.

    PubMed

    Redondo, E; Gázquez, A; Vadillo, S; García, A; Franco, A; Masot, A J

    2014-05-01

    This study aimed to determine the immunohistochemical expression of interleukin (IL)-1β, tumour necrosis factor alpha (TNF)-α, interferon (IFN)-γ, IL-4, IL-6, IL-8, IL-10 and IL-12 and to measure the concentrations of these cytokines in lung tissue from lambs infected experimentally with bovine respiratory syncytial virus (BRSV). Lambs (n = 15) were inoculated at 2 days of age with 20 ml of viral inoculum (1.26 × 10(6) TCID50 per ml) or sterile medium (n = 15). Rectal temperature, pulse and respiratory rates were monitored daily in control and infected lambs. Lambs were killed and subject to necropsy examination at 1, 3, 5, 7 and 15 days post inoculation (dpi). There was a temporal association between pulmonary expression of these cytokines and lung pathology in BRSV-infected lambs. The cytokines IL-4 and IL-10 were not elevated, but there was a significant increase in IL-1β, TNF-α, IFN-γ and IL-6 proteins and labelled cells, suggesting that these cytokines may play a role in the biological response to BRSV infection and contribute to the development of lung lesions. There was also a significant increase in the cytokine concentration and number of immunolabelled cells expressing IL-8 and IL-12 in infected lungs, suggesting that these cytokines might be used as therapeutic targets in the management of BRSV, in conjunction with measures to combat the causative pathogen and prophylactic methods aimed at preventing infection.

  9. Clinical improvement in feline herpesvirus 1 infected cats by oral low dose of interleukin-12 plus interferon-gamma.

    PubMed

    Fiorito, Filomena; Cantiello, Antonietta; Granato, Giovanna Elvira; Navas, Luigi; Diffidenti, Carmine; De Martino, Luisa; Maharajan, Veeramani; Olivieri, Fabio; Pagnini, Ugo; Iovane, Giuseppe

    2016-10-01

    Feline herpesvirus 1 (FHV-1) is a widespread cat pathogen inducing rhinitis, conjunctivitis and corneal ulcers. To alleviate acute FHV-1-induced disease, antiviral agents are used often with antibiotics. But sometimes, these treatments, as well as conventional doses of cytokines have moderate efficacy and/or collateral effects. Herein we have investigated the effects of low dose interleukin (IL)-12 plus interferon (IFN)-gamma, prepared by Sequential Kinetic Activated (SKA), on the treatment of FHV-1 infection. Twenty-five, unvaccinated FHV-1-positive cats were recruited into a prospective, randomized, placebo-controlled, double-blinded clinical trial. Fifteen cats were treated for 6 months with oral low doses of SKA IL-12 plus IFN-gamma and 10 cats were treated with placebo. At 1, 6 and 12 months (follow-up) after the beginning of treatment, clinical assessment, PCR assay and blood count were carried out. At follow-up, in treated group, we observed significant (p<0.05) improvements in clinical signs and PCR became negative in 12/15 cats (80%). In placebo, 10/10 cats were PCR-positive, with improvements (30%) or worsening (70%) in clinical signs. Blood values were normal in both groups. Our results show that the low dose therapy, based on activated solutions of IL-12 plus IFN-gamma, represents a novel approach to treat FHV-1 infection in cats.

  10. Transgenic tomato expressing interleukin-12 has a therapeutic effect in a murine model of progressive pulmonary tuberculosis.

    PubMed

    Elías-López, A L; Marquina, B; Gutiérrez-Ortega, A; Aguilar, D; Gomez-Lim, M; Hernández-Pando, R

    2008-10-01

    Host control of mycobacterial infection, in both human and mouse models, has been shown to be associated with the production of interferon (IFN)-gamma by CD4(+) T cells. Interleukin (IL)-12 is known to be a crucial cytokine in the differentiation of IFN-gamma-producing T helper 1 (Th1) cells. To determine whether continuous administration of IL-12 expressed in transgenic tomato (TT-IL-12) has therapeutic efficacy in a murine model of pulmonary tuberculosis, BALB/c mice were infected with either Mycobacterium tuberculosis H37Rv strain or a multi-drug-resistant clinical isolate (MDR) and treated with a daily oral dose of TT-IL12 crude fruit extracts. For the early H37Rv infection, TT-IL-12 administration was started 1 day before infection and continued for 60 days. In the H37Rv or MDR late infection, treatment was started 60 days after infection and continued for another 60 days. In both phases of infection, TT-IL-12 administration resulted in a reduction of bacterial loads and tissue damage compared with wild-type tomato (non-TT). The Th1 response was increased and the Th2 response was reduced. In the late infection, a long-term treatment with TT-IL-12 was necessary. We demonstrate that TT-IL-12 increases resistance to infection and reduces lung tissue damage during early and late drug-sensitive and drug-resistant mycobacterial infection.

  11. Effects of interleukin-12 in the long-term protection conferred by a Mycobacterium avium subunit vaccine.

    PubMed

    Silva, R A; Pais, T F; Appelberg, R

    2000-12-01

    The effects of the addition of recombinant interleukin (IL)-12 to a mycobacterial subunit vaccine were analyzed in terms of the longevity of the protective immunity generated. BALB/c mice were immunized with culture filtrate proteins from Mycobacterium avium with dimethyl-dioctadecilammonium bromide (DDA) as an adjuvant. This subunit vaccine induced protection against a challenge by M. avium which lasted for at least 6 months while waning with time until 1 year postvaccination. Whereas the addition of IL-12 enhanced the initial protective efficacy of this subunit vaccine during the first 6 months, it accelerated the loss of protective efficacy observed at 1 year postvaccination. These data confirm the adjuvant properties of IL-12 in vaccines against mycobacteria and raise the possibility of late counter-protective untoward effects.

  12. Non-heat pipe receiver/p-40 Stirling engine

    NASA Technical Reports Server (NTRS)

    Haglund, R. A.

    1981-01-01

    The technology for a full-up hybrid dish-Stirling Solar Thermal Power system is discussed. Overall solar-to-electric efficiency for the dish-Stirling system demonstration is approximately 30%. Hybrid operation is provided by fossil fuel combustion augmentation, which enables the Stirling engine to operate continuously at constant speed and power, regardless of insolation level, thus providing the capability to operate on cloudy days and at night.

  13. Effect of preoperative topical diclofenac on intraocular interleukin-12 concentration and macular edema after cataract surgery in patients with diabetic retinopathy: a randomized controlled trial

    PubMed Central

    Medić, Aleksej; Jukić, Tomislav; Matas, Anita; Vukojević, Katarina; Sapunar, Ada; Znaor, Ljubo

    2017-01-01

    Aim To determine if preoperative treatment with a topical non-steroidal anti-inflammatory drug (NSAID) lowers the concentration of intraocular interleukin (IL)-12 and the incidence of postoperative macular edema in patients with non-proliferative diabetic retinopathy undergoing cataract surgery. Methods A total of 55 patients were randomized to diclofenac (n = 27) or placebo (n = 28). Patients receiving diclofenac started preoperative treatment with 0.1% topical diclofenac four times a day 7 days before cataract surgery and the therapy was discontinued 30 days after surgery. Patients in the control group were administered placebo 7 days preoperatively and a standard postoperative therapy with 0.1% topical dexamethasone four times a day for 30 days after surgery. All patients received postoperative antibiotic prophylaxis with tobramycin eye drops four times daily for 30 days. Seven days before the cataract surgery, on the day of surgery, and 1, 7, 30, and 90 days after surgery, central foveal thickness (CFT) was measured with optical coherence tomography (OCT) and the aqueous humor was sampled at the beginning of cataract surgery for the analysis of IL-12 concentration. Due to loss to follow-up and insufficient aqueous humor samples, the data of 3 patients treated with diclofenac and 8 patients receiving placebo were not analyzed. Results The aqueous humor IL-12 concentration was significantly lower in the diclofenac group than in the placebo group (t=−2.85, P = 0.007). The diclofenac group had a significantly smaller increase in CFT after phacoemulsification (F = 13.57, p<0.001). Conclusion Patients preoperatively treated with diclofenac had significantly lower intraocular levels of IL-12 and a lower increase in CFT, which indicates that a combination of preoperative and postoperative treatment with a topical NSAID may lower the incidence of postoperative macular edema in patients with diabetic retinopathy. ClinicalTrials.gov trial registration number MZJ-2106 PMID:28252875

  14. Augmentation by interleukin-18 of MHC-nonrestricted killer activity of human peripheral blood mononuclear cells in response to interleukin-12.

    PubMed

    Singh, S M; Yanagawa, H; Hanibuchi, M; Miki, T; Okamura, H; Sone, S

    2000-01-01

    Interleukin (IL)-18 is a novel cytokine with pleiotropic functions. In the present study, we examined the induction of the killer activity of peripheral blood mononuclear cells (MNC) against lung cancer cell lines upon treatment with IL-18 in combination with IL-12. Cytotoxic activity was measured by standard (51)Cr release assay. IL-18 (100 ng/ml) was found to significantly augment IL-12-induced killer activity in a MHC-nonrestricted manner against allogeneic NK-resistant Daudi cells and lung cancer cell lines: SBC-3, RERF-LC-AI and A549. IL-18 could augment IL-12-induced killer activity both at the optimal as well as suboptimal doses of the latter. However, IL-18 was found to have little effect on the killer activity of MNC induced by optimal or suboptimal dose of IL-2 or IL-15. Treatment of MNC with IL-18 in combination with IL-12 for a period of more than 4 days was observed to optimally induce the killer activity. As for induction of IFN-gamma production by MNC, IL-18 augmented that induced by IL-2 and IL-15, as well as that induced by IL-12. These results show the potential of IL-18 in combination with IL-12 for clinical application in treatment of cancer.

  15. Dendritic Cell Activation and Cytokine Production Induced by Group B Neisseria meningitidis: Interleukin-12 Production Depends on Lipopolysaccharide Expression in Intact Bacteria

    PubMed Central

    Dixon, Garth L. J.; Newton, Phillippa J.; Chain, Benjamin M.; Katz, David; Andersen, Svein Rune; Wong, Simon; van der Ley, Peter; Klein, Nigel; Callard, Robin E.

    2001-01-01

    Interactions between dendritic cells (DCs) and microbial pathogens are fundamental to the generation of innate and adaptive immune responses. Upon stimulation with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of in vitro-generated human DCs to a serogroup B strain of Neisseria meningitidis compared to an isogenic mutant lpxA strain totally deficient in LPS and purified LPS from the same strain. We show that the parent strain, lpxA mutant, and meningococcal LPS all induce DC maturation as measured by increased surface expression of costimulatory molecules and HLA class I and II molecules. Both the parent and lpxA strains induced production of tumor necrosis factor alpha (TNF-α), interleukin-1α (IL-1α), and IL-6 in DCs, although the parent was the more potent stimulus. In contrast, high-level IL-12 production was only seen with the parent strain. Compared to intact bacteria, purified LPS was a very poor inducer of IL-1α, IL-6, and TNF-α production and induced no detectable IL-12. Addition of exogenous LPS to the lpxA strain only partially restored cytokine production and did not restore IL-12 production. These data show that non-LPS components of N. meningitidis induce DC maturation, but that LPS in the context of the intact bacterium is required for high-level cytokine production, especially that of IL-12. These findings may be useful in assessing components of N. meningitidis as potential vaccine candidates. PMID:11401973

  16. Dendritic cell activation and cytokine production induced by group B Neisseria meningitidis: interleukin-12 production depends on lipopolysaccharide expression in intact bacteria.

    PubMed

    Dixon, G L; Newton, P J; Chain, B M; Katz, D; Andersen, S R; Wong, S; van der Ley, P; Klein, N; Callard, R E

    2001-07-01

    Interactions between dendritic cells (DCs) and microbial pathogens are fundamental to the generation of innate and adaptive immune responses. Upon stimulation with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of in vitro-generated human DCs to a serogroup B strain of Neisseria meningitidis compared to an isogenic mutant lpxA strain totally deficient in LPS and purified LPS from the same strain. We show that the parent strain, lpxA mutant, and meningococcal LPS all induce DC maturation as measured by increased surface expression of costimulatory molecules and HLA class I and II molecules. Both the parent and lpxA strains induced production of tumor necrosis factor alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), and IL-6 in DCs, although the parent was the more potent stimulus. In contrast, high-level IL-12 production was only seen with the parent strain. Compared to intact bacteria, purified LPS was a very poor inducer of IL-1alpha, IL-6, and TNF-alpha production and induced no detectable IL-12. Addition of exogenous LPS to the lpxA strain only partially restored cytokine production and did not restore IL-12 production. These data show that non-LPS components of N. meningitidis induce DC maturation, but that LPS in the context of the intact bacterium is required for high-level cytokine production, especially that of IL-12. These findings may be useful in assessing components of N. meningitidis as potential vaccine candidates.

  17. Interleukin-12 requires initial CD80-mediated T-cell activation to support immune responses toward human breast and ovarian carcinoma.

    PubMed

    Gückel, B; Meyer, G C; Rudy, W; Batrla, R; Meuer, S C; Bastert, G; Wallwiener, D; Moebius, U

    1999-01-01

    One possible reason for the poor immunogenicity of tumors is the induction of peripheral tolerance by tumor cells that fail to deliver costimulatory signals. Furthermore, T cells stimulated with wild-type tumor cells often fail to secrete cytokines. The present study has been undertaken to identify cytokines that cooperate with CD80 in T-cell activation in vitro toward human breast and ovarian carcinoma cell lines. Tumor cell-mediated T-lymphocyte activation was analyzed directly in allogeneic mixed lymphocyte/tumor cell cultures as proliferation and effector functions were assessed in cytotoxic T-cell assays. Interleukin-7 (IL-7) amplified the proliferative response toward CD80-transfected breast and ovarian carcinomas and stimulated predominantly CD4+ T lymphocytes. IL-12 represses the proliferative response of naive T cells but cooperates with CD80-mediated activation during secondary stimulations. In long-term T-cell cultures, IL-12 synergizes with CD80 expression to stimulate cytolytic CD8+ T-cell lines, which recognize a breast carcinoma line in a human histocompatibility leukocyte antigen-restricted manner. These studies illustrate that costimulation is necessary for tumor cells to function as alloantigen-presenting cells. Furthermore, when added after the priming of T cells with CD80-transfected tumor cells, IL-12 could be helpful in propagating sufficient T-cell numbers to be used in adoptive transfers during cellular immunotherapy.

  18. Interleukin 12 exerts a differential effect on the maturation of neonatal and adult human CD45R0- CD4 T cells.

    PubMed Central

    Shu, U; Demeure, C E; Byun, D G; Podlaski, F; Stern, A S; Delespesse, G

    1994-01-01

    It is now recognized that IL-12 plays a predominant role in protective immunity against intracellular pathogens by promoting the development of T helper type 1 (Th1) responses. We here report the unexpected observations that IL-12 exerts differential effects on the maturation of "native" human CD4 T cells isolated from umbilical cord blood or from the blood of healthy adults. After priming in the presence of IL-12, naive cells of adult donors, defined as CD45R0- CD4+ T cells, acquire a Th1 phenotype whereas neonatal cells develop into effector cells producing high levels of IL-4 in addition to IFN-gamma. This effect of IL-12 on neonatal T cells is direct inasmuch as it is observed on highly purified CD4 T cells, however, it is not inhibited by CD8 T cells and natural killer cells. Unstimulated neonatal T cells which have been preincubated with IL-12 before the priming behave like adult T cells and acquire a Th1 phenotype after stimulation in the presence of IL-12. Given that IL-4 is a potent antagonist of Th1 responses, the finding that IL-12 promotes the maturation of neonatal T cells into IL-4 producers may explain the increased susceptibility of neonates to intracellular pathogens and should be taken into account for the development of vaccines to be used in the perinatal period. Images PMID:7929809

  19. Wrestlers’ immune cells produce higher interleukin-6 and lower interleukin-12 and interleukin-13 in response to in vitro mitogen activation

    PubMed Central

    Zamani, Alireza; Omidi, Mostafa; Hemmatfar, Ahmad; Salehi, Iraj; Bazmamoun, Hassan

    2014-01-01

    Objective(s): Although recent investigations have shown chronic inflammation and inflammation-associated diseases might be ameliorated by exercise; little is known about the relation between exercise training with anti/pro-inflammatory cytokines. Materials and Methods: This cross sectional study was conducted to compare interleukin-4 (IL-4), IL-6, IL-10, IL-12, IL-13, interferon gamma (IFN-γ ) levels in serum, and their in vitro production by whole blood (WB) cells and by peripheral blood mononuclear cells (PBMCs) in response to mitogens lipopolysaccharide and phytohemagglutinin. Twelve elite wrestlers with history of three times per week exercise training for about 9.5 years, and thirteen healthy silent controls were recruited. To analysis the cytokines by enzyme linked immunosorbent assay (ELISA), the blood samples were taken 24 hr after the last training session from the wrestlers. Results: Serum analysis for IL-4, IL-6, IL-10, IL-12, IL-13 and IFN-γ indicated no statistical difference between the two groups. Meanwhile, 48 hr in vitro activation of WB and PBMCs by the mitogens revealed that IL-6 production was elevated in both WB and PBMCs. Whereas, IL-12 and IL-13 were decreased in supernatant of PBMCs and WB cells cultures, respectively. Conclusion: It seems that wrestling cause immune system cells to produce anti-inflammatory myokine IL-6 and decrease production of pro-inflammatory cytokine IL-12 and IL-13. PMID:25691935

  20. Differential effects of tumour necrosis factor-α and interleukin-12 on isopentenyl pyrophosphate-stimulated interferon-γ production by cord blood Vγ9 T cells

    PubMed Central

    Alberto, Eduardo Jose Campos; Shimojo, Naoki; Aoyagi, Masahiko; Kohno, Yoichi

    2009-01-01

    Lower numbers of Vγ9Vδ2 T cells in cord blood (CB) than in adult peripheral blood (PB), as well as their impaired ability to produce interferon-γ (IFN-γ) in response to stimulation, are associated with functional deficiency in the immune system in newborns. In this study, we stimulated CB Vγ9 T cells with their T-cell receptor-specific ligand, isopentenyl pyrophosphate (IPP), plus exogenous costimulatory cytokines such as interleukin-2 (IL-2), IL-12 and tumour necrosis factor-α (TNF-α), which are known to play important roles in the activation of PB γδ T cells. Our data show that CB Vγ9 T cells are able to produce IFN-γ at levels comparable to PB Vγ9 T cells by the addition of TNF-α in the presence of IPP and IL-2; however, under the same culture conditions, IL-12 does not efficiently activate CB Vγ9 T cells to produce IFN-γ. The frequency of TNF-α receptor II-positive Vγ9T cells and the expression levels of TNF-α receptor II are similar in CB and PB; in contrast, the frequency of IL-12 receptor βI (IL-12RβI)-positive Vγ9T cells and expression levels of IL-12RβI are significantly lower in CB than PB. TNF-α but not IL-12 increases the expression of IL-2Rβ on CB Vγ9 T cells. These results provide new insights into the role of TNF-α in the activation of CB Vγ9 T cells. PMID:19019091

  1. Association of the interleukin-12 polymorphic variants with the development of antibodies to surface antigen of hepatitis B virus in hemodialysis patients in response to vaccination or infection.

    PubMed

    Grzegorzewska, Alicja E; Wobszal, Piotr M; Sowińska, Anna; Mostowska, Adrianna; Jagodziński, Paweł P

    2013-12-01

    Cytokines, involved in the T-helper 1 system, play a role in the regulation of hepatitis B virus (HBV) clearance and the immune response to HBV antigens during natural infection or planned vaccination. Our aim was to examine whether the polymorphic variants of IL-12 are equally associated with development of antibodies to HBV surface antigen (anti-HBs) in hemodialysis (HD) patients in the case of HBV vaccination or HBV infection. The IL-12A rs568408 and IL-12B rs3212227 polymorphisms were analyzed in relation to anti-HBs development in 602 HD patients with negative antibodies to HBV core antigen (anti-HBc) who were hepatitis B vaccinated (group I) as well as in 237 anti-HBc positive HD patients who were infected with HBV in the past (group II). In group I, 199 patients did not develop an anti-HBs titre >10 IU/L (subgroup Ia), whereas in group II, 55 patients did not develop an anti-HBs titre >10 IU/L (subgroup IIa). Patients of groups I and II that developed an anti-HBs >10 IU/L were included into subgroups Ib and IIb, respectively. In hepatitis B vaccinated HD patients, development of a protective anti-HBs titre was positively associated with vintage of renal replacement therapy (RRT), chronic glomerulonephritis as a cause of RRT, and GA rs 568408 IL-12A (OR 1.6, 95 % CI 1.0-2.5, P = 0.035), but a frequency distribution of this genotype between responders and non-responders was not significant when the Bonferroni correction was applied. In HBV infected HD patients, anti-HBs development was positively associated with AC rs3212227 IL-12B (OR 8.0, 95 % CI 2.6-24.9, P < 0.001), whereas HBsAg positivity, AA rs3212227 IL-12B (OR 0.3, 95 % CI 0.1-0.7, P = 0.007), and CC rs3212227 IL-12B (OR 0.1, 95 % CI 0.03-0.6, P = 0.011) were negative predictors of positive anti-HBs phenotype. When the Bonferroni correction was applied, if appropriate, these associations remained significant. In HD patients, the studied IL-12 polymorphic variants seem to be associated with the anti-HBs phenotype (a) with borderline significance for IL-12A in hepatitis B vaccinated patients, and (b) significantly for IL-12B in patients who underwent natural HBV infection.

  2. The effect of interleukin-10 and of interleukin-12 on the in vitro production of anti-double-stranded DNA antibodies from patients with systemic lupus erythematosus

    PubMed Central

    Tyrrell-Price, J; Lydyard, P M; Isenberg, D A

    2001-01-01

    IL-10 and IL-12 are cytokines which are important in regulating immune responses. Plasma levels of IL-10 and autoantibodies against double-stranded DNA (dsDNA) often mirror disease activity in patients with SLE. IL-12 secretion from SLE patients' blood mononuclear cells also correlates with disease activity, but has an inverse relationship. The aim of this study was to measure the effect of IL-10 and of IL-12 on the production of IgG autoantibodies from patients with SLE, both cross-sectionally and longitudinally. Peripheral blood mononuclear cells (PBMC) were cultured with IL-10 (at 20 ng/ml or 2 ng/ml) or IL-12 (at 2 ng/ml or 0·2 ng/ml) or without cytokine and the supernatanants tested for the production of double-stranded DNA antibodies (dsDNA abs), single-stranded DNA antibodies (ssDNA abs) and total IgG antibodies (IgG abs) by ELISA. The BILAG disease activity index was recorded at each patient visit (a global score of six or more is regarded as active disease). In general, treatment with IL-10 caused PBMCs from patients with inactive disease to increase their antissDNA and dsDNA ab production (by upto 354% and 186%, respectively) while patients with active disease decreased their antibody production (by upto 91% and 97%, respectively). Overall there was a correlation between disease activity and change in antissDNA and dsDNA ab production (r = − 0·51; P = 0·03 and r = − 0·48; P = 0·042, respectively). Treatment with IL-12 at 0·2 ng/ml inhibited antissDNA and dsDNA antibody production, having the greatest effect on patients with active disease (decreasing antissDNA and dsDNA antibody production by upto 75% and 73%, respectively). This resulted in a significant correlation between disease activity and change in antissDNA antibody production (r = − 0·76; P = 0·03), but significance was not reached with antidsDNA antibody production (P = 0·06). Together these data suggest that the effect of these cytokines on antibody production by SLE PBMCs involves several factors; one of which is disease activity. PMID:11359450

  3. Interleukin 12 acts directly on CD4+ T cells to enhance priming for interferon gamma production and diminishes interleukin 4 inhibition of such priming.

    PubMed Central

    Seder, R A; Gazzinelli, R; Sher, A; Paul, W E

    1993-01-01

    Naive CD4+ T cells produce interleukin 2 (IL-2) but little IL-4 or interferon gamma (IFN-gamma). In vitro, they develop into IL-4 or IFN-gamma producers depending on the conditions of the priming culture. Using T-cell receptor transgenic CD4+ T cells, the role of IL-12 and IL-4 in antigen-specific priming was examined. IL-12 substantially enhanced the ability of naive CD4+ T cells to develop into cells that produced IFN-gamma upon restimulation. However, it was not essential since anti-IL-12 antibodies failed to block the priming for IFN-gamma observed in the absence of exogenous IL-12. When both IL-12 and IL-4 were present in the priming culture, IL-12 did not inhibit priming for IL-4 production. In contrast, IL-4 diminished but did not abolish priming for IFN-gamma production. In an accessory cell-independent priming system, IL-12 strikingly augmented priming for IFN-gamma production, indicating that it acts directly on T cells. IFN-gamma itself did not enhance priming for IFN-gamma production in either accessory cell-dependent or independent systems. In an accessory cell-dependent system, the IL-12-mediated enhancement was not blocked by adding neutralizing anti-IFN-gamma monoclonal antibody. However, in an accessory cell-independent system, anti-IFN-gamma antibody did inhibit priming for IFN-gamma production leaving open a role for IFN-gamma in the priming process. These data indicate that IL-12 has a major effect on the inductive phase of T-cell priming by enhancing commitment to IFN-gamma production and thus can profoundly influence the state of immunity that develops. Images Fig. 4 PMID:7901851

  4. The Wnt5a-Ror2 axis promotes the signaling circuit between interleukin-12 and interferon-γ in colitis

    PubMed Central

    Sato, Akira; Kayama, Hisako; Shojima, Kensaku; Matsumoto, Shinji; Koyama, Hirofumi; Minami, Yasuhiro; Nojima, Satoshi; Morii, Eiichi; Honda, Hiroaki; Takeda, Kiyoshi; Kikuchi, Akira

    2015-01-01

    Wnt5a, which regulates various cellular functions in Wnt signaling, is involved in inflammatory responses, however the mechanism is not well understood. We examined the role of Wnt5a signaling in intestinal immunity using conditional knockout mice for Wnt5a and its receptor Ror2. Removing Wnt5a or Ror2 in adult mice suppressed dextran sodium sulfate (DSS)-induced colitis. It also attenuated the DSS-dependent increase in inflammatory cytokine production and decreased interferon-γ (IFN-γ)-producing CD4+ Th1 cell numbers in the colon. Wnt5a was highly expressed in stromal fibroblasts in ulcerative lesions in the DSS-treated mice and inflammatory bowel disease patients. Dendritic cells (DCs) isolated from the colon of Wnt5a and Ror2 deficient mice reduced the ability to differentiate naïve CD4+ T cells to IFN-γ-producing CD4+ Th1 cells. In vitro experiments demonstrated that the Wnt5a-Ror2 signaling axis augmented the DCs priming effect of IFN-γ, leading to enhanced lipopolysaccharide (LPS)-induced interleukin (IL)-12 expression. Taken together, these results suggest that Wnt5a promotes IFN-γ signaling, leading to IL-12 expression in DCs, and thereby inducing Th1 differentiation in colitis. PMID:26030277

  5. Early coupled up-regulation of interleukin-12 receptor beta-1 in CD8+ central memory and effector T cells for better clinical outcomes in liver transplant recipients.

    PubMed

    Uemoto, S; Ozawa, K; Kaido, T; Mori, A; Fujimoto, Y; Ogawa, K

    2015-08-01

    This study aimed to investigate the role of initial priming of interleukin (IL)-12 receptor beta-1 in CD8(+) central memory T cells (initial IL-12RTCM priming) and CCR7-negative subsets (CNS) in effector cell expansion and clinical outcome after living donor liver transplantation (LDLT). One hundred and six patients who underwent LDLT were classified into the following three groups according to hierarchical clustering of CD8(+) CD45 isoforms before LDLT: I, naive-dominant; II, effector memory-dominant; and III, effector-dominant. The pre-existing CD8(+) effector cells (TE ) and activated immune status increased progressively from group I to group II to group III. Groups I, II and III received tacrolimus (Tac)/glucocorticoid (GC) regimens. Eighteen group III recipients received Tac/mycophenolate mofetil (MMF) and were defined as group IV. Initial IL-12RTCM priming was slightly, moderately and markedly decreased in droups I, II, and III, respectively. Initial priming of IL-12Rβ1 in CNS was decreased markedly in the three groups with marked decreases of TE , perforin and interferon (IFN)-γ; all parameters were restored by up-regulation of IL-12Rβ1(+) TCM through the self-renewal of TCM . The lag time required until coupled up-regulation of IL-12Rβ1 of TCM and CNS to above baseline was 12, 20 and 32 days in groups I, II and III, respectively. Inferior clinical outcomes were associated with increasing lag time. In contrast, the initial priming of IL-12Rβ1 in TCM and CNS remained above baseline in group IV due to MMF-mediated increase of IL-12Rβ1. Early coupled up-regulation of TCM and CNS leads to efficient TE differentiation and optimal clinical outcomes.

  6. Enhanced immunomodulatory activity and stability in simulated digestive juices of Lactobacillus plantarum L-137 by heat treatment.

    PubMed

    Fujiki, Takashi; Hirose, Yoshitaka; Yamamoto, Yoshihiro; Murosaki, Shinji

    2012-01-01

    This study reports the effect of heat treating Lactobacillus plantarum L-137 on its in vitro cytokine-inducing activity, on the stability of this activity in simulated digestive juices, and on its in vivo immunomodulatory properties. L-137 cells were harvested at the stationary phase with or without the subsequent heat treatment and then lyophilized. Heat-killed L-137 cells stimulated mouse spleen cells to produce more interleukin-12p40 than unheated L-137. The interleukin-12p40-inducing activity of unheated L-137 was significantly lower when incubated with simulated intestinal juice, but the activity of heat-killed L-137 cells was maintained. Furthermore, heat-killed L-137 was more protective than unheated L-137 in a mouse model of dextran sulfate sodium-induced colitis. A heat treatment may therefore be effective for enhancing the immunomodulatory activity of L-137 cells.

  7. Activated d16HER2 homodimers and SRC kinase mediate optimal efficacy for trastuzumab.

    PubMed

    Castagnoli, Lorenzo; Iezzi, Manuela; Ghedini, Gaia C; Ciravolo, Valentina; Marzano, Giulia; Lamolinara, Alessia; Zappasodi, Roberta; Gasparini, Patrizia; Campiglio, Manuela; Amici, Augusto; Chiodoni, Claudia; Palladini, Arianna; Lollini, Pier Luigi; Triulzi, Tiziana; Menard, Sylvie; Nanni, Patrizia; Tagliabue, Elda; Pupa, Serenella M

    2014-11-01

    A splice isoform of the HER2 receptor that lacks exon 16 (d16HER2) is expressed in many HER2-positive breast tumors, where it has been linked with resistance to the HER2-targeting antibody trastuzumab, but the impact of d16HER2 on tumor pathobiology and therapeutic response remains uncertain. Here, we provide genetic evidence in transgenic mice that expression of d16HER2 is sufficient to accelerate mammary tumorigenesis and improve the response to trastuzumab. A comparative analysis of effector signaling pathways activated by d16HER2 and wild-type HER2 revealed that d16HER2 was optimally functional through a link to SRC activation (pSRC). Clinically, HER2-positive breast cancers from patients who received trastuzumab exhibited a positive correlation in d16HER2 and pSRC abundance, consistent with the mouse genetic results. Moreover, patients expressing high pSRC or an activated "d16HER2 metagene" were found to derive the greatest benefit from trastuzumab treatment. Overall, our results establish the d16HER2 signaling axis as a signature for decreased risk of relapse after trastuzumab treatment.

  8. Synthesis and Evaluation of Chloramphenicol Homodimers: Molecular Target, Antimicrobial Activity, and Toxicity against Human Cells

    PubMed Central

    Kostopoulou, Ourania N.; Magoulas, George E.; Papadopoulos, Georgios E.; Mouzaki, Athanasia; Dinos, George P.; Papaioannou, Dionissios; Kalpaxis, Dimitrios L.

    2015-01-01

    As fight against antibiotic resistance must be strengthened, improving old drugs that have fallen in reduced clinical use because of toxic side effects and/or frequently reported resistance, like chloramphenicol (CAM), is of special interest. Chloramphenicol (CAM), a prototypical wide-spectrum antibiotic has been shown to obstruct protein synthesis via binding to the bacterial ribosome. In this study we sought to identify features intensifying the bacteriostatic action of CAM. Accordingly, we synthesized a series of CAM-dimers with various linker lengths and functionalities and compared their efficiency in inhibiting peptide-bond formation in an Escherichia coli cell-free system. Several CAM-dimers exhibited higher activity, when compared to CAM. The most potent of them, compound 5, containing two CAM bases conjugated via a dicarboxyl aromatic linker of six successive carbon-bonds, was found to simultaneously bind both the ribosomal catalytic center and the exit-tunnel, thus revealing a second, kinetically cryptic binding site for CAM. Compared to CAM, compound 5 exhibited comparable antibacterial activity against MRSA or wild-type strains of Staphylococcus aureus, Enterococcus faecium and E. coli, but intriguingly superior activity against some CAM-resistant E. coli and Pseudomonas aeruginosa strains. Furthermore, it was almost twice as active in inhibiting the growth of T-leukemic cells, without affecting the viability of normal human lymphocytes. The observed effects were rationalized by footprinting tests, crosslinking analysis, and MD-simulations. PMID:26267355

  9. The X-ray Structure of a BAK Homodimer Reveals an Inhibitory Zinc Binding Site

    SciTech Connect

    Modoveanu,T.; Liu, Q.; Tocilj, A.; Watson, M.; Shore, G.; Gehring, K.

    2006-01-01

    BAK/BAX-mediated mitochondrial outer-membrane permeabilization (MOMP) drives cell death during development and tissue homeostasis from zebrafish to humans. In most cancers, this pathway is inhibited by BCL-2 family antiapoptotic members, which bind and block the action of proapoptotic BCL proteins. We report the 1.5 {angstrom} crystal structure of calpain-proteolysed BAK, cBAK, to reveal a zinc binding site that regulates its activity via homodimerization. cBAK contains an occluded BH3 peptide binding pocket that binds a BID BH3 peptide only weakly . Nonetheless, cBAK requires activation by truncated BID to induce cytochrome c release in mitochondria isolated from bak/bax double-knockout mouse embryonic fibroblasts. The BAK-mediated MOMP is inhibited by low micromolar zinc levels. This inhibition is alleviated by mutation of the zinc-coordination site in BAK. Our results link directly the antiapoptotic effects of zinc to BAK.

  10. Adoptive transfer of experimental allergic encephalomyelitis after in vitro treatment with recombinant murine interleukin-12. Preferential expansion of interferon-gamma-producing cells and increased expression of macrophage-associated inducible nitric oxide synthase as immunomodulatory mechanisms.

    PubMed Central

    Waldburger, K. E.; Hastings, R. C.; Schaub, R. G.; Goldman, S. J.; Leonard, J. P.

    1996-01-01

    In an adoptive transfer model of experimental allergic encephalomyelitis, stimulation of lymph node cells with proteolipid protein and recombinant murine interleukin (rmIL)-12 before cell transfer accelerated the onset and exacerbates clinical disease. In vitro stimulation with proteolipid protein in the presence of rmIL-12 was associated with an increase in interferon-gamma-producing cells and a decrease in IL-4-producing cells, indicating a preferential expansion of Th1 effector cells. This was supported by the finding that severe disease with rapid onset could be transferred with as few as 10 x 10(6) rmIL-12-stimulated lymph node cells. Immunohistochemical analysis confirmed that the accelerated onset of disease after in vitro stimulation with rmIL-12 coincided with an acute inflammatory response in the central nervous system. At peak disease, both control and rmIL-12 treatment groups exhibited extensive cellular infiltration with characteristic perivascular cuffing. No notable differences in either the cellular composition or cytokine expression within the lesions were seen between groups. However, the frequency of macrophages that stained positively for inducible nitric oxide synthase was increased in animals challenged with rmIL-12-treated lymph node cells. The results suggest that, in addition to promoting the preferential expansion of interferon-gamma-producing cells by rmIL-12 in vitro, secondary in vivo effects leading to macrophage activation and inducible nitric oxide synthase expression may contribute to the severe and protracted course of central nervous system inflammation in this model. Images Figure 2 PMID:8579100

  11. Immunogenicity and protective efficacy conferred by a novel recombinant Mycobacterium bovis bacillus Calmette-Guérin strain expressing interleukin-12p70 of human cytokine and Ag85A of Mycobacterium tuberculosis fusion protein.

    PubMed

    Deng, Y H; He, H Y; Zhang, F J

    2013-12-01

    Mycobacterium bovis bacillus Calmette-Guérin (BCG) immunization provides protection against tuberculosis (TB) in infants, but the antituberculosis protective immunity wanes gradually after initial immunization and lasts less than 15 years. Therefore, more efficacious vaccines are urgently needed. In this study, we constructed a new tuberculosis vaccine of recombinant BCG strain (rBCG-IA), which could express IL-12p70 of human cytokine and Ag85A of M. tuberculosis fusion protein, and investigated its immunogenicity in BALB/c mice by measuring antibody titres, proliferation rate of splenocytes, ratios of CD4(+) T and CD8(+) T cells stimulated by specific antigens and levels of IFN-γ production in antigen-stimulated splenocyte cultures. Meanwhile, we evaluated its protective efficacy against M. tuberculosis H37Rv infection through detecting lung histopathology, organ bacterial loads and lung acid-fast stain. Immunogenicity experiments illustrated that from 2nd to 8th week after immunization, the rBCG-IA vaccine was able to induce the highest level of antibody titres, proliferation rate of splenocytes and IFN-γ production among groups and gained improved ratio of CD4(+) T and CD8(+) T cells from 6th to 8th week after vaccination. And from 2nd to 8th week after M. tuberculosis H37Rv infection, the score of pathology and bacterial loads in the rBCG-IA group were obviously lower than that in rBCG-I group, rBCG-A group or control group (PBST group), but similar to that in BCG group. This study suggested that rBCG-IA was able to elicit stronger humoral and cellular immune responses, but could only confer similar protective efficacy compared with its parental BCG vaccine.

  12. Binding of nitrogen-containing bisphosphonates (N-BPs) to the Trypanosoma cruzi farnesyl diphosphate synthase homodimer

    SciTech Connect

    Huang, Chuan-Hsiang; Gabelli, Sandra B.; Oldfield, Eric; Amzel, L. Mario

    2010-11-15

    Bisphosphonates (BPs) are a class of compounds that have been used extensively in the treatment of osteoporosis and malignancy-related hypercalcemia. Some of these compounds act through inhibition of farnesyl diphosphate synthase (FPPS), a key enzyme in the synthesis of isoprenoids. Recently, nitrogen-containing bisphosphonates (N-BPs) used in bone resorption therapy have been shown to be active against Trypanosoma cruzi, the parasite that causes American trypanosomiasis (Chagas disease), suggesting that they may be used as anti-trypanosomal agents. The crystal structures of TcFPPS in complex with substrate (isopentenyl diphosphate, IPP) and five N-BP inhibitors show that the C-1 hydroxyl and the nitrogen-containing groups of the inhibitors alter the binding of IPP and the conformation of two TcFPPS residues, Tyr94 and Gln167. Isothermal titration calorimetry experiments suggest that binding of the first N-BPs to the homodimeric TcFPPS changes the binding properties of the second site. This mechanism of binding of N-BPs to TcFPPS is different to that reported for the binding of the same compounds to human FPPS.

  13. Regulation of the PI3K pathway through a p85α monomer–homodimer equilibrium | Office of Cancer Genomics

    Cancer.gov

    The canonical action of the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is to associate with the p110α catalytic subunit to allow stimuli-dependent activation of the PI3K pathway. We elucidate a p110α-independent role of homodimerized p85α in the positive regulation of PTEN stability and activity.

  14. The Vesicle Priming Factor CAPS Functions as a Homodimer via C2 Domain Interactions to Promote Regulated Vesicle Exocytosis*

    PubMed Central

    Petrie, Matt; Esquibel, Joseph; Maciuba, Stephanie; Takahashi, Hirohide

    2016-01-01

    Neurotransmitters and peptide hormones are secreted by regulated vesicle exocytosis. CAPS (also known as CADPS) is a 145-kDa cytosolic and peripheral membrane protein required for vesicle docking and priming steps that precede Ca2+-triggered vesicle exocytosis. CAPS binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and SNARE proteins and is proposed to promote SNARE protein complex assembly for vesicle docking and priming. We characterized purified soluble CAPS as mainly monomer in equilibrium with small amounts of dimer. However, the active form of CAPS bound to PC12 cell membranes or to liposomes containing PI(4,5)P2 and Q-SNARE proteins was mainly dimer. CAPS dimer formation required its C2 domain based on mutation or deletion studies. Moreover, C2 domain mutations or deletions resulted in a loss of CAPS function in regulated vesicle exocytosis, indicating that dimerization is essential for CAPS function. Comparison of the CAPS C2 domain to a structurally defined Munc13-1 C2A domain dimer revealed conserved residues involved in CAPS dimerization. We conclude that CAPS functions as a C2 domain-mediated dimer in regulated vesicle exocytosis. The unique tandem C2-PH domain of CAPS may serve as a PI(4,5)P2-triggered switch for dimerization. CAPS dimerization may be coupled to oligomeric SNARE complex assembly for vesicle docking and priming. PMID:27528604

  15. N15 Cro And Gamma Cro Orthologous DNA-Binding Domains With Completely Different But Equally Effective Homodimer Interfaces

    SciTech Connect

    Dubrava, M.S.; Ingram, W.M.; Roberts, S.A.; Weichsel, A.; Montfort, W.R.; Cordes, M.H.J.

    2009-05-18

    Bacteriophage Cro proteins bind to target DNA as dimers but do not all dimerize with equal strength, and differ in fold in the region of the dimer interface. We report the structure of the Cro protein from Enterobacteria phage N15 at 1.05 {angstrom} resolution. The subunit fold contains five alpha-helices and is closely similar to the structure of P22 Cro (1.3 {angstrom} backbone room mean square difference over 52 residues), but quite different from that of lambda Cro, a structurally diverged member of this family with a mixed alpha-helix/beta-sheet fold. N15 Cro crystallizes as a biological dimer with an extensive interface (1303 {angstrom}{sub 2} change in accessible surface area per dimer) and also dimerizes in solution with a K(d) of 5.1 {+-} 1.5 {micro}M. Its dimerization is much stronger than that of its structural homolog P22 Cro, which does not self-associate detectably in solution. Instead, the level of self-association and interfacial area for N15 Cro is similar to that of lambda Cro, even though these two orthologs do not share the same fold and have dimer interfaces that are qualitatively different in structure. The common Cro ancestor is thought to be an all-helical monomer similar to P22 Cro. We propose that two Cro descendants independently developed stronger dimerization by entirely different mechanisms.

  16. Histone deacetylase inhibitors decrease Toll-like receptor-mediated activation of proinflammatory gene expression by impairing transcription factor recruitment

    PubMed Central

    Bode, Konrad A; Schroder, Kate; Hume, David A; Ravasi, Timothy; Heeg, Klaus; Sweet, Matthew J; Dalpke, Alexander H

    2007-01-01

    Post-translational modifications of histone proteins are major mechanisms that modify chromatin structure and regulate gene expression in eukaryotes. Activation of histone acetyltransferases or inhibition of histone deacetylases (HDACs) is generally believed to allow chromatin to assume a more open state, permitting transcriptional activity. We report here the surprising observation that treatment of murine dendritic cells with the HDAC inhibitors trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) in non-apoptotic concentrations strongly inhibited induction of both interleukin-12 protein p40 (IL-12p40) mRNA and protein upon stimulation of Toll-like receptors (TLRs). Moreover, TLR-mediated up-regulation of costimulatory molecules was also inhibited. Up-regulation of tumour necrosis factor-α mRNA and protein in response to TLR agonists was only affected upon prolonged exposure to HDAC inhibitors and regulation of IL-1β was not affected. Similar effects were apparent in murine and human macrophages. Regarding the mode of action, HDAC inhibition increased the acetylation status at the IL-12p40 locus. Nevertheless, IL-12p40 chromatin remodelling, binding of Rel-A and IRF1 to the IL-12p40 promoter and transcriptional activation were abrogated. In contrast, HDAC inhibitors had no effects on upstream nuclear factor-κB and mitogen-activated protein kinase activation. Thus HDACs positively regulate the expression of a subset of cytokine genes by enabling transcription factor recruitment. PMID:17635610

  17. Recombinant p35 from bacteria can form Interleukin (IL-)12, but Not IL-35.

    PubMed

    Aparicio-Siegmund, Samadhi; Moll, Jens M; Lokau, Juliane; Grusdat, Melanie; Schröder, Jutta; Plöhn, Svenja; Rose-John, Stefan; Grötzinger, Joachim; Lang, Philipp A; Scheller, Jürgen; Garbers, Christoph

    2014-01-01

    The Interleukin (IL)-12 family contains several heterodimeric composite cytokines which share subunits among each other. IL-12 consists of the subunits p40 (shared with IL-23) and p35. p35 is shared with the composite cytokine IL-35 which comprises of the p35/EBI3 heterodimer (EBI3 shared with IL-27). IL-35 signals via homo- or heterodimers of IL-12Rβ2, gp130 and WSX-1, which are shared with IL-12 and IL-27 receptor complexes, respectively. p35 was efficiently secreted in complex with p40 as IL-12 but not with EBI3 as IL-35 in several transfected cell lines tested which complicates the analysis of IL-35 signal transduction. p35 and p40 but not p35 and EBI3 form an inter-chain disulfide bridge. Mutation of the responsible cysteine residue (p40C197A) reduced IL-12 formation and activity only slightly. Importantly, the p40C197A mutation prevented the formation of antagonistic p40 homodimers which enabled the in vitro reconstitution of biologically active IL-12 with p35 produced in bacteria (p35bac). Reconstitution of IL-35 with p35bac and EBI3 did, however, fail to induce signal transduction in Ba/F3 cells expressing IL-12Rβ2 and gp130. In summary, we describe the in vitro reconstitution of IL-12, but fail to produce recombinant IL-35 by this novel approach.

  18. SU-E-P-40: Dosimetric Characteristics of Field Aperture Margin Design in Stereotactic Body Radiation Therapy (SBRT)

    SciTech Connect

    Zhu, J

    2015-06-15

    Purpose: To characterize the dosimetric effects of field aperture margin design in Stereotactic Body Radiation Therapy (SBRT). Methods: Three artificial spherical PTVs, with diameter of 10mm, 20mm and 30mm, were created on CT images of a human body thoracic phantom. Seven non-coplanar isocentric fields were used for treatment planning. For each PTV, treatment plans with margins 0mm, 1mm, 2mm and 3mm were planned. Dosimetric comparison among plans was done considering the following parameters: prescribed isodose line for target coverage, maximum dose, mean dose as well as dose spillages of V80, V50, and V20. Results: Corresponding to aperture margins of 0mm, 1mm,2m and 3mm used in the treatment planning, the percentage of isodose line chosen for dose prescription increases from 65% to 93% for 10mm PTV, 70% to 92% for 20mm PTV, and 75% to 92% for 30mm PTV. The maximum dose decrease accordingly from 155.7% to 109.5% for 10mm PTV, 145% to 111.6% for 20mm PTV, 137% to 112.2% for 30mm PTV. The mean dose decrease from 138.% to 104.4% for 10mm PTV, 122.8% to 106.1% for 20mm PTV, 121.3% to 106% for 30mm PTV. Dose spillages (mm3) increase (V80−2.6 to 4.02, V50−4.55 to 9.3, V20–87.86 to 101.71) for 10 mm PTV, (V80−6.78 to 9.89, V50–13.46 to 20.4, V20-119.16 to 219.1) for 20 mm PTV, (V80–22.01 to 28.59, V50–41.56 to 52.66, V20-532.71 to 551.84) for 30 mm PTV. Conclusion: In SBRT treatment planning, tight field aperture margin requires prescribing dose to lower isodose line that leading to higher dose inhomogeneity and higher mean dose to PTV. Loose margin allows prescribing dose to higher isodose line, therefore improves the dose homogeneity. However, it increases dose spillages. Clinician could try different margins according to the PTV size and location of surrounding critical organs to optimize the dose delivered to the patient.

  19. CDK-activating kinase (Ee;CDKF;1) of leafy spurge (Euphorbia esula) forms both homo-dimers and homo-trimers in its native state

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leafy spurge is a deep rooted perennial weed that propagates both by seeds and underground adventitious buds located on the crown and roots (crown and root buds). As buds develop during the normal growing season, they are maintained in a quiescent state through correlative inhibition. To enhance our...

  20. Arginine mutations within a transmembrane domain of Tar, an Escherichia coli aspartate receptor, can drive homodimer dissociation and heterodimer association in vivo

    PubMed Central

    2004-01-01

    The interactions between the TM (transmembrane) domains of many membrane proteins are important for their proper functioning. Mutations of residues into positively charged ones within TM domains were reported to be involved in many genetic diseases, possibly because these mutations affect the self- and/or hetero-assembly of the corresponding proteins. To our knowledge, despite significant progress in understanding the role of various amino acids in TM–TM interactions in vivo, the direct effect of positively charged residues on these interactions has not been studied. To address this issue, we employed the N-terminal TM domain of the aspartate receptor (Tar-1) as a dimerization model system. We expressed within the ToxR TM assembly system several Tar-1 constructs that dimerize via polar- or non-polar amino acid motifs, and mutated these by replacement with a single arginine residue. Our results have revealed that a mutation in each of the motifs significantly reduced the ability of the TMs to dimerize. Furthermore, a Tar-1 construct that contained two arginine residues was unable to correctly integrate itself into the membrane. Nevertheless, an exogenous synthetic Tar-1 peptide containing these two arginine residues was able to inhibit in vivo the marked dimerization of a mutant Tar-1 construct that contained two glutamate residues at similar positions. This indicates that hetero-assembly of TM domains can be mediated by the interaction of two oppositely charged residues, probably by formation of ion pairs. This study broadens our knowledge regarding the effect of positively charged residues on TM–TM interactions in vivo, and provides a potential therapeutic approach to inhibit uncontrolled dimerization of TM domains caused by mutations of polar amino acids. PMID:15330757

  1. The Yersinia enterocolitica phage shock proteins B and C can form homodimers and heterodimers in vivo with the possibility of close association between multiple domains.

    PubMed

    Gueguen, Erwan; Flores-Kim, Josué; Darwin, Andrew J

    2011-10-01

    The Yersinia enterocolitica phage shock protein (Psp) stress response is essential for virulence and for survival during the mislocalization of outer membrane secretin proteins. The cytoplasmic membrane proteins PspB and PspC are critical components involved in regulating psp gene expression and in facilitating tolerance to secretin-induced stress. Interactions between PspB and PspC monomers might be important for their functions and for PspC stability. However, little is known about these interactions and there are conflicting reports about the ability of PspC to dimerize. To address this, we have used a combination of independent approaches to systematically analyze the ability of PspB and PspC to form dimers in vivo. Formaldehyde cross-linking of the endogenous chromosomally encoded proteins in Y. enterocolitica revealed discrete complexes corresponding in size to PspB-PspB, PspC-PspC, and PspB-PspC. Bacterial two-hybrid analysis corroborated these protein associations, but an important limitation of the two-hybrid approach was uncovered for PspB. A series of PspB and PspC proteins with unique cysteine substitutions at various positions was constructed. In vivo disulfide cross-linking experiments with these proteins further supported close association between PspB and PspC monomers. Detailed cysteine substitution analysis of predicted leucine zipper-like amphipathic helices in both PspB and PspC suggested that their hydrophobic faces could form homodimerization interfaces.

  2. KBF1 (p50 NF-kappa B homodimer) acts as a repressor of H-2Kb gene expression in metastatic tumor cells

    PubMed Central

    1993-01-01

    Downregulation of major histocompatibility complex class I expression is causally related to high malignancy and low immunogenicity of certain murine tumors. In this study, we have analyzed the roles of the nuclear factors KBF1/p50 and p65 in regulation of class I expression in high and low metastatic tumor cells. Low class I-expressing cells show at higher levels of KBF1/p50 and NF-kappa B (p50/p65) binding activity than high class I-expressing cells. However, an excess of KBF1 over NF- kappa B is observed in low expressing cells, while an excess of NF- kappa B over KBF1 is observed in high expressing cells. Stable transfection of a p65 expression vector into low class I-expressing cells activated H-2 transcription and cell surface expression, while stable transfection of p50 expression vector into high expressing cells suppressed H-2Kb transcription and cell surface expression. Our studies suggest that KBF1 has the potential of downregulating class I gene expression, whereas dimers containing the p65 subunit are activators of class I gene expression. PMID:8496683

  3. Expression of bioactive single-chain murine IL-12 in transgenic plants.

    PubMed

    Liu, Jianyun; Dolan, Maureen C; Reidy, Michael; Cramer, Carole L

    2008-06-01

    Interleukin-12 (IL-12), an important immunomodulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anticancer therapeutic. However, its clinical application is limited in part by lack of an effective bioproduction system for this complex heterodimeric glycoprotein. Transgenic plants show promise as scalable bioproduction platforms for challenging biopharmaceutical proteins. To test the potential of plants to effectively produce bioactive IL-12, we developed transgenic tobacco plant lines and derived root cultures yielding high levels of mouse IL-12 (MuIL-12). Functional IL-12 is a heterodimer consisting of two disulfide-linked subunits, p35 and p40. To ensure the stoichiometric expression and assembly of p35 and p40, we expressed a single-chain version of MuIL-12. Plant-derived single-chain MuIL-12 was characterized and purified for in vitro bioactivity assays. Our results demonstrated precise cleavage of the endogenous mouse p40 signal peptide in plants as well as addition of N-linked glycans. Plant-derived MuIL-12 triggered induction of interferon-gamma (IFN-gamma) secretion from mouse splenocytes and stimulated splenocyte proliferation with comparable activities to those observed for commercially available animal cell-derived MuIL-12. These studies indicate that plants produce fully functional MuIL-12 at levels compatible with commercial production and may serve as an effective bioproduction platform for bioactive IL-12s from other species for human or veterinary vaccine and therapeutic applications.

  4. Infectious Dose Dictates the Host Response during Staphylococcus aureus Orthopedic-Implant Biofilm Infection

    PubMed Central

    Vidlak, Debbie

    2016-01-01

    Staphylococcus aureus is a leading cause of prosthetic joint infections (PJIs) that are typified by biofilm formation. Given the diversity of S. aureus strains and their propensity to cause community- or hospital-acquired infections, we investigated whether the immune response and biofilm growth during PJI were conserved among distinct S. aureus clinical isolates. Three S. aureus strains representing USA200 (UAMS-1), USA300 (LAC), and USA400 (MW2) lineages were equally effective at biofilm formation in a mouse model of PJI and elicited similar leukocyte infiltrates and cytokine/chemokine profiles. Another factor that may influence the course of PJI is infectious dose. In particular, higher bacterial inocula could accelerate biofilm formation and alter the immune response, making it difficult to discern underlying pathophysiological mechanisms. To address this issue, we compared the effects of two bacterial doses (103 or 105 CFU) on inflammatory responses in interleukin-12p40 (IL-12p40) knockout mice that were previously shown to have reduced myeloid-derived suppressor cell recruitment concomitant with bacterial clearance after low-dose challenge (103 CFU). Increasing the infectious dose of LAC to 105 CFU negated these differences in IL-12p40 knockout animals, demonstrating the importance of bacterial inoculum on infection outcome. Collectively, these observations highlight the importance of considering infectious dose when assessing immune responsiveness, whereas biofilm formation during PJI is conserved among clinical isolates commonly used in mouse S. aureus infection models. PMID:27091926

  5. Definition of a natural killer NKR-P1A+/CD56-/CD16- functionally immature human NK cell subset that differentiates in vitro in the presence of interleukin 12 [published erratum appears in J Exp Med 1997 Mar 17;185(6):1150-1

    PubMed Central

    1996-01-01

    Human natural killer (NK) cell differentiation from immature lineage negative (Lin-) umbilical cord blood cells was examined in vitro. Cells expressing differentiation antigens of mature NK cells (CD56, CD16, CD2, CD8, NKR-P1A) were generated from Lin- cells cultured with interleukin (IL)-2 and a murine bone marrow stromal cell line expressing the human membrane-bound form of stem cell factor. Two subsets of NK cells were identified in these cultures: one expressed both NKR-P1A and CD56 and, in variable proportions, all other NK cell differentiation antigens; the second subset expressed only NKR-P1A and, unlike the former, was not cytotoxic. Neither subset expressed interferon (IFN)-gamma mRNA even after stimulation with phorbol di- ester and Ca2+ ionophore, but both expressed tumor necrosis factor alpha mRNA and the cytotoxic granule-associated proteins TIA-1, perforin, and serine esterase-1. After 10-d culture with IL-2, IL-12, and irradiated B lymphoblastoid cells, approximately 45% of the NKR- P1A+/ CD56- cells became CD56+, and the same cultures contained cells capable of cytotoxicity and of IFN-gamma production. These results indicate that NKR-P1A expression in the absence of other NK cell markers defines an intermediate, functionally immature stage of NK cell differentiation, and that effector functions develop in these cells, concomitantly with CD56 expression, in the presence of IL-12. These cells likely represent the counterpart of a CD3-/NKR-P1A+/ CD56-/CD16- cell subset that, as shown here, is present both in adult and neonatal circulating lymphocytes. PMID:8920872

  6. Rotavirus Infection Activates Dendritic Cells from Peyer's Patches in Adult Mice ▿ †

    PubMed Central

    Lopez-Guerrero, Delia V.; Meza-Perez, Selene; Ramirez-Pliego, Oscar; Santana-Calderon, Maria A.; Espino-Solis, Pavel; Gutierrez-Xicotencatl, Lourdes; Flores-Romo, Leopoldo; Esquivel-Guadarrama, Fernando R.

    2010-01-01

    This study used an in vivo mouse model to analyze the response of dendritic cells (DCs) in Peyer's patches (PPs) within the first 48 h of infection with the wild-type murine rotavirus EDIM (EDIMwt). After the infection, the absolute number of DCs was increased by 2-fold in the PPs without a modification of their relative percentage of the total cell number. Also, the DCs from PPs of infected mice showed a time-dependent migration to the subepithelial dome (SED) and an increase of the surface activation markers CD40, CD80, and CD86. This response was more evident at 48 h postinfection (p.i.) and depended on viral replication, since DCs from PPs of mice inoculated with UV-treated virus did not show this phenotype. As a result of the activation, the DCs showed an increase in the expression of mRNA for the proinflammatory cytokines interleukin-12/23p40 (IL-12/23p40), tumor necrosis factor alpha (TNF-α), and beta interferon (IFN-β), as well as for the regulatory cytokine IL-10. These results suggest that, a short time after rotavirus infection, the DCs from PPs play a critical role in controlling the infection and, at the same time, avoiding an excessive inflammatory immune response. PMID:20007263

  7. Infection with Theiler's murine encephalomyelitis virus directly induces proinflammatory cytokines in primary astrocytes via NF-kappaB activation: potential role for the initiation of demyelinating disease.

    PubMed

    Palma, JoAnn P; Kwon, Daeho; Clipstone, Neil A; Kim, Byung S

    2003-06-01

    Theiler's virus infection in the central nervous system (CNS) induces a demyelinating disease very similar to human multiple sclerosis. We have assessed cytokine gene activation upon Theiler's murine encephalomyelitis virus (TMEV) infection and potential mechanisms in order to delineate the early events in viral infection that lead to immune-mediated demyelinating disease. Infection of SJL/J primary astrocyte cultures induces selective proinflammatory cytokine genes (interleukin-12p40 [IL-12p40], IL-1, IL-6, tumor necrosis factor alpha, and beta interferon [IFN-beta]) important in the innate immune response to infection. We find that TMEV-induced cytokine gene expression is mediated by the NF-kappaB pathway based on the early nuclear NF-kappaB translocation and suppression of cytokine activation in the presence of specific inhibitors of the NF-kappaB pathway. Further studies show this to be partly independent of dsRNA-dependent protein kinase (PKR) and IFN-alpha/beta pathways. Altogether, these results demonstrate that infection of astrocytes and other CNS-resident cells by TMEV provides the early NF-kappaB-mediated signals that directly activate various proinflammatory cytokine genes involved in the initiation and amplification of inflammatory responses in the CNS known to be critical for the development of immune-mediated demyelination.

  8. Toll-like Receptor 7 Mitigates Lethal West Nile Encephalitis via Interleukin 23-Dependent Immune Cell Infiltration and Homing

    PubMed Central

    Town, Terrence; Bai, Fengwei; Wang, Tian; Kaplan, Amber T.; Qian, Feng; Montgomery, Ruth R.; Anderson, John F.; Flavell, Richard A.; Fikrig, Erol

    2009-01-01

    SUMMARY West Nile virus (WNV), a mosquito-transmitted single-stranded RNA (ssRNA) flavivirus, causes human disease of variable severity. We investigated Toll-like receptor 7-deficient (Tlr7−/−) and myeloid differentiation factor 88-deficient (Myd88−/−) mice, which both have defective recognition of ssRNA, and found increased viremia and susceptibility to lethal WNV infection. Despite increased tissue concentrations of most innate cytokines, CD45+ leukocytes and CD11b+ macrophages failed to home to WNV-infected cells and infiltrate into target organs of Tlr7−/− mice. Tlr7−/− mice and macrophages had reduced interleukin-12 (IL-12) and IL-23 responses after WNV infection, and mice deficient in IL-12 p40 and IL-23 p40 (Il12b−/−) or IL-23 p19 (Il23a−/−), but not IL-12 p35 (Il12a−/−), responded similarly to Tlr7−/− mice, with increased susceptibility to lethal WNV encephalitis. Collectively, these results demonstrate that TLR7 andIL-23-dependent WNV responses representa vital host defense mechanism that operates by affecting immune cell homing to infected target cells. PMID:19200759

  9. Studies on nonidet P40 lysis of murine lymphoid cells. I. Use of cholera toxin and cell surface Ig to determine degree of dissociation of the plasma membrane.

    PubMed

    Hart, D A

    1975-09-01

    Lymphoid cells from A/J mice were iodinated (125I) by the lactoperoxidase lysed with the non-ionic detergent NP-40. The plasma membrane glycolipid receptor for cholera toxin and cell surface immunoglobulin were utilized in immune precipitation systems to characterize the degree of dissociation of the plasma membrane under various conditions. It was found that at 0.1% NP-40 and at cell concentration from 5 to 10 times 10(7) cells/ml, lipid-protein and protein-lipid-protein complexes formed in NP-40 which were soluble after centrifugation at 10(5) times G. Column chromatography of 125I-cell lysates on agarose A-0.5 M in 0.1% or 0.5% NP-40/PBS indicated that the majority of iodinated cell surface material existed as aggregates in detergent micelles. The availability of the oligosaccharide moiety of the glycolipid to interact with the cholera toxin was dependent on both the detergent concentration and the cell concentration used for cell lysis. However, the cell surface immunoglobulin was immunoprecipitable under all conditions of lysis tested.

  10. The 3'UTR 1188A/C polymorphism of IL-12p40 is not associated with susceptibility for developing plaque psoriasis in Mestizo population from western Mexico.

    PubMed

    Sandoval-Talamantes, Ana Karen; Brito-Luna, Myrian Johanna; Fafutis-Morris, Mary; Villanueva-Quintero, Delfina Guadalupe; Graciano-Machuca, Omar; Ramírez-Dueñas, María Guadalupe; Alvarado-Navarro, Anabell

    2015-02-01

    Psoriasis is a chronic autoimmune inflammatory disease that affects the skin and the joints. Psoriasis is characterized by the keratinocyte proliferation, which is induced by cytokines Th1 and Th17. Patients with plaque psoriasis present a chronic inflammatory response with high levels of interleukin (IL)-12 and IL-23. Various single-nucleotide polymorphisms (SNP) have been identified in the IL12B gene, such as SNP 3' UTR 1188 A/C (SNP rs3212227), which has been associated with susceptibility to developing plaque psoriasis and with the production of IL-12 and IL-23 in individuals of different ethnic groups. In this study, we determined whether there is an association of SNP rs3212227 with the susceptibility of developing plaque psoriasis and with serum levels of IL-12 and IL-23 in Mestizo population in western Mexico. We included 112 patients with psoriasis and 112 clinical healthy individuals in the study. The frequencies of genotypes A/A, A/C, and C/C in patients with plaque psoriasis were 41, 53, and 6%, respectively, while in the control group, these were 37, 53, and 10%, respectively, without finding statistically significant differences between both groups (p>0.05). Although IL-12 and IL-23 serum levels were higher in patients than in controls, we found no significant differences. The group of patients with genotype CC presented the highest levels of IL-23 (p<0.05). These data suggest that the SNP rs3212227 phenotype is not associated with the risk of developing plaque psoriasis or with IL-12 and IL-23 levels in Mestizo population in western Mexico.

  11. IL-12 promotes myeloid-derived suppressor cell recruitment and bacterial persistence during Staphylococcus aureus orthopedic implant infection.

    PubMed

    Heim, Cortney E; Vidlak, Debbie; Scherr, Tyler D; Hartman, Curtis W; Garvin, Kevin L; Kielian, Tammy

    2015-04-15

    Staphylococcus aureus is a leading cause of human prosthetic joint infections (PJIs) typified by biofilm formation. We recently identified a critical role for myeloid-derived suppressor cells (MDSCs) in S. aureus biofilm persistence. Proinflammatory signals induce MDSC recruitment and activation in tumor models; however, the mechanisms responsible for MDSC homing to sites of biofilm infection are unknown. In this study, we report that several cytokines (IL-12p40, IL-1β, TNF-α, and G-CSF) and chemokines (CXCL2, CCL5) were significantly elevated in a mouse model of S. aureus PJI. This coincided with significantly increased MDSC infiltrates concomitant with reduced monocyte, macrophage, and T cell influx compared with uninfected animals. Of the cytokines detected, IL-12 was of particular interest based on its ability to possess either pro- or anti-inflammatory effects mediated through p35-p40 heterodimers or p40 homodimers, respectively. MDSC recruitment was significantly reduced in both p40 and p35 knockout mice, which resulted in enhanced monocyte and neutrophil influx and bacterial clearance. Adoptive transfer of wild-type MDSCs into infected p40 knockout animals worsened disease outcome, as evidenced by the return of S. aureus burdens to levels typical of wild-type mice. Tissues obtained from patients undergoing revision surgery for PJI revealed similar patterns of immune cell influx, with increased MDSC-like cells and significantly fewer T cells compared with aseptic revisions. These findings reveal a critical role for IL-12 in shaping the anti-inflammatory biofilm milieu by promoting MDSC recruitment.

  12. An ethanol extract of Piper betle Linn. mediates its anti-inflammatory activity via down-regulation of nitric oxide.

    PubMed

    Ganguly, Sudipto; Mula, Soumyaditya; Chattopadhyay, Subrata; Chatterjee, Mitali

    2007-05-01

    The leaves of Piper betle (locally known as Paan) have long been in use in the Indian indigenous system of medicine for the relief of pain; however, the underlying molecular mechanisms of this effect have not been elucidated. The anti-inflammatory and immunomodulatory effects of an ethanolic extract of the leaves of P. betle (100 mg kg(-1); PB) were demonstrated in a complete Freund's adjuvant-induced model of arthritis in rats with dexamethasone (0.1 mg kg(-1)) as the positive control. At non-toxic concentrations of PB (5-25 microg mL(-1)), a dose-dependent decrease in extracellular production of nitric oxide in murine peritoneal macrophages was measured by the Griess assay and corroborated by flow cytometry using the nitric oxide specific probe, 4,5-diaminofluorescein-2 diacetate. This decreased generation of reactive nitrogen species was mediated by PB progressively down-regulating transcription of inducible nitric oxide synthase in macrophages, and concomitantly causing a dose-dependent decrease in the expression of interleukin-12 p40, indicating the ability of PB to down-regulate T-helper 1 pro-inflammatory responses. Taken together, the anti-inflammatory and anti-arthrotic activity of PB is attributable to its ability to down-regulate the generation of reactive nitrogen species, thus meriting further pharmacological investigation.

  13. Effects of tumor necrosis factor alpha on host immune response in chronic persistent tuberculosis: possible role for limiting pathology.

    PubMed

    Mohan, V P; Scanga, C A; Yu, K; Scott, H M; Tanaka, K E; Tsang, E; Tsai, M M; Flynn, J L; Chan, J

    2001-03-01

    Reactivation of latent tuberculosis contributes significantly to the incidence of disease caused by Mycobacterium tuberculosis. The mechanisms involved in the containment of latent tuberculosis are poorly understood. Using the low-dose model of persistent murine tuberculosis in conjunction with MP6-XT22, a monoclonal antibody that functionally neutralizes tumor necrosis factor alpha (TNF-alpha), we examined the effects of TNF-alpha on the immunological response of the host in both persistent and reactivated tuberculous infections. The results confirm an essential role for TNF-alpha in the containment of persistent tuberculosis. TNF-alpha neutralization resulted in fatal reactivation of persistent tuberculosis characterized by a moderately increased tissue bacillary burden and severe pulmonic histopathological deterioration that was associated with changes indicative of squamous metaplasia and fluid accumulation in the alveolar space. Analysis of pulmonic gene and protein expression of mice in the low-dose model revealed that nitric oxide synthase was attenuated during MP6-XT22-induced reactivation, but was not totally suppressed. Interleukin-12p40 and gamma interferon gene expression in TNF-alpha-neutralized mice was similar to that in control mice. In contrast, interleukin-10 expression was augmented in the TNF-alpha-neutralized mice. In summary, results of this study suggest that TNF-alpha plays an essential role in preventing reactivation of persistent tuberculosis, modulates the pulmonic expression of specific immunologic factors, and limits the pathological response of the host.

  14. Ustekinumab for the treatment of psoriatic arthritis.

    PubMed

    Wofford, Jay; Menter, Alan

    2014-02-01

    Ustekinumab is a fully human monoclonal antibody directed against the p40 subunit shared by interleukin 12 and interleukin 23, two naturally occurring protein regulators that play an important role in immune-mediated inflammatory diseases, including psoriatic arthritis (PsA). In September of 2009, the US FDA approved ustekinumab for the treatment of adult patients with moderate to severe plaque psoriasis. Beginning in November of 2009, Janssen Biotech (formerly Centocor Biotech), the developer of ustekinumab, initiated clinical trials to investigate the efficacy of ustekinumab in the treatment of other inflammatory disorders, including PsA. Phase II and Phase III studies showed both a good safety profile and significant efficacy for ustekinumab in the treatment of PsA, leading to the drug's approval in both Europe and the USA. In an immunotherapy market currently dominated by anti-TNF-α drugs for the treatment of PsA, ustekinumab offers an alternative option for patients with PsA, including those unresponsive to methotrexate and the TNF-α inhibitory agents currently approved for this potentially debilitating disease.

  15. EssE Promotes Staphylococcus aureus ESS-Dependent Protein Secretion To Modify Host Immune Responses during Infection.

    PubMed

    Anderson, Mark; Ohr, Ryan Jay; Aly, Khaled A; Nocadello, Salvatore; Kim, Hwan K; Schneewind, Chloe E; Schneewind, Olaf; Missiakas, Dominique

    2017-01-01

    Staphylococcus aureus, an invasive pathogen of humans and animals, requires a specialized ESS pathway to secrete proteins (EsxA, EsxB, EsxC, and EsxD) during infection. Expression of ess genes is required for S. aureus establishment of persistent abscess lesions following bloodstream infection; however, the mechanisms whereby effectors of the ESS pathway implement their virulence strategies were heretofore not known. Here, we show that EssE forms a complex with other members of the ESS secretion pathway and its substrates, promoting the secretion of EsxA, EsxB, EsxC, EsxD, and EssD. During bloodstream infection of mice, the S. aureus essE mutant displays defects in host cytokine responses, specifically in the production of interleukin-12 (IL-12) (p40/p70) and the suppression of RANTES (CCL5), activators of TH1 T cell responses and immune cell chemotaxis, respectively. Thus, essE-mediated secretion of protein effectors via the ESS pathway may enable S. aureus to manipulate host immune responses by modifying the production of cytokines.

  16. IL-12 Family Cytokines: General Characteristics, Pathogenic Microorganisms, Receptors, and Signalling Pathways.

    PubMed

    Behzadi, Payam; Behzadi, Elham; Ranjbar, Reza

    2016-03-01

    Among a wide range of cytokines, the Interleukin 12 (IL-12) family has its unique structural, functional, and immunological characteristics that have made this family as important immunological playmakers. Because of the importance of IL-12 heterodimeric cytokines in microbial infections, autoimmune diseases, and cancers, the authors of this literature discuss about the general characteristics of IL-12 family members, the interactions between IL-12 cytokines and pathogenic microorganisms, the interleukins receptors and their strategies for selecting different signalling pathways. IL-12 and IL-23 are similar in p40 subunits and both are involved in proinflammatory responses while, IL-27 and IL-35 contribute to anti-inflammatory activities; however, IL-27 is also involved in pro-inflammatory responses. There are some similarities and dissimilarities among IL-12 family members which make them a unique bridge between innate and adaptive immune systems. The bioactivities of IL-12 family indicate a brilliant promise for their applications in different fields of medicine. The members of IL-12 family are candidate for several therapeutics including gene therapy, cancer therapy, tumour therapy, and vaccination. To have an accurate diagnostic technique and definite treatment regarding to infectious diseases, the playmakers of IL-12 family as effective criteria together with microarray technology are the best choices for current and future applications.

  17. Enhanced natural killer activity and production of pro-inflammatory cytokines in mice selected for high acute inflammatory response (AIRmax).

    PubMed

    Castoldi, Lindsey; Golim, Marjorie Assis; Filho, Orlando Garcia Ribeiro; Romagnoli, Graziela Gorete; Ibañez, Olga Célia Martinez; Kaneno, Ramon

    2007-03-01

    Strains of mice with maximal and minimal acute inflammatory responsiveness (AIRmax and AIRmin, respectively) were developed through selective breeding based on their high- or low-acute inflammatory responsiveness. Previous reports have shown that AIRmax mice are more resistant to the development of a variety of tumours than AIRmin mice, including spontaneous metastasis of murine melanoma. Natural killer activity is involved in immunosurveillance against tumour development, so we analysed the number and activity of natural killer cells (CD49b(+)), T-lymphocyte subsets and in vitro cytokine production by spleen cells of normal AIRmax and AIRmin mice. Analysis of lymphocyte subsets by flow cytometry showed that AIRmax mice had a higher relative number of CD49b(+) cells than AIRmin mice, as well as cytolytic activity against Yac.1 target cells. The number of CD3(+) CD8(+) cells was also higher in AIRmax mice. These findings were associated with the ability of spleen cells from AIRmax mice in vitro to produce higher levels of the pro-inflammatory cytokines tumour necrosis factor-alpha, interleukin-12p40 and interferon-gamma but not the anti-inflammatory interleukin-10. Taken together, our data suggest that the selective breeding to achieve the AIRmax and AIRmin strains was able to polarize the genes associated with cytotoxic activity, which can be responsible for the antitumour resistance observed in AIRmax mice.

  18. Ustekinumab in treatment of Crohn’s disease: design, development, and potential place in therapy

    PubMed Central

    Deepak, Parakkal; Loftus, Edward V

    2016-01-01

    Crohn’s disease is characterized by a dysregulation of both innate and adaptive immunity responses. Interleukin-12/23 (IL-12/23) pathway has been found to be a major driver of inflammation in adaptive immune responses. Ustekinumab is a fully human immunoglobulin G1 kappa monoclonal antibody that blocks the p40 subunit of IL-12 and IL-23 and prevents their interaction with their cell surface receptor and further cytokine activation. It is currently approved in the management of plaque psoriasis and psoriatic arthritis. Very promising data have emerged through phase II and phase III trials (UNITI-1, UNITI-2, and IM-UNITI) for both induction and maintenance of clinical response and remission in moderate-to-severe Crohn’s disease, resulting in approval by the Food and Drug Administration for this condition. This article reviews the immunology of the IL-12/23 pathway, available data regarding the initial designing of ustekinumab, drug development through clinical trials including pharmacokinetics, efficacy, and safety, and its potential place in the treatment of Crohn’s disease. PMID:27956825

  19. Interleukin-35: odd one out or part of the family?

    PubMed Central

    Collison, Lauren W.; Vignali, Dario A. A.

    2008-01-01

    Summary Advances in cytokine biology have helped us understand the complex communication that takes place between antigen-presenting cells and cells of the adaptive immune system, such as T cells, which collectively mediate an appropriate immune response to a plethora of pathogens while maintaining tolerance to self-antigens. The interleukin-12 (IL-12) cytokine family remains one of the most important and includes IL-12, IL-23, IL-27, and the recently identified IL-35. All four are heterodimeric cytokines, composed of an α chain (p19, p28, or p35) and a β chain (p40 or Ebi3), and signal through unique pairings of five receptor chains (IL-12Rβ1, IL-12Rβ2, IL-23R, gp130, and WSX-1). Despite the interrelationship between the cytokines themselves and their receptors, their source, activity, and kinetics of expression are quite different. Studies using genetically deficient mice have greatly enhanced our understanding of the biology of these cytokines. However, interpretation of these data has been complicated by the recent realization that p40−/−, p35−/−, and Ebi3−/− mice all lack more than one cytokine (IL-12/IL-23, IL-12/IL-35, IL-27/IL-35, respectively). In this review, we compare and contrast the biology of this expanded IL-12 family and re-evaluate data derived from the analysis of these dual cytokine-deficient mice. We also discuss how the opposing characteristics of the IL-12 family siblings may help to promote a balanced immune response. PMID:19161429

  20. Protective effects of intranasal losartan in the APP/PS1 transgenic mouse model of Alzheimer disease.

    PubMed

    Danielyan, Lusine; Klein, Roman; Hanson, Leah R; Buadze, Marine; Schwab, Matthias; Gleiter, Christoph H; Frey, William H

    2010-01-01

    The local renin-angiotensin system (RAS) in the brain is a multitasking system controlling a plethora of essential functions such as neurogenic hypertension, baroreflexes, and sympathetic activity. Aside from its vasoactive actions, brain angiotensin II (AT-II) has also been implicated in the pathogenesis of cognitive decline, and beneficial effects of angiotensin receptor blockers (ARBs) in Alzheimer (AD) and Parkinson diseases (PD) are suggested. However, the use of ARBs at antihypertensive dosages would lead to unwanted hypotensive reactions in AD patients. Here we treated the APP/PS1 transgenic mouse model of AD with the ARB losartan (10 mg/kg body weight) to determine whether blockade of the AT-II receptor subtype 1 (AT1-R) with intranasal losartan, using at a dosage far below its systemic antihypertensive dose, could maintain its neuroprotective effects independent of its systemic vasoactive action. Intranasal losartan treatment (10 mg/kg every other day for 2 months) of APP/PS1 mice decreased amyloid beta (Abeta) plaques 3.7-fold. Blood serum levels of interleukin-12 (IL-12)p40/p70, IL-1beta, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in the vehicle-treated APP/PS1 mice. Intranasal losartan not only decreased IL-12p40/p70, IL-1beta, and GM-CSF, but also increased IL-10, which suppresses inflammation. Furthermore, losartan markedly increased tyrosine hydroxylase expression in the striatum and locus coeruleus. In conclusion, losartan exerts direct neuroprotective effects via its Abeta-reducing and antiinflammatory effects in the central nervous system (CNS). Therefore, intranasal losartan and potentially other ARBs, at concentrations below their threshold for altering systemic blood pressure, offer a new approach for the treatment of AD.

  1. The immunostimulatory activity of CpG oligonucleotides on microglial N9 cells is affected by a polyguanosine motif.

    PubMed

    Zhang, Zhiren; Guo, Ketai; Schluesener, Hermann J

    2005-04-01

    Oligonucleotides (ODN) with hexameric motifs containing central unmethylated CpG dinucleotides are immunostimulatory. Also ODN with continuous guanosines (polyG motif) show a wide range of immunological activity. Depending on the position, the chemical property of the ODN backbone and the cell type, polyG motifs have either an enhancing or a suppressing effect on the immunostimulatory activity of the CpG-ODN. Microglial cells are central components of the innate immune system of the brain and are activated by CpG-ODN in vitro and in vivo. Here we present the analysis of the immunomodulatory effects of CpG-ODN carrying a polyG motif on the microglial cell line N9. Our data show that N9 cells express Toll-like receptor 9 (TLR9) and are activated by CpG-ODN, which leads to expression of interleukin-12p40 (IL12p40), tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). A 3'-end polyG motif inhibits phosphothioate (PS) CpG-ODN immunostimulatory activity but enhances the immunostimulatory activity of phosphodiester (PE) CpG-ODN. Correspondingly, a 3'-end polyG motif improves the cellular uptake of PE CpG-ODN but does not change their cellular distribution pattern. Furthermore, PE CpG-ODN with a 3'-end polyG motif interact with a much higher number of cellular proteins than PE CpG-ODN. These data indicate that the 3'-end polyG motif could enhance the immunostimulatory activity of PE CpG-ODN in microglial N9 cells through increasing interaction with cellular proteins. Therefore PE CpG-ODN containing a 3'-end polyG motif resulting in increased immunostimulatory activity might be promising alternate analogues for studies in the central nervous system.

  2. In Vitro Evaluation of the Biological Responses of Canine Macrophages Challenged with PLGA Nanoparticles Containing Monophosphoryl Lipid A

    PubMed Central

    Guldner, Delphine; Hwang, Julianne K.; Cardieri, Maria Clara D.; Eren, Meaghan; Ziaei, Parissa; Norton, M. Grant; Souza, Cleverson D.

    2016-01-01

    Poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) have been considerably studied as a promising biodegradable delivery system to induce effective immune responses and to improve stability, safety, and cost effectiveness of vaccines. The study aimed at evaluating early inflammatory effects and cellular safety of PLGA NPs, co-encapsulating ovalbumin (PLGA/OVA NPs), as a model antigen and the adjuvant monophosphoryl lipid A (PLGA/MPLA NPs) as an adjuvant, on primary canine macrophages. The PLGA NPs constructs were prepared following the emulsion-solvent evaporation technique and further physic-chemically characterized. Peripheral blood mononuclear cells were isolated from canine whole blood by magnetic sorting and further cultured to generate macrophages. The uptake of PLGA NP constructs by macrophages was demonstrated by flow cytometry, transmission electron microscopy and confocal microscopy. Macrophage viability and morphology were evaluated by trypan blue exclusion and light microscopy. Macrophages were immunophenotyped for the expression of MHC-I and MHC-II and gene expression of Interleukin-10 (IL-10), Interleukin-12 (IL-12p40), and tumor necrosis factor alpha (TNF-α) were measured. The results showed that incubation of PLGA NP constructs with macrophages revealed effective early uptake of the PLGA NPs without altering the viability of macrophages. PLGA/OVA/MPLA NPs strongly induced TNF-α and IL-12p40 expression by macrophages as well as increase relative expression of MHC-I but not MHC-II molecules. Taken together, these results indicated that PLGA NPs with addition of MPLA represent a good model, when used as antigen carrier, for further, in vivo, work aiming to evaluate their potential to induce strong, specific, immune responses in dogs. PMID:27835636

  3. Mycobacterium indicus pranii and Mycobacterium bovis BCG lead to differential macrophage activation in Toll-like receptor-dependent manner.

    PubMed

    Kumar, Pawan; Tyagi, Rohit; Das, Gobardhan; Bhaskar, Sangeeta

    2014-10-01

    Mycobacterium indicus pranii (MIP) is an atypical mycobacterial species possessing strong immunomodulatory properties. It is a potent vaccine candidate against tuberculosis, promotes Th1 immune response and protects mice from tumours. In previous studies, we demonstrated higher protective efficacy of MIP against experimental tuberculosis as compared with bacillus Calmette-Guérin (BCG). Since macrophages play an important role in the pathology of mycobacterial diseases and cancer, in the present study, we evaluated the MIP in live and killed form for macrophage activation potential, compared it with BCG and investigated the underlying mechanisms. High levels of tumour necrosis factor-α, interleukin-12p40 (IL-12p40), IL-6 and nitric oxide were produced by MIP-stimulated macrophages as compared with BCG-stimulated macrophages. Prominent up-regulation of co-stimulatory molecules CD40, CD80 and CD86 was also observed in response to MIP. Loss of response in MyD88-deficient macrophages showed that both MIP and BCG activate the macrophages in a MyD88-dependent manner. MyD88 signalling pathway culminates in nuclear factor-κB/activator protein-1 (NF-κB/AP-1) activation and higher activation of NF-κB/AP-1 was observed in response to MIP. With the help of pharmacological inhibitors and Toll-like receptor (TLR) -deficient macrophages, we observed the role of TLR2, TLR4 and intracellular TLRs in MIP-mediated macrophage activation. Stimulation of HEK293 cells expressing TLR2 in homodimeric or heterodimeric form showed that MIP has a distinctly higher level of TLR2 agonist activity compared with BCG. Further experiments suggested that TLR2 ligands are well exposed in MIP whereas they are obscured in BCG. Our findings establish the higher macrophage activation potential of MIP compared with BCG and delineate the underlying mechanism.

  4. Nramp1 Is Not a Major Determinant in the Control of Brucella melitensis Infection in Mice

    PubMed Central

    Guilloteau, Laurence A.; Dornand, Jacques; Gross, Antoine; Olivier, Michel; Cortade, Fabienne; Vern, Yves Le; Kerboeuf, Dominique

    2003-01-01

    Brucella, the causative agent of brucellosis in animals and humans, can survive and proliferate within macrophages. Macrophages mediate mouse resistance to various pathogens through the expression of the Nramp1 gene. The role of this gene in the control of Brucella infection was investigated. When BALB/c mice (Nramp1s) and C.CB congenic mice (Nramp1r) were infected with Brucella melitensis, the number of Brucella organisms per spleen was significantly larger in the C.CB mice than in the BALB/c mice during the first week postinfection (p.i.). This Nramp1-linked susceptibility to Brucella was temporary, since similar numbers of Brucella were recovered from the two strains of mice 2 weeks p.i. The effect of Nramp1 expression occurred within splenocytes intracellularly infected by Brucella. However, there was no difference between in vitro replication rates of Brucella in macrophages isolated from the two strains of mice infected in vivo or in Nramp1 RAW264 transfectants. In mice, infection with Brucella induced an inflammatory response, resulting in splenomegaly and recruitment of phagocytes in the spleen, which was amplified in C.CB mice. Reverse transcription-PCR (RT-PCR), performed 5 days p.i., showed that inducible nitric oxide synthase, tumor necrosis factor alpha (TNF-α), interleukin-12 p40 (IL-12p40), gamma interferon (IFN-γ), and IL-10 mRNAs were similarly induced in spleens of the two strains. In contrast, the mRNA of KC, a C-X-C chemokine, was induced only in infected C.CB mice at this time. This pattern of mRNA expression was maintained at 14 days p.i., with IFN-γ and IL-12p40 mRNAs being more intensively induced in the infected C.CB mice, but TNF-α mRNA was no longer induced. The higher recruitment of neutrophils observed in the spleens of infected C.CB mice could explain the temporary susceptibility of C.CB mice to B. melitensis infection. In contrast to infections with Salmonella, Leishmania, and Mycobacterium, the expression of the Nramp1 gene

  5. Anti-Tumor Effects after Adoptive Transfer of IL-12 Transposon-Modified Murine Splenocytes in the OT-I-Melanoma Mouse Model.

    PubMed

    Galvan, Daniel L; O'Neil, Richard T; Foster, Aaron E; Huye, Leslie; Bear, Adham; Rooney, Cliona M; Wilson, Matthew H

    2015-01-01

    Adoptive transfer of gene modified T cells provides possible immunotherapy for patients with cancers refractory to other treatments. We have previously used the non-viral piggyBac transposon system to gene modify human T cells for potential immunotherapy. However, these previous studies utilized adoptive transfer of modified human T cells to target cancer xenografts in highly immunodeficient (NOD-SCID) mice that do not recapitulate an intact immune system. Currently, only viral vectors have shown efficacy in permanently gene-modifying mouse T cells for immunotherapy applications. Therefore, we sought to determine if piggyBac could effectively gene modify mouse T cells to target cancer cells in a mouse cancer model. We first demonstrated that we could gene modify cells to express murine interleukin-12 (p35/p40 mIL-12), a transgene with proven efficacy in melanoma immunotherapy. The OT-I melanoma mouse model provides a well-established T cell mediated immune response to ovalbumin (OVA) positive B16 melanoma cells. B16/OVA melanoma cells were implanted in wild type C57Bl6 mice. Mouse splenocytes were isolated from C57Bl6 OT-I mice and were gene modified using piggyBac to express luciferase. Adoptive transfer of luciferase-modified OT-I splenocytes demonstrated homing to B16/OVA melanoma tumors in vivo. We next gene-modified OT-I cells to express mIL-12. Adoptive transfer of mIL-12-modified mouse OT-I splenocytes delayed B16/OVA melanoma tumor growth in vivo compared to control OT-I splenocytes and improved mouse survival. Our results demonstrate that the piggyBac transposon system can be used to gene modify splenocytes and mouse T cells for evaluating adoptive immunotherapy strategies in immunocompetent mouse tumor models that may more directly mimic immunotherapy applications in humans.

  6. Epithelial-cell-intrinsic IKK-beta expression regulates intestinal immune homeostasis.

    PubMed

    Zaph, Colby; Troy, Amy E; Taylor, Betsy C; Berman-Booty, Lisa D; Guild, Katherine J; Du, Yurong; Yost, Evan A; Gruber, Achim D; May, Michael J; Greten, Florian R; Eckmann, Lars; Karin, Michael; Artis, David

    2007-03-29

    Intestinal epithelial cells (IECs) provide a primary physical barrier against commensal and pathogenic microorganisms in the gastrointestinal (GI) tract, but the influence of IECs on the development and regulation of immunity to infection is unknown. Here we show that IEC-intrinsic IkappaB kinase (IKK)-beta-dependent gene expression is a critical regulator of responses of dendritic cells and CD4+ T cells in the GI tract. Mice with an IEC-specific deletion of IKK-beta show a reduced expression of the epithelial-cell-restricted cytokine thymic stromal lymphopoietin in the intestine and, after infection with the gut-dwelling parasite Trichuris, fail to develop a pathogen-specific CD4+ T helper type 2 (T(H)2) response and are unable to eradicate infection. Further, these animals show exacerbated production of dendritic-cell-derived interleukin-12/23p40 and tumour necrosis factor-alpha, increased levels of CD4+ T-cell-derived interferon-gamma and interleukin-17, and develop severe intestinal inflammation. Blockade of proinflammatory cytokines during Trichuris infection ablates the requirement for IKK-beta in IECs to promote CD4+ T(H)2 cell-dependent immunity, identifying an essential function for IECs in tissue-specific conditioning of dendritic cells and limiting type 1 cytokine production in the GI tract. These results indicate that the balance of IKK-beta-dependent gene expression in the intestinal epithelium is crucial in intestinal immune homeostasis by promoting mucosal immunity and limiting chronic inflammation.

  7. TLR ligands of Lactobacillus sakei LK-117 isolated from seed mash for brewing sake are potent inducers of IL-12.

    PubMed

    Masuda, Yasuyuki; Takahashi, Toshinari; Yoshida, Kazutoshi; Nishitani, Yosuke; Mizuno, Masashi; Mizoguchi, Haruhiko

    2011-10-01

    Many studies have investigated the immunostimulatory effects of bacteria, such as the anti-allergic effects of lactic acid bacteria (LAB) and LAB-fermented milk. Importantly, these anti-allergic effects have been observed for both viable and nonviable bacteria. However, there are no reported immunological effects of LAB isolated from kimoto, the traditional yeast starter culture used for brewing sake, which also involves spontaneous lactate fermentation. In this study, we determined whether the Leuconostoc mesenteroides and Lactobacillus sakei bacterial strains obtained from kimoto affected the production of interleukin-12 (IL-12), an inducer of the T-helper type-1 immune response. By incubating autoclaved bacteria with J774.1 macrophage-like cells, we found that L. sakei LK-117 induced a sustained increase in IL-12p40 production. The IL-12-inducing ability of LK-117 was unaffected by anti-TLR2 neutralization and was entirely inhibited when the LK-117 cells were treated with RNase. When LK-117 cells were treated with M-1, an N-acetylmuramidase, at varying concentrations and for different periods of time, the ability of the bacteria to induce IL-12 decreased quickly. Although an active fraction could be prepared by chromatography from the soluble products of enzymolysis, the fraction's induction ability was <2% of that of intact organisms, and induction ability disappeared completely upon anti-TLR2 neutralization after treating the active fraction with RNase. These results suggest that single-stranded RNA released from cells that were disrupted by autoclaving might act as a TLR ligand and provide a novel mechanism in which heat-killed LAB could be used to regulate immune activity.

  8. Deregulation of Fas ligand expression as a novel cause of autoimmune lymphoproliferative syndrome-like disease.

    PubMed

    Nabhani, Schafiq; Ginzel, Sebastian; Miskin, Hagit; Revel-Vilk, Shoshana; Harlev, Dan; Fleckenstein, Bernhard; Hönscheid, Andrea; Oommen, Prasad T; Kuhlen, Michaela; Thiele, Ralf; Laws, Hans-Jürgen; Borkhardt, Arndt; Stepensky, Polina; Fischer, Ute

    2015-09-01

    Autoimmune lymphoproliferative syndrome is frequently caused by mutations in genes involved in the Fas death receptor pathway, but for 20-30% of patients the genetic defect is unknown. We observed that treatment of healthy T cells with interleukin-12 induces upregulation of Fas ligand and Fas ligand-dependent apoptosis. Consistently, interleukin-12 could not induce apoptosis in Fas ligand-deficient T cells from patients with autoimmune lymphoproliferative syndrome. We hypothesized that defects in the interleukin-12 signaling pathway may cause a similar phenotype as that caused by mutations of the Fas ligand gene. To test this, we analyzed 20 patients with autoimmune lymphoproliferative syndrome of unknown cause by whole-exome sequencing. We identified a homozygous nonsense mutation (c.698G>A, p.R212*) in the interleukin-12/interleukin-23 receptor-component IL12RB1 in one of these patients. The mutation led to IL12RB1 protein truncation and loss of cell surface expression. Interleukin-12 and -23 signaling was completely abrogated as demonstrated by deficient STAT4 phosphorylation and interferon γ production. Interleukin-12-mediated expression of membrane-bound and soluble Fas ligand was lacking and basal expression was much lower than in healthy controls. The patient presented with the classical symptoms of autoimmune lymphoproliferative syndrome: chronic non-malignant, non-infectious lymphadenopathy, splenomegaly, hepatomegaly, elevated numbers of double-negative T cells, autoimmune cytopenias, and increased levels of vitamin B12 and interleukin-10. Sanger sequencing and whole-exome sequencing excluded the presence of germline or somatic mutations in genes known to be associated with the autoimmune lymphoproliferative syndrome. Our data suggest that deficient regulation of Fas ligand expression by regulators such as the interleukin-12 signaling pathway may be an alternative cause of autoimmune lymphoproliferative syndrome-like disease.

  9. Lymphopenia associated with highly virulent H5N1 virus infection due to plasmacytoid dendritic cell mediated apoptosis of T cells

    PubMed Central

    Boonnak, Kobporn; Vogel, Leatrice; Feldmann, Friederike; Feldmann, Heinz; Legge, Kevin L.; Subbarao, Kanta

    2014-01-01

    Although lymphopenia is a hallmark of severe infection with highly pathogenic H5N1 and the newly emerged H7N9 influenza viruses in humans, the mechanism(s) by which lethal H5N1 viruses cause lymphopenia in mammalian hosts remains poorly understood. Because influenza-specific T cell responses are initiated in the lung draining lymph nodes, and lymphocytes subsequently traffic to the lungs or peripheral circulation, we compared the immune responses in the lung draining lymph nodes following infection with a lethal A/HK/483/97 or non-lethal A/HK/486/97 (H5N1) virus in a mouse model. We found that lethal H5N1, but not non-lethal H5N1 virus infection in mice enhances Fas ligand (FasL) expression on plasmacytoid dendritic cells (pDCs), resulting in apoptosis of influenza-specific CD8+ T cells via a Fas-FasL mediated pathway. We also found that pDCs, but not other DC subsets, preferentially accumulate in the lung draining lymph nodes of lethal H5N1 virus-infected mice and that the induction of FasL expression on pDCs correlates with high levels of IL-12p40 monomer/homodimer in the lung draining lymph nodes. Our data suggest that one of the mechanisms of lymphopenia associated with lethal H5N1 virus infection involves a deleterious role for pDCs. PMID:24829418

  10. Glutathione Depletion Is Linked with Th2 Polarization in Mice with a Retrovirus-Induced Immunodeficiency Syndrome, Murine AIDS: Role of Proglutathione Molecules as Immunotherapeutics

    PubMed Central

    Brundu, Serena; Palma, Linda; Picceri, Giusi Giada; Ligi, Daniela; Orlandi, Chiara; Galluzzi, Luca; Chiarantini, Laura; Casabianca, Anna; Schiavano, Giuditta Fiorella; Santi, Martina; Mannello, Ferdinando; Smietana, Michaël; Magnani, Mauro

    2016-01-01

    ABSTRACT Injection of the LP-BM5 murine leukemia virus into mice causes murine AIDS, a disease characterized by many dysfunctions of immunocompetent cells. To establish whether the disease is characterized by glutathione imbalance, reduced glutathione (GSH) and cysteine were quantified in different organs. A marked redox imbalance, consisting of GSH and/or cysteine depletion, was found in the lymphoid organs, such as the spleen and lymph nodes. Moreover, a significant decrease in cysteine and GSH levels in the pancreas and brain, respectively, was measured at 5 weeks postinfection. The Th2 immune response was predominant at all times investigated, as revealed by the expression of Th1/Th2 cytokines. Furthermore, investigation of the activation status of peritoneal macrophages showed that the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arginase1, was induced. Conversely, expression of inducible nitric oxide synthase, a marker of classical activation of macrophages, was detected only when Th1 cytokines were expressed at high levels. In vitro studies revealed that during the very early phases of infection, GSH depletion and the downregulation of interleukin-12 (IL-12) p40 mRNA were correlated with the dose of LP-BM5 used to infect the macrophages. Treatment of LP-BM5-infected mice with N-(N-acetyl-l-cysteinyl)-S-acetylcysteamine (I-152), an N-acetyl-cysteine supplier, restored GSH/cysteine levels in the organs, reduced the expression of alternatively activated macrophage markers, and increased the level of gamma interferon production, while it decreased the levels of Th2 cytokines, such as IL-4 and IL-5. Our findings thus establish a link between GSH deficiency and Th1/Th2 disequilibrium in LP-BM5 infection and indicate that I-152 can be used to restore the GSH level and a balanced Th1/Th2 response in infected mice. IMPORTANCE The first report of an association between Th2 polarization and alteration of the redox state in LP-BM5

  11. The Helicobacter pylori chemotaxis receptor TlpB (HP0103) is required for pH taxis and for colonization of the gastric mucosa.

    PubMed

    Croxen, Matthew A; Sisson, Gary; Melano, Roberto; Hoffman, Paul S

    2006-04-01

    The location of Helicobacter pylori in the gastric mucosa of mammals is defined by natural pH gradients within the gastric mucus, which are more alkaline proximal to the mucosal epithelial cells and more acidic toward the lumen. We have used a microscope slide-based pH gradient assay and video data collection system to document pH-tactic behavior. In response to hydrochloric acid (HCl), H. pylori changes its swimming pattern from straight-line random swimming to arcing or circular patterns that move the motile population away from the strong acid. Bacteria in more-alkaline regions did not swim toward the acid, suggesting the pH taxis is a form of negative chemotaxis. To identify the chemoreceptor(s) responsible for the transduction of pH-tactic signals, a vector-free allelic replacement strategy was used to construct mutations in each of the four annotated chemoreceptor genes (tlpA, tlpB, tlpC, and tlpD) in H. pylori strain SS1 and a motile variant of strain KE26695. All deletion mutants were motile and displayed normal chemotaxis in brucella soft agar, but only tlpB mutants were defective for pH taxis. tlpD mutants exhibited more tumbling and arcing swimming, while tlpC mutants were hypermotile and responsive to acid. While tlpA, tlpC, and tlpD mutants colonized mice to near wild-type levels, tlpB mutants were defective for colonization of highly permissive C57BL/6 interleukin-12 (IL-12) (p40-/-)-deficient mice. Complementation of the tlpB mutant (tlpB expressed from the rdxA locus) restored pH taxis and infectivity for mice. pH taxis, like motility and urease activity, is essential for colonization and persistence in the gastric mucosa, and thus TlpB function might represent a novel target in the development of therapeutics that blind tactic behavior.

  12. A proinflammatory cytokine interleukin-32beta promotes the production of an anti-inflammatory cytokine interleukin-10.

    PubMed

    Kang, Jeong-Woo; Choi, Seung-Chul; Cho, Min-Chul; Kim, Hee-Jong; Kim, Jae-Hwa; Lim, Jong-Seok; Kim, Soo-Hyun; Han, Jae-Yong; Yoon, Do-Young

    2009-09-01

    A new proinflammatory cytokine interleukin-32 (IL-32) has six isoforms. Although IL-32 can be detected in sera from patients suffering from Crohn's disease and rheumatoid arthritis, it is unclear which isoforms are involved. To this end, we investigated the functions of the most abundant IL-32beta by generating K562-IL-32beta stable cell lines. This report confirms, using IL-32 small interfering RNA, that IL-32beta induces an anti-inflammatory cytokine IL-10 in K562-IL-32beta cells and U937 promonocytic cells, which express endogenous IL-32beta upon phorbol 12-myristate 13-acetate (PMA) treatment, and monocyte-derived dendritic cells (DC) upon lipopolysaccharide (LPS) treatment. Interleukin-32beta was induced in monocyte-derived macrophages by LPS and in monocyte-derived DC by LPS, poly(I:C), or anti-CD40 antibody, but was not induced by PMA. We showed that IL-32beta expression was increased in a time-dependent manner in monocyte-derived DC upon LPS treatment and peaked at 24 hr. Production of IL-10 was exactly coincident with IL-32beta expression, but IL-1beta and tumour necrosis factor-alpha production peaked at 6 hr after LPS treatment, then steeply declined. Interleukin-12 p40 was induced at 9 hr and gradually increased until 48 hr, at which time IL-32beta and IL-10 were no longer increased. Knock-down of IL-32beta by IL-32 small interfering RNA led to the decrease of IL-10, but the increase of IL-12 in monocyte-derived DC, which means that IL-32beta promotes IL-10 production, but limits IL-12 production. We also showed that IL-10 neutralization increases IL-12, IL-1beta and tumour necrosis factor-alpha production, which implies that IL-10 suppresses such proinflammatory cytokines. Taken together, our results suggest that IL-32beta upregulates the production of an anti-inflammatory cytokine IL-10, and then IL-10 suppresses proinflammatory cytokines.

  13. The Helicobacter pylori Chemotaxis Receptor TlpB (HP0103) Is Required for pH Taxis and for Colonization of the Gastric Mucosa

    PubMed Central

    Croxen, Matthew A.; Sisson, Gary; Melano, Roberto; Hoffman, Paul S.

    2006-01-01

    The location of Helicobacter pylori in the gastric mucosa of mammals is defined by natural pH gradients within the gastric mucus, which are more alkaline proximal to the mucosal epithelial cells and more acidic toward the lumen. We have used a microscope slide-based pH gradient assay and video data collection system to document pH-tactic behavior. In response to hydrochloric acid (HCl), H. pylori changes its swimming pattern from straight-line random swimming to arcing or circular patterns that move the motile population away from the strong acid. Bacteria in more-alkaline regions did not swim toward the acid, suggesting the pH taxis is a form of negative chemotaxis. To identify the chemoreceptor(s) responsible for the transduction of pH-tactic signals, a vector-free allelic replacement strategy was used to construct mutations in each of the four annotated chemoreceptor genes (tlpA, tlpB, tlpC, and tlpD) in H. pylori strain SS1 and a motile variant of strain KE26695. All deletion mutants were motile and displayed normal chemotaxis in brucella soft agar, but only tlpB mutants were defective for pH taxis. tlpD mutants exhibited more tumbling and arcing swimming, while tlpC mutants were hypermotile and responsive to acid. While tlpA, tlpC, and tlpD mutants colonized mice to near wild-type levels, tlpB mutants were defective for colonization of highly permissive C57BL/6 interleukin-12 (IL-12) (p40−/−)-deficient mice. Complementation of the tlpB mutant (tlpB expressed from the rdxA locus) restored pH taxis and infectivity for mice. pH taxis, like motility and urease activity, is essential for colonization and persistence in the gastric mucosa, and thus TlpB function might represent a novel target in the development of therapeutics that blind tactic behavior. PMID:16547053

  14. Risk Factors for Disseminated Coccidioidomycosis, United States

    PubMed Central

    Odio, Camila D.; Marciano, Beatriz E.; Galgiani, John N.

    2017-01-01

    Of 150,000 new coccidioidomycosis infections that occur annually in the United States, ≈1% disseminate; one third of those cases are fatal. Immunocompromised hosts have higher rates of dissemination. We identified 8 patients with disseminated coccidioidomycosis who had defects in the interleukin-12/interferon-γ and STAT3 axes, indicating that these are critical host defense pathways. PMID:28098554

  15. Mycobacterium avium Complex Cervical Lymphadenitis in an Immunocompetent Adult▿

    PubMed Central

    Christensen, Joshua B.; Koeppe, John

    2010-01-01

    Nontuberculosis mycobacterial cervical lymphadenitis is a relatively common disease in immunocompetent children but a rare disease in immunocompetent adults. We report the diagnosis and treatment of Mycobacterium avium complex cervical lymphadenitis in an adult female. Our evaluation of immune competence, including gamma interferon (IFN-γ) and interleukin-12 (IL-12) signaling, found no evidence of deficiency. PMID:20668140

  16. The transcription factor GATA3 actively represses RUNX3 protein-regulated production of interferon-gamma

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The transcription factor GATA3 is crucial for the differentiation of naive CD4+ T cells into T helper 2 (Th2) cells. Here, we show that deletion of Gata3 allowed the appearance of interferon-g (IFN-g)-producing cells in the absence of interleukin-12 (IL-12) and IFN-g. Such IFN-g production was tra...

  17. GDNF — EDRN Public Portal

    Cancer.gov

    The GDNF gene encodes a neurotrophic factor that enhances survival and morphological differentiation of dopaminergic neurons. This protein is a mature secreted homodimer. Several transcript variants encoding multiple isoforms have been identified.

  18. Reactive oxygen species derived from xanthine oxidase interrupt dimerization of breast cancer resistance protein, resulting in suppression of uric acid excretion to the intestinal lumen.

    PubMed

    Ogura, Jiro; Kuwayama, Kaori; Sasaki, Shunichi; Kaneko, Chihiro; Koizumi, Takahiro; Yabe, Keisuke; Tsujimoto, Takashi; Takeno, Reiko; Takaya, Atsushi; Kobayashi, Masaki; Yamaguchi, Hiroaki; Iseki, Ken

    2015-09-01

    The prevalence of hyperuricemia/gout increases with aging. However, the effect of aging on function for excretion of uric acid to out of the body has not been clarified. We found that ileal uric acid clearance in middle-aged rats (11-12 months) was decreased compared with that in young rats (2 months). In middle-aged rats, xanthine oxidase (XO) activity in the ileum was significantly higher than that in young rats. Inosine-induced reactive oxygen species (ROS), which are derived from XO, also decreased ileal uric acid clearance. ROS derived from XO decreased the active homodimer level of breast cancer resistance protein (BCRP), which is a uric acid efflux transporter, in the ileum. Pre-administration of allopurinol recovered the BCRP homodimer level, resulting in the recovering ileal uric acid clearance. Moreover, we investigated the effects of ROS derived from XO on BCRP homodimer level directly in Caco-2 cells using hypoxanthine. Treatment with hypoxanthine decreased BCRP homodimer level. Treatment with hypoxanthine induced mitochondrial dysfunction, suggesting that the decreasing BCRP homodimer level might be caused by mitochondrial dysfunction. In conclusion, ROS derived from XO decrease BCRP homodimer level, resulting in suppression of function for uric acid excretion to the ileal lumen. ROS derived from XO may cause the suppression of function of the ileum for the excretion of uric acid with aging. The results of our study provide a new insight into the causes of increasing hyperuricemia/gout prevalence with aging.

  19. The subunit interfaces of weakly associated homodimeric proteins.

    PubMed

    Dey, Sucharita; Pal, Arumay; Chakrabarti, Pinak; Janin, Joël

    2010-04-23

    We analyzed subunit interfaces in 315 homodimers with an X-ray structure in the Protein Data Bank, validated by checking the literature for data that indicate that the proteins are dimeric in solution and that, in the case of the "weak" dimers, the homodimer is in equilibrium with the monomer. The interfaces of the 42 weak dimers, which are smaller by a factor of 2.4 on average than in the remainder of the set, are comparable in size with antibody-antigen or protease-inhibitor interfaces. Nevertheless, they are more hydrophobic than in the average transient protein-protein complex and similar in amino acid composition to the other homodimer interfaces. The mean numbers of interface hydrogen bonds and hydration water molecules per unit area are also similar in homodimers and transient complexes. Parameters related to the atomic packing suggest that many of the weak dimer interfaces are loosely packed, and we suggest that this contributes to their low stability. To evaluate the evolutionary selection pressure on interface residues, we calculated the Shannon entropy of homologous amino acid sequences at 60% sequence identity. In 93% of the homodimers, the interface residues are better conserved than the residues on the protein surface. The weak dimers display the same high degree of interface conservation as other homodimers, but their homologs may be heterodimers as well as homodimers. Their interfaces may be good models in terms of their size, composition, and evolutionary conservation for the labile subunit contacts that allow protein assemblies to share and exchange components, allosteric proteins to undergo quaternary structure transitions, and molecular machines to operate in the cell.

  20. Involvement of the cytoplasmic cysteine-238 of CD40 in its up-regulation of CD23 expression and its enhancement of TLR4-triggered responses.

    PubMed

    Nadiri, Amal; Jundi, Malek; El Akoum, Souhad; Hassan, Ghada S; Yacoub, Daniel; Mourad, Walid

    2015-11-01

    CD40, a member of the tumor necrosis factor receptor superfamily, plays a key role in both adaptive and innate immunity. Engagement of CD40 with its natural trimeric ligand or with cross-linked antibodies results in disulfide-linked CD40 (dl-CD40) homodimer formation, a process mediated by the cysteine-238 residues of the cytoplasmic tail of CD40. The present study was designed to elucidate the biological relevance of cysteine-238-mediated dl-CD40 homodimers to the expression of CD23 on B cells and to investigate its possible involvement in the innate response. Our results indicate that cysteine-238-mediated dl-CD40 homodimerization is required for CD40-induced activation of PI3-kinase/Akt signaling and the subsequent CD23 expression, as inhibition of dl-CD40 homodimer formation through a point mutation-approach specifically impairs these responses. Interestingly, cysteine-238-mediated dl-CD40 homodimers are also shown to play a crucial role in Toll-like receptor 4-induced CD23 expression, further validating the importance of this system in bridging innate and adaptive immune responses. This process also necessitates the activation of the PI3-kinase/Akt cascade. Thus, our results highlight new roles for CD40 and cysteine-238-mediated CD40 homodimers in cell biology and identify a potential new target for therapeutic strategies against CD40-associated chronic inflammatory diseases.

  1. A CpG oligonucleotide can protect mice from a low aerosol challenge dose of Burkholderia mallei.

    PubMed

    Waag, David M; McCluskie, Michael J; Zhang, Ningli; Krieg, Arthur M

    2006-03-01

    Treatment with an oligodeoxynucleotide (ODN) containing CPG motifs (CpG ODN 7909) was found to protect BALB/c mice from lung infection or death after aerosol challenge with Burkholderia mallei. Protection was associated with enhanced levels of gamma interferon (IFN-gamma)-inducible protein 10, interleukin-12 (IL-12), IFN-gamma, and IL-6. Preexposure therapy with CpG ODNs may protect victims of a biological attack from glanders.

  2. Crystal structure of the MazE/MazF complex: molecular bases of antidote-toxin recognition.

    PubMed

    Kamada, Katsuhiko; Hanaoka, Fumio; Burley, Stephen K

    2003-04-01

    A structure of the Escherichia coli chromosomal MazE/MazF addiction module has been determined at 1.7 A resolution. Addiction modules consist of stable toxin and unstable antidote proteins that govern bacterial cell death. MazE (antidote) and MazF (toxin) form a linear heterohexamer composed of alternating toxin and antidote homodimers (MazF(2)-MazE(2)-MazF(2)). The MazE homodimer contains a beta barrel from which two extended C termini project, making interactions with flanking MazF homodimers that resemble the plasmid-encoded toxins CcdB and Kid. The MazE/MazF heterohexamer structure documents that the mechanism of antidote-toxin recognition is common to both chromosomal and plasmid-borne addiction modules, and provides general molecular insights into toxin function, antidote degradation in the absence of toxin, and promoter DNA binding by antidote/toxin complexes.

  3. Genetically engineered photoinducible homodimerization system with improved dimer-forming efficiency.

    PubMed

    Nihongaki, Yuta; Suzuki, Hideyuki; Kawano, Fuun; Sato, Moritoshi

    2014-03-21

    Vivid (VVD) is a photoreceptor derived from Neurospora Crassa that rapidly forms a homodimer in response to blue light. Although VVD has several advantages over other photoreceptors as photoinducible homodimerization system, VVD has a critical limitation in its low dimer-forming efficiency. To overcome this limitation of wild-type VVD, here we conduct site-directed saturation mutagenesis in the homodimer interface of VVD. We have found that the Ile52Cys mutation of VVD (VVD-52C) substantially improves its homodimer-forming efficiency up to 180%. We have demonstrated the utility of VVD-52C for making a light-inducible gene expression system more robust. In addition, using VVD-52C, we have developed photoactivatable caspase-9, which enables optical control of apoptosis of mammalian cells. The present genetically engineered photoinducible homodimerization system can provide a powerful tool to optically control a broad range of molecular processes in the cell.

  4. Evidence for Homodimerization of the c-Fos Transcription Factor in Live Cells Revealed by Fluorescence Microscopy and Computer Modeling.

    PubMed

    Szalóki, Nikoletta; Krieger, Jan Wolfgang; Komáromi, István; Tóth, Katalin; Vámosi, György

    2015-11-01

    The c-Fos and c-Jun transcription factors, members of the activator protein 1 (AP-1) complex, form heterodimers and bind to DNA via a basic leucine zipper and regulate the cell cycle, apoptosis, differentiation, etc. Purified c-Jun leucine zipper fragments could also form stable homodimers, whereas c-Fos leucine zipper homodimers were found to be much less stable in earlier in vitro studies. The importance of c-Fos overexpression in tumors and the controversy in the literature concerning c-Fos homodimerization prompted us to investigate Fos homodimerization. Förster resonance energy transfer (FRET) and molecular brightness analysis of fluorescence correlation spectroscopy data from live HeLa cells transfected with fluorescent-protein-tagged c-Fos indicated that c-Fos formed homodimers. We developed a method to determine the absolute concentrations of transfected and endogenous c-Fos and c-Jun, which allowed us to determine dissociation constants of c-Fos homodimers (Kd = 6.7 ± 1.7 μM) and c-Fos-c-Jun heterodimers (on the order of 10 to 100 nM) from FRET titrations. Imaging fluorescence cross-correlation spectroscopy (SPIM-FCCS) and molecular dynamics modeling confirmed that c-Fos homodimers were stably associated and could bind to the chromatin. Our results establish c-Fos homodimers as a novel form of the AP-1 complex that may be an autonomous transcription factor in c-Fos-overexpressing tissues and could contribute to tumor development.

  5. Evolutionary and structural analyses of heterodimeric proteins composed of subunits with same fold.

    PubMed

    Sudha, Govindarajan; Naveenkumar, Nagarajan; Srinivasan, Narayanaswamy

    2015-10-01

    Heterodimeric proteins with homologous subunits of same fold are involved in various biological processes. The objective of this study is to understand the evolution of structural and functional features of such heterodimers. Using a non-redundant dataset of 70 such heterodimers of known 3D structure and an independent dataset of 173 heterodimers from yeast, we note that the mean sequence identity between interacting homologous subunits is only 23-24% suggesting that, generally, highly diverged paralogues assemble to form such a heterodimer. We also note that the functional roles of interacting subunits/domains are generally quite different. This suggests that, though the interacting subunits/domains are homologous, the high evolutionary divergence characterize their high functional divergence which contributes to a gross function for the heterodimer considered as a whole. The inverse relationship between sequence identity and RMSD of interacting homologues in heterodimers is not followed. We also addressed the question of formation of homodimers of the subunits of heterodimers by generating models of fictitious homodimers on the basis of the 3D structures of the heterodimers. Interaction energies associated with these homodimers suggests that, in overwhelming majority of the cases, such homodimers are unlikely to be stable. Majority of the homologues of heterodimers of known structures form heterodimers (51.8%) and a small proportion (14.6%) form homodimers. Comparison of 3D structures of heterodimers with homologous homodimers suggests that interfacial nature of residues is not well conserved. In over 90% of the cases we note that the interacting subunits of heterodimers are co-localized in the cell.

  6. On the Pyrazine and Pyrazine-Pyrimidine Dimers.

    DTIC Science & Technology

    1986-06-01

    Lennard - Jones -hydrogen-bonding (LJ-HB) potential energy calculations. The pyrazine isotopic hetero- and homo-dimers possess nearly identical spectra with the exception that the perpendicular dimer features are displaced to the red by approx. 11 cm. Exchange or exciton interactions in this system are vanishingly small (less than 1/cm). The geometrics suggested by the isotopically substituted pyrazine dimer spectra are the same as those found for the pyrazine-h sub 4 homo-dimer: a parallel planar hydrogen bonded and a perpendicular dimer. The pyrazine-h sub 4 and pyrazine-h

  7. Synthesis and STM imaging of symmetric and dissymmetric ethynyl-bridged dimers of boron-subphthalocyanine bowl-shaped nanowheels.

    PubMed

    Jacquot de Rouville, Henri-Pierre; Garbage, Romain; Ample, Francisco; Nickel, Anja; Meyer, Joerg; Moresco, Francesca; Joachim, Christian; Rapenne, Gwénaël

    2012-07-16

    The future's wheel: A new class of wheels, based on subphthalocyanine fragments, for future incorporation in functional nanovehicles is reported (see figure). The syntheses of a symmetric wheel, a nitrogen-tagged wheel, and their ethynyl-bridged homodimers are presented. Theoretical calculations and STM imaging demonstrate the advantage of a bowl-shaped structure and the efficiency of the tag for STM imaging.

  8. The neuropeptide bursicon acts in cuticle metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bursicon is a heterodimeric neuropeptide formed of bursicon a (burs a) and bursicon B (burs B) that controls cuticle tanning and wing expansion in insects. Burs a-a and burs B-B homodimers are also formed; they act via an unknown receptor to induce expression of prophylactic immune and stress genes ...

  9. Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex

    PubMed Central

    Ohashi, Yohei; Soler, Nicolas; García Ortegón, Miguel; Zhang, Lufei; Kirsten, Marie L.; Perisic, Olga; Masson, Glenn R.; Burke, John E.; Jakobi, Arjen J.; Apostolakis, Apostolos A.; Johnson, Christopher M.; Ohashi, Maki; Ktistakis, Nicholas T.; Sachse, Carsten; Williams, Roger L.

    2016-01-01

    ABSTRACT The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies. PMID:27630019

  10. More than meets the dimer: What is the quaternary structure of the glucocorticoid receptor?

    PubMed Central

    Hager, Gordon L.

    2017-01-01

    ABSTRACT It is widely accepted that the glucocorticoid receptor (GR), a ligand-regulated transcription factor that triggers anti-inflammatory responses, binds specific response elements as a homodimer. Here, we will discuss the original primary data that established this model and contrast it with a recent report characterizing the GR–DNA complex as a tetramer. PMID:27764575

  11. Substitution of specific cysteine residues in E1 glycoprotein of classical swine fever virus strain Brescia affects formation of E1-E2 heterodimers and alters virulence in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E1, along with E^rns and E2, is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). E1 and E2 are anchored to the virus envelope at their carboxyl termini and E^rns loosely associates with the viral envelope. In infected cells, E2 forms homodimers and heterodimers with E1,...

  12. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E2, along with E^rns and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions including cell attachment, host range susceptibility and virulence in natural hosts. In infected cells, E2 forms homodimers as well as heterodimers with E1, media...

  13. ADH5 — EDRN Public Portal

    Cancer.gov

    ADH5 is a member of the alcohol dehydrogenase family. This protein forms a homodimer. ADH5 is ineffective in oxidizing ethanol, but exhibits high activity for oxidation of long-chain primary alcohols and for oxidation of S-hydroxymethyl-glutathione, a spontaneous adduct between formaldehyde and glutathione. There are several non-transcribed pseuodogenes related to this gene.

  14. The cytoplasmic PASC domain of the sensor kinase DcuS of Escherichia coli: role in signal transduction, dimer formation, and DctA interaction

    PubMed Central

    Monzel, Christian; Degreif-Dünnwald, Pia; Gröpper, Christina; Griesinger, Christian; Unden, Gottfried

    2013-01-01

    The cytoplasmic PASC domain of the fumarate responsive sensor kinase DcuS of Escherichia coli links the transmembrane to the kinase domain. PASC is also required for interaction with the transporter DctA serving as a cosensor of DcuS. Earlier studies suggested that PASC functions as a hinge and transmits the signal to the kinase. Reorganizing the PASC dimer interaction and, independently, removal of DctA, converts DcuS to the constitutive ON state (active without fumarate stimulation). ON mutants were categorized with respect to these two biophysical interactions and the functional state of DcuS: type I-ON mutations grossly reorganize the homodimer, and decrease interaction with DctA. Type IIA-ON mutations create the ON state without grossly reorganizing the homodimer, whereas interaction with DctA is decreased. The type IIB-ON mutations were neither in PASC/PASC, nor in DctA/DcuS interaction affected, similar to fumarate activated wild-typic DcuS. OFF mutations never affected dimer stability. The ON mutations provide novel mechanistic insight: PASC dimerization is essential to silence the kinase. Reorganizing the homodimer and its interaction with DctA activate the kinase. The study suggests a novel ON homo-dimer conformation (type IIB) and an OFF conformation for PASC. Type IIB-ON corresponds to the fumarate induced wild-type conformation, representing an interesting target for structural biology. PMID:24039243

  15. A Non-Nuclear Role of the Estrogen Receptor Alpha in the Regulation of Cell-Cell Interactions

    DTIC Science & Technology

    2007-08-01

    the absence of E2, staining for E-cadherin/j3-catenin and ERu did not overlap, suggesting that ERu does not interact with these junctions (Fig. 5...vitro expressed ERa. As shown in figure 7 A, ERa interacted preferably with the a-catenin homodimer. A role of ERu in F-actin cytoskeleton remodeling

  16. Biological Sensors Using DNA Functionalized Multiwalled Carbon Nanotubes

    DTIC Science & Technology

    2009-10-01

    hydrodynamic voltammetry and the results have been discussed. 5 2. Experimental Methods Reagents GOD (EC 1.1.3.4, Aspergillus niger , >100 U...is of practical use, stable and inexpensive. GOD from Aspergillus , is a homodimer containing two tightly bound flavine adenine dinucleotide (FAD

  17. Diversification of Paralogous α-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae

    PubMed Central

    Quezada, Héctor; Duhne, Mariana; González, James; Lezama, Mijail; El-Hafidi, Mohammed; Colón, Maritrini; Martínez de la Escalera, Ximena; Flores-Villegas, Mirelle Citlali; Scazzocchio, Claudio; DeLuna, Alexander; González, Alicia

    2015-01-01

    Production of α-isopropylmalate (α-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeast Saccharomyces cerevisiae has two paralogous genes, LEU4 and LEU9, that encode α-IPM synthase (α-IPMS) isozymes. Little is known about the biochemical differences between these two α-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. The leu4Δ mutant, which expresses only the feedback-resistant Leu9 homodimer, grows slowly with either glucose or ethanol and accumulates elevated pools of leucine; this phenotype is alleviated by the addition of leucine. Transformation of the leu4Δ mutant with a centromeric plasmid carrying LEU4 restored the wild-type phenotype. Bimolecular fluorescent complementation analysis showed that Leu4-Leu9 heterodimeric isozymes are formed in vivo. Purification and kinetic analysis showed that the hetero-oligomeric isozyme has a distinct leucine sensitivity behavior. Determination of α-IPMS activity in ethanol-grown cultures showed that α-IPM biosynthesis and growth under these respiratory conditions depend on the feedback-sensitive Leu4 homodimer. We conclude that retention and further diversification of two yeast α-IPMSs have resulted in a specific regulatory system that controls the leucine–α-IPM biosynthetic pathway by selective feedback sensitivity of homomeric and heterodimeric isoforms. PMID:25841022

  18. Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies.

    PubMed

    Herrera, Cristina; Tremblay, Jacqueline M; Shoemaker, Charles B; Mantis, Nicholas J

    2015-11-13

    Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax, and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC50 values in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors.

  19. Crystal structure and assembly of the functional Nanoarchaeum equitans tRNA splicing endonuclease

    SciTech Connect

    Mitchell, Michelle; Xue, Song; Erdman, Rachel; Randau, Lennart; Söll, Dieter; Li, Hong

    2009-10-27

    The RNA splicing and processing endonuclease from Nanoarchaeum equitans (NEQ) belongs to the recently identified ({alpha}{beta}){sub 2} family of splicing endonucleases that require two different subunits for splicing activity. N. equitans splicing endonuclease comprises the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Here, we report the crystal structure of the functional NEQ enzyme at 2.1 {angstrom} containing both subunits, as well as that of the NEQ261 subunit alone at 2.2 {angstrom}. The functional enzyme resembles previously known {alpha}{sub 2} and {alpha}{sub 4} endonucleases but forms a heterotetramer: a dimer of two heterodimers of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Surprisingly, NEQ261 alone forms a homodimer, similar to the previously known homodimer of the catalytic subunit. The homodimers of isolated subunits are inhibitory to heterodimerization as illustrated by a covalently linked catalytic homodimer that had no RNA cleavage activity upon mixing with the structural subunit. Detailed structural comparison reveals a more favorable hetero- than homodimerization interface, thereby suggesting a possible regulation mechanism of enzyme assembly through available subunits. Finally, the uniquely flexible active site of the NEQ endonuclease provides a possible explanation for its broader substrate specificity.

  20. Unwinding the differences of the mammalian PERIOD clock proteins from crystal structure to cellular function.

    PubMed

    Kucera, Nicole; Schmalen, Ira; Hennig, Sven; Öllinger, Rupert; Strauss, Holger M; Grudziecki, Astrid; Wieczorek, Caroline; Kramer, Achim; Wolf, Eva

    2012-02-28

    The three PERIOD homologues mPER1, mPER2, and mPER3 constitute central components of the mammalian circadian clock. They contain two PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B), which mediate homo- and heterodimeric mPER-mPER interactions as well as interactions with transcription factors and kinases. Here we present crystal structures of PAS domain fragments of mPER1 and mPER3 and compare them with the previously reported mPER2 structure. The structures reveal homodimers, which are mediated by interactions of the PAS-B β-sheet surface including a highly conserved tryptophan (Trp448(mPER1), Trp419(mPER2), Trp359(mPER3)). mPER1 homodimers are additionally stabilized by interactions between the PAS-A domains and mPER3 homodimers by an N-terminal region including a predicted helix-loop-helix motive. We have verified the existence of these homodimer interfaces in solution and inside cells using analytical gel filtration and luciferase complementation assays and quantified their contributions to homodimer stability by analytical ultracentrifugation. We also show by fluorescence recovery after photobleaching analyses that destabilization of the PAS-B/tryptophan dimer interface leads to a faster mobility of mPER2 containing complexes in human U2OS cells. Our study reveals structural and quantitative differences between the homodimeric interactions of the three mouse PERIOD homologues, which are likely to contribute to their distinct clock functions.

  1. Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium.

    PubMed

    Banerji, Suneale; Lawrance, William; Metcalfe, Clive; Briggs, David C; Yamauchi, Akira; Dushek, Omer; van der Merwe, P Anton; Day, Anthony J; Jackson, David G

    2016-11-25

    The lymphatic vessel endothelial receptor LYVE-1 is implicated in the uptake of hyaluronan (HA) and trafficking of leukocytes to draining lymph nodes. Yet LYVE-1 has only weak affinity for hyaluronan and depends on receptor clustering and higher order ligand organization for durable binding in lymphatic endothelium. An unusual feature of LYVE-1 not found in other HA receptors is the potential to form disulfide-linked homodimers. However, their influence on function has not been investigated. Here we show LYVE-1 homodimers are the predominant configuration in lymphatic endothelium in vitro and in vivo, and formation solely requires the unpaired cysteine residue Cys-201 within the membrane-proximal domain, yielding a 15-fold higher HA binding affinity and an ∼67-fold slower off-rate than the monomer. Moreover, we show non-dimerizing LYVE-1 mutants fail to bind HA even when expressed at high densities in lymphatic endothelial cells or artificially cross-linked with antibody. Consistent with these findings, small angle X-ray scattering (SAXS) indicates the Cys-201 interchain disulfide forms a hinge that maintains the homodimer in an "open scissors" conformation, likely allowing arrangement of the two HA binding domains for mutual engagement with ligand. Finally, we demonstrate the Cys-201 interchain disulfide is highly labile, and selective reduction with TCEP-HCl disrupts LYVE-1 homodimers, ablating HA binding. These findings reveal binding is dependent not just on clustering but also on the biochemical properties of LYVE-1 homodimers. They also mark LYVE-1 as the first Link protein superfamily member requiring covalent homodimerization for function and suggest the interchain disulfide acts as a redox switch in vivo.

  2. Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium*

    PubMed Central

    Banerji, Suneale; Lawrance, William; Metcalfe, Clive; Briggs, David C.; Yamauchi, Akira; Dushek, Omer; van der Merwe, P. Anton

    2016-01-01

    The lymphatic vessel endothelial receptor LYVE-1 is implicated in the uptake of hyaluronan (HA) and trafficking of leukocytes to draining lymph nodes. Yet LYVE-1 has only weak affinity for hyaluronan and depends on receptor clustering and higher order ligand organization for durable binding in lymphatic endothelium. An unusual feature of LYVE-1 not found in other HA receptors is the potential to form disulfide-linked homodimers. However, their influence on function has not been investigated. Here we show LYVE-1 homodimers are the predominant configuration in lymphatic endothelium in vitro and in vivo, and formation solely requires the unpaired cysteine residue Cys-201 within the membrane-proximal domain, yielding a 15-fold higher HA binding affinity and an ∼67-fold slower off-rate than the monomer. Moreover, we show non-dimerizing LYVE-1 mutants fail to bind HA even when expressed at high densities in lymphatic endothelial cells or artificially cross-linked with antibody. Consistent with these findings, small angle X-ray scattering (SAXS) indicates the Cys-201 interchain disulfide forms a hinge that maintains the homodimer in an “open scissors” conformation, likely allowing arrangement of the two HA binding domains for mutual engagement with ligand. Finally, we demonstrate the Cys-201 interchain disulfide is highly labile, and selective reduction with TCEP-HCl disrupts LYVE-1 homodimers, ablating HA binding. These findings reveal binding is dependent not just on clustering but also on the biochemical properties of LYVE-1 homodimers. They also mark LYVE-1 as the first Link protein superfamily member requiring covalent homodimerization for function and suggest the interchain disulfide acts as a redox switch in vivo. PMID:27733683

  3. Activation of P13K/PKB Signaling in Breast Cancer may Inhibit TGFB-Induced G1 Arrest through Changes in p27 Function

    DTIC Science & Technology

    2004-10-01

    reactions were then diluted with 500 [1l of ice-cold Nonidet P-40 lysis buffer (0.5% Nondet P-40, 50mM Tris [pH7.5], 150 mM NaCl, 1 mM Phenylmethylsulfonyl...lysed in an ice-cold Nondet P40 lysis buffer. Cell lysates were then precleared with normal rabbit IgG prior to immunoprecipitation of either cyclin D1

  4. Development and Evaluation of an Enzyme-Linked Immunosorbent Assay for Plasmodium vivax-VK247 Sporozoites

    DTIC Science & Technology

    1992-01-01

    0.05% Nonidet P40 (Sigma Chemical Co., St. search, Washington. DC 20307-5100. Louis, MO). The blocking buffer was prepared 3 Tropical Health Program...MAbs were conjugated to peptide or Nonidet P-40 treated sporozoites. horseradish peroxidase, aliquoted, and lyophyl- When testing sporozoite... Nonidet P-40-treated sporozoites, synthetic VK247, and recombinant VK210 circumsporo- zoite proteins (Monoclonal antibody concentrations: VK247

  5. Transcriptional Inducers of Acetylcholinesterase Expression as Novel Antidotes for Protection Against Chemical Warfare Agents

    DTIC Science & Technology

    2005-10-01

    7 days. B. Extracellular AChE expression determined by microassay of 20 µl culture supernatant. C. Cells were lysed with 1% nonidet - P40 and the...culture supernatant. B. Cells were lysed with 1% nonidet - P40 and the intracellular BChE expression was determined by using 50 µl cell lysate. All...was removed and the cells were washed with phosphate buffered saline (PBS) and lysed using ice-cold phosphate buffer containing 1% Nonidet P-40 or

  6. A LGG-derived protein promotes IgA production through up-regulation of APRIL expression in intestinal epithelial cells

    PubMed Central

    Wang, Yang; Liu, Liping; Moore, Daniel J; Shen, Xi; Peek, Richard M.; Acra, Sari A; Li, Hui; Ren, Xiubao; Polk, D Brent; Yan, Fang

    2016-01-01

    p40, a Lactobacillus rhamnosus GG (LGG)-derived protein, transactivates epidermal growth factor receptor (EGFR) in intestinal epithelial cells, leading to amelioration of intestinal injury and inflammation. To elucidate mechanisms by which p40 regulates mucosal immunity to prevent inflammation, this study aimed to determine the effects and mechanisms of p40 on regulation of a proliferation-inducing ligand (APRIL) expression in intestinal epithelial cells for promoting IgA production. p40 up-regulated April gene expression and protein production in mouse small intestine epithelial (MSIE) cells, which were inhibited by blocking EGFR expression and kinase activity. Enteroids from Egfrfl/fl , but not Egfrfl/fl-Vil-Cre mice with EGFR specifically deleted in intestinal epithelial cells, exhibited increased April gene expression by p40 treatment. p40-conditioned media from MSIE cells increased B cell class switching to IgA+ cells and IgA production, which was suppressed by APRIL receptor neutralizing antibodies. Treatment of B cells with p40 did not show any effects on IgA production. p40 treatment increased April gene expression and protein production in small intestinal epithelial cells, fecal IgA levels, IgA+B220+, IgA+CD19+, and IgA+ plasma cells in lamina propria of Egfrfl/fl, but not Egfrfl/fl-Vil-Cre mice. Thus, p40 up-regulates EGFR-dependent APRIL production in intestinal epithelial cells, which may contribute to promoting IgA production. PMID:27353252

  7. Mucin (MUC1) Expression and Function in Prostate Cancer Cells

    DTIC Science & Technology

    2004-03-01

    P40 containing 1 mM EDTA, and a cocktail of serine IP: C1 tr Ct tr t c1 Itf Ci protease inhibitors for 5min, 0.5% Nonidet P40 "" II...containing no inhibitors at 4°C for 1 h or 0.5% Nonidet "" .0 P40 containing 1 mM EDTA and a cocktail of serine protease inhibitors at 4VC for 1 h. Neither...intracellular were prepared from confluent cultures by addition of 0.5% (v/v) processing and delivery to the cell surface. Although the Nonidet P-40 in

  8. Regulation of Intracellular Signaling Leading to Gene Expression in Lipopolysaccharide Stimulated Murine Macrophages

    DTIC Science & Technology

    1995-09-20

    oxide N02- - nitrite NP-40 - Nonidet P-40 p40 - inducible chain of Interleukin-l2 PAGE - polyacrylamide gel electrophoresis PKC - Protein kinase...treated as indicated with 150 nM okadaic acid, and/or 100 ng/ml LPS Figure 26. RT-PCR analysis of lFN-P, IL-6, !L-10, p40 and GAPDH gene 114 expression...major cellular source of IL-1P, lL-6, lL-10, lL-12 (the inducible chain, p40 ), and TNF-a mRNA produced in the liver (Salkowski et al., 1995

  9. Decreased serum IL-27 and IL-35 levels are associated with disease severity in neuromyelitis optica spectrum disorders.

    PubMed

    Zhang, Da-Qi; Jia, Kun; Wang, Rong; Li, Ting; Zhao, Ning; Yang, Li-Na; Yang, Li

    2016-04-15

    The interleukin 12 (IL-12) family plays important roles in autoimmune diseases. To explore the roles of the IL-12 family members IL-27 and IL-35 in the pathogenesis of neuromyelitis optica spectrum disorders (NMOSD), we determined their serum and cerebral spinal fluid levels and assessed potential correlations with clinical characteristics. Serum IL-27 levels were negatively correlated with disease severity and spinal cord lesion length, while serum IL-35 levels were negatively correlated with disease severity and annual relapse rate. Thus, IL-27 and IL-35 may be important biomarkers of NMOSD severity and these molecules might represent potential therapeutic cytokines for treating NMOSD.

  10. Plasmid IL-12 electroporation in melanoma

    PubMed Central

    Cha, Edward; Daud, Adil

    2012-01-01

    Intratumoral gene electroporation uses electric charges to facilitate entry of plasmid DNA into cells in a reproducible and highly efficient manner, especially to accessible sites such as cutaneous and subcutaneous melanomas. Effective for locally treated disease, electroporation of plasmid DNA encoding interleukin-12 can also induce responses in untreated distant disease, suggesting that adaptive immune responses are being elicited that can target melanoma-associated antigens. In vivo electroporation with immunomodulatory cytokine DNA is a promising approach that can trigger systemic anti-tumor immune responses without the systemic toxicity associated with intravenous cytokine delivery and potentially offer complete long-term tumor regression. PMID:23151447

  11. Immunologic Approaches for Oncolytic Viral Therapy of Prostate Cancer

    DTIC Science & Technology

    2005-02-01

    12 and interferon alfa - 2b : phase I trial of patients with metastatic renal cell carcinoma or malignant melanoma. J Clin Oncol. 22: 2891-2900. 6...Alatrash G., et al. (2004). Clinical and immunologic effects of subcutaneously administered interleukin-12 and interferon alfa - 2b : phase I trial of...tumor growth monitored. As illustrated in Fig 2B , NV1023 was significantly more effective than G207, G47A or mock (pɘ.05, student’s t test; days

  12. Interleukin-17 inhibitors. A new era in treatment of psoriasis and other skin diseases

    PubMed Central

    Wasilewska, Agnieszka; Winiarska, Marta; Olszewska, Małgorzata

    2016-01-01

    Psoriasis is a chronic skin disease caused by the excessive secretion of inflammatory cytokines. Available therapeutic options include biologic drugs such as tumor necrosis factor alpha inhibitors and interleukin 12/23 (IL-12/23) inhibitors. The recent discovery of IL-17, which contributes to development of psoriasis, opened new possibilities for further treatment modalities. Currently, one anti-IL17 biological agent is approved for the treatment – a fully human monoclonal antibody that targets IL-17A (secukinumab). Further clinical trials, including a humanized IgG4 specific for IL-17 (ixekizumab) and a fully human antibody that targets the IL-17 receptor A (brodalumab). PMID:27605893

  13. Getting from the bench to the patient: biotechnology strategies.

    PubMed

    Youssoufian, Hagop; Lewis, Jonathan

    2013-10-01

    Despite significant advances in understanding of molecular pathobiology, the adoption of this knowledge and its application to drug development is sporadic. Consequently, the drug development process has remained intrinsically laborious and inefficient. Translational research seeks to improve this process by integrating scientific advances into well-defined clinical challenges and opportunities. The focus of this article is on cancer therapeutics, specifically, 2 biotechnology organizations' advances in oncology: (1) optimizing the safety and efficacy of a novel DNA cross-linking small molecule, palifosfamide, and (2) bringing synthetic biology into clinical practice using a controllable gene therapy strategy with intratumorally injected interleukin-12 DNA, a potent cytokine.

  14. Identification and expression analysis of two interleukin-23α (p19) isoforms, in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar.

    PubMed

    Jiang, Yousheng; Husain, Mansourah; Qi, Zhitao; Bird, Steve; Wang, Tiehui

    2015-08-01

    Interleukin (IL)-23 is a heterodimeric IL-12 family cytokine composed of a p19 α-chain, linked to a p40 β-chain that is shared with IL-12. IL-23 is distinguished functionally from IL-12 by its ability to induce the production of IL-17, and differentiation of Th17 cells in mammals. Three isoforms of p40 (p40a, p40b and p40c) have been found in some 3R teleosts. Salmonids also possess three p40 isoforms (p40b1, p40b2 and p40c) although p40a is missing, and two copies (paralogues) of p40b are present that have presumably been retained following the 4R duplication in this fish lineage. Teleost p19 has been discovered recently in zebrafish, but to date there is limited information on expression and modulation of this molecule. In this report we have cloned two p19 paralogues (p19a and p19b) in salmonids, suggesting that a salmonid can possess six potential IL-23 isoforms. Whilst Atlantic salmon has two active p19 genes, the rainbow trout p19b gene may have been pseudogenized. The salmonid p19 translations share moderate identities (22.8-29.9%) to zebrafish and mammalian p19 molecules, but their identity was supported by structural features, a conserved 4 exon/3 intron gene organisation, and phylogenetic tree analysis. The active salmonid p19 genes are highly expressed in blood and gonad. Bacterial (Yersinia ruckeri) and viral infection in rainbow trout induces the expression of p19a, suggesting pathogen-specific induction of IL-23 isoforms. Trout p19a expression was also induced by PAMPs (poly IC and peptidoglycan) and the proinflammatory cytokine IL-1β in primary head kidney macrophages. These data may indicate diverse functional roles of trout IL-23 isoforms in regulating the immune response in fish.

  15. IL12B expression is sustained by a heterogenous population of myeloid lineages during tuberculosis

    PubMed Central

    Reeme, Allison E.; Miller, Halli E.; Robinson, Richard T.

    2015-01-01

    Summary IL12B is required for resistance to Mycobacterium tuberculosis (Mtb) infection, promoting the initiation and maintenance of Mtb-specific effector responses. While this makes the IL12-pathway an attractive target for experimental tuberculosis (TB) therapies, data regarding what lineages express IL12B after infection is established are limited. This is not obvious in the lung, an organ in which both hematopoietic and non-hematopoietic lineages produce IL12p40 upon pathogen encounter. Here, we use radiation bone marrow chimeras and Yet40 reporter mice to determine what lineages produce IL12p40 during experimental TB. We observed that hematopoietic IL12p40-production was sufficient to control Mtb, with no contribution by non-hematopoietic lineages. Furthermore, rather than being produced by a single subset, IL12p40 was produced by cells that were heterogenous in their size, granularity, autofluorescence and expression of CD11c, CD11b and CD8α. While depending on the timepoint and tissue examined, the surface phenotype of IL12p40-producers most closely resembled macrophages based on previous surveys of lung myeloid lineages. Importantly, depletion of CDllchi cells during infection had no affect on lung IL12p40-concentrations. Collectively, our data demonstrate that IL12p40 production is sustained by a heterogenous population of myeloid lineages during experimental TB, and that redundant mechanisms of IL12p40-production exist when CD11chi lineages are absent. PMID:23491716

  16. Actions of Tamoxifen and Estrogen on Osteoblast Protein Kinase C Expression.

    DTIC Science & Technology

    1996-07-01

    phosphate buffered saline (PBS), 1% Nonidet - P40 (NP40), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.1 mg/ml phenylmethylsulfonyl...saline (PBS), 1% Nonidet - P40 (NP40), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.1 mg/ml phenylmethylsulfonyl fluoride (PMSF), 0.15

  17. IL12B expression is sustained by a heterogenous population of myeloid lineages during tuberculosis.

    PubMed

    Reeme, Allison E; Miller, Halli E; Robinson, Richard T

    2013-05-01

    IL12B is required for resistance to Mycobacterium tuberculosis (Mtb) infection, promoting the initiation and maintenance of Mtb-specific effector responses. While this makes the IL12-pathway an attractive target for experimental tuberculosis (TB) therapies, data regarding what lineages express IL12B after infection is established are limited. This is not obvious in the lung, an organ in which both hematopoietic and non-hematopoietic lineages produce IL12p40 upon pathogen encounter. Here, we use radiation bone marrow chimeras and Yet40 reporter mice to determine what lineages produce IL12p40 during experimental TB. We observed that hematopoietic IL12p40-production was sufficient to control Mtb, with no contribution by non-hematopoietic lineages. Furthermore, rather than being produced by a single subset, IL12p40 was produced by cells that were heterogenous in their size, granularity, autofluorescence and expression of CD11c, CD11b and CD8α. While depending on the timepoint and tissue examined, the surface phenotype of IL12p40-producers most closely resembled macrophages based on previous surveys of lung myeloid lineages. Importantly, depletion of CD11c(hi) cells during infection had no affect on lung IL12p40-concentrations. Collectively, our data demonstrate that IL12p40 production is sustained by a heterogenous population of myeloid lineages during experimental TB, and that redundant mechanisms of IL12p40-production exist when CD11c(hi) lineages are absent.

  18. Differential Growth Inhibition of Cerebral Metastases by Anti-angiogenic Compounds

    PubMed Central

    MARTIN, DANIEL K.; UCKERMANN, ORTRUD; BERTRAM, AIKO; LIEBNER, CORINA; HENDRUSCHK, SANDY; SITOCI-FICICI, KERIM HAKAN; SCHACKERT, GABRIELE; LORD, EDITH M.; TEMME, ACHIM; KIRSCH, MATTHIAS

    2015-01-01

    Background The formation of brain metastases is intrinsically linked to concomitant angiogenesis. The purpose of the present study was to investigate the combined effects of interleukin-12 (IL-12) and EMD121974 on the growth and distribution of melanoma brain metastases since both substances may interact with important steps in the cascade of brain metastases formation. Materials and Methods Brain metastases were induced by either stereotactic implantation of cells to the brain parenchyma or by injection of the melanoma cells into the internal carotid artery to mimic hematogenous metastatic spread in mice. Naive or IL-12-overexpressing murine K1735 melanoma cells were used either alone or in combination with intraperitoneal anti-integrin treatment using EMD121974. Results Solid melanoma metastases were more susceptible to daily low-dose treatment of EMD121974 than multiple hematogenous metastases. Interleukin-12 had a profound effect on both types of brain metastases. After 21 days, a marked reduction of vascularity was observed in both tumor types. Conclusion The combination of endogenous IL-12 production with integrin blockade resulted in additive effects for murine hematogenous brain metastases but not for focal brain metastases. PMID:24982333

  19. Mannoproteins from Cryptococcus neoformans promote dendritic cell maturation and activation.

    PubMed

    Pietrella, Donatella; Corbucci, Cristina; Perito, Stefano; Bistoni, Giovanni; Vecchiarelli, Anna

    2005-02-01

    Our previous data show that mannoproteins (MPs) from Cryptococcus neoformans are able to induce protective responses against both C. neoformans and Candida albicans. Here we provide evidence that MPs foster maturation and activation of human dendritic cells (DCs). Maturation was evaluated by the ability of MPs to facilitate expression of costimulatory molecules such as CD40, CD86, CD83, and major histocompatibility complex classes I and II and to inhibit receptors such as CD14, CD16, and CD32. Activation of DCs was measured by the capacity of MPs to promote interleukin-12 and tumor necrosis factor alpha secretion. DC-induced maturation and interleukin-12 induction are largely mediated by engagement of mannose receptors and presume MP internalization and degradation. DC activation leads to IkappaBalpha phosphorylation, which is necessary for nuclear factor kappaB transmigration into the nucleus. MP-loaded DCs are efficient stimulators of T cells and show a remarkable capacity to promote CD4 and CD8 proliferation. In conclusion, we have evidenced a novel regulatory role of MPs that promotes their candidacy as a vaccine against fungi.

  20. Mannoproteins from Cryptococcus neoformans Promote Dendritic Cell Maturation and Activation

    PubMed Central

    Pietrella, Donatella; Corbucci, Cristina; Perito, Stefano; Bistoni, Giovanni; Vecchiarelli, Anna

    2005-01-01

    Our previous data show that mannoproteins (MPs) from Cryptococcus neoformans are able to induce protective responses against both C. neoformans and Candida albicans. Here we provide evidence that MPs foster maturation and activation of human dendritic cells (DCs). Maturation was evaluated by the ability of MPs to facilitate expression of costimulatory molecules such as CD40, CD86, CD83, and major histocompatibility complex classes I and II and to inhibit receptors such as CD14, CD16, and CD32. Activation of DCs was measured by the capacity of MPs to promote interleukin-12 and tumor necrosis factor alpha secretion. DC-induced maturation and interleukin-12 induction are largely mediated by engagement of mannose receptors and presume MP internalization and degradation. DC activation leads to IκBα phosphorylation, which is necessary for nuclear factor κB transmigration into the nucleus. MP-loaded DCs are efficient stimulators of T cells and show a remarkable capacity to promote CD4 and CD8 proliferation. In conclusion, we have evidenced a novel regulatory role of MPs that promotes their candidacy as a vaccine against fungi. PMID:15664921

  1. Lovastatin dose-dependently potentiates the pro-inflammatory activity of lipopolysaccharide both in vitro and in vivo.

    PubMed

    Zanin, Valentina; Marcuzzi, Annalisa; Kleiner, Giulio; Piscianz, Elisa; Monasta, Lorenzo; Zacchigna, Serena; Crovella, Sergio; Zauli, Giorgio

    2013-12-01

    Since contradictory findings have been reported on potential effects of statins in modulating the inflammatory response, we have analysed the biological activity of lovastatin both in vitro using the Raw 264.7 murine macrophagic cell line and in vivo using BALB/c mice. When added to Raw 264.7 cells in combination with lipopolysaccharide, lovastatin significantly potentiated the release of interleukin-1β, interleukin-6 and interleukin-12 with respect to lipopolysaccharide alone and showed an additive effect on the release of nitric oxide. Similarly, when lovastatin was intraperitoneally administrated to BALB/c mice, it did not induce any pro-inflammatory effect when used alone, but it significantly potentiated the pro-inflammatory activity of lipopolysaccharide, in terms of number of intraperitoneal cells and serum levels of serum amyloid A, interleukin-1β, interleukin-6 and interleukin-12. A potential clinical implication of our study is that lovastatin might exert a pro-inflammatory activity in subjects affected by inflammatory processes, with clinically evident or subclinical infections.

  2. Thermodynamics of the beta(2) association in light-harvesting complex I of Rhodospirillum rubrum. Implication of peptide identity in dimer stability.

    PubMed

    Seguin, Jérôme; Mayer, Claudine; Robert, Bruno; Arluison, Véronique

    2008-03-01

    The core light-harvesting LH1 protein from Rhodospirillum rubrum can dissociate reversibly in the presence of n-octyl-beta-D-glucopyranoside into smaller subunit forms, exhibiting a dramatic blue-shift in absorption. During this process, two main species are observed: a dimer that absorbs at 820 nm (B820) and a monomer absorbing at 777 nm (B777). In the presence of n-octyl-beta-D-glucopyranoside, we have previously shown that the B820 form is not only constituted by the alphabeta heterodimer alone, but that it exists in an equilibrium between the alphabeta heterodimer and beta(2) homodimer states. We investigated the dissociation equilibrium for both oligomeric B820 forms. Using a theoretical model for alphabeta and beta(2), we conclude that the B820 homodimer is stabilized by both hydrophobic effects (entropy) and non-covalent bonds (enthalpy). We discuss a possible interpretation of the energy changes.

  3. Carboxylation of cytosine (5caC) in the CG dinucleotide in the E-box motif (CGCAG|GTG) increases binding of the Tcf3|Ascl1 helix-loop-helix heterodimer 10-fold.

    PubMed

    Golla, Jaya Prakash; Zhao, Jianfei; Mann, Ishminder K; Sayeed, Syed K; Mandal, Ajeet; Rose, Robert B; Vinson, Charles

    2014-06-27

    Three oxidative products of 5-methylcytosine (5mC) occur in mammalian genomes. We evaluated if these cytosine modifications in a CG dinucleotide altered DNA binding of four B-HLH homodimers and three heterodimers to the E-Box motif CGCAG|GTG. We examined 25 DNA probes containing all combinations of cytosine in a CG dinucleotide and none changed binding except for carboxylation of cytosine (5caC) in the strand CGCAG|GTG. 5caC enhanced binding of all examined B-HLH homodimers and heterodimers, particularly the Tcf3|Ascl1 heterodimer which increased binding ~10-fold. These results highlight a potential function of the oxidative products of 5mC, changing the DNA binding of sequence-specific transcription factors.

  4. Attractant Signaling by an Aspartate Chemoreceptor Dimer with a Single Cytoplasmic Domain

    NASA Astrophysics Data System (ADS)

    Gardina, Paul J.; Manson, Michael D.

    1996-10-01

    Signal transduction across cell membranes often involves interactions among identical receptor subunits, but the contribution of individual subunits is not well understood. The chemoreceptors of enteric bacteria mediate attractant responses by interrupting a phosphotransfer circuit initiated at receptor complexes with the protein kinase CheA. The aspartate receptor (Tar) is a homodimer, and oligomerized cytoplasmic domains stimulate CheA activity much more than monomers do in vitro. Intragenic complementation was used to show in Escherichia coli that heterodimers containing one full-length and one truncated Tar subunit mediated responses to aspartate in the presence of full-length Tar homodimers that could not bind aspartate. Thus, a Tar dimer containing only one cytoplasmic domain can initiate an attractant (inhibitory) signal, although it may not be able to stimulate kinase activity of CheA.

  5. The Dimerization State of the Mammalian High Mobility Group Protein AT-Hook 2 (HMGA2)

    PubMed Central

    Frost, Lorraine; Baez, Maria A. M.; Harrilal, Christopher; Garabedian, Alyssa; Fernandez-Lima, Francisco; Leng, Fenfei

    2015-01-01

    The mammalian high mobility group protein AT-hook 2 (HMGA2) is a chromosomal architectural transcription factor involved in cell transformation and oncogenesis. It consists of three positively charged “AT-hooks” and a negatively charged C-terminus. Sequence analyses, circular dichroism experiments, and gel-filtration studies showed that HMGA2, in the native state, does not have a defined secondary or tertiary structure. Surprisingly, using combined approaches of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemical cross-linking, analytical ultracentrifugation, fluorescence resonance energy transfer (FRET), and mass spectrometry, we discovered that HMGA2 is capable of self-associating into homodimers in aqueous buffer solution. Our results showed that electrostatic interactions between the positively charged “AT-hooks” and the negatively charged C-terminus greatly contribute to the homodimer formation. PMID:26114780

  6. An improved zinc-finger nuclease architecture for highly specific genome editing.

    PubMed

    Miller, Jeffrey C; Holmes, Michael C; Wang, Jianbin; Guschin, Dmitry Y; Lee, Ya-Li; Rupniewski, Igor; Beausejour, Christian M; Waite, Adam J; Wang, Nathaniel S; Kim, Kenneth A; Gregory, Philip D; Pabo, Carl O; Rebar, Edward J

    2007-07-01

    Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.

  7. Snyder-Robinson Syndrome: Rescuing the Disease-Causing Effect of G56S mutant by Small Molecule Binding

    NASA Astrophysics Data System (ADS)

    Zhang, Zhe; Martiny, Virginie; Lagorce, David; Alexov, Emil; Miteva, Maria; Clemson University Team; Université Paris Diderot Team

    2013-03-01

    Snyder-Robinson Syndrome (SRS) is an X-linked mental retardation disorder, which is caused by defects in a particular gene coding for the spermine synthase (SMS) protein. Among the missense mutations known to be disease-causing is the G56S, which is positioned at the interface of the SMS homo-dimer. Previous computational and experimental investigations have shown that G56S mutation destabilizes the homo-dimer and thus greatly reduces the SMS enzymatic activity. In this study, we explore the possibility of mitigating the effect of G56S mutation by binding small molecules to suitable pockets around the mutation site. It is done by combined efforts of molecular dynamics simulations and in silico screening. The binding of selected molecules was calculated to fully compensate the effect of the mutation and rescue the wild type dimer affinity. This work was supported by NIH, NLM grant. No. 1R03LM009748

  8. Asymmetric conformational maturation of HIV-1 reverse transcriptase.

    PubMed

    Zheng, Xunhai; Perera, Lalith; Mueller, Geoffrey A; DeRose, Eugene F; London, Robert E

    2015-06-03

    HIV-1 reverse transcriptase utilizes a metamorphic polymerase domain that is able to adopt two alternate structures that fulfill catalytic and structural roles, thereby minimizing its coding requirements. This ambiguity introduces folding challenges that are met by a complex maturation process. We have investigated this conformational maturation using NMR studies of methyl-labeled RT for the slower processes in combination with molecular dynamics simulations for rapid processes. Starting from an inactive conformation, the p66 precursor undergoes a unimolecular isomerization to a structure similar to its active form, exposing a large hydrophobic surface that facilitates initial homodimer formation. The resulting p66/p66' homodimer exists as a conformational heterodimer, after which a series of conformational adjustments on different time scales can be observed. Formation of the inter-subunit RH:thumb' interface occurs at an early stage, while maturation of the connection' and unfolding of the RH' domains are linked and occur on a much slower time scale.

  9. Crystal structures of the coil 2B fragment and the globular tail domain of human lamin B1

    SciTech Connect

    Ruan, Jianbin; Xu, Chao; Bian, Chuanbing; Lam, Robert; Wang, Jia-Pey; Kania, Joanna; Min, Jinrong; Zang, Jianye

    2012-07-18

    We present here the crystal structures of human lamin B1 globular tail domain and coiled 2B domain, which adopt similar folds to Ig-like domain and coiled-coil domain of lamin A, respectively. Despite the overall similarity, we found an extra intermolecular disulfide bond in the lamin B1 coil 2B domain, which does not exist in lamin A/C. In addition, the structural analysis indicates that interactions at the lamin B1 homodimer interface are quite different from those of lamin A/C. Thus our research not only reveals the diversely formed homodimers among lamin family members, but also sheds light on understanding the important roles of lamin B1 in forming the nuclear lamina matrix.

  10. Structure of D-lactate dehydrogenase from Aquifex aeolicus complexed with NAD(+) and lactic acid (or pyruvate).

    PubMed

    Antonyuk, Svetlana V; Strange, Richard W; Ellis, Mark J; Bessho, Yoshitaka; Kuramitsu, Seiki; Inoue, Yumiko; Yokoyama, Shigeyuki; Hasnain, S Samar

    2009-12-01

    The crystal structure of D-lactate dehydrogenase from Aquifex aeolicus (aq_727) was determined to 2.12 A resolution in space group P2(1)2(1)2(1), with unit-cell parameters a = 90.94, b = 94.43, c = 188.85 A. The structure was solved by molecular replacement using the coenzyme-binding domain of Lactobacillus helveticus D-lactate dehydrogenase and contained two homodimers in the asymmetric unit. Each subunit of the homodimer was found to be in a ;closed' conformation with the NADH cofactor bound to the coenzyme-binding domain and with a lactate (or pyruvate) molecule bound at the interdomain active-site cleft.

  11. Asymmetric DNA binding by a homodimeric bHLH protein.

    PubMed

    Winston, R L; Ehley, J A; Baird, E E; Dervan, P B; Gottesfeld, J M

    2000-08-08

    Protein-DNA interactions that lie outside of the core recognition sequence for the Drosophila bHLH transcription factor Deadpan (Dpn) were investigated using minor groove binding pyrrole-imidazole polyamides. Electrophoretic mobility shift assays and DNase I footprinting demonstrate that hairpin polyamides bound immediately upstream, but not immediately downstream of the Dpn homodimer selectively inhibit protein-DNA complex formation. Mutation of the Dpn consensus binding site from the asymmetric sequence 5'-CACGCG-3' to the palindromic sequence 5'-CACGTG-3' abolishes asymmetric inhibition. A Dpn mutant containing the unnatural amino acid norleucine in place of lysine at position 80 in the bHLH loop region is not inhibited by the polyamide, suggesting that the epsilon amino group at this position is responsible for DNA contacts outside the major groove. We conclude that the nonpalindromic Dpn recognition site imparts binding asymmetry by providing unique contacts to the basic region of each monomer in the bHLH homodimer.

  12. Bivalent Ligands Targeting Chemokine Receptor Dimerization: Molecular Design and Functional Studies

    PubMed Central

    Arnatt, Christopher Kent; Zhang, Yan

    2015-01-01

    Increasing evidence has shown that chemokine receptors may form functional dimers with unique pharmacological profiles. A common practice to characterize such G protein-coupled receptor dimerization processes is to apply bivalent ligands as chemical probes which can interact with both receptors simultaneously. Currently, two chemokine receptor dimers have been studied by applying bivalent compounds: the CXCR4-CXCR4 homodimer and the CCR5-MOR heterodimer. These bivalent compounds have revealed how dimerization influences receptor function and may lead to novel therapeutics. Future design of bivalent ligands for chemokine receptor dimers may be aided with the recently available CXCR4 homodimer, and CCR5 monomer crystal structures by more accurately simulating chemokine receptors and their dimers. PMID:25159160

  13. Functional and Structural Characterization of the Antiphagocytic Properties of a Novel Transglutaminase from Streptococcus suis*

    PubMed Central

    Yu, Jie; Pian, Yaya; Ge, Jingpeng; Guo, Jie; Zheng, Yuling; Jiang, Hua; Hao, Huaijie; Yuan, Yuan; Jiang, Yongqiang; Yang, Maojun

    2015-01-01

    Streptococcus suis serotype 2 (Ss2) is an important swine and human zoonotic pathogen. In the present study, we identified a novel secreted immunogenic protein, SsTGase, containing a highly conserved eukaryotic-like transglutaminase (TGase) domain at the N terminus. We found that inactivation of SsTGase significantly reduced the virulence of Ss2 in a pig infection model and impaired its antiphagocytosis in human blood. We further solved the crystal structure of the N-terminal portion of the protein in homodimer form at 2.1 Å. Structure-based mutagenesis and biochemical studies suggested that disruption of the homodimer directly resulted in the loss of its TGase activity and antiphagocytic ability. Characterization of SsTGase as a novel virulence factor of Ss2 by acting as a TGase would be beneficial for developing new therapeutic agents against Ss2 infections. PMID:26085092

  14. [Glutathione S-transferase of alpha class from pike liver].

    PubMed

    Borvinskaia, E V; Smirnov, L P; Nemova, N N

    2013-01-01

    In this study, glutathione S-transferase (GST) was isolated from the liver of pike Esox lucius, which was homogenous according to SDS-PAGE and isoelectrofocusing. It is a homodimer with subunits mass 25235.36 Da (according to HPLC-MS/MS) and pI about 6.4. Substrate specificity, thermostability, some kinetic characteristics and optimum pH were determined. The enzyme was identified as Alpha class GST.

  15. The Atypical Response Regulator Protein ChxR Has Structural Characteristics and Dimer Interface Interactions That Are Unique within the OmpR/PhoB Subfamily

    SciTech Connect

    Hickey, John M.; Lovell, Scott; Battaile, Kevin P.; Hu, Lei; Middaugh, C. Russell; Hefty, P. Scott

    2013-05-29

    Typically as a result of phosphorylation, OmpR/PhoB response regulators form homodimers through a receiver domain as an integral step in transcriptional activation. Phosphorylation stabilizes the ionic and hydrophobic interactions between monomers. Recent studies have shown that some response regulators retain functional activity in the absence of phosphorylation and are termed atypical response regulators. The two currently available receiver domain structures of atypical response regulators are very similar to their phospho-accepting homologs, and their propensity to form homodimers is generally retained. An atypical response regulator, ChxR, from Chlamydia trachomatis, was previously reported to form homodimers; however, the residues critical to this interaction have not been elucidated. We hypothesize that the intra- and intermolecular interactions involved in forming a transcriptionally competent ChxR are distinct from the canonical phosphorylation (activation) paradigm in the OmpR/PhoB response regulator subfamily. To test this hypothesis, structural and functional studies were performed on the receiver domain of ChxR. Two crystal structures of the receiver domain were solved with the recently developed method using triiodo compound I3C. These structures revealed many characteristics unique to OmpR/PhoB subfamily members: typical or atypical. Included was the absence of two {alpha}-helices present in all other OmpR/PhoB response regulators. Functional studies on various dimer interface residues demonstrated that ChxR forms relatively stable homodimers through hydrophobic interactions, and disruption of these can be accomplished with the introduction of a charged residue within the dimer interface. A gel shift study with monomeric ChxR supports that dimerization through the receiver domain is critical for interaction with DNA.

  16. The roles of the RIIβ linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of the type IIβ protein kinase A: a small angle x-ray and neutron scattering study.

    PubMed

    Blumenthal, Donald K; Copps, Jeffrey; Smith-Nguyen, Eric V; Zhang, Ping; Heller, William T; Taylor, Susan S

    2014-10-10

    Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.

  17. PDGFA — EDRN Public Portal

    Cancer.gov

    PDGFA, or platelet-derived growth factor alpha polypeptide, is a member of the platelet-derived growth factor family. PDGFA is a growth factor that plays an essential role in the regulation of embryonic development, cell proliferation, cell migration, survival and chemotaxis. There are four members of the PDGF family, characterized by a motif of eight cysteines. PDGFA exists as either a homodimer or a heterodimer with PDGFB (platelet-derived growth factor beta polypeptide).

  18. HSP90a — EDRN Public Portal

    Cancer.gov

    The HSP90AA1 protein, also known as HSP90a, is a molecular chaperone that promotes the maturation, structural maintenance and proper regulation of specific target proteins involved in cell cycle control and signal transduction. HSP90AA1 is a homodimer that assists in the proper folding of specific target proteins by use of an ATPase activity that is modulated by co-chaperones. Two transcript variants encoding different isoforms have been found for this gene.

  19. Towards an exact theory of linear absorbance and circular dichroism of pigment-protein complexes: Importance of non-secular contributions

    SciTech Connect

    Dinh, Thanh-Chung; Renger, Thomas

    2015-01-21

    A challenge for the theory of optical spectra of pigment-protein complexes is the equal strength of the pigment-pigment and the pigment-protein couplings. Treating both on an equal footing so far can only be managed by numerically costly approaches. Here, we exploit recent results on a normal mode analysis derived spectral density that revealed the dominance of the diagonal matrix elements of the exciton-vibrational coupling in the exciton state representation. We use a cumulant expansion technique that treats the diagonal parts exactly, includes an infinite summation of the off-diagonal parts in secular and Markov approximations, and provides a systematic perturbative way to include non-secular and non-Markov corrections. The theory is applied to a model dimer and to chlorophyll (Chl) a and Chl b homodimers of the reconstituted water-soluble chlorophyll-binding protein (WSCP) from cauliflower. The model calculations reveal that the non-secular/non-Markov effects redistribute oscillator strength from the strong to the weak exciton transition in absorbance and they diminish the rotational strength of the exciton transitions in circular dichroism. The magnitude of these corrections is in a few percent range of the overall signal, providing a quantitative explanation of the success of time-local convolution-less density matrix theory applied earlier. A close examination of the optical spectra of Chl a and Chl b homodimers in WSCP suggests that the opening angle between Q{sub y} transition dipole moments in Chl b homodimers is larger by about 9{sup ∘} than for Chl a homodimers for which a crystal structure of a related WSCP complex exists. It remains to be investigated whether this change is due to a different mutual geometry of the pigments or due to the different electronic structures of Chl a and Chl b.

  20. Towards an exact theory of linear absorbance and circular dichroism of pigment-protein complexes: Importance of non-secular contributions

    NASA Astrophysics Data System (ADS)

    Dinh, Thanh-Chung; Renger, Thomas

    2015-01-01

    A challenge for the theory of optical spectra of pigment-protein complexes is the equal strength of the pigment-pigment and the pigment-protein couplings. Treating both on an equal footing so far can only be managed by numerically costly approaches. Here, we exploit recent results on a normal mode analysis derived spectral density that revealed the dominance of the diagonal matrix elements of the exciton-vibrational coupling in the exciton state representation. We use a cumulant expansion technique that treats the diagonal parts exactly, includes an infinite summation of the off-diagonal parts in secular and Markov approximations, and provides a systematic perturbative way to include non-secular and non-Markov corrections. The theory is applied to a model dimer and to chlorophyll (Chl) a and Chl b homodimers of the reconstituted water-soluble chlorophyll-binding protein (WSCP) from cauliflower. The model calculations reveal that the non-secular/non-Markov effects redistribute oscillator strength from the strong to the weak exciton transition in absorbance and they diminish the rotational strength of the exciton transitions in circular dichroism. The magnitude of these corrections is in a few percent range of the overall signal, providing a quantitative explanation of the success of time-local convolution-less density matrix theory applied earlier. A close examination of the optical spectra of Chl a and Chl b homodimers in WSCP suggests that the opening angle between Qy transition dipole moments in Chl b homodimers is larger by about 9∘ than for Chl a homodimers for which a crystal structure of a related WSCP complex exists. It remains to be investigated whether this change is due to a different mutual geometry of the pigments or due to the different electronic structures of Chl a and Chl b.

  1. The structure of the GemC1 coiled coil and its interaction with the Geminin family of coiled-coil proteins.

    PubMed

    Caillat, Christophe; Fish, Alexander; Pefani, Dafni Eleftheria; Taraviras, Stavros; Lygerou, Zoi; Perrakis, Anastassis

    2015-11-01

    GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the crystal structure of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and the Geminin-Idas heterodimer structures. It is also shown that both in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using circular dichroism to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells.

  2. The Roles of the RIIβ Linker and N-terminal Cyclic Nucleotide-binding Domain in Determining the Unique Structures of the Type IIβ Protein Kinase A

    PubMed Central

    Blumenthal, Donald K.; Copps, Jeffrey; Smith-Nguyen, Eric V.; Zhang, Ping; Heller, William T.; Taylor, Susan S.

    2014-01-01

    Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA. PMID:25112875

  3. Towards an exact theory of linear absorbance and circular dichroism of pigment-protein complexes: importance of non-secular contributions.

    PubMed

    Dinh, Thanh-Chung; Renger, Thomas

    2015-01-21

    A challenge for the theory of optical spectra of pigment-protein complexes is the equal strength of the pigment-pigment and the pigment-protein couplings. Treating both on an equal footing so far can only be managed by numerically costly approaches. Here, we exploit recent results on a normal mode analysis derived spectral density that revealed the dominance of the diagonal matrix elements of the exciton-vibrational coupling in the exciton state representation. We use a cumulant expansion technique that treats the diagonal parts exactly, includes an infinite summation of the off-diagonal parts in secular and Markov approximations, and provides a systematic perturbative way to include non-secular and non-Markov corrections. The theory is applied to a model dimer and to chlorophyll (Chl) a and Chl b homodimers of the reconstituted water-soluble chlorophyll-binding protein (WSCP) from cauliflower. The model calculations reveal that the non-secular/non-Markov effects redistribute oscillator strength from the strong to the weak exciton transition in absorbance and they diminish the rotational strength of the exciton transitions in circular dichroism. The magnitude of these corrections is in a few percent range of the overall signal, providing a quantitative explanation of the success of time-local convolution-less density matrix theory applied earlier. A close examination of the optical spectra of Chl a and Chl b homodimers in WSCP suggests that the opening angle between Qy transition dipole moments in Chl b homodimers is larger by about 9(∘) than for Chl a homodimers for which a crystal structure of a related WSCP complex exists. It remains to be investigated whether this change is due to a different mutual geometry of the pigments or due to the different electronic structures of Chl a and Chl b.

  4. Advancing the Capabilities of an Authentic Ex Vivo Model of Primary Human Prostate Cancer

    DTIC Science & Technology

    2014-10-01

    After five days in culture, slices were incubated in PBS with the ethidium homodimer-1 and calcein AM reagents that emit red fluorescence in dead...cells and green in viable cells, respectively. Visualization under a fluorescent microscope revealed primarily red (dead) cells in the benign slices...of the benign and malignant human prostate. Lab Invest 94, 208 (Feb, 2014). 3. S. A. Bigler, R. E. Deering , M. K. Brawer, Comparison of microscopic

  5. SOD1 — EDRN Public Portal

    Cancer.gov

    SOD1, superoxide dismutase, is one of two isozymes that destroy free superoxide radicals that are normally produced within the cells and which are toxic to biological systems. The SOD1 isozyme is a soluble homodimer found in the cytoplasm. It converts naturally-occuring but harmful superoxide radicals to molecular oxygen and hydrogen peroxide. The other isozyme is a mitochondrial protein. Defects in SOD1 are the cause of amyotrophic lateral sclerosis type 1, a familial form of amyotrophic lateral sclerosis.

  6. TMEM16A(a)/anoctamin-1 Shares a Homodimeric Architecture with CLC Chloride Channels*

    PubMed Central

    Fallah, Ghada; Römer, Thomas; Detro-Dassen, Silvia; Braam, Ursula; Markwardt, Fritz; Schmalzing, Günther

    2011-01-01

    TMEM16A/anoctamin-1 has been identified as a protein with the classic properties of a Ca2+-activated chloride channel. Here, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking to assess the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac) splice variants as well as a genetically concatenated TMEM16A(a) homodimer. The constructs carried hexahistidyl (His) tags to allow for their purification using a nondenaturing metal affinity resin. Neither His-tagging nor head-to-tail concatenation of two copies of TMEM16A(a) noticeably affected Ca2+-induced measured macroscopic Cl− currents compared with the wild-type TMEM16A(a) channel. The digitonin-solubilized, nondenatured TMEM16A(a) protein migrated in the BN-PAGE gel as a homodimer, as judged by comparison with the concatenated TMEM16A(a) homodimer and channel proteins of known oligomeric structures (e.g. the voltage-gated Cl− channel CLC-1). Cross-linking with glutaraldehyde corroborated the homodimeric structure of TMEM16A(a). The TMEM16A(a) homodimer detected in Xenopus laevis oocytes and HEK 293 cells dissociated into monomers following denaturation with SDS, and reducing versus nonreducing SDS-PAGE provided no evidence for the presence of intersubunit disulfide bonds. Together, our data demonstrate that the Ca2+-activated chloride channel member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel. PMID:20974900

  7. Crystal structure of calpain-3 penta-EF-hand (PEF) domain - a homodimerized PEF family member with calcium bound at the fifth EF-hand

    SciTech Connect

    Partha, Sarathy K.; Ravulapalli, Ravikiran; Allingham, John S.; Campbell, Robert L.; Davies, Peter L.

    2014-08-21

    Calpains are Ca2+dependent intracellular cysteine proteases that cleave a wide range of protein substrates to help implement Ca2+ signaling in the cell. The major isoforms of this enzyme family, calpain-1 and calpain-2, are heterodimers of a large and a small subunit, with the main dimer interface being formed through their C-terminal penta-EF hand (PEF) domains. Calpain-3, or p94, is a skeletal muscle-specific isoform that is genetically linked to limb-girdle muscular dystrophy. Biophysical and modeling studies with the PEF domain of calpain-3 support the suggestion that full-length calpain-3 exists as a homodimer. Here, we report the crystallization of calpain-3's PEF domain and its crystal structure in the presence of Ca2+, which provides evidence for the homodimer architecture of calpain-3 and supports the molecular model that places a protease core at either end of the elongated dimer. Unlike other calpain PEF domain structures, the calpain-3 PEF domain contains a Ca2+ bound at the EF5-hand used for homodimer association. Three of the four Ca2+-binding EF-hands of the PEF domains are concentrated near the protease core, and have the potential to radically change the local charge within the dimer during Ca2+ signaling. Examination of the homodimer interface shows that there would be steric clashes if the calpain-3 large subunit were to try to pair with a calpain small subunit.

  8. The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study

    SciTech Connect

    Blumenthal, Donald K.; Copps, Jeffrey; Smith-Nguyen, Eric V.; Zhang, Ping; Heller, William T.; Taylor, Susan S.

    2014-08-11

    Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. Moreover, the PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. These results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.

  9. The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study

    DOE PAGES

    Blumenthal, Donald K.; Copps, Jeffrey; Smith-Nguyen, Eric V.; ...

    2014-08-11

    Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. Moreover, the PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme ismore » much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. These results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.« less

  10. The structure of the GemC1 coiled coil and its interaction with the Geminin family of coiled-coil proteins

    SciTech Connect

    Caillat, Christophe; Fish, Alexander; Pefani, Dafni-Eleftheria; Taraviras, Stavros; Lygerou, Zoi; Perrakis, Anastassis

    2015-10-31

    The GemC1 coiled-coil structure has subtle differences compared with its homologues Geminin and Idas. Co-expression experiments in cells and biophysical stability analysis of the Geminin-family coiled coils suggest that the GemC1 coiled coil alone is unstable. GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the crystal structure of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and the Geminin–Idas heterodimer structures. It is also shown that both in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using circular dichroism to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells.

  11. [RXR, a key member of the oncogenic complex in acute promyelocytic leukemia].

    PubMed

    Halftermeyer, Juliane; Le Bras, Morgane; De Thé, Hugues

    2011-11-01

    Acute promyelocytic leukaemia (APL) is induced by fusion proteins always implying the retinoic acid receptor RARa. Although PML-RARa and other fusion oncoproteins are able to bind DNA as homodimers, in vivo they are always found in association with the nuclear receptor RXRa (Retinoid X Receptor). Thus, RXRa is an essential cofactor of the fusion protein for the transformation. Actually, RXRa contributes to several aspects of in vivo -transformation: RARa fusion:RXRa hetero-oligomeric complexes bind DNA with a much greater affinity than RARa fusion homodimers. Besides, PML-RARa:RXRa recognizes an enlarged repertoire of DNA binding sites. Thus the association between fusion proteins and RXRa regulates more genes than the homodimer alone. Titration of RXRa by the fusion protein may also play a role in the transformation process, as well as post-translational modifications of RXRa in the complex. Finally, RXRa is required for rexinoid-induced APL differentiation. Thus, RXRa is a key member of the oncogenic complex.

  12. G Protein–Coupled Receptor Oligomerization Revisited: Functional and Pharmacological Perspectives

    PubMed Central

    Casadó, Vicent; Devi, Lakshmi A.; Filizola, Marta; Jockers, Ralf; Lohse, Martin J.; Milligan, Graeme; Pin, Jean-Philippe; Guitart, Xavier

    2014-01-01

    Most evidence indicates that, as for family C G protein–coupled receptors (GPCRs), family A GPCRs form homo- and heteromers. Homodimers seem to be a predominant species, with potential dynamic formation of higher-order oligomers, particularly tetramers. Although monomeric GPCRs can activate G proteins, the pentameric structure constituted by one GPCR homodimer and one heterotrimeric G protein may provide a main functional unit, and oligomeric entities can be viewed as multiples of dimers. It still needs to be resolved if GPCR heteromers are preferentially heterodimers or if they are mostly constituted by heteromers of homodimers. Allosteric mechanisms determine a multiplicity of possible unique pharmacological properties of GPCR homomers and heteromers. Some general mechanisms seem to apply, particularly at the level of ligand-binding properties. In the frame of the dimer-cooperativity model, the two-state dimer model provides the most practical method to analyze ligand–GPCR interactions when considering receptor homomers. In addition to ligand-binding properties, unique properties for each GPCR oligomer emerge in relation to different intrinsic efficacy of ligands for different signaling pathways (functional selectivity). This gives a rationale for the use of GPCR oligomers, and particularly heteromers, as novel targets for drug development. Herein, we review the functional and pharmacological properties of GPCR oligomers and provide some guidelines for the application of discrete direct screening and high-throughput screening approaches to the discovery of receptor-heteromer selective compounds. PMID:24515647

  13. Exploring Symmetry as an Avenue to the Computational Design of Large Protein Domains

    SciTech Connect

    Fortenberry, Carie; Bowman, Elizabeth Anne; Proffitt, Will; Dorr, Brent; Combs, Steven; Harp, Joel; Mizoue, Laura; Meiler, Jens

    2012-03-15

    It has been demonstrated previously that symmetric, homodimeric proteins are energetically favored, which explains their abundance in nature. It has been proposed that such symmetric homodimers underwent gene duplication and fusion to evolve into protein topologies that have a symmetric arrangement of secondary structure elements - 'symmetric superfolds'. Here, the ROSETTA protein design software was used to computationally engineer a perfectly symmetric variant of imidazole glycerol phosphate synthase and its corresponding symmetric homodimer. The new protein, termed FLR, adopts the symmetric ({beta}{alpha}){sub 8} TIM-barrel superfold. The protein is soluble and monomeric and exhibits two-fold symmetry not only in the arrangement of secondary structure elements but also in sequence and at atomic detail, as verified by crystallography. When cut in half, FLR dimerizes readily to form the symmetric homodimer. The successful computational design of FLR demonstrates progress in our understanding of the underlying principles of protein stability and presents an attractive strategy for the in silico construction of larger protein domains from smaller pieces.

  14. A crystallographic study of human NONO (p54(nrb)): overcoming pathological problems with purification, data collection and noncrystallographic symmetry.

    PubMed

    Knott, Gavin J; Panjikar, Santosh; Thorn, Andrea; Fox, Archa H; Conte, Maria R; Lee, Mihwa; Bond, Charles S

    2016-06-01

    Non-POU domain-containing octamer-binding protein (NONO, a.k.a. p54(nrb)) is a central player in nuclear gene regulation with rapidly emerging medical significance. NONO is a member of the highly conserved Drosophila behaviour/human splicing (DBHS) protein family, a dynamic family of obligatory dimeric nuclear regulatory mediators. However, work with the NONO homodimer has been limited by rapid irreversible sample aggregation. Here, it is reported that L-proline stabilizes purified NONO homodimers, enabling good-quality solution small-angle X-ray structure determination and crystallization. NONO crystallized in the apparent space group P21 with a unique axis (b) of 408.9 Å and with evidence of twinning, as indicated by the cumulative intensity distribution L statistic, suggesting the possibility of space group P1. Structure solution by molecular replacement shows a superhelical arrangement of six NONO homodimers (or 12 in P1) oriented parallel to the long axis, resulting in extensive noncrystallographic symmetry. Further analysis revealed that the crystal was not twinned, but the collected data suffered from highly overlapping reflections that obscured the L-test. Optimized data collection on a new crystal using higher energy X-rays, a smaller beam width and an increased sample-to-detector distance produced non-overlapping reflections to 2.6 Å resolution. The steps taken to analyse and overcome this series of practical difficulties and to produce a biologically informative structure are discussed.

  15. Structural basis of ultraviolet-B perception by UVR8.

    PubMed

    Wu, Di; Hu, Qi; Yan, Zhen; Chen, Wen; Yan, Chuangye; Huang, Xi; Zhang, Jing; Yang, Panyu; Deng, Haiteng; Wang, Jiawei; Deng, XingWang; Shi, Yigong

    2012-02-29

    The Arabidopsis thaliana protein UVR8 is a photoreceptor for ultraviolet-B. Upon ultraviolet-B irradiation, UVR8 undergoes an immediate switch from homodimer to monomer, which triggers a signalling pathway for ultraviolet protection. The mechanism by which UVR8 senses ultraviolet-B remains largely unknown. Here we report the crystal structure of UVR8 at 1.8 Å resolution, revealing a symmetric homodimer of seven-bladed β-propeller that is devoid of any external cofactor as the chromophore. Arginine residues that stabilize the homodimeric interface, principally Arg 286 and Arg 338, make elaborate intramolecular cation-π interactions with surrounding tryptophan amino acids. Two of these tryptophans, Trp 285 and Trp 233, collectively serve as the ultraviolet-B chromophore. Our structural and biochemical analyses identify the molecular mechanism for UVR8-mediated ultraviolet-B perception, in which ultraviolet-B radiation results in destabilization of the intramolecular cation-π interactions, causing disruption of the critical intermolecular hydrogen bonds mediated by Arg 286 and Arg 338 and subsequent dissociation of the UVR8 homodimer.

  16. Transcription Factor WRKY62 Plays a Role in Pathogen Defense and Hypoxia-Responsive Gene Expression in Rice.

    PubMed

    Fukushima, Setsuko; Mori, Masaki; Sugano, Shoji; Takatsuji, Hiroshi

    2016-12-01

    WRKY62 is a transcriptional repressor regulated downstream of WRKY45, a central transcription factor of the salicylic acid signaling pathway in rice. Previously, WRKY62 was reported to regulate defense negatively. However, our expressional analysis using WRKY62-knockdown rice indicated that WRKY62 positively regulates defense genes, including diterpenoid phytoalexin biosynthetic genes and their transcriptional regulator DPF. Blast and leaf blight resistance tests also showed that WRKY62 is a positive defense regulator. Yeast two-hybrid, co-immunoprecipitation and gel-shift assays showed that WRKY45 and WRKY62 can form a heterodimer, as well as homodimers, that bind to W-boxes in the DPF promoter. In transient assays in rice sheaths, the simultaneous introduction of WRKY45 and WRKY62 as effectors resulted in a strong activation of the DPF promoter:hrLUC reporter gene, whereas the activity declined with excessive WRKY62. Thus, the WRKY45-WRKY62 heterodimer acts as a strong activator, while the WRKY62 homodimer acts as a repressor. While benzothiadiazole induced equivalent numbers of WRKY45 and WRKY62 transcripts, consistent with heterodimer formation and DPF activation, submergence and nitrogen replacement induced only WRKY62 transcripts, consistent with WRKY62 homodimer formation and DPF repression. Moreover, WRKY62 positively regulated hypoxia genes, implying a role forWRKY62 in the modulation of the 'trade-off' between defense and hypoxia responses.

  17. Prm1 Functions as a Disulfide-linked Complex in Yeast Mating*

    PubMed Central

    Olmo, Valerie N.; Grote, Eric

    2010-01-01

    Prm1 is a pheromone-induced membrane glycoprotein that promotes plasma membrane fusion in yeast mating pairs. HA-Prm1 migrates at twice its expected molecular weight on non-reducing SDS-PAGE gels and coprecipitates with Prm1-TAP, indicating that Prm1 is a disulfide-linked homodimer. The N terminus of a plasma membrane-localized GFP-Prm1 endocytic mutant projects into the cytoplasm, where it is protected from low pH quenching in live cells and from external protease in spheroplasts. In a revised topological map, Prm1 has four transmembrane domains and two large extracellular loops. Mutation of all four cysteines in the extracellular loops blocked disulfide bond formation and destabilized the Prm1 homodimer without preventing Prm1 transport to contact sites in mating pairs. Cys120 in loop 1 and Cys545 in loop 2 form disulfide cross-links in the Prm1 homodimer and are required for fusion activity. Cys120 lies between a hydrophobic segment formerly thought to be a transmembrane domain and an amphipathic helix. An interaction between either of these regions and the opposing membrane could promote fusion. PMID:19933274

  18. Exploring symmetry as an avenue to the computational design of large protein domains.

    PubMed

    Fortenberry, Carie; Bowman, Elizabeth Anne; Proffitt, Will; Dorr, Brent; Combs, Steven; Harp, Joel; Mizoue, Laura; Meiler, Jens

    2011-11-16

    It has been demonstrated previously that symmetric, homodimeric proteins are energetically favored, which explains their abundance in nature. It has been proposed that such symmetric homodimers underwent gene duplication and fusion to evolve into protein topologies that have a symmetric arrangement of secondary structure elements--"symmetric superfolds". Here, the ROSETTA protein design software was used to computationally engineer a perfectly symmetric variant of imidazole glycerol phosphate synthase and its corresponding symmetric homodimer. The new protein, termed FLR, adopts the symmetric (βα)(8) TIM-barrel superfold. The protein is soluble and monomeric and exhibits two-fold symmetry not only in the arrangement of secondary structure elements but also in sequence and at atomic detail, as verified by crystallography. When cut in half, FLR dimerizes readily to form the symmetric homodimer. The successful computational design of FLR demonstrates progress in our understanding of the underlying principles of protein stability and presents an attractive strategy for the in silico construction of larger protein domains from smaller pieces.

  19. Genomic redistribution of GR monomers and dimers mediates transcriptional response to exogenous glucocorticoid in vivo.

    PubMed

    Lim, Hee-Woong; Uhlenhaut, N Henriette; Rauch, Alexander; Weiner, Juliane; Hübner, Sabine; Hübner, Norbert; Won, Kyoung-Jae; Lazar, Mitchell A; Tuckermann, Jan; Steger, David J

    2015-06-01

    Glucocorticoids (GCs) are commonly prescribed drugs, but their anti-inflammatory benefits are mitigated by metabolic side effects. Their transcriptional effects, including tissue-specific gene activation and repression, are mediated by the glucocorticoid receptor (GR), which is known to bind as a homodimer to a palindromic DNA sequence. Using ChIP-exo in mouse liver under endogenous corticosterone exposure, we report here that monomeric GR interaction with a half-site motif is more prevalent than homodimer binding. Monomers colocalize with lineage-determining transcription factors in both liver and primary macrophages, and the GR half-site motif drives transcription, suggesting that monomeric binding is fundamental to GR's tissue-specific functions. In response to exogenous GC in vivo, GR dimers assemble on chromatin near ligand-activated genes, concomitant with monomer evacuation of sites near repressed genes. Thus, pharmacological GCs mediate gene expression by favoring GR homodimer occupancy at classic palindromic sites at the expense of monomeric binding. The findings have important implications for improving therapies that target GR.

  20. A structural dissection of large protein-protein crystal packing contacts.

    PubMed

    Luo, Jiesi; Liu, Zhongyu; Guo, Yanzhi; Li, Menglong

    2015-09-15

    With the rapid increase in crystal structures of protein-protein complexes deposited in the Protein Data Bank (PDB), more and more crystal contacts have been shown to have similar or even larger interface areas than biological interfaces. However, little attention has been paid to these large crystal packing contacts and their structural principles remain unknown. To address this issue, we used a comparative feature analysis to analyze the geometric and physicochemical properties of large crystal packing contacts by comparing two types of specific protein-protein interactions (PPIs), weak transient complexes and permanent homodimers. Our results show that although large crystal packing contacts have a similar interface area and contact size as permanent homodimers, they tend to be more planar, loosely packed and less hydrophobic than permanent homodimers and cannot form a central core region that is fully buried during interaction. However, the properties of large crystal packing contacts, except for the interface area and contact size, more closely resemble those of weak transient complexes. The large overlap between biological and large crystal packing contacts indicates that interface properties are not efficient indicators for classification of biological interfaces from large crystal packing contacts and finding other specific features urgently needed.

  1. Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site.

    PubMed Central

    Swanson, H I; Yang, J H

    1999-01-01

    Basic helix-loop-helix proteins that interact with the DNA recognition site CACGTG include the c-Myc/Max heterodimer and the ARNT (Ahreceptornucleartranslocator) homodimer. We have utilized a PCR-based protocol to identify high affinity binding sites of either the c-Myc/Max or ARNT/ARNT dimers and analyzed the ability of these dimers to interact with their derived consensus sequences and activate genes. chi(2)analysis of the selected DNA recognition sites revealed that DNA binding of the ARNT homodimer is symmetric, resulting in the consensus sequence RTCACGTGAY. Gel shift analysis demonstrated that the flanking nucleotides play an important role in dictating DNA binding affinity of the ARNT homodimer. These flanking sequences also regulate the ability of ARNT to competitively displace the c-Myc/Max heterodimer from a CACGTG-containing sequence. However, transient transfection analyses in CV-1 cells revealed that ARNT and c-Myc/Max exhibited similar abilities to activate transcription through each other's consensus sequences. Taken together, these results indicate that although binding affinity of these dimers for the CACGTG core sequences may be differentially influenced by flanking nucleotides, transcriptional activity may also be determined by other factors, such as cellular concentrations of these proteins and their co-activators. PMID:10454619

  2. Formation of Functional Heterodimers by TREK-1 and TREK-2 Two-pore Domain Potassium Channel Subunits.

    PubMed

    Lengyel, Miklós; Czirják, Gábor; Enyedi, Péter

    2016-06-24

    Two-pore domain (K2P) potassium channels are the major molecular correlates of the background (leak) K(+) current in a wide variety of cell types. They generally play a key role in setting the resting membrane potential and regulate the response of excitable cells to various stimuli. K2P channels usually function as homodimers, and only a few examples of heteromerization have been previously reported. Expression of the TREK (TWIK-related K(+) channel) subfamily members of K2P channels often overlaps in neurons and in other excitable cells. Here, we demonstrate that heterologous coexpression of TREK-1 and TREK-2 subunits results in the formation of functional heterodimers. Taking advantage of a tandem construct (in which the two different subunits were linked together to enforce heterodimerization), we characterized the biophysical and pharmacological properties of the TREK-1/TREK-2 current. The heteromer was inhibited by extracellular acidification and by spadin similarly to TREK-1, and its ruthenium red sensitivity was intermediate between TREK-1 and TREK-2 homodimers. The heterodimer has also been distinguished from the homodimers by its unique single channel conductance. Assembly of the two different subunits was confirmed by coimmunoprecipitation of epitope-tagged TREK-1 and TREK-2 subunits, coexpressed in Xenopus oocytes. Formation of TREK-1/TREK-2 channels was also demonstrated in native dorsal root ganglion neurons indicating that heterodimerization may provide greater diversity of leak K(+) conductances also in native tissues.

  3. A Variable Light Domain Fluorogen Activating Protein Homodimerizes To Activate Dimethylindole Red

    SciTech Connect

    Senutovitch, Nina; Stanfield, Robyn L.; Bhattacharyya, Shantanu; Rule, Gordon S.; Wilson, Ian A.; Armitage, Bruce A.; Waggoner, Alan S.; Berget, Peter B.

    2012-07-11

    Novel fluorescent tools such as green fluorescent protein analogues and fluorogen activating proteins (FAPs) are useful in biological imaging for tracking protein dynamics in real time with a low fluorescence background. FAPs are single-chain variable fragments (scFvs) selected from a yeast surface display library that produce fluorescence upon binding a specific dye or fluorogen that is normally not fluorescent when present in solution. FAPs generally consist of human immunoglobulin variable heavy (V{sub H}) and variable light (V{sub L}) domains covalently attached via a glycine- and serine-rich linker. Previously, we determined that the yeast surface clone, V{sub H}-V{sub L} M8, could bind and activate the fluorogen dimethylindole red (DIR) but that the fluorogen activation properties were localized to the M8V{sub L} domain. We report here that both nuclear magnetic resonance and X-ray diffraction methods indicate the M8V{sub L} forms noncovalent, antiparallel homodimers that are the fluorogen activating species. The M8V{sub L} homodimers activate DIR by restriction of internal rotation of the bound dye. These structural results, together with directed evolution experiments with both V{sub H}-V{sub L} M8 and M8V{sub L}, led us to rationally design tandem, covalent homodimers of M8V{sub L} domains joined by a flexible linker that have a high affinity for DIR and good quantum yields.

  4. Secreted CXCL12 (SDF-1) Forms Dimers under Physiologic Conditions

    PubMed Central

    Ray, Paramita; Lewin, Sarah A.; Mihalko, Laura Anne; Lesher-Perez, Sasha Cai; Takayama, Shuichi; Luker, Kathryn E.; Luker, Gary D.

    2015-01-01

    Chemokine CXCL12 signaling through receptors CXCR4 and CXCR7 has essential functions in development and underlies diseases including cancer, atherosclerosis, and autoimmunity. Chemokines may form homodimers that regulate receptor binding and signaling, but previous studies with synthetic CXCL12 have produced conflicting evidence for homodimerization. We used bioluminescence imaging with Gaussia luciferase fusions to investigate dimerization of CXCL12 secreted from mammalian cells. By column chromatography and Gaussia luciferase complementation, we established that CXCL12 was secreted from mammalian cells as both monomers and dimers. Secreted CXCL12 also formed homodimers in the extracellular space. Monomeric CXCL12 preferentially activated CXCR4 signaling through Gαi and AKT, while dimeric CXCL12 more effectively promoted recruitment of β-arrestin 2 to CXCR4 and chemotaxis of CXCR4-expressing breast cancer cells. We also showed that CXCR7 preferentially sequestered monomeric CXCL12 from the extracellular space and had minimal effects on dimeric CXCL12 in cell-based assays and an orthotopic tumor xenograft model of human breast cancer. These studies establish that CXCL12 secreted from mammalian cells forms homodimers under physiologic conditions. Since monomeric and dimeric CXCL12 have distinct effects on cell signaling and function, our results have important implications for ongoing efforts to target CXCL12 pathways for therapy. PMID:22142194

  5. Crenomytilus grayanus 40kDa calponin-like protein: cDNA cloning, sequence analysis, tissue expression, and post-translational modifications.

    PubMed

    Matusovsky, Oleg S; Dobrzhanskaya, Anna V; Pankova, Victoria V; Kiselev, Konstantin V; Girich, Ulyana V; Shelud'ko, Nikolay S

    2017-03-02

    Calponin-like protein (CaP-40), a third major protein after actin and tropomyosin, has recently been identified by us in the Ca(2+)-regulated thin filaments of mussel Crenomytilus grayanus. It contains calponin homology domain, five calponin family repeats and possesses similar biochemical properties as vertebrate smooth muscle calponin. In this paper, we report a full-length cDNA sequence of CaP-40, study its expression pattern on mRNA and protein levels, evaluate CaP-40 post-translational modifications and perform protein-protein interaction analysis. The full-length sequence of CaP-40 consists of 398 amino acids and has high similarity to calponins among molluscan species. CaP-40 gene is widely expressed in mussel tissues, with the highest expression in adductor and mantle. Comparison of these data with protein content established by mass-spectrometry analysis revealed that the high mRNA content is mirrored by high protein levels for adductor smooth muscles. To provide unbiased insight into the function of CaP-40 and effect of its over-expression in adductor smooth muscle, we built protein-protein interaction network of identified Crenomytilus grayanus proteome. In addition, we showed that CaP-40 is subjected to post-translational N- and C-terminal acetylation at N127, G229 and G349 sites which potentially regulates its function in vivo.

  6. Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism

    PubMed Central

    Seth, A.; Yan, Fang; Polk, D.Brent; Rao, R. K.

    2009-01-01

    Probiotics promote intestinal epithelial integrity and reduce infection and diarrhea. We evaluated the effect of Lactobacillus rhamnosus GG-produced soluble proteins (p40 and p75) on the hydrogen peroxide-induced disruption of tight junctions and barrier function in Caco-2 cell monolayers. Pretreatment of cell monolayers with p40 or p75 attenuated the hydrogen peroxide-induced decrease in transepithelial resistance and increase in inulin permeability in a time- and dose-dependent manner. p40 and p75 also prevented hydrogen peroxide-induced redistribution of occludin, ZO-1, E-cadherin, and β-catenin from the intercellular junctions and their dissociation from the detergent-insoluble fractions. Both p40 and p75 induced a rapid increase in the membrane translocation of PKCβI and PKCε. The attenuation of hydrogen peroxide-induced inulin permeability and redistribution of tight junction proteins by p40 and p75 was abrogated by Ro-32-0432, a PKC inhibitor. p40 and p75 also rapidly increased the levels of phospho-ERK1/2 in the detergent-insoluble fractions. U0126 (a MAP kinase inhibitor) attenuated the p40- and p75-mediated reduction of hydrogen peroxide-induced tight junction disruption and inulin permeability. These studies demonstrate that probiotic-secretory proteins protect the intestinal epithelial tight junctions and the barrier function from hydrogen peroxide-induced insult by a PKC- and MAP kinase-dependent mechanism. PMID:18292183

  7. Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism.

    PubMed

    Seth, A; Yan, Fang; Polk, D Brent; Rao, R K

    2008-04-01

    Probiotics promote intestinal epithelial integrity and reduce infection and diarrhea. We evaluated the effect of Lactobacillus rhamnosus GG-produced soluble proteins (p40 and p75) on the hydrogen peroxide-induced disruption of tight junctions and barrier function in Caco-2 cell monolayers. Pretreatment of cell monolayers with p40 or p75 attenuated the hydrogen peroxide-induced decrease in transepithelial resistance and increase in inulin permeability in a time- and dose-dependent manner. p40 and p75 also prevented hydrogen peroxide-induced redistribution of occludin, ZO-1, E-cadherin, and beta-catenin from the intercellular junctions and their dissociation from the detergent-insoluble fractions. Both p40 and p75 induced a rapid increase in the membrane translocation of PKCbetaI and PKCepsilon. The attenuation of hydrogen peroxide-induced inulin permeability and redistribution of tight junction proteins by p40 and p75 was abrogated by Ro-32-0432, a PKC inhibitor. p40 and p75 also rapidly increased the levels of phospho-ERK1/2 in the detergent-insoluble fractions. U0126 (a MAP kinase inhibitor) attenuated the p40- and p75-mediated reduction of hydrogen peroxide-induced tight junction disruption and inulin permeability. These studies demonstrate that probiotic-secretory proteins protect the intestinal epithelial tight junctions and the barrier function from hydrogen peroxide-induced insult by a PKC- and MAP kinase-dependent mechanism.

  8. In vitro proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

    PubMed

    Maxwell, S; Arlinghaus, R B

    1981-09-01

    Moloney murine leukemia virus, disrupted in concentrations of 0.1 to 0.5% Nonidet P-40, catalyzed the cleavage of p65, the gag gene polyprotein of the Gazdar strain of murine sarcoma virus, into polypeptides with sizes and antigenic determinants of murine leukemia virus-specified p30, p15, pp12, and p10. Cleavage performed in the presence of 0.15% Nonidet P-40 in water yielded polypeptides of approximately 40,000 (P40) and 25,000 (P25) Mr. In vitro cleavage performed in a buffered solution containing dithiothreitol in addition to 0.1% Nonidet P-40 allowed the efficient processing of P40 to p30 and a band migrating with p10. Immunoprecipitation with monospecific sera indicated that P40 contained p30 and p10, whereas P25 contained p15 and pp12 determinants. P40 and P25 are similar in size and antigenic properties to Pr40gag and Pr25gag observed in infected cells (Naso et al, J. Virol. 32:187-198, 1979).

  9. An LGG-derived protein promotes IgA production through upregulation of APRIL expression in intestinal epithelial cells.

    PubMed

    Wang, Y; Liu, L; Moore, D J; Shen, X; Peek, R M; Acra, S A; Li, H; Ren, X; Polk, D B; Yan, F

    2017-03-01

    p40, a Lactobacillus rhamnosus GG (LGG)-derived protein, transactivates epidermal growth factor receptor (EGFR) in intestinal epithelial cells, leading to amelioration of intestinal injury and inflammation. To elucidate mechanisms by which p40 regulates mucosal immunity to prevent inflammation, this study aimed to determine the effects and mechanisms of p40 on regulation of a proliferation-inducing ligand (APRIL) expression in intestinal epithelial cells for promoting immunoglobulin A (IgA) production. p40 upregulated April gene expression and protein production in mouse small intestine epithelial (MSIE) cells, which were inhibited by blocking EGFR expression and kinase activity. Enteroids from Egfr(fl/fl), but not Egfr(fl/fl)-Vil-Cre mice with EGFR specifically deleted in intestinal epithelial cells, exhibited increased April gene expression by p40 treatment. p40-conditioned media from MSIE cells increased B-cell class switching to IgA(+) cells and IgA production, which was suppressed by APRIL receptor-neutralizing antibodies. Treatment of B cells with p40 did not show any effects on IgA production. p40 treatment increased April gene expression and protein production in small intestinal epithelial cells, fecal IgA levels, IgA(+)B220(+), IgA(+)CD19(+), and IgA(+) plasma cells in lamina propria of Egfr(fl/fl), but not of Egfr(fl/fl)-Vil-Cre, mice. Thus p40 upregulates EGFR-dependent APRIL production in intestinal epithelial cells, which may contribute to promoting IgA production.

  10. Dimerization of Nitrophorin 4 at Low pH and Comparison to the K1A Mutant of Nitrophorin 1

    PubMed Central

    2015-01-01

    Nitrophorin 4, one of the four NO-carrying heme proteins from the salivary glands of Rhodnius prolixus, forms a homodimer at pH 5.0 with a Kd of ∼8 μM. This dimer begins to dissociate at pH 5.5 and is completely dissociated to monomer at pH 7.3, even at 3.7 mM. The dimer is significantly stabilized by binding NO to the heme and at pH 7.3 would require dilution to well below 0.2 mM to completely dissociate the NP4-NO homodimer. The primary techniques used for investigating the homodimer and the monomer–dimer equilibrium were size-exclusion fast protein liquid chromatography at pH 5.0 and 1H{15N} heteronuclear single-quantum coherence spectroscopy as a function of pH and concentration. Preparation of site-directed mutants of NP4 (A1K, D30A, D30N, V36A/D129A/L130A, K38A, R39A, K125A, K125E, D132A, L133V, and K38Q/R39Q/K125Q) showed that the N-terminus, D30, D129, D132, at least one heme propionate, and, by association, likely also E32 and D35 are involved in the dimerization. The “closed loop” form of the A–B and G–H flexible loops of monomeric NP4, which predominates in crystal structures of the monomeric protein reported at pH 5.6 but not at pH 7.5 and which involves all of the residues listed above except D132, is required for dimer formation. Wild-type NP1 does not form a homodimer, but NP1(K1A) and native N-terminal NP1 form dimers in the presence of NO. The homodimer of NP1, however, is considerably less stable than that of NP4 in the absence of NO. This suggests that additional aspartate or glutamate residues present in the C-terminal region of NP4, but not NP1, are also involved in stabilizing the dimer. PMID:25489673

  11. Discovery and Testing of Ricin Therapeutics

    DTIC Science & Technology

    2011-06-01

    Immunoprecipitation, SDS-PAGE, and Endoglyco- sidase H and Peptide N-Glycanase Assays—Briefly, cells (1 " 106) were lysed in 0.5% Nonidet P - 40 lysis mixture...methionine (25 mM) (22). RTA proteins were recov- ered from Nonidet P - 40 cell lysates using anti-HA antibodies and resolved by SDS-PAGE (12.5%). The...sample buffer (57% (w/v) urea, 2% (v/v) Nonidet P - 40 , 0.02% ampholytes (pH 3.5–10; Amersham Biosciences), and 0.025%2-mercaptoethanol) and resolved on a

  12. Analysis of a Novel Paralogue of SWI/SNF Member p270, Which is Frequently Down-Regulated in Breast Cancer

    DTIC Science & Technology

    2005-07-01

    GST, glutathione S-transferase; mAb, monoclonal antibody; NP40, Nonidet P40 ; poly(A)+, polyadenylated, ’ Present address: TVW Telethon Institute for...EKPVDLQNFGLRTDIYSK(C), cor- [250 mM NaCI, 0.1 % NP40 ( Nonidet P40 ),40 mM Tris (pH 7.4) responding to residues 591-608 in the BAF155 sequence. The and...lysed in p300 lysis buffer 6 [0.1% Nonidet P-40, 250 mM sodium chloride, 20 mM sodium phosphate (pH 7.0), 30 mM sodium 7 pyrophosphate, 5 mM

  13. Dendritic Cell-Based Genetic Immunotherapy for Ovarian Cancer

    DTIC Science & Technology

    2008-12-01

    post-infection. The concentrations of IL-1A, IL-1B, IL-2, IL-3, IL-4, IL-6, IL-12 ( p40 ), IL-12 (p70), IL-13, IL-17, TNF-α, KC and MCP-1 were analyzed...chemokines whose secretion was up regulated was mixed, and included the Th-1 type cytokines, IL-1α, IL-1β, IL-3, IL-12 ( p40 ), IL-12 (p70), IL-17 and TNF-α...were maximally secreted at 48 h post-infection, and IL-6, IL-12 ( p40 ), IL-12 (p70) and KC were maximally secreted by the DCs at 72 h post-infection

  14. Biochemical Characterization of Complexes with p21, a CDK Inhibitor

    DTIC Science & Technology

    1998-08-01

    additional experiments to further characterize p28 and p40 , two potentially novel proteins that co-fractionated with p21 on glycerol gradients, sizing...well as with amino- and carboxy-terminal fragments of p21. Neither p28 nor p40 was captured in preliminary binding experiments, suggesting that these...an additional step of 1.0 HMGNB (25 mM Z 75 HEPES [pH 7.6], 1 M NaCI, 10% glycerol, 0.1% Nonidet P-40 [NP-40], 5 mM U P-mercaptoethanol, and 0.2 mM

  15. Forskolin, an Inducer of Camp, Up-Regulates Acetylcholinesterase Expression and Protects Against Organophosphate Exposure in Neuro 2A Cells

    DTIC Science & Technology

    2004-11-16

    of dbcAMP for 7 days. B. Extracellular AChE expression determined by microassay of 20 µl culture supernatant. C. Cells were lysed with 1% nonidet - P40 ...determined by microassay using 20 µl culture supernatant. C. Intracellular AChE expression. Cells were lysed with 1% nonidet - P40 and the...phosphate buffer containing 1% Nonidet P-40 or with a lysis buffer, T- PER, purchased from Sigma. The homogenate was centrifuged for 10 minutes at 3,000 x g

  16. Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27.

    PubMed

    Freshney, N W; Rawlinson, L; Guesdon, F; Jones, E; Cowley, S; Hsuan, J; Saklatvala, J

    1994-09-23

    An IL-1-stimulated protein kinase cascade resulting in phosphorylation of the small heat shock protein hsp27 has been identified in KB cells. It is distinct from the p42 MAP kinase cascade. An upstream activator kinase phosphorylated a 40 kDa kinase (p40) upon threonine and tyrosine residues, which in turn phosphorylated a 50 kDa kinase (p50) upon threonine (and some serine) residues. p50 phosphorylated hsp27 upon serine. p40 and p50 were purified to near homogeneity. All three components were inactivated by protein phosphatase 2A, and p40 was inactivated by protein tyrosine phosphatase 1B. The substrate specificity of p40 differed from that of p42 and p54 MAP kinases. The upstream activator was not a MAP kinase kinase. p50 resembled MAPKAPK-2 and may be identical.

  17. CHD8, A Novel Beta-Catenin Associated Chromatin Remodeling Enzyme, Regulates Androgen Receptor Mediated Gene Transcription

    DTIC Science & Technology

    2010-03-01

    NP- 40 , Nonidet P - 40 ; PPAR, peroxisome prolif- erator-activated receptor; PSA, prostate-specific antigen; SDS, sodium dodecyl sulfate; siRNA, small... Nonidet P - 40 (NP- 40 ). Cell lysates were cleared by centrifugation at 20,800 g for 10 min at 4 C and used for protein interaction studies as...or therapeutic target in prostate cancer. REFERENCES 1. Mulholland, D. J., Cheng, H., Reid, K., Rennie, P . S., and Nelson, C. C. (2002) J Biol

  18. Analysis of hRad1, a Human G2 Checkpoint Control Gene

    DTIC Science & Technology

    2002-03-01

    Phosphatase (CIP) Treatment—Cells were lysed in 1 ml of NETN lysis buffer (250 nut NaCl, 1 it« EDTA, 20 mM Tris, pH 8.0, 0.5% Nonidet P-40...smaller subunits (p36, p37, p38 and p40 , 36, 37, 38 and 40 kDa respectively) (176). RFC serves to load the homotrimeric proliferating cell nuclear anigen

  19. Implications of Protein Alkyation and Proteolysis on Vesication Caused by Sulfur Mustard

    DTIC Science & Technology

    1999-10-01

    150 mM NaCI, 3 mM EDTA and 0.1% nonidet - P40 (NP40), pH 7.4) and then for 10 min at 4 °C with an ice-cold high salt buffer (10 mM Tris-base, 150 mM...kDa kilodalton KGM keratinocyte growth medium MMP matrix metalloproteinase MT-i MMP membrane type-i matrix metalloproteinase -58- NP40 nonidet - P40

  20. Regulation of Glutathione in a Rat Diploid Hepatic Epithelial Cell Line

    DTIC Science & Technology

    1990-06-01

    pelleted and resuspended in Hoechst 33258 staining solution (0.154 M NaCl, 0.1 M Tris, 0.5 mM MgCl3 , 0.2% bovine serum albumin, 0.1% Nonidet - P40 and 1.2...trypsinized by adding 500 VI trypsin-EDTA and incubated for 2 minutes at 370C. Then 500 Vl of Nonidet P 40 (NP-40, Sigma, St. Louis, MO) was added to each

  1. Effects of Radiation on Proteasome Function in Prostate Cancer Cells

    DTIC Science & Technology

    2009-02-01

    Transcription ribosomal protein L19, L24l L26, L27a, S13, S3a similar to 40S ribosomal protein SA ( p40 ) (34/67 kDa laminin receptor...mM ATP, 0.2% [vol/vol] Nonidet P-40 and 20% glycerol) were added to the cells, and the mixtures were vortexed for 1 minute. Beads and cell debris

  2. Functional Analysis of the Beclin-1 Tumor Suppressor Interaction With hVps34 (Type-III P13-Kinase) in Breast Cancer Cells

    DTIC Science & Technology

    2006-06-01

    1% Nonidet P40 , 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA. Insoluble material was removed by centrifugation at 100,000 g for 45 minutes at 4°C...7.4, 2 mM EDTA, 0.5% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with complete mini protease inhibitors (Roche). Insoluble material

  3. The Role of Heparin-Binding EGF-Like Growth Factor in Breast Cancer

    DTIC Science & Technology

    1996-10-01

    analysis: Human breast cancer cells were lysed using lysis buffer containing 20 mM Tris pH 7.5, 100 mM NaCl, 1% Nonidet - P40 , 0.5% deoxycholate, 5 mM...protein (MBP) assay: MCF-10A nontransformed and transformed cells were lysed in a buffer containing 1% Nonidet - P40 , 0.5 % deoxycholate, 20 mM Tris, pH 7.5

  4. A Structure-Function Analysis of Shiga-Like Toxin Type 2 of Enterohemorrhagic Escherichia Coli

    DTIC Science & Technology

    1990-05-07

    40 solution (10 mM Tris pH 7.3; 0.5 M NaCI; 0.5% Nonidet P40 ) by centrlfugation (5,000 x g for 5 minutes), and the pellet was resuspended to the...urea, 0.1% Nonidet P-40, and 0.0125% bromophenol blue). The lysed bacterial cells were boiled for 5 minutes and subjected to sodium dodecyl sulfate

  5. Thyroid dysfunction in patients with diffuse large B-cell lymphoma receiving lenalidomide is mediated by TNF-α

    PubMed Central

    Iams, Wade T.; Hames, Megan L.; Tsai, Judy P.; Dahlman, Kimberly B.; Talbott, Mahsa S.; Richards, Kristy L.; Reddy, Nishitha M.

    2016-01-01

    As the use of lenalidomide expands, the poorly understood phenomenon of lenalidomide-induced thyroid abnormalities will increase. In this study we compared rates of therapy-induced hypothyroidism in 329 patients with DLBCL treated with conventional chemotherapy (DLBCL-c) or conventional chemotherapy plus lenalidomide (DLBCL-len). We measured serum levels of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-6 (IL-6), interleukin-12 (IL-12), and interleukin-15 (IL-15) before and after treatment. We found a significantly higher rate of therapy-induced hypothyroidism in the DLBCL-len group (25.8% vs 1.3%), and we found a statistically significant increase in serum TNF-α in patients with lenalidomide-induced hypothyroidism. PMID:25448491

  6. Monoclonal gammopathy of undetermined significance in patients with psoriasis: is it really a side effect of biological therapy?

    PubMed

    Conti, Andrea; Esposito, Ilaria; Lasagni, Claudia; Miglietta, Roberta; Padalino, Claudia; Fabiano, Antonella; Pellacani, Giovanni

    2014-11-01

    Moderate-to-severe psoriasis is treated using biological drugs targeting cytokines involved in the pathogenesis of the disease, such as tumor necrosis factor alpha (TNF-α) (adalimumab, infliximab, etanercept) and interleukin 12/23 (IL 12/23) (ustekinumab). There is a slight risk of developing hematological malignancies, such as monoclonal gammopathy of undetermined significance (MGUS) with anti TNF-α agents. There are no data available on anti-IL12/23 drugs. This retrospective study of data from 191 patients describes the appearance and follow-up of MGUS in three patients with psoriasis receiving long-term biological therapy. Since the appearance of MGUS occurred after about 6 years of anti-TNFα treatment in only three subjects, it was deemed unlikely to be due to the biological treatment. The decision not to suspend biological therapy after the appearance of MGUS was taken after careful assessment of the possible risks and benefits.

  7. The risk of herpes zoster during biological therapy for psoriasis and other inflammatory conditions.

    PubMed

    Adelzadeh, L; Jourabchi, N; Wu, J J

    2014-07-01

    Recent advances in biological therapies have proved highly effective in treating psoriasis and other inflammatory conditions, including psoriatic arthritis, rheumatoid arthritis, inflammatory bowel disease and ankylosing spondylitis. However, adverse effects related to their immunosuppression have been observed, including an increased propensity to viral infections. This review evaluates the evidence of herpes zoster (HZ) risk from biologics based on clinical reports, cohort studies and randomized controlled studies. The risk of HZ associated with these agents remains controversial, especially when comparing their risk with non-biological therapy used to treat the same inflammatory conditions. This review specifically assesses the risk of the TNF inhibitors etanercept, adalimumab and infliximab, as well as interleukin-12/23 inhibitor ustekinumab. We found multiple cohort studies, randomized controlled trials and case reports that suggest infliximab increases risk of HZ, whereas adalimumab, etanercept and ustekinumab HZ risk remain controversial. Nevertheless, HZ vaccination should be considered prior to initiation of biological therapy, particularly infliximab.

  8. An animal model for open femur fracture and osteomyelitis: Part I.

    PubMed

    Lindsey, Brock A; Clovis, Nina B; Smith, E Suzanne; Salihu, Sydha; Hubbard, David F

    2010-01-01

    Infection is an everyday problem in orthopaedics and is quite common in open fracture management. To study this process and provide a basis to prevent infection, we developed a model that includes trauma (blunt fracture in the fashion of Bonnarens and Einhorn), surgical stabilization (standardized intramedullary K-wire fixation), and infection (Staphylococcus aureus inoculum). In this two-part study, we found that 10(2) colony-forming units of inoculum produced an optimal infection rate of 90-100%, which substantially challenged the immune system without overwhelming sepsis. We hypothesized that, in traumatic fractures, there is a specific immunological response that may lead to an increased rate of infection. In Part 2, we demonstrated immunosuppression (decreased Interleukin-12 levels) at days 6, 10, and 12 after fracture fixation versus nonfractured control groups (p < 0.05). This study describes a rat model of femur factures with osteomyelitis that allows investigation of posttraumatic immunosuppression.

  9. Successful treatment with ustekinumab of psoriasis vulgaris in a patient undergoing hemodialysis.

    PubMed

    Nimmannitya, Kulsupa; Tateishi, Chiharu; Mizukami, Yukari; Hamamoto, Kae; Yamada, Shinsuke; Goto, Hitoshi; Okada, Shigeki; Tsuruta, Daisuke

    2016-01-01

    Psoriasis is a common chronic inflammatory skin disease but psoriasis patients with renal impairment undergoing dialysis are not frequently seen. Furthermore, the published work contains little information on the treatment with biologic drugs of patients with end-stage renal disease. We describe a 57-year-old man with refractory plaque-type psoriasis and end-stage renal disease due to polycystic kidney disease undergoing hemodialysis. He had tried topical medications and ultraviolet therapy for many years and was then treated with ustekinumab (an interleukin-12 and interleukin-23 blocker), which resulted in good clinical response along with stable renal function. After a few years of therapy, no side-effects have been observed. Our experience with this patient expands the spectrum of ustekinumab to include psoriasis patients with renal failure undergoing hemodialysis.

  10. Molecular immunity to mycobacteria: knowledge from the mutation and phenotype spectrum analysis of Mendelian susceptibility to mycobacterial diseases

    PubMed Central

    Qu, Hui-Qi; Fisher-Hoch, Susan P.; McCormick, Joseph B.

    2011-01-01

    Summary Understanding molecular immunity against mycobacterial infection is critical for the development of effective strategies to control tuberculosis (TB), which is a major health issue in the developing world. Host immunogenetic studies represent an indispensable approach to understand the molecular mechanisms against mycobacterial infection. A superb paradigm is the identification of rare mutations causing Mendelian susceptibility to mycobacterial diseases (MSMD). Mutations in the interferon-gamma (IFN-γ) receptor genes are highly specific (although not exclusive) for mycobacterial infection. Only dominant negative mutations of STAT1 have specific susceptibility to mycobacterial infection. Mutations in the interleukin-12 (IL-12) signaling genes have phenotypes with non-specificity. Current studies highlight a complex molecular network in antimycobacterial immunity, centered on IFN-γ signaling. PMID:21330176

  11. EssD, a Nuclease Effector of the Staphylococcus aureus ESS Pathway.

    PubMed

    Ohr, Ryan Jay; Anderson, Mark; Shi, Miaomiao; Schneewind, Olaf; Missiakas, Dominique

    2017-01-01

    Specialized secretion systems of bacteria evolved for selective advantage, either killing microbial competitors or implementing effector functions during parasitism. Earlier work characterized the ESAT-6 secretion system (ESS) of Staphylococcus aureus and demonstrated its contribution to persistent staphylococcal infection of vertebrate hosts. Here, we identify a novel secreted effector of the ESS pathway, EssD, that functions as a nuclease and cleaves DNA but not RNA. EssI, a protein of the DUF600 family, binds EssD to block its nuclease activity in the staphylococcal cytoplasm. An essD knockout mutant or a variant lacking nuclease activity, essD(L546P), elicited a diminished interleukin-12 (IL-12) cytokine response following bloodstream infection of mice, suggesting that the effector function of EssD stimulates immune signaling to support the pathogenesis of S. aureus infections.

  12. Successful gene transfer into dendritic cells with cationized gelatin and plasmid DNA complexes via a phagocytosis-dependent mechanism.

    PubMed

    Inada, Satoshi; Fujiwara, Hitoshi; Atsuji, Kiyoto; Takashima, Kazuhiro; Araki, Yasunobu; Kubota, Takeshi; Tabata, Yasuhiko; Yamagishi, Hisakazu

    2006-01-01

    The use of gene-modified dendritic cells (DC) is a powerful tool to enhance antitumor immune responses stimulated by these cells in cancer immunotherapy. Cationized gelatin is preferably incorporated via phagocytosis and is gradually degraded by proteolysis while buffering lysosomal activity. This may be appropriate for gene transfer into phagocytic cells, such as immature DC. In the present study, successful transfection into monocyte-derived immature DC was demonstrated using cationized gelatin and plasmid DNA complexes. A high transfection efficiency, approaching 16%, was obtained upon transfection of the enhanced green fluorescent protein (EGFP) gene as evaluated by flow cytometry. Transgene expression of EGFP and murine interleukin 12 were also detected by RT-PCR. The antigen-presenting capacity of the transfected DC was equal to that of untransfected DC as evaluated by the allogeneic mixed lymphocyte reaction. Cationized gelatin has the potential to be a unique non-viral vector for gene transfer into DC.

  13. TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function.

    PubMed

    Krug, Anne; French, Anthony R; Barchet, Winfried; Fischer, Jens A A; Dzionek, Andrzej; Pingel, Jeanette T; Orihuela, Michael M; Akira, Shizuo; Yokoyama, Wayne M; Colonna, Marco

    2004-07-01

    Natural interferon-producing cells (IPC) respond to viruses by secreting type I interferon (IFN) and interleukin-12 (IL-12). Toll-like receptor (TLR) 9 mediates IPC recognition of some of these viruses in vitro. However, whether TLR9-induced activation of IPC is necessary for an effective antiviral response in vivo is not clear. Here, we demonstrate that IPC and dendritic cells (DC) recognize murine cytomegalovirus (MCMV) through TLR9. TLR9-mediated cytokine secretion promotes viral clearance by NK cells that express the MCMV-specific receptor Ly49H. Although depletion of IPC leads to a drastic reduction of the IFN-alpha response, this allows other cell types to secrete IL-12, ensuring normal IFN-gamma and NK cell responses to MCMV. We conclude that the TLR9/MyD88 pathway mediates antiviral cytokine responses by IPC, DC, and possibly other cell types, which are coordinated to promote effective NK cell function and MCMV clearance.

  14. Cytokine gene-mediated immunotherapy: current status and future perspectives.

    PubMed

    Jinushi, Masahisa; Tahara, Hideaki

    2009-08-01

    Recent understanding of the molecular events crucial in overcoming immunosuppressive tumor microenvironments and generating effective antitumor immunity provides us with the wreath opportunity to manipulate genes that have a key role in antitumor immune responses. Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-12 (IL-12) are two indispensable cytokines for activating dendritic cells and boosting the strong immune responses against cancer. In this review, we describe the antitumor mechanisms and clinical application of gene-modified tumor cells and dendritic cells to secrete GM-CSF or IL-12, respectively, in various preclinical and clinical settings. The principles operative in these vaccination strategies may prove applicable to other immunotherapy strategies, especially in combination with other therapeutic modalities, such as chemotherapy and targeted therapy.

  15. Role of leptin in reverse epidemiology in chronic kidney disease.

    PubMed

    Scholze, Alexandra; Tepel, Martin

    2007-01-01

    Leptin is mainly produced by adipocytes and metabolized in the kidney. Leptin is taken up into the central nervous system by a saturable transport system, and controls appetite in rodents and in healthy subjects. Leptin acts on peripheral tissue and increases the inflammatory response by stimulating the production of tumor necrosis factor alpha, interleukin-6 and interleukin-12. In healthy humans, serum leptin concentration is related to the size of adipose tissue mass in the body. The majority of obese subjects have inappropriately high levels of circulating plasma leptin concentrations, indicating leptin resistance. In healthy subjects increased leptin concentration constitutes a biomarker for increased cardiovascular risk. On the other hand, a recent prospective long-term study in patients with chronic kidney disease stage 5 on hemodialysis therapy showed that reduced serum leptin concentration is an independent risk factor for mortality in these patients.

  16. Congenital IL-12R1β receptor deficiency and thrombophilia in a girl homozygous for an IL12RB1 mutation and compound heterozygous for MTFHR mutations: A case report and literature review

    PubMed Central

    Kose, M.; Ceylan, O.; Patiroglu, T.; Bustamante, J.; Casanova, J. L.; Akyildiz, B. N.; Doganay, S.

    2014-01-01

    Interleukin-12 (IL-12) plays an important role in the production of interferon gamma from T cells and natural killer cells and is essential for protection against intra-macrophagic pathogens such as Mycobacterium and Salmonella. Here, we describe a 16-year-old girl with homozygous mutation in exon 12 of the IL12RB1 gene, which causes complete IL-12Rβ1 deficiency in association with heterozygous mutation (C677T and A1298C) in the methylenetetrahydrofolate reductase gene. She presented with disseminated Mycobacterium tuberculosis complex infection, retroperitoneal fungal abscess and also thrombosis in the superior mesenteric–portal vein junction. This is the first case report of a primary immunodeficiency associated with a genetically determined venous thrombosis. PMID:24678409

  17. Lung airway-surveilling CXCR3(hi) memory CD8(+) T cells are critical for protection against influenza A virus.

    PubMed

    Slütter, Bram; Pewe, Lecia L; Kaech, Susan M; Harty, John T

    2013-11-14

    Inducing memory CD8(+) T cells specific for conserved antigens from influenza A virus (IAV) is a potential strategy for broadly protective vaccines. Here we show that memory CD8(+) T cells in the airways played an important role in early control of IAV. Expression of the chemokine receptor CXCR3 was critical for memory CD8(+) T cells to populate the airways during the steady state and vaccination approaches were designed to favor the establishment of memory CD8(+) T cells in the airways. Specifically, we found that interleukin-12 (IL-12) signaling shortly after immunization limited CXCR3 expression on memory CD8(+) T cells. Neutralization of IL-12 or adjuvants that did not induce high amounts of IL-12 enhanced CXCR3 expression, sustained airway localization of memory CD8(+) T cells, and resulted in superior protection against IAV.

  18. New insights into IL-12-mediated tumor suppression

    PubMed Central

    Tugues, S; Burkhard, S H; Ohs, I; Vrohlings, M; Nussbaum, K; vom Berg, J; Kulig, P; Becher, B

    2015-01-01

    During the past two decades, interleukin-12 (IL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models. Through pleiotropic effects on different immune cells that form the tumor microenvironment, IL-12 establishes a link between innate and adaptive immunity that involves different immune effector cells and cytokines depending on the type of tumor or the affected tissue. The robust antitumor response exerted by IL-12, however, has not yet been successfully translated into the clinics. The majority of clinical trials involving treatment with IL-12 failed to show sustained antitumor responses and were associated to toxic side effects. Here we discuss the therapeutic effects of IL-12 from preclinical to clinical studies, and will highlight promising strategies to take advantage of the antitumor activity of IL-12 while limiting adverse effects. PMID:25190142

  19. New insights into IL-12-mediated tumor suppression.

    PubMed

    Tugues, S; Burkhard, S H; Ohs, I; Vrohlings, M; Nussbaum, K; Vom Berg, J; Kulig, P; Becher, B

    2015-02-01

    During the past two decades, interleukin-12 (IL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models. Through pleiotropic effects on different immune cells that form the tumor microenvironment, IL-12 establishes a link between innate and adaptive immunity that involves different immune effector cells and cytokines depending on the type of tumor or the affected tissue. The robust antitumor response exerted by IL-12, however, has not yet been successfully translated into the clinics. The majority of clinical trials involving treatment with IL-12 failed to show sustained antitumor responses and were associated to toxic side effects. Here we discuss the therapeutic effects of IL-12 from preclinical to clinical studies, and will highlight promising strategies to take advantage of the antitumor activity of IL-12 while limiting adverse effects.

  20. Cyclophilin-40 has a cellular role in the aryl hydrocarbon receptor signaling.

    PubMed

    Luu, Tony C; Bhattacharya, Pompeya; Chan, William K

    2008-09-22

    Cyclophilin-40 (CyP40) promotes the formation of the gel shift complex that contains the aryl hydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and dioxin response element (DRE) using baculovirus expressed proteins. Here we reported that CyP40 plays a role in the AhR signaling. When the CyP40 content in MCF-7 cells is reduced, up-regulation of cyp1a1 and cyp1b1 by 3-methylchloranthrene (3MC) is also reduced, suggesting that CyP40 is essential for maximal AhR function. The CyP40 region containing amino acids 186-215, but not the peptidyl-prolyl cis-trans isomerase and tetratricopeptide repeat domains, is essential for forming the AhR/Arnt/DRE complex. CyP40 is found in the cell nucleus after 3MC treatment and appears to promote the DRE binding form of the AhR/Arnt heterodimer.

  1. A comparison of surface proteins in embryonal carcinoma cells and their differentiated derivatives.

    PubMed

    Keil-Dlouha, V; Paulin, D; Bagilet, L K; Keil, B

    1980-03-27

    Surface proteins from five cell lines (three embryonal carcinoma cell lines (F9, PCC4 and PCC3), teratocarcinoma-derived endodermal cells (PYS) and fibroblasts (line 3/A/1-D-3 differentiated from PCC3) were compared by two-dimensional polyacrylamide gel electrophoresis after selective iodination with 125I in the presence of lactoperoxidase. The labeled proteins were solubilized either in Nonidet P40/urea/ampholyte/mercaptoethanol solution or in Nonidet P40 only. In total, about thirty major 125I-labeled surface proteins were identified by their isoelectric point and molecular weight. 14 proteins are present in all five cell types, although their quantity or accessibility for labeling differs between differentiated and undifferentiated cells. Three proteins (200, 160 and 150 kilodaltons) are present in undifferentiated cells only. Two of them (160 and 150 kilodaltons) were solubilized by Nonidet P40/urea/ampholyte/mercaptoethanol, but not by Nonidet P40. One protein (50 kilodaltons) was found in nullipotent F9 cells only. About 14--15 proteins (including fibronectin) were released by Nonidet P40/urea/ampholyte/mercaptoethanol but not by Nonidet P40. They are presumably bound to submembrane or cytoskeleton structures by non-covalent bonds.

  2. Folate-conjugated immunoglobulin targets melanoma tumor cells for NK cell effector functions.

    PubMed

    Skinner, Cassandra C; McMichael, Elizabeth L; Jaime-Ramirez, Alena C; Abrams, Zachary B; Lee, Robert J; Carson, William E

    2016-08-01

    The folate receptor (FR) is overexpressed on the vascular side of cancerous cells including those of the breast, ovaries, testes, and cervix. We hypothesized that a folate-conjugated immunoglobulin (F-IgG) would bind to the FR that is overexpressed on melanoma tumor cells to target these cells for lysis by natural killer (NK) cells. Folate receptor expression was confirmed in the Mel-39 (human melanoma) cell line by flow cytometry and immunoblot analysis using KB (human oral epithelial) and F01 (human melanoma) as a positive and a negative control, respectively. FR-positive and FR-negative cell lines were treated with F-IgG or control immunoglobulin G in the presence or absence of cytokines to determine NK cell ability to lyse FR-positive cell lines. NK cell activation was significantly upregulated and lysis of Mel 39 tumor cells increased following treatment with F-IgG compared with control immunoglobulin G at all effector : target (E : T) ratios (P<0.01). This trend further increased by NK cell stimulation with the activating cytokine interleukin-12. NK cell production of cytokines such as interferon-gamma, macrophage inflammatory protein 1α, and regulated on activation normal T-cell expressed and secreted (RANTES) was also significantly increased in response to costimulation with interleukin-12 stimulation and F-IgG-coated Mel 39 target cells compared with controls (P<0.01). In contrast, F-IgG did not bind to the FR-negative cell line F01 and had no significant effect on NK cell lysis or cytokine production. This research indicates the potential use of F-IgG for its ability to induce an immune response from NK cells against FR-positive melanoma tumor cells, which can be further increased by the addition of cytokines.

  3. Immunohistochemical analysis of the expression of cellular transcription NFκB (p65), AP-1 (c-Fos and c-Jun), and JAK/STAT in leprosy.

    PubMed

    Silva, Luciana Mota; Hirai, Kelly Emi; de Sousa, Jorge Rodrigues; de Souza, Juarez; Fuzii, Hellen Thais; Dias, Leonidas Braga; Carneiro, Francisca Regina Oliveira; de Souza Aarão, Tinara Leila; Quaresma, Juarez Antonio Simões

    2015-05-01

    Leprosy is a disease whose clinical spectrum depends on the cytokine patterns produced during the early stages of the immune response. The main objective of this study was to describe the activation pattern of cellular transcription factors and to correlate these factors with the clinical forms of leprosy. Skin samples were obtained from 16 patients with the tuberculoid (TT) form and 14 with the lepromatous (LL) form. The histologic sections were immunostained with anti-c-Fos and anti-c-Jun monoclonal antibodies for investigation of AP-1, anti-NFκB p65 for the study of NFκB, and anti-JAK2, STAT1, STAT3, and STAT4 for investigation of the JAK/STAT pathway. Cells expressing STAT1 were more frequent in the TT form than in LL lesions (P = .0096), in agreement with the protective immunity provided by IFN-γ. STAT4 was also more highly expressed in the TT form than in the LL form (P = .0098). This transcription factor is essential for the development of a Th1 response because it is associated with interleukin-12. NFκB (p65) and STAT4 expression in the TT form showed a strong and significant correlation (r = 0.7556 and P = .0007). A moderate and significant correlation was observed between JAK2 and STAT4 in the TT form (r = 0.6637 and P = .0051), with these factors responding to interleukin-12 in Th1 profiles. The results suggest that STAT1, JAK2, and NFκB, together with STAT4, contribute to the development of cell-mediated immunity, which is able to contain the proliferation of Mycobacterium leprae.

  4. The restoration of the antitumor T cell response from stress-induced suppression using a traditional Chinese herbal medicine Hochu-ekki-to (TJ-41:Bu-Zhong-Yi-Qi-Tang).

    PubMed

    Li, T; Tamada, K; Abe, K; Tada, H; Onoe, Y; Tatsugami, K; Harada, M; Kubo, C; Nomoto, K

    1999-06-01

    We previously reported that restraint stress impairs the antitumor immune responses through its suppressive effect on the Th1-type cytokine production from CD4+ T cells. In this study, we investigated a potential of Hochu-ekki-to (TJ-41:Bu-Zhong-Yi-Qi-Tang) to restore stress-induced immunosuppression. The oral administration of TJ-41 was able to improve a decreased cellularity in the lymph node and spleen and to improve an inhibition of tumor-specific Th1-type cytokine production, both of which were induced by repeated restraint stress in tumor-bearing mice. The oral administration of TJ-41 also induced a partial recovery of the antitumor cytolytic activity in the stress-burdened tumor-bearing mice. More importantly, the growth of tumors in stress-burdened preimmunized mice was obviously inhibited by TJ-41, and resulted in tumor-free state in 75% of the mice. Regarding the mechanisms by which TJ-41 restored the antitumor responses in stress-burdened mice, we found that the serum levels of corticosterone and interleukin-12 were normalized by TJ-41. In addition, the expression of CD80 and CD86, which both decreased in the stress-burdened mice, was restored to the normal level by TJ-41. Taken together, our results indicate that the oral administration of TJ-41 is able to restore the antitumor T cell responses in stress-burdened tumor-bearing mice by normalizing the serum corticosterone, interleukin-12 and the expression of costimulatory molecules.

  5. A novel approach to investigation of the pathogenesis of active minimal-change nephrotic syndrome using subtracted cDNA library screening.

    PubMed

    Sahali, Djillali; Pawlak, André; Valanciuté, Asta; Grimbert, Philippe; Lang, Philippe; Remy, Philippe; Bensman, Albert; Guellaën, Georges

    2002-05-01

    Clinical and experimental observations suggest that minimal-change nephrotic syndrome (MCNS) results from T cell dysfunction, via unknown mechanisms. For the identification of genes that are potentially involved in MCNS, a subtractive cDNA library was constructed from cDNA from T cell-enriched peripheral blood mononuclear cells obtained from the same patient during relapse versus remission ("relapse minus remission"). This library was screened by differential hybridization with forward ("relapse minus remission") and reverse ("remission minus relapse") subtractive cDNAs probes, as well as unsubtracted probes from relapse and remission, and irrelevant nephrotic syndrome (membranous nephropathy). A total of 84 transcripts were isolated, of which 12 matched proteins of unknown function and 30 were unknown clones. Among the 42 known transcripts, at least 18 are closely involved in the T cell receptor-mediated complex signaling cascade, including genes encoding components of the T cell receptor and proteins associated with the cytoskeletal scaffold, as well as transcription factors. In particular, it was demonstrated that the expression levels of Fyb/Slap, L-plastin, and grancalcin were increased during relapse, suggesting that the integration of proximal signaling after T cell engagement involves the preferential recruitment of these cytoskeleton-associated proteins in MCNS. Because very low levels of interleukin-12 receptor beta2 mRNA were detected in relapse samples, the interleukin-12 signaling pathway might be defective, suggesting that, in MCNS, T cell activation evolves toward a T helper 2 phenotype. Therefore, the combination of subtractive cloning and differential screening constitutes an efficient approach to the identification of genes that are likely to be involved in the pathophysiologic processes of MCNS.

  6. Activation and inhibition of erythropoietin receptor function: role of receptor dimerization.

    PubMed Central

    Watowich, S S; Hilton, D J; Lodish, H F

    1994-01-01

    Members of the cytokine receptor superfamily have structurally similar extracellular ligand-binding domains yet diverse cytoplasmic regions lacking any obvious catalytic domains. Many of these receptors form ligand-induced oligomers which are likely to participate in transmembrane signaling. A constitutively active (factor-independent) mutant of the erythropoietin receptor (EPO-R), R129C in the exoplasmic domain, forms disulfide-linked homodimers, suggesting that the wild-type EPO-R is activated by ligand-induced homodimerization. Here, we have taken two approaches to probe the role EPO-R dimerization plays in signal transduction. First, on the basis of the crystal structure of the ligand-bound, homodimeric growth hormone receptor (GH-R) and sequence alignment between the GH-R and EPO-R, we identified residues of the EPO-R which may be involved in intersubunit contacts in an EPO-R homodimer. Residue 129 of the EPO-R corresponds to a residue localized to the GH-R dimer interface region. Alanine or cysteine substitutions were introduced at four other residues of the EPO-R predicted to be in the dimer interface region. Substitution of residue E-132 or E-133 with cysteine renders the EPO-R constitutively active. Like the arginine-to-cysteine mutation at position 129 in the exoplasmic domain (R129C), E132C and E133C form disulfide-linked homodimers, suggesting that constitutive activity is due to covalent dimerization. In the second approach, we have coexpressed the wild-type EPO-R with inactive mutants of the receptor missing all or part of the cytosolic domain. These truncated receptors have a dominant inhibitory effect on the proliferative action of the wild-type receptor. Taken together, these results strengthen the hypothesis that an initial step in EPO- and EPO-R-mediated signal transduction is ligand-induced receptor dimerization. Images PMID:8196600

  7. Light-induced disulfide dimerization of recoverin under ex vivo and in vivo conditions.

    PubMed

    Zernii, Evgeni Yu; Nazipova, Aliya A; Gancharova, Olga S; Kazakov, Alexey S; Serebryakova, Marina V; Zinchenko, Dmitry V; Tikhomirova, Natalya K; Senin, Ivan I; Philippov, Pavel P; Permyakov, Eugene A; Permyakov, Sergei E

    2015-06-01

    Despite vast knowledge of the molecular mechanisms underlying photochemical damage of photoreceptors, linked to progression of age-related macular degeneration, information on specific protein targets of the light-induced oxidative stress is scarce. Here, we demonstrate that prolonged intense illumination (halogen bulb, 1500 lx, 1-5 h) of mammalian eyes under ex vivo (cow) or in vivo (rabbit) conditions induces disulfide dimerization of recoverin, a Ca(2+)-dependent inhibitor of rhodopsin kinase. Western blotting and mass spectrometry analysis of retinal extracts reveals illumination time-dependent accumulation of disulfide homodimers of recoverin and its higher order disulfide cross-linked species, including a minor fraction of mixed disulfides with intracellular proteins (tubulins, etc.). Meanwhile, monomeric bovine recoverin remains mostly reduced. These effects are accompanied by accumulation of disulfide homodimers of visual arrestin. Histological studies demonstrate that the light-induced oxidation of recoverin and arrestin occurs in intact retina (illumination for 2 h), while illumination for 5 h is associated with damage of the photoreceptor layer. A comparison of ex vivo levels of disulfide homodimers of bovine recoverin with redox dependence of its in vitro thiol-disulfide equilibrium (glutathione redox pair) gives the lowest estimate of redox potential in rod outer segments under illumination from -160 to -155 mV. Chemical crosslinking and dynamic light scattering data demonstrate an increased propensity of disulfide dimer of bovine recoverin to multimerization/aggregation. Overall, the oxidative stress caused by the prolonged intense illumination of retina might affect rhodopsin desensitization via concerted disulfide dimerization of recoverin and arrestin. The developed herein models of eye illumination are useful for studies of the light-induced thiol oxidation of visual proteins.

  8. Evaluation of one-dimensional potential energy surfaces for prediction of spectroscopic properties of hydrogen bonds in linear bonded complexes.

    PubMed

    Jouypazadeh, Hamidreza; Farrokhpour, Hossein; Solimannejad, Mohammad

    2017-05-01

    This work evaluated the reliability of the one-dimensional potential energy surface for calculating the spectroscopic properties (rovibrational constants and rotational line energies) of hydrogen bonds in linear bonded complexes by comparing theoretical results with the corresponding experimental results. For this purpose, two hydrogen bonded complexes were selected: the HCN···HCN homodimer and the HCN···HF heterodimer. The one-dimensional potential energy surfaces related to the hydrogen bonds in these complexes were calculated using different computational methods and basis sets. The calculated potential curve of each complex was fitted to an analytical one-dimensional potential function to obtain the potential parameters. The obtained analytical potential function of each complex was used in a two-particle Schrödinger equation to obtain the rovibrational energy levels of the hydrogen bond. Using the calculated rovibrational levels, the rovibrational spectra and constants of each complex were calculated and compared with experimental data available from the literature. Compared with experimental data, the calculated one-dimensional potential energy surface at the QCISD/aug-cc-pVDZ level of theory was found to predict the spectroscopic properties of hydrogen bonds better than the potential curves obtained using other computational methods, especially for the HCN···HCN homodimer complex. Generally, the results obtained for the HCN···HCN homodimer complex were closer to experimental data than those obtained for the HCN···HF heterodimer complex. The investigation performed in this work showed that the one-dimensional potential curve related to the hydrogen bond between two linear molecules can be used to predict the spectroscopic constants of hydrogen bonds. Graphical abstract Potential energy curves of HCN···HCN and HCN···HF complexes calculated at the different computational levels.

  9. Mapping the Interaction of B Cell Leukemia 3 (BCL-3) and Nuclear Factor κB (NF-κB) p50 Identifies a BCL-3-mimetic Anti-inflammatory Peptide*

    PubMed Central

    Collins, Patricia E.; Grassia, Gianluca; Colleran, Amy; Kiely, Patrick A.; Ialenti, Armando; Maffia, Pasquale; Carmody, Ruaidhrí J.

    2015-01-01

    The NF-κB transcriptional response is tightly regulated by a number of processes including the phosphorylation, ubiquitination, and subsequent proteasomal degradation of NF-κB subunits. The IκB family protein BCL-3 stabilizes a NF-κB p50 homodimer·DNA complex through inhibition of p50 ubiquitination. This complex inhibits the binding of the transcriptionally active NF-κB subunits p65 and c-Rel on the promoters of NF-κB target genes and functions to suppress inflammatory gene expression. We have previously shown that the direct interaction between p50 and BCL-3 is required for BCL-3-mediated inhibition of pro-inflammatory gene expression. In this study we have used immobilized peptide array technology to define regions of BCl-3 that mediate interaction with p50 homodimers. Our data show that BCL-3 makes extensive contacts with p50 homodimers and in particular with ankyrin repeats (ANK) 1, 6, and 7, and the N-terminal region of Bcl-3. Using these data we have designed a BCL-3 mimetic peptide based on a region of the ANK1 of BCL-3 that interacts with p50 and shares low sequence similarity with other IκB proteins. When fused to a cargo carrying peptide sequence this BCL-3-derived peptide, but not a mutated peptide, inhibited Toll-like receptor-induced cytokine expression in vitro. The BCL-3 mimetic peptide was also effective in preventing inflammation in vivo in the carrageenan-induced paw edema mouse model. This study demonstrates that therapeutic strategies aimed at mimicking the functional activity of BCL-3 may be effective in the treatment of inflammatory disease. PMID:25922067

  10. Rational design of small-molecule stabilizers of spermine synthase dimer by virtual screening and free energy-based approach.

    PubMed

    Zhang, Zhe; Martiny, Virginie; Lagorce, David; Ikeguchi, Yoshihiko; Alexov, Emil; Miteva, Maria A

    2014-01-01

    Snyder-Robinson Syndrome (SRS) is a rare mental retardation disorder which is caused by the malfunctioning of an enzyme, the spermine synthase (SMS), which functions as a homo-dimer. The malfunctioning of SMS in SRS patients is associated with several identified missense mutations that occur away from the active site. This investigation deals with a particular SRS-causing mutation, the G56S mutation, which was shown computationally and experimentally to destabilize the SMS homo-dimer and thus to abolish SMS enzymatic activity. As a proof-of-concept, we explore the possibility to restore the enzymatic activity of the malfunctioning SMS mutant G56S by stabilizing the dimer through small molecule binding at the mutant homo-dimer interface. For this purpose, we designed an in silico protocol that couples virtual screening and a free binding energy-based approach to identify potential small-molecule binders on the destabilized G56S dimer, with the goal to stabilize it and thus to increase SMS G56S mutant activity. The protocol resulted in extensive list of plausible stabilizers, among which we selected and tested 51 compounds experimentally for their capability to increase SMS G56S mutant enzymatic activity. In silico analysis of the experimentally identified stabilizers suggested five distinctive chemical scaffolds. This investigation suggests that druggable pockets exist in the vicinity of the mutation sites at protein-protein interfaces which can be used to alter the disease-causing effects by small molecule binding. The identified chemical scaffolds are drug-like and can serve as original starting points for development of lead molecules to further rescue the disease-causing effects of the Snyder-Robinson syndrome for which no efficient treatment exists up to now.

  11. Differential endocytic routing of homo- and hetero-dimeric ErbB tyrosine kinases confers signaling superiority to receptor heterodimers.

    PubMed Central

    Lenferink, A E; Pinkas-Kramarski, R; van de Poll, M L; van Vugt, M J; Klapper, L N; Tzahar, E; Waterman, H; Sela, M; van Zoelen, E J; Yarden, Y

    1998-01-01

    Both homo- and hetero-dimers of ErbB receptor tyrosine kinases mediate signaling by a large group of epidermal growth factor (EGF)-like ligands. However, some ligands are more potent than others, although they bind to the same direct receptor. In addition, signaling by receptor heterodimers is superior to homodimers. We addressed the mechanism underlying these two features of signal tuning by using three ligands: EGF; transforming growth factor alpha (TGFalpha); and their chimera, denoted E4T, which act on cells singly expressing ErbB-1 as a weak, a strong, and a very strong agonist, respectively. Co-expression of ErbB-2, a developmentally important co-receptor whose expression is frequently elevated in human cancers, specifically potentiated EGF signaling to the level achieved by TGFalpha, an effect that was partially mimicked by ErbB-3. Analysis of the mechanism underlying this trans-potentiation implied that EGF-driven homodimers of ErbB-1 are destined for intracellular degradation, whereas the corresponding heterodimers with ErbB-2 or with ErbB-3, dissociate in the early endosome. As a consequence, in the presence of either co-receptor, ErbB-1 is recycled to the cell surface and its signaling is enhanced. This latter route is followed by TGFalpha-driven homodimers of ErbB-1, and also by E4T-bound receptors, whose signaling is further enhanced by repeated cycles of binding and dissociation from the receptors. We conclude that alternative endocytic routes of homo- and hetero-dimeric receptor complexes may contribute to tuning and diversification of signal transduction. In addition, the ability of ErbB-2 to shunt ligand-activated receptors to recycling may explain, in part, its oncogenic potential. PMID:9628875

  12. Characterization of IRA/IRB hybrid insulin receptors using bioluminescence resonance energy transfer.

    PubMed

    Blanquart, Christophe; Achi, Josepha; Issad, Tarik

    2008-10-01

    The insulin receptor (IR) is composed of two alpha-chains that bind ligands and two beta-chains that possess an intracellular tyrosine kinase activity. The IR is expressed in cells as two isoforms containing or not exon 11 (IRB and IRA, respectively). Several mRNA studies have demonstrated that the two isoforms are co-expressed in different tissues and in several cancer cells. IRA/IRB hybrid receptors, constituting of an alphabeta-chain from IRA and an alphabeta-chain from IRB, are likely to occur in cells co-expressing both isoforms, but their study has been hampered by the lack of specific tools. In previous work, we used BRET to study IR and IGF1R homodimers and heterodimers. Here, we have used BRET to characterize IRA/IRB hybrids. BRET saturation experiments showed that IRA/IRB hybrids are randomly formed in cells. Moreover, by co-transfecting HEK-293 cells with a luciferase-tagged kinase-dead version of one isoform and a wild-type untagged version of the other isoform, we showed that IRA/IRB hybrids can recruit, upon ligand stimulation, a YFP-tagged intracellular partner. Finally, using BRET, we have studied ligand-induced conformational changes within IRA/IRB hybrids. Dose-response experiments showed that hybrid receptors bind IGF-2 with the same affinity than IRA homodimers, whereas they bind IGF-1 with a lower affinity. Altogether, our data indicate that IRA/IRB hybrid receptors can form in cells co-expressing both IR isoforms, that they are capable of recruiting intracellular partners upon ligand stimulation, and that they have pharmacological properties more similar to those of IRA than those of IRB homodimers with regards to IGF-2.

  13. Structural reorganization of the interleukin-7 signaling complex

    SciTech Connect

    McElroy, Craig A.; Holland, Paul J.; Zhao, Peng; Lim, Jae-Min; Wells, Lance; Eisenstein, Edward; Walsh, Scott T.R.

    2012-06-29

    We report here an unliganded receptor structure in the common gamma-chain ({gamma}{sub c}) family of receptors and cytokines. The crystal structure of the unliganded form of the interleukin-7 alpha receptor (IL-7R{alpha}) extracellular domain (ECD) at 2.15 {angstrom} resolution reveals a homodimer forming an 'X' geometry looking down onto the cell surface with the C termini of the two chains separated by 110 {angstrom} and the dimer interface comprising residues critical for IL-7 binding. Further biophysical studies indicate a weak association of the IL-7R{alpha} ECDs but a stronger association between the {gamma}{sub c}/IL-7R{alpha} ECDs, similar to previous studies of the full-length receptors on CD4{sup +} T cells. Based on these and previous results, we propose a molecular mechanism detailing the progression from the inactive IL-7R{alpha} homodimer and IL-7R{alpha}-{gamma}{sub c} heterodimer to the active IL-7-IL-7R{alpha}-{gamma}{sub c} ternary complex whereby the two receptors undergo at least a 90{sup o} rotation away from the cell surface, moving the C termini of IL-7R{alpha} and {gamma}{sub c} from a distance of 110 {angstrom} to less than 30 {angstrom} at the cell surface. This molecular mechanism can be used to explain recently discovered IL-7- and {gamma}{sub c}-independent gain-of-function mutations in IL-7R{alpha} from B- and T-cell acute lymphoblastic leukemia patients. The mechanism may also be applicable to other {gamma}{sub c} receptors that form inactive homodimers and heterodimers independent of their cytokines.

  14. Bacterial superantigen toxins induce a lethal cytokine storm by enhancing B7-2/CD28 costimulatory receptor engagement, a critical immune checkpoint

    PubMed Central

    Kaempfer, Raymond; Popugailo, Andrey; Levy, Revital; Arad, Gila; Hillman, Dalia; Rotfogel, Ziv

    2017-01-01

    Formation of the costimulatory axis between the B7-2 and CD28 coreceptors is critical for T-cell activation. Superantigens, Gram-positive bacterial virulence factors, cause toxic shock and sepsis by hyperinducing inflammatory cytokines. We report a novel role for costimulatory receptors CD28 and B7-2 as obligatory receptors for superantigens, rendering them therapeutic targets. We show that by engaging not only CD28 but also its coligand B7-2 directly, superantigens potently enhance the interaction between B7-2 and CD28, inducing thereby T-cell hyperactivation. Using a conserved twelve amino-acid domain, superantigens engage both B7-2 and CD28 at their homodimer interfaces, sites far removed from where these receptors interact, implying that inflammatory signaling can be controlled through the receptor homodimer interfaces. Short B7-2 and CD28 dimer interface mimetic peptides bind diverse superantigens, prevent superantigen binding to cell-surface B7-2 or CD28, attenuate inflammatory cytokine overexpression, and protect mice from lethal superantigen challenge. Thus, superantigens induce a cytokine storm by mediating not only the interaction between MHC-II molecule and T-cell receptor but critically, by promoting B7-2/CD28 coreceptor engagement, forcing the principal costimulatory axis to signal excessively. Our findings highlight the B7/CD28 interaction as a bottleneck in signaling for expression of inflammatory cytokines. B7-2 and CD28 homodimer interface mimetic peptides prevent superantigen lethality by blocking the superantigen-host costimulatory receptor interaction. PMID:28286804

  15. New structural determinants for c-Myc specific heterodimerization with Max and development of a novel homodimeric c-Myc b-HLH-LZ.

    PubMed

    Beaulieu, Marie-Eve; McDuff, François-Olivier; Frappier, Vincent; Montagne, Martin; Naud, Jean-François; Lavigne, Pierre

    2012-07-01

    c-Myc must heterodimerize with Max to accomplish its functions as a transcription factor. This specific heterodimerization occurs through the b-HLH-LZ (basic region, helix 1-loop-helix 2-leucine zipper) domains. In fact, many studies have shown that the c-Myc b-HLH-LZ (c-Myc'SH) preferentially forms a heterodimer with the Max b-HLH-LZ (Max'SH). The primary mechanism underlying the specific heterodimerization lies on the destabilization of both homodimers and the formation of a more stable heterodimer. In this regard, it has been widely reported that c-Myc'SH has low solubility and homodimerizes poorly and that repulsions within the LZ domain account for the homodimer instability. Here, we show that replacing one residue in the basic region and one residue in Helix 1 (H(1)) of c-Myc'SH with corresponding residues conserved in b-HLH proteins confers to c-Myc'SH a higher propensity to form a stable homodimer in solution. In stark contrast to the wild-type protein, this double mutant (L362R, R367L) of the c-Myc b-HLH-LZ (c-Myc'RL) shows limited heterodimerization with Max'SH in vitro. In addition, c-Myc'RL forms highly stable and soluble complexes with canonical as well as non-canonical E-box probes. Altogether, our results demonstrate for the first time that structural determinants driving the specific heterodimerization of c-Myc and Max are embedded in the basic region and H(1) of c-Myc and that these can be exploited to engineer a novel homodimeric c-Myc b-HLH-LZ with the ability of binding the E-box sequence autonomously and with high affinity.

  16. Quantifying the topography of the intrinsic energy landscape of flexible biomolecular recognition.

    PubMed

    Chu, Xiakun; Gan, Linfeng; Wang, Erkang; Wang, Jin

    2013-06-25

    Biomolecular functions are determined by their interactions with other molecules. Biomolecular recognition is often flexible and associated with large conformational changes involving both binding and folding. However, the global and physical understanding for the process is still challenging. Here, we quantified the intrinsic energy landscapes of flexible biomolecular recognition in terms of binding-folding dynamics for 15 homodimers by exploring the underlying density of states, using a structure-based model both with and without considering energetic roughness. By quantifying three individual effective intrinsic energy landscapes (one for interfacial binding, two for monomeric folding), the association mechanisms for flexible recognition of 15 homodimers can be classified into two-state cooperative "coupled binding-folding" and three-state noncooperative "folding prior to binding" scenarios. We found that the association mechanism of flexible biomolecular recognition relies on the interplay between the underlying effective intrinsic binding and folding energy landscapes. By quantifying the whole global intrinsic binding-folding energy landscapes, we found strong correlations between the landscape topography measure Λ (dimensionless ratio of energy gap versus roughness modulated by the configurational entropy) and the ratio of the thermodynamic stable temperature versus trapping temperature, as well as between Λ and binding kinetics. Therefore, the global energy landscape topography determines the binding-folding thermodynamics and kinetics, crucial for the feasibility and efficiency of realizing biomolecular function. We also found "U-shape" temperature-dependent kinetic behavior and a dynamical cross-over temperature for dividing exponential and nonexponential kinetics for two-state homodimers. Our study provides a unique way to bridge the gap between theory and experiments.

  17. Glucose-Sensing Receptor T1R3: A New Signaling Receptor Activated by Glucose in Pancreatic β-Cells.

    PubMed

    Kojima, Itaru; Nakagawa, Yuko; Hamano, Kunihisa; Medina, Johan; Li, Longfei; Nagasawa, Masahiro

    2015-01-01

    Subunits of the sweet taste receptors T1R2 and T1R3 are expressed in pancreatic β-cells. Compared with T1R3, mRNA expression of T1R2 is considerably lower. At the protein level, expression of T1R2 is undetectable in β-cells. Accordingly, a major component of the sweet taste-sensing receptor in β-cells may be a homodimer of T1R3 rather than a heterodimer of T1R2/T1R3. Inhibition of this receptor by gurmarin or deletion of the T1R3 gene attenuates glucose-induced insulin secretion from β-cells. Hence the T1R3 homodimer functions as a glucose-sensing receptor (GSR) in pancreatic β-cells. When GSR is activated by the T1R3 agonist sucralose, elevation of intracellular ATP concentration ([ATP]i) is observed. Sucralose increases [ATP]i even in the absence of ambient glucose, indicating that sucralose increases [ATP]i not simply by activating glucokinase, a rate-limiting enzyme in the glycolytic pathway. In addition, sucralose augments elevation of [ATP]i induced by methylsuccinate, suggesting that sucralose activates mitochondrial metabolism. Nonmetabolizable 3-O-methylglucose also increases [ATP]i and knockdown of T1R3 attenuates elevation of [ATP]i induced by high concentration of glucose. Collectively, these results indicate that the T1R3 homodimer functions as a GSR; this receptor is involved in glucose-induced insulin secretion by activating glucose metabolism probably in mitochondria.

  18. NF-kappaB-Mediated Repression of GADD153/CHOP: A Role in Breast Cancer Initiation

    DTIC Science & Technology

    2005-08-01

    normal versus neoplastic mammary epithelial cells. J Cell Physiol 2001;189(1):91-105. 18 . Bundy LM, Sealy L. CCAAT/enhancer binding protein beta (C...cytoplasm of resting cells through its association with inhibitor-of- kappaB (IKB) proteins and translocates to nucleus upon exposure of cells to cytokines...of proteins to C/EBP homodimers binding elements as well as to C/EBP/CHOP heterodimer binding elements was reduced in MCF IOA/p65 cells compared to

  19. ALDH3A1 — EDRN Public Portal

    Cancer.gov

    ALDH3A1 is a member of the aldehyde dehydrogenase family. They are involved in the metabolism of corticosteroids, biogenic amines, neurotransmitters, and lipid peroxidation. They are also involved in the detoxification of alcohol-derived acetaldehyde. The ALDH3A1 protein is an enzyme that forms a cytoplasmic homodimer that oxidizes aromatic and medium-chain (6 carbons or more) saturated and unsaturated aldehyde substrates. It is thought to promote resistance to UV and 4-hydroxy-2-nonenal-induced oxidative damage in the cornea. There are several splice variants that encode the same protein.

  20. CEBPG — EDRN Public Portal

    Cancer.gov

    The CEBPG protein is a transcription factor that binds to the enhancer element PRE-I (positive regulatory element-I) of the IL-4 gene.. The C/EBP family of transcription factors regulate viral and cellular CCAAT/enhancer element-mediated transcription. This family includes several related proteins: C/EBP alpha, C/EBP beta, C/EBP gamma, and C/EBP delta, that form homodimers and that form heterodimers with each other. There are two transcript variants that encode the same protein.

  1. The acetate kinase of Clostridum acetobutylicum strain P262.

    PubMed

    Diez-Gonzalez, F; Russell, J B; Hunter, J B

    1996-12-01

    Clostridum acetobutylicum strain P262 fermented glucose, pyruvate, or lactate, and the butyrate production was substrate-dependent. Differences in butyrate yield could not be explained by changes in butyrate kinase activities, but the butyrate production was inversely related to acetate kinase activity. The acetate kinase had a pH optimum of 8.0, a Km for acetate of 160 mM, and a kcat of 16, 800 min-1. The enyzme had a native molecular mass of 78 kDa; the size of 42 kDa on SDS-PAGE indicated that the acetate kinase of strain P262 was a homodimer.

  2. S100P — EDRN Public Portal

    Cancer.gov

    The S100P protein is a member of the S100 family of proteins containing 2 EF-hand calcium-binding motifs. S100P is a homodimer, and it interacts with S S100PBP and S100Z. S100 proteins are located in the cytoplasm and/or the nucleus and are involved in regulating many different processes, including cell cycle progression and differentiation. S100 genes include at least 13 members which are located as a cluster on chromosome 1q21; however, the S100P gene is located at 4p16. This protein may play a role in prostate cancer.

  3. Homo-dimerization and ligand binding by the leucine-rich repeat domain at RHG1/RFS2 underlying resistance to two soybean pathogens

    PubMed Central

    2013-01-01

    Background The protein encoded by GmRLK18-1 (Glyma_18_02680 on chromosome 18) was a receptor like kinase (RLK) encoded within the soybean (Glycine max L. Merr.) Rhg1/Rfs2 locus. The locus underlies resistance to the soybean cyst nematode (SCN) Heterodera glycines (I.) and causal agent of sudden death syndrome (SDS) Fusarium virguliforme (Aoki). Previously the leucine rich repeat (LRR) domain was expressed in Escherichia coli. Results The aims here were to evaluate the LRRs ability to; homo-dimerize; bind larger proteins; and bind to small peptides. Western analysis suggested homo-dimers could form after protein extraction from roots. The purified LRR domain, from residue 131–485, was seen to form a mixture of monomers and homo-dimers in vitro. Cross-linking experiments in vitro showed the H274N region was close (<11.1 A) to the highly conserved cysteine residue C196 on the second homo-dimer subunit. Binding constants of 20–142 nM for peptides found in plant and nematode secretions were found. Effects on plant phenotypes including wilting, stem bending and resistance to infection by SCN were observed when roots were treated with 50 pM of the peptides. Far-Western analyses followed by MS showed methionine synthase and cyclophilin bound strongly to the LRR domain. A second LRR from GmRLK08-1 (Glyma_08_g11350) did not show these strong interactions. Conclusions The LRR domain of the GmRLK18-1 protein formed both a monomer and a homo-dimer. The LRR domain bound avidly to 4 different CLE peptides, a cyclophilin and a methionine synthase. The CLE peptides GmTGIF, GmCLE34, GmCLE3 and HgCLE were previously reported to be involved in root growth inhibition but here GmTGIF and HgCLE were shown to alter stem morphology and resistance to SCN. One of several models from homology and ab-initio modeling was partially validated by cross-linking. The effect of the 3 amino acid replacements present among RLK allotypes, A87V, Q115K and H274N were predicted to alter domain

  4. Crystal structure of the Rasputin NTF2-like domain from Drosophila melanogaster.

    PubMed

    Vognsen, Tina; Kristensen, Ole

    2012-03-30

    The crystal structure of the NTF2-like domain of the Drosophila homolog of Ras GTPase SH3 Binding Protein (G3BP), Rasputin, was determined at 2.7Å resolution. The overall structure is highly similar to nuclear transport factor 2: It is a homodimer comprised of a β-sheet and three α-helices forming a cone-like shape. However, known binding sites for RanGDP and FxFG containing peptides show electrostatic and steric differences compared to nuclear transport factor 2. A HEPES molecule bound in the structure suggests a new, and possibly physiologically relevant, ligand binding site.

  5. Crystal structure of enolase from Drosophila melanogaster.

    PubMed

    Sun, Congcong; Xu, Baokui; Liu, Xueyan; Zhang, Zhen; Su, Zhongliang

    2017-04-01

    Enolase is an important enzyme in glycolysis and various biological processes. Its dysfunction is closely associated with diseases. Here, the enolase from Drosophila melanogaster (DmENO) was purified and crystallized. A crystal of DmENO diffracted to 2.0 Å resolution and belonged to space group R32. The structure was solved by molecular replacement. Like most enolases, DmENO forms a homodimer with conserved residues in the dimer interface. DmENO possesses an open conformation in this structure and contains conserved elements for catalytic activity. This work provides a structural basis for further functional and evolutionary studies of enolase.

  6. Estimation on the individual hydrogen-bond strength in molecules with multiple hydrogen bonds.

    PubMed

    Dong, Hao; Hua, Weijie; Li, Shuhua

    2007-04-19

    A simple atom-replacement approach is proposed for estimating the individual contributions of each intermolecular hydrogen bond (HB) in multiple hydrogen-bonded systems. The approach is validated by calculations on the homodimer of formylformamide and then applied to nucleic acid base pairs (adenine-thymine and guanine-cytosine) and some quadruply hydrogen-bonded dimers. With the help of this method, it is easy to distinguish the relative strength of each HB, and identify the main factors contributing to the total binding energies of multiple HBs.

  7. Green flavoprotein from P. leiognathi: purification, characterization and identification as the product of the lux G(N) gene.

    PubMed

    Raibekas, A A

    1991-01-01

    A green flavoprotein (GFP) was isolated and purified to homogeneity from Photobacterium leiognathi, strain 208. GFP is a homodimer of molecular weight 54,000 and contains two molecules of an unusual flavin per molecule of protein. Various biochemical characteristics including isoelectric point, trypsin and chymotrypsin degradation, SDS and temperature influence on subunit dissociation and the dissociation of the flavin chromophore, were investigated. The sequence of 23 N-terminal amino acids was determined and found to be concurrent with the N-terminal amino acid sequence encoded by the lux G(N) gene of P. leiognathi. This fact suggests that GFP is a structural component of the Photobacterium luminescence system.

  8. Dimerization and DNA-binding of ASR1, a small hydrophilic protein abundant in plant tissues suffering from water loss

    SciTech Connect

    Maskin, Laura; Frankel, Nicolas; Gudesblat, Gustavo; Demergasso, Maria J.; Pietrasanta, Lia I.; Iusem, Norberto D. . E-mail: norbius@fbmc.fcen.uba.ar

    2007-01-26

    The Asr gene family is present in Spermatophyta. Its members are generally activated under water stress. We present evidence that tomato ASR1, one of the proteins of the family, accumulates in seed during late stages of embryogenesis, a physiological process characterized by water loss. In vitro, electrophoretic assays show a homo-dimeric structure for ASR1 and highlight strong non-covalent interactions between monomers prone to self-assemble. Direct visualization of single molecules by atomic force microscopy (AFM) confirms that ASR1 forms homodimers and that uncovers both monomers and dimers bind double stranded DNA.

  9. The ESCRT Complexes

    PubMed Central

    Hurley, James H.

    2010-01-01

    The ESCRT machinery consists of the peripheral membrane protein complexes, ESCRT-0, -I, -II, -III, and Vps4-Vta1, and the ALIX homodimer. The ESCRT system is required for degradation of unneeded or dangerous plasma membrane proteins; biogenesis of the lysosome and the yeast vacuole; the budding of most membrane enveloped viruses; the membrane abscission step in cytokinesis; macroautophagy; and several other processes. From their initial discovery in 2001-2002, the literature on ESCRTs has grown exponentially. This review will describe the structure and function of the six complexes noted above and summarizes current knowledge of their mechanistic roles in cellular pathways and in disease. PMID:20653365

  10. CKM — EDRN Public Portal

    Cancer.gov

    CKM is a cytoplasmic enzyme involved in energy homeostasis and is an important serum marker for myocardial infarction. It reversibly catalyzes the transfer of phosphate between ATP and various phosphogens, such as creatine phosphate. Creatine kinase isoenzymes play a central role in energy transduction in tissues with large, fluctuating energy demands, such as skeletal muscle, heart, brain and spermatozoa. Structurally, CKM exists primarily as a homodimer in striated muscle and myocardium, and as a heterodimer in brain and in heart, as well as other tissues. CKM is a member of the ATP:guanido phosphotransferase family.

  11. Overview and discovery of IgNARs and generation of VNARs.

    PubMed

    Nuttall, Stewart D

    2012-01-01

    Immunoglobulin new antigen receptors (IgNARs) from sharks are a distinct class of immune receptors, consisting of homodimers with no associated light chains. Antigen binding is encapsulated within single VNAR immunoglobulin domains of 13-14 kDa in size. This small size and single domain format means that they exhibit considerable stability and are readily produced in heterologous protein expression systems. In this chapter, I describe the history and discovery of IgNARs, the development of VNAR biotechnology, and highlight important factors in VNAR protein production.

  12. Backbone NMR assignments of a topologically knotted protein in urea-denatured state.

    PubMed

    Hsieh, Shu-Ju Micky; Mallam, Anna L; Jackson, Sophie E; Hsu, Shang-Te Danny

    2014-10-01

    YbeA is a 3-methylpseudoridine methyltransferase from Escherichia coli that forms a stable homodimer in solution. It is one of the deeply trefoil 31 knotted proteins, of which the knot encompasses the C-terminal helix that threads through a long loop. Recent studies on the knotted protein folding pathways using YbeA have suggested that the protein knot remains present under chemically denaturing conditions. Here, we report (1)H, (13)C and (15)N chemical shift assignments for urea-denatured YbeA, which will serve as the basis for further structural characterisations using solution state NMR spectroscopy with paramagnetic spin labeled and partial alignment media.

  13. Tissue protection by erythropoietin: new findings in a moving field.

    PubMed

    Nangaku, Masaomi

    2013-09-01

    Two groups elucidate novel mechanisms of tissue protection by erythropoietin (EPO). Hu et al. demonstrate that Klotho's protective effect against oxidant-induced cytotoxicity is partially mediated by an increase in the endogenous expression of the classical EPO receptor (EpoR). While erythropoiesis is stimulated by the canonical EpoR homodimer, the tissue-protective effects of EPO are mediated through a heterodimeric 'tissue-protective' receptor. Coldewey et al. demonstrate a protective role of the 'tissue-protective' EpoR against acute kidney injury.

  14. Avidity-mediated enhancement of in vivo tumor targeting by single-chain Fv dimers.

    PubMed

    Adams, Gregory P; Tai, Mei-Sheng; McCartney, John E; Marks, James D; Stafford, Walter F; Houston, L L; Huston, James S; Weiner, Louis M

    2006-03-01

    Radiolabeled single-chain Fv (sFv) molecules display highly specific tumor retention in the severe combined immunodeficient (SCID) mouse model; however, the absolute quantity of sFv retained in the tumors is diminished by the rapid renal elimination resulting from the small size of the sFv molecules (Mr 27,000) and by dissociation of the monovalent sFv from tumor-associated antigen. We previously reported significant improvement in tumor retention without a loss of targeting specificity on converting monovalent sFv into divalent [(sFv')2] dimers, linked by a disulfide bond between COOH-terminal cysteinyl peptides engineered into the sFv'. However, our data for enhanced dimer localization in tumors could not distinguish between the contributions of enhanced avidity and increased systemic retention associated with the larger size of 54 kDa [(sFv')2] dimers relative to 27-kDa sFv. In this investigation, we have compared tumor targeting of divalent anti-c-erbB-2/HER2/neu 741F8-1 (sFv')2 homodimers with monovalent 741F8/26-10 (sFv')2 heterodimers (Mr 54,000) and 741F8 sFv monomers (741F8 sFv has binding specificity for erbB-2/HER2/neu and 26-10 sFv specificity for digoxin and related cardiac glycosides). These studies allowed us to distinguish the dominant effect of valency over molecular weight in accounting for the superior tumor retention of 741F8-1 (sFv')2 homodimers. Each of the radioiodinated species was administered i.v. to SCID mice bearing SK-OV-3 human tumor xenografts and tumor localization at 24 hours post i.v. injection was determined for 125I-741F8-1 (sFv')2 (3.57 %ID/g), 125I-741F8/26-10 (sFv')2 (1.13 %ID/g), and 125I-741F8-1 sFv (1.25 %ID/g). These findings substantiate that the improved tumor retention of (sFv')2 homodimers over sFv monomers results from the availability of dual binding sites rather than from the slower systemic clearance of homodimers.

  15. De Novo Fragment Design for Drug Discovery and Chemical Biology.

    PubMed

    Rodrigues, Tiago; Reker, Daniel; Welin, Martin; Caldera, Michael; Brunner, Cyrill; Gabernet, Gisela; Schneider, Petra; Walse, Björn; Schneider, Gisbert

    2015-12-07

    Automated molecular de novo design led to the discovery of an innovative inhibitor of death-associated protein kinase 3 (DAPK3). An unprecedented crystal structure of the inactive DAPK3 homodimer shows the fragment-like hit bound to the ATP pocket. Target prediction software based on machine learning models correctly identified additional macromolecular targets of the computationally designed compound and the structurally related marketed drug azosemide. The study validates computational de novo design as a prime method for generating chemical probes and starting points for drug discovery.

  16. Comparison of uptake between PureSorb-Q40 and regular hydrophobic coenzyme Q10 in rats and humans after single oral intake.

    PubMed

    Nukui, Kazuki; Yamagishi, Toshihiko; Miyawaki, Hiromi; Kettawan, Aikkarach; Okamoto, Tadashi; Sato, Kiyoshi

    2007-04-01

    Coenzyme Q10 (CoQ10) is a lipid-soluble antioxidant and essential component of the mitochondrial electron transfer system in the body, and is in wide use as a functional food material and cosmetic raw material. However, as CoQ10 is extremely lipid-soluble, absorption by the body is not easy. In general, people use soft-gel capsules in which CoQ10 is suspended in oil, and take these capsules with food. PureSorb-Q40 (P40) was developed to improve CoQ10 processability and absorption when taken without food, and the present study compared the effects of food on absorption between P40 and conventional lipid-soluble CoQ10 in rats and humans. The results of a rat study showed higher uptake when P40 was administered in the fasting state or with food compared to lipid-soluble CoQ10. The results of a human study showed that uptake was favorable when P40 was administered in the fasting state, and even when administered postprandially, a significant difference was noted in uptake rate up to 6 h after intake and uptake volume up to 8 h after intake when compared to lipid-soluble CoQ10. These results show that any CoQ10 product using P40 can be quickly and reliably absorbed by the body regardless of dosage form or intake time.

  17. Serum amyloid A is an endogenous ligand that differentially induces IL-12 and IL-23.

    PubMed

    He, Rong; Shepard, Larry W; Chen, Jia; Pan, Zhixing K; Ye, Richard D

    2006-09-15

    The acute-phase proteins, C-reactive protein and serum amyloid A (SAA), are biomarkers of infection and inflammation. However, their precise role in immunity and inflammation remains undefined. We report in this study a novel property of SAA in the differential induction of Th1-type immunomodulatory cytokines IL-12 and IL-23. In peripheral blood monocytes and the THP-1 monocytic cell line, SAA induces the expression of IL-12p40, a subunit shared by IL-12 and IL-23. SAA-stimulated expression of IL-12p40 was rapid (< or = 4 h), sustainable (> or = 20 h), potent (up to 3380 pg/ml/10(6) cells in 24 h), and insensitive to polymyxin B treatment. The SAA-stimulated IL-12p40 secretion required de novo protein synthesis and was accompanied by activation of the transcription factors NF-kappaB and C/EBP. Expression of IL-12p40 required activation of the p38 MAPK and PI3K. Interestingly, the SAA-induced IL-12p40 production was accompanied by a sustained expression of IL-23p19, but not IL-12p35, resulting in preferential secretion of IL-23, but not IL-12. These results identify SAA as an endogenous ligand that potentially activates the IL-23/IL-17 pathway and present a novel mechanism for regulation of inflammation and immunity by an acute-phase protein.

  18. A 40 kd protein binds specifically to the 5'-untranslated regions of yeast mitochondrial mRNAs.

    PubMed Central

    Papadopoulou, B; Dekker, P; Blom, J; Grivell, L A

    1990-01-01

    Using a gel mobility shift assay we show that a 40 kd protein (p40), present in extracts of yeast mitochondria, binds specifically to the 5'-untranslated leader of cytochrome c oxidase subunit II mRNA. Binding of p40 to coxII RNA protects an 8-10 nucleotide segment from diethylpyocarbonate modification, indicating that the protein interacts with only a restricted region of the 5'-leader. This segment is located at position -12 with respect to the initiation AUG. Deletion of 10 nucleotides encompassing this site completely abolishes protein binding. Nevertheless, Bal31 deletion analysis within the coxII leader shows that a major part of the leader is essential for p40 binding, suggesting that binding of the protein is also dependent on secondary structural features. p40 binds to other mitochondrial leader mRNAs including those for coxI, coxIII and cyt b. p40 is present in a cytoplasmic (rho0) petite mutant lacking mitochondrial protein synthesis. It is therefore presumably nuclear encoded. The possible biological function of the protein is discussed. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 8. Fig. 9. PMID:1701144

  19. Characterization of a probiotic-derived soluble protein which reveals a mechanism of preventive and treatment effects of probiotics on intestinal inflammatory diseases.

    PubMed

    Yan, Fang; Polk, D Brent

    2012-01-01

    The beneficial effects of probiotics have been demonstrated in many diseases, such as inflammatory bowel disease. The known mechanisms for probiotic action include blocking pathogenic bacterial effects, enhancing the innate immunity and decreasing pathogen-induced inflammation, and promoting intestinal epithelial cell survival, barrier function, and protective responses. We purified and cloned a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, p40. This protein ameliorated cytokine-induced apoptosis in intestinal epithelial cells through activation of the EGF receptor and its down-stream target, Akt. By using special hydrogel beads to protect p40 from degradation, we showed that p40 reduced intestinal epithelial apoptosis and preserved barrier function in the colon epithelium in an EGF receptor-dependent manner, thereby preventing and treating intestinal inflammation in mouse models of colitis. Further works regarding structural analysis of p40, regulation of EGF receptor activation and immunoregulatory effects by p40 are discussed. These results may provide insights into the clinical application of probiotics for intestinal inflammatory disorders.

  20. Post-translational regulation of rice MADS29 function: homodimerization or binary interactions with other seed-expressed MADS proteins modulate its translocation into the nucleus.

    PubMed

    Nayar, Saraswati; Kapoor, Meenu; Kapoor, Sanjay

    2014-10-01

    OsMADS29 is a seed-specific MADS-box transcription factor that affects embryo development and grain filling by maintaining hormone homeostasis and degradation of cells in the nucellus and nucellar projection. Although it has a bipartite nuclear localization signal (NLS) sequence, the transiently expressed OsMADS29 monomer does not localize specifically in the nucleus. Dimerization of the monomers alters the intracellular localization fate of the resulting OsMADS29 homodimer, which then translocates into the nucleus. By generating domain-specific deletions/mutations, we show that two conserved amino acids (lysine(23) and arginine(24)) in the NLS are important for nuclear localization of the OsMADS29 homodimer. Furthermore, the analyses involving interaction of OsMADS29 with 30 seed-expressed rice MADS proteins revealed 19 more MADS-box proteins, including five E-class proteins, which interacted with OsMADS29. Eleven of these complexes were observed to be localized in the nucleus. Deletion analysis revealed that the KC region (K-box and C-terminal domain) plays a pivotal role in homodimerization. These data suggest that the biological function of OsMADS29 may not only be regulated at the level of transcription and translation as reported earlier, but also at the post-translational level by way of the interaction between OsMADS29 monomers, and between OsMADS29 and other MADS-box proteins.

  1. Pnc1 piggy-back import into peroxisomes relies on Gpd1 homodimerisation

    PubMed Central

    Saryi, Nadal A. Al; Hutchinson, John D.; Al-hejjaj, Murtakab Y.; Sedelnikova, Svetlana; Baker, Patrick; Hettema, Ewald H.

    2017-01-01

    Peroxisomes are eukaryotic organelles that posttranslationally import proteins via one of two conserved peroxisomal targeting signal (PTS1 or 2) mediated pathways. Oligomeric proteins can be imported via these pathways but evidence is accumulating that at least some PTS1-containing monomers enter peroxisomes before they assemble into oligomers. Some proteins lacking a PTS are imported by piggy-backing onto PTS-containing proteins. One of these proteins is the nicotinamidase Pnc1, that is co-imported with the PTS2-containing enzyme Glycerol-3-phosphate dehydrogenase 1, Gpd1. Here we show that Pnc1 co-import requires Gpd1 to form homodimers. A mutation that interferes with Gpd1 homodimerisation does not prevent Gpd1 import but prevents Pnc1 co-import. A suppressor mutation that restores Gpd1 homodimerisation also restores Pnc1 co-import. In line with this, Pnc1 interacts with Gpd1 in vivo only when Gpd1 can form dimers. Redirection of Gpd1 from the PTS2 import pathway to the PTS1 import pathway supports Gpd1 monomer import but not Gpd1 homodimer import and Pnc1 co-import. Our results support a model whereby Gpd1 may be imported as a monomer or a dimer but only the Gpd1 dimer facilitates co-transport of Pnc1 into peroxisomes. PMID:28209961

  2. IR/UV and UV/UV double-resonance study of guaiacol and eugenol dimers

    NASA Astrophysics Data System (ADS)

    Longarte, Asier; Redondo, Carolina; Fernández, José A.; Castaño, Fernando

    2005-04-01

    Guaiacol (2-methoxyphenol) and eugenol (4-allyl-2-methoxyphenol) molecules are biologically active phenol derivatives with an intramolecular -OH⋯OCH3 hydrogen bond (H bond). Pulsed supersonic expansions of mixtures of either of the two molecules with He yield weakly bound homodimers as well as other higher-order complexes. A number of complementary and powerful laser spectroscopic techniques, including UV-UV and IR-UV double resonances, have been employed to interrogate the species formed in the expansion in order to get information on their structures and spectroscopic properties. The interpretation of the spectra of eugenol dimer is complex and required a previous investigation on a similar but simpler molecule both to gain insight into the possible structures and support the conclusions. Guaiacol (2-methoxyphenol) has been used for that purpose. The combination of the broad laser study combined with ab initio calculations at the Becke 3 Lee-Yang-Parr/6-31+G(d) level has provided the isomer structures, the potential-energy wells, and shed light on the inter- and intramolecular interactions involved. Guaiacol homodimer has been shown to have a single isomer whereas eugenol dimer has at least two. The comparison between the computed geometries of the dimers, their respective energies, and the vibrational normal modes permits the identification of the spectra.

  3. Early expression of the receptor for advanced glycation end products in a toxic model produced by 6-hydroxydopamine in the rat striatum.

    PubMed

    Serratos, Iris N; Castellanos, Pilar; Pastor, Nina; Millán-Pacheco, César; Colín-González, Ana Laura; Rembao, Daniel; Pérez-Montfort, Ruy; Cabrera, Nallely; Sánchez-García, Aurora; Gómez, Isabel; Rangel-López, Edgar; Santamaria, Abel

    2016-04-05

    The receptor for advanced glycation end products (RAGE) is commonly involved in different neurodegenerative and inflammatory disorders. The cellular signaling associated to RAGE activation may occur upon binding to different ligands. In this study we investigated whether the toxic model produced by 6-hydroxydopamine (6-OHDA) in rats comprises early noxious responses related to RAGE-mediated signaling cascades. In order to explore a possible interaction between 6-OHDA and RAGE, affinity parameters of RAGE with 6-OHDA were estimated by different means. The possible binding sites of 6-OHDA with the VC1 homodimer for both rat and human RAGE were also modeled. Our results show that the striatal infusion of 6-OHDA recruits RAGE upregulation, as evidenced by an early expression of the receptor. 6-OHDA was also found to bind the VC1 homodimer, although its affinity was moderate when compared to other ligands. This work contributes to the understanding of the role of RAGE activation for 6-OHDA-induced neurotoxicity.

  4. Post-translational regulation of rice MADS29 function: homodimerization or binary interactions with other seed-expressed MADS proteins modulate its translocation into the nucleus

    PubMed Central

    Nayar, Saraswati; Kapoor, Meenu; Kapoor, Sanjay

    2014-01-01

    OsMADS29 is a seed-specific MADS-box transcription factor that affects embryo development and grain filling by maintaining hormone homeostasis and degradation of cells in the nucellus and nucellar projection. Although it has a bipartite nuclear localization signal (NLS) sequence, the transiently expressed OsMADS29 monomer does not localize specifically in the nucleus. Dimerization of the monomers alters the intracellular localization fate of the resulting OsMADS29 homodimer, which then translocates into the nucleus. By generating domain-specific deletions/mutations, we show that two conserved amino acids (lysine23 and arginine24) in the NLS are important for nuclear localization of the OsMADS29 homodimer. Furthermore, the analyses involving interaction of OsMADS29 with 30 seed-expressed rice MADS proteins revealed 19 more MADS-box proteins, including five E-class proteins, which interacted with OsMADS29. Eleven of these complexes were observed to be localized in the nucleus. Deletion analysis revealed that the KC region (K-box and C-terminal domain) plays a pivotal role in homodimerization. These data suggest that the biological function of OsMADS29 may not only be regulated at the level of transcription and translation as reported earlier, but also at the post-translational level by way of the interaction between OsMADS29 monomers, and between OsMADS29 and other MADS-box proteins. PMID:25096923

  5. Thermodynamic and mechanical effects of disulfide bonds in CXCLl7 chemokine

    NASA Astrophysics Data System (ADS)

    Singer, Christopher

    Chemokines are a family of signaling proteins mainly responsible for the chemotaxis of leukocytes, where their biological activity is modulated by their oligomerization state. Here, the dynamics and thermodynamic stability are characterized in monomer and homodimer structures of CXCL7, one of the most abundant platelet chemokines. The effects of dimerization and disulfide bond formation are investigated using computational methods that include molecular dynamics (MD) simulations and the Distance Constraint Model (DCM). A consistent picture emerges for the effect of dimerization and role of the Cys5-Cys31 and Cys7- Cys47 disulfide bonds. Surprisingly, neither disulfide bond is critical for maintaining structural stability in the monomer or dimer, although the monomer is destabilized more than the dimer upon removal of disulfide bonds. Instead, it is found that disulfide bonds influence the native state dynamics as well as modulates the relative stability between monomer and dimer. The combined analysis elucidates how CXCL7 is mechanically stable as a monomer, and how upon dimerization flexibly correlated motions are induced between the 30s and 50s loop within each monomer and across the dimer interface. Interestingly, the greatest gain in flexibility upon dimerization occurs when both disulfide bonds are present in each domain, and the homodimer is least stable relative to its two monomers. These results suggest the highly conserved disulfide bonds in chemokines facilitate a structural mechanism for distinguishing functional characteristics between monomer and dimer.

  6. The 1.9 A Structure of the Branched-Chain Amino-Acid Transaminase (IlvE) from Mycobacterium tuberculosis

    SciTech Connect

    Tremblay, L.; Blanchard, J

    2009-01-01

    Unlike mammals, bacteria encode enzymes that synthesize branched-chain amino acids. The pyridoxal 5'-phosphate-dependent transaminase performs the final biosynthetic step in these pathways, converting keto acid precursors into {alpha}-amino acids. The branched-chain amino-acid transaminase from Mycobacterium tuberculosis (MtIlvE) has been crystallized and its structure has been solved at 1.9 {angstrom} resolution. The MtIlvE monomer is composed of two domains that interact to form the active site. The biologically active form of IlvE is a homodimer in which each monomer contributes a substrate-specificity loop to the partner molecule. Additional substrate selectivity may be imparted by a conserved N-terminal Phe30 residue, which has previously been observed to shield the active site in the type IV fold homodimer. The active site of MtIlvE contains density corresponding to bound PMP, which is likely to be a consequence of the presence of tryptone in the crystallization medium. Additionally, two cysteine residues are positioned at the dimer interface for disulfide-bond formation under oxidative conditions. It is unknown whether they are involved in any regulatory activities analogous to those of the human mitochondrial branched-chain amino-acid transaminase.

  7. Independent Mechanisms Target SMCHD1 to Trimethylated Histone H3 Lysine 9-Modified Chromatin and the Inactive X Chromosome

    PubMed Central

    Brideau, Nicholas J.; Coker, Heather; Gendrel, Anne-Valerie; Siebert, C. Alistair; Bezstarosti, Karel; Demmers, Jeroen; Poot, Raymond A.; Nesterova, Tatyana B.

    2015-01-01

    The chromosomal protein SMCHD1 plays an important role in epigenetic silencing at diverse loci, including the inactive X chromosome, imprinted genes, and the facioscapulohumeral muscular dystrophy locus. Although homology with canonical SMC family proteins suggests a role in chromosome organization, the mechanisms underlying SMCHD1 function and target site selection remain poorly understood. Here we show that SMCHD1 forms an active GHKL-ATPase homodimer, contrasting with canonical SMC complexes, which exist as tripartite ring structures. Electron microscopy analysis demonstrates that SMCHD1 homodimers structurally resemble prokaryotic condensins. We further show that the principal mechanism for chromatin loading of SMCHD1 involves an LRIF1-mediated interaction with HP1γ at trimethylated histone H3 lysine 9 (H3K9me3)-modified chromatin sites on the chromosome arms. A parallel pathway accounts for chromatin loading at a minority of sites, notably the inactive X chromosome. Together, our results provide key insights into SMCHD1 function and target site selection. PMID:26391951

  8. T-cell receptor. gamma. chain-CD3 complex: implication in the cytotoxic activity of a CD3/sup +/ CD4/sup -/ CD8/sup -/ human natural killer clone

    SciTech Connect

    Alarcon, B.; De Vries, J.; Pettey, C.; Boylston, A.; Yssel, H.; Terhorst, C.; Spits, H.

    1987-06-01

    A subset of human T cells has recently been described. These cells express the CD3 complex but they do not carry the classical T-cell receptor (TCR)-..gamma../-..beta.. heterodimer on their surface (WT31/sup -/ CD3/sup +/). Instead, they express a TCR-..gamma.. chain associated with another type of polypeptide termed TCR-delta. The authors report here that a T-cell clone with natural killer (NK)-like activity, WM-14, had a disulfide bridged TCR-..gamma.. homodimer associated with CD3 on its surface. The TCR-..gamma.. chains of WM-14 cells were present in three different glycosylation forms of 43, 40, and 38 kDa, but they appeared to contain the same polypeptide backbone. Since cytotoxicity by WM-14 could be inhibited by anti-CD3 antibodies, they concluded that the TCR-..gamma..-CD3 complex was involved in the NK-like unrestricted killer activity. Although normal CD3-..gamma.., CD3-delta, and CD3-element of chains were present in this clone, the association with the TCR-..gamma.. homodimer may be the cause of a complete processing of the N-linked oligosaccharides attached to the CD3-delta chain.

  9. Unusual domain pairing in a mutant of bovine lens gammaB-crystallin.

    PubMed

    Palme, S; Jaenicke, R; Slingsby, C

    1998-06-26

    beta gamma-Crystallins from the eye lens are proteins consisting of two domains joined by a short linker. All 3D structures solved so far reveal a similar pseudo-2-fold pairing of the domains, reflecting their presumed ancient origin from a single-domain homodimer. Here we report the 2.2 A X-ray structure of the N-terminal domain of gammaB-crystallin, bearing a mutation of a residue involved in domain contacts in the native molecule (Phe56Ala). It forms a crystallographic homodimer, yet the domain orientation is different from native beta gamma-crystallins. It is considered that the new orientation derives from two structural features. (1) The replacement of the bulky phenylalanine 56 by an alanine results in a different optimal hydrophobic packing of interface residues between identical domains. (2) The paired domains have extensions derived from the domain linker, each containing a proline conserved in gamma-crystallins, and the resulting steric constraints preclude a native-like pairing but support the new arrangement. These data highlight the pivotal role of interface residues and sequence extensions in overall domain assembly.

  10. Naturally occurring amino acid substitutions at Arg1174 in the human insulin receptor result in differential effects on receptor biosynthesis and hybrid formation, leading to discordant clinical phenotypes.

    PubMed

    Rau, H; Kocova, M; O'Rahilly, S; Whitehead, J P

    2000-07-01

    Missense mutations in the tyrosine kinase domain of the human insulin receptor frequently result in a dominantly inherited form of insulin resistance. We noted a marked disparity in the clinical phenotypes of our study subjects with different missense mutations at the same residue (Arg1174) of the insulin receptor. Subjects with a tryptophan substitution (W) were only moderately hyperinsulinemic, whereas those with a glutamine substitution (Q) had severe clinical and biochemical insulin resistance. Studies were undertaken to explore the molecular mechanisms underlying these differences. Both W and Q mutant receptors bound insulin normally but were kinase inactive. The W mutation resulted in more rapid degradation of newly synthesized mutant receptor, which contrasted with the near-normal biosynthesis of the Q receptor. The propensity of the W receptor to form hybrids with the cotransfected wild-type (WT) receptor was also markedly impaired compared with the Q receptor, to an extent greater than could be explained by lower steady-state expression. Thus, the more clinically benign consequences of the heterozygous W mutant receptor are likely to relate to its impaired biosynthesis and/or reduced capacity to form hybrids with WT receptors. In addition to providing an explanation for the milder phenotype of 1174W versus 1174Q carriers, these studies provide further support for the notion that the dominant-negative effect of insulin receptor tyrosine kinase mutations involves the competition between inactive mutant homodimers and WT/mutant hybrids with active WT homodimers for both ligands and intracellular substrates.

  11. Integrating ELF4 into the circadian system through combined structural and functional studies

    PubMed Central

    Kolmos, Elsebeth; Nowak, Monika; Werner, Maria; Fischer, Katrin; Schwarz, Guenter; Mathews, Sarah; Schoof, Heiko; Nagy, Ferenc; Bujnicki, Janusz M.; Davis, Seth J.

    2009-01-01

    The circadian clock is a timekeeping mechanism that enables anticipation of daily environmental changes. In the plant Arabidopsis thaliana, the circadian system is a multiloop series of interlocked transcription-translation feedbacks. Several genes have been arranged in these oscillation loops, but the position of the core-clock gene ELF4 in this network was previously undetermined. ELF4 lacks sequence similarity to known domains, and functional homologs have not yet been identified. Here we show that ELF4 is functionally conserved within a subclade of related sequences, and forms an alpha-helical homodimer with a likely electrostatic interface that could be structurally modeled. We support this hypothesis by expression analysis of new elf4 hypomorphic alleles. These weak mutants were found to have expression level phenotypes of both morning and evening clock genes, implicating multiple entry points of ELF4 within the multiloop network. This could be mathematically modeled. Furthermore, morning-expression defects were particular to some elf4 alleles, suggesting predominant ELF4 action just preceding dawn. We provide a new hypothesis about ELF4 in the oscillator—it acts as a homodimer to integrate two arms of the circadian clock. PMID:20357892

  12. Unfolding the HIV-1 reverse transcriptase RNase H domain – how to lose a molecular tug-of-war

    PubMed Central

    Zheng, Xunhai; Pedersen, Lars C.; Gabel, Scott A.; Mueller, Geoffrey A.; DeRose, Eugene F.; London, Robert E.

    2016-01-01

    Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66′ homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66′ subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of the isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH′ domain unfolding behavior of the p66/p66′ homodimer. This study demonstrates the feasibility of directly targeting RT maturation with therapeutics. PMID:26773054

  13. Molecular basis of sidekick-mediated cell-cell adhesion and specificity

    PubMed Central

    Goodman, Kerry M; Yamagata, Masahito; Jin, Xiangshu; Mannepalli, Seetha; Katsamba, Phinikoula S; Ahlsén, Göran; Sergeeva, Alina P; Honig, Barry; Sanes, Joshua R; Shapiro, Lawrence

    2016-01-01

    Sidekick (Sdk) 1 and 2 are related immunoglobulin superfamily cell adhesion proteins required for appropriate synaptic connections between specific subtypes of retinal neurons. Sdks mediate cell-cell adhesion with homophilic specificity that underlies their neuronal targeting function. Here we report crystal structures of Sdk1 and Sdk2 ectodomain regions, revealing similar homodimers mediated by the four N-terminal immunoglobulin domains (Ig1–4), arranged in a horseshoe conformation. These Ig1–4 horseshoes interact in a novel back-to-back orientation in both homodimers through Ig1:Ig2, Ig1:Ig1 and Ig3:Ig4 interactions. Structure-guided mutagenesis results show that this canonical dimer is required for both Sdk-mediated cell aggregation (via trans interactions) and Sdk clustering in isolated cells (via cis interactions). Sdk1/Sdk2 recognition specificity is encoded across Ig1–4, with Ig1–2 conferring the majority of binding affinity and differential specificity. We suggest that competition between cis and trans interactions provides a novel mechanism to sharpen the specificity of cell-cell interactions. DOI: http://dx.doi.org/10.7554/eLife.19058.001 PMID:27644106

  14. Structural features of the KPI domain control APP dimerization, trafficking, and processing.

    PubMed

    Ben Khalifa, Naouel; Tyteca, Donatienne; Marinangeli, Claudia; Depuydt, Mathieu; Collet, Jean-François; Courtoy, Pierre J; Renauld, Jean-Christophe; Constantinescu, Stefan; Octave, Jean-Noël; Kienlen-Campard, Pascal

    2012-02-01

    The two major isoforms of human APP, APP695 and APP751, differ by the presence of a Kunitz-type protease inhibitor (KPI) domain in the extracellular region. APP processing and function is thought to be regulated by homodimerization. We used bimolecular fluorescence complementation (BiFC) to study dimerization of different APP isoforms and mutants. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain did not affect fluorescence complementation, but native folding of KPI is critical for APP751 homodimerization. APP751 and APP695 dimers were mostly localized at steady state in the Golgi region, suggesting that most of the APP751 and 695 dimers are in the secretory pathway. Mutation of the KPI led to the retention of the APP homodimers in the endoplasmic reticulum. We finally showed that APP751 is more efficiently processed through the nonamyloidogenic pathway than APP695. These findings provide new insight on the particular role of KPI domain in APP dimerization. The correlation observed between dimerization, subcellular localization, and processing suggests that dimerization acts as an efficient regulator of APP trafficking in the secretory compartments that has major consequences on its processing.

  15. Evaluation of effects of bivalent cations on the formation of purine-rich triple-helix DNA by ESI-FT-MS.

    PubMed

    Wan, Cuihong; Cui, Meng; Song, Fengrui; Liu, Zhiqiang; Liu, Shuying

    2009-07-01

    The GGA triplet repeats are widely dispersed throughout eukaryotic genomes. (GGA)n or (GGT)n oligonucleotides can interact with double-stranded DNA containing (GGA:CCT)n to form triple-stranded DNA. The effects of 8 divalent metal ions (3 alkaline-earth metals and 5 transition metals) on formation of these purine-rich triple-helix DNA were investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-MS). In the absence of metal ions, no triplex but single-strand, duplex, and purine homodimer ions were observed in mass spectra. The triple-helix DNA complexes were observed only in the presence of certain divalent ions. The effects of different divalent cations on the formation of purine-rich triplexes were compared. Transition-metal ions, especially Co(2+) and Ni(2+), significantly boost the formation of triple-helix DNA, whereas alkaline-earth metal ions have no positive effects on triplex formation. In addition, Ba(2+) is notably beneficial to the formation of homodimer instead of triplex.

  16. Hetero- and homodimerization of Arabidopsis thaliana arginine decarboxylase AtADC1 and AtADC2.

    PubMed

    Maruri-López, Israel; Jiménez-Bremont, Juan F

    2017-03-11

    The arginine decarboxylase enzyme (ADC) carries out the production of agmatine from arginine, which is the precursor of the first polyamine (PA) known as putrescine; subsequently, putrescine is turned into the higher PAs, spermidine and spermine. In Arabidopsis thaliana PA production occurs only from arginine and this step is initiated by two ADC paralogues, AtADC1 and AtADC2. PA production is essential for A. thaliana life cycle. Here, we analyzed the sub-cellular localization of AtADC1 and AtADC2 enzymes through GFP translational fusions. Our data revealed that the A. thaliana arginine decarboxylase enzymes exhibit a dual sub-cellular localization both in the cytosol and chloroplast. Moreover, we examined the protein dimer assembly using a Bimolecular Fluorescence Complementation (BiFC) approach, which showed that AtADC1 and AtADC2 proteins were able to form homodimers in the cytosol and chloroplast. Interestingly, we found the formation of AtADC1/AtADC2 heterodimers with similar sub-cellular localization than homodimers. This study reveals that both ADC proteins are located in the same cell compartments, and they are able to form protein interaction complexes with each other.

  17. Structure of the apo form of the catabolite control protein A (CcpA) from Bacillus megaterium with a DNA-binding domain

    SciTech Connect

    Singh, Rajesh Kumar; Palm, Gottfried J.; Panjikar, Santosh

    2007-04-01

    Crystal structure analysis of the apo form of catabolite control protein A reveals the three-helix bundle of the DNA-binding domain. In the crystal packing, this domain interacts with the binding site for the corepressor protein. Crystal structure determination of catabolite control protein A (CcpA) at 2.6 Å resolution reveals for the first time the structure of a full-length apo-form LacI-GalR family repressor protein. In the crystal structures of these transcription regulators, the three-helix bundle of the DNA-binding domain has only been observed in cognate DNA complexes; it has not been observed in other crystal structures owing to its mobility. In the crystal packing of apo-CcpA, the protein–protein contacts between the N-terminal three-helix bundle and the core domain consisted of interactions between the homodimers that were similar to those between the corepressor protein HPr and the CcpA N-subdomain in the ternary DNA complex. In contrast to the DNA complex, the apo-CcpA structure reveals large subdomain movements in the core, resulting in a complete loss of contacts between the N-subdomains of the homodimer.

  18. An unusual dimeric structure and assembly for TLR4 regulator RP105-MD-1

    SciTech Connect

    Yoon, Sung-il; Hong, Minsun; Wilson, Ian A

    2011-11-16

    RP105-MD-1 modulates the TLR4-MD-2-mediated, innate immune response against bacterial lipopolysaccharide (LPS). The crystal structure of the bovine 1:1 RP105-MD-1 complex bound to a putative endogenous lipid at 2.9 Å resolution shares a similar overall architecture to its homolog TLR4-MD-2 but assembles into an unusual 2:2 homodimer that differs from any other known TLR-ligand assembly. The homodimer is assembled in a head-to-head orientation that juxtaposes the N-terminal leucine-rich repeats (LRRs) of the two RP105 chains, rather than the usual tail-to-tail configuration of C-terminal LRRs in ligand-activated TLR dimers, such as TLR1-TRL2, TLR2-TLR6, TLR3-TLR3 and TLR4-TLR4. Another unusual interaction is mediated by an RP105-specific asparagine-linked glycan, which wedges MD-1 into the co-receptor binding concavity on RP105. This unique mode of assembly represents a new paradigm for TLR complexes and suggests a molecular mechanism for regulating LPS responses.

  19. Theoretical prediction of familial amyotrophic lateral sclerosis missense mutation effects on Cu/Zn superoxide dismutase structural stability

    SciTech Connect

    Potier, M.; Tu, Y.

    1994-09-01

    Cu/Zn superoxide dismutase (SOD) deficiency is associated with the progressive paralytic disorder familial amyotrophic lateral sclerosis (FALS). Fifteen missense mutations in the SOD gene were identified in several patients. These mutations may prevent correct promoter folding or hamper homodimer formation necessary for SOD activity. To understand the effect of the missense mutations on SOD structure and function, we used a theoretical analysis of structural effects based on two predictive methods using the modeled tertiary structure of human SOD. The first method uses the TORSO program which optimizes amino acid side-chains repacking in both wild-type and mutant SODs and calculates protein internal packing energy. The second method uses a hydrophobicity scale of the amino acid residues and considers both solvent accessibility and hydrophobic nature of residue substitutions to compute a stabilization energy change ({delta}E). These predictive methods have been tested in 187 single and multiple missense mutants of 8 proteins (T4 lysozyme, human carbonic anhydrase II, chymotrypsin inhibitor 2, f1 gene V protein, barnase, {lambda}-repressor, chicken and human lysozymes) with experimentally determined thermostability. The overall prediction accuracy with these proteins was 88%. Analysis of FALS missense mutations {delta}E predicts that 14 of 15 mutations destabilize the SOD structure. The other missense mutation is located at the homodimer interface and may hinder dimer formation. This approach is applicable to any protein with known tertiary structure to predict missense mutation effects on protein stability.

  20. In vivo reconstitution of a homodimeric cytochrome b559 like structure: The role of the N-terminus α-subunit from Synechocystis sp. PCC 6803.

    PubMed

    Luján, María A; Martínez, Jesús I; Alonso, Pablo J; Torrado, Alejandro; Roncel, Mercedes; Ortega, José M; Sancho, Javier; Picorel, Rafael

    2015-11-01

    The cytochrome b559 is a heme-bridged heterodimeric protein with two subunits, α and β. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the β-subunit (β/β) but not with the full length α-subunit. In the present work, reconstitution of a homodimer (α/α) cytochrome b559 like structure was possible using a chimeric N-terminus α-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic α-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the α-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric α-subunit cytochrome b559 like structure. The features of the new structure were determined by UV-Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the cytochrome b559 maturation in the bacterial cytoplasmic membrane.

  1. Spidroin N-terminal Domain Promotes a pH-dependent Association of Silk Proteins during Self-assembly*

    PubMed Central

    Gaines, William A.; Sehorn, Michael G.; Marcotte, William R.

    2010-01-01

    Spider silks are spun from concentrated solutions of spidroin proteins. The appropriate timing of spidroin assembly into organized fibers must be highly regulated to avoid premature fiber formation. Chemical and physical signals presented to the silk proteins as they pass from the ampulle and through the tapered duct include changes in ionic environment and pH as well as the introduction of shear forces. Here, we show that the N-terminal domain of spidroins from the major ampullate gland (MaSp-NTDs) for both Nephila and Latrodectus spiders associate noncovalently as homodimers. The MaSp-NTDs are highly pH-responsive and undergo a structural transition in the physiological pH range of the spider duct. Tryptophan fluorescence of the MaSp-NTDs reveals a change in conformation when pH is decreased, and the pH at which the transition occurs is determined by the amount and type of salt present. Size exclusion chromatography and pulldown assays both indicate that the lower pH conformation is associated with a significantly increased MaSp-NTD homodimer stability. By transducing the duct pH signal into specific protein-protein interactions, this conserved spidroin domain likely contributes significantly to the silk-spinning process. Based on these results, we propose a model of spider silk assembly dynamics as mediated through the MaSp-NTD. PMID:20959449

  2. Phylogenetic analysis and homology modelling of Paracentrotus lividus nectin.

    PubMed

    Costa, Caterina; Cavalcante, Carmela; Zito, Francesca; Yokota, Yukio; Matranga, Valeria

    2010-11-01

    The extracellular matrix protein Pl-nectin, a 210-kDa homodimer originally purified from sea urchin eggs, plays a crucial role in cell adhesion and embryonic morphogenesis. The compiled cDNA sequence, obtained by RT-PCR primer walking and 3' RACE, identified a 984aa product containing a 23aa signal peptide and including all six internal peptides identified by protein microsequencing. The protein is a new member of the galactose-binding protein superfamily as it consists of six 151-156aa-long tandemly repeated domains (D1-D6), homologous to the discoidin-like domains, also known as F5/8-type C domains. Based on homology modelling, we present a three-dimensional structure (3D) for D5, identified as the prototype domain. The molecular modelling of the assembled Pl-nectin homodimer accounts for a Pl-nectin quaternary structure composed of two 105-kDa C-