Sample records for b2 receptor b2r

  1. Downregulation of kinin B1 receptor function by B2 receptor heterodimerization and signaling.

    PubMed

    Zhang, Xianming; Brovkovych, Viktor; Zhang, Yongkang; Tan, Fulong; Skidgel, Randal A

    2015-01-01

    Signaling through the G protein-coupled kinin receptors B1 (kB1R) and B2 (kB2R) plays a critical role in inflammatory responses mediated by activation of the kallikrein-kinin system. The kB2R is constitutively expressed and rapidly desensitized in response to agonist whereas kB1R expression is upregulated by inflammatory stimuli and it is resistant to internalization and desensitization. Here we show that the kB1R heterodimerizes with kB2Rs in co-transfected HEK293 cells and natively expressing endothelial cells, resulting in significant internalization and desensitization of the kB1R response in cells pre-treated with kB2R agonist. However, pre-treatment of cells with kB1R agonist did not affect subsequent kB2R responses. Agonists of other G protein-coupled receptors (thrombin, lysophosphatidic acid) had no effect on a subsequent kB1R response. The loss of kB1R response after pretreatment with kB2R agonist was partially reversed with kB2R mutant Y129S, which blocks kB2R signaling without affecting endocytosis, or T342A, which signals like wild type but is not endocytosed. Co-endocytosis of the kB1R with kB2R was dependent on β-arrestin and clathrin-coated pits but not caveolae. The sorting pathway of kB1R and kB2R after endocytosis differed as recycling of kB1R to the cell surface was much slower than that of kB2R. In cytokine-treated human lung microvascular endothelial cells, pre-treatment with kB2R agonist inhibited kB1R-mediated increase in transendothelial electrical resistance (TER) caused by kB1R stimulation (to generate nitric oxide) and blocked the profound drop in TER caused by kB1R activation in the presence of pyrogallol (a superoxide generator). Thus, kB1R function can be downregulated by kB2R co-endocytosis and signaling, suggesting new approaches to control kB1R signaling in pathological conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Downregulation of kinin B1 receptor function by B2 receptor heterodimerization and signaling

    PubMed Central

    Zhang, Xianming; Brovkovych, Viktor; Zhang, Yongkang; Tan, Fulong; Skidgel, Randal A.

    2014-01-01

    Signaling through the G protein-coupled kinin receptors B1 (kB1R) and B2 (kB2R) plays a critical role in inflammatory responses mediated by activation of the kallikrein-kinin system. The kB2R is constitutively expressed and rapidly desensitized in response to agonist whereas kB1R expression is upregulated by inflammatory stimuli and it is resistant to internalization and desensitization. Here we show that the kB1R heterodimerizes with kB2Rs in co-transfected HEK293 cells and natively expressing endothelial cells, resulting in significant internalization and desensitization of the kB1R response in cells pre-treated with kB2R agonist. However, pre-treatment of cells with kB1R agonist did not affect subsequent kB2R responses. Agonists of other G protein-coupled receptors (thrombin, lysophosphatidic acid) had no effect on a subsequent kB1R response. The loss of kB1R response after pretreatment with kB2R agonist was partially reversed with kB2R mutant Y129S, which blocks kB2R signaling without affecting endocytosis, or T342A, which signals like wild type but is not endocytosed. Co-endocytosis of the kB1R with kB2R was dependent on β-arrestin and clathrin-coated pits but not caveolae. The sorting pathway of kB1R and kB2R after endocytosis differed as recycling of kB1R to the cell surface was much slower than that of kB2R. In cytokine-treated human lung microvascular endothelial cells, pre-treatment with kB2R agonist inhibited kB1R-mediated increase in transendothelial electrical resistance (TER) caused by kB1R stimulation (to generate nitric oxide) and blocked the profound drop in TER caused by kB1R activation in the presence of pyrogallol (a superoxide generator). Thus, kB1R function can be downregulated by kB2R co-endocytosis and signaling, suggesting new approaches to control kB1R signaling in pathological conditions. PMID:25289859

  3. Vasopressor meets vasodepressor: The AT1-B2 receptor heterodimer.

    PubMed

    Quitterer, Ursula; AbdAlla, Said

    2014-04-01

    The AT1 receptor for the vasopressor angiotensin II is one of the most important drug targets for the treatment of cardiovascular diseases. Sensitization of the AT1 receptor system is a common feature contributing to the pathogenesis of many cardiovascular disorders but underlying mechanisms are not fully understood. More than a decade ago, evidence was provided for control of AT1R activation by heterodimerization with the B2 receptor for the vasodepressor peptide, bradykinin, a physiological counterpart of the vasoconstrictor angiotensin II. AT1-B2 receptor heterodimerization was shown to enhance AT1R-stimulated signaling under pathophysiological conditions such as experimental and human pregnancy hypertension. Notably, AT1R signal sensitization of patients with preeclampsia hypertension was attributed to AT1R-B2R heterodimerization. Vice versa, transgenic mice lacking the AT1-B2 receptor heterodimer due to targeted deletion of the B2R gene showed a significantly reduced AT1R-stimulated vasopressor response compared to transgenic mice with abundant AT1R-B2R heterodimerization. Biophysical methods such as BRET and FRET confirmed those data by demonstrating efficient AT1-B2 receptor heterodimerization in transfected cells and transgenic mice. Recently, a study on AT1R-specific biased agonism directed the focus to the AT1-B2 receptor heterodimer again. The β-arrestin-biased [Sar1,Ile4,Ile8]-angiotensin II promoted not only the recruitment of β-arrestin to the AT1R but also stimulated the down-regulation of the AT1R-associated B2 receptor by co-internalization. Thereby specific targeting of the AT1R-B2R heterodimer became feasible and could open the way to a new class of drugs, which specifically interfere with pathological angiotensin II-AT1 receptor system activation. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Decoy receptor 3 suppresses TLR2-mediated B cell activation by targeting NF-κB.

    PubMed

    Huang, Zi-Ming; Kang, Jhi-Kai; Chen, Chih-Yu; Tseng, Tz-Hau; Chang, Chien-Wen; Chang, Yung-Chi; Tai, Shyh-Kuan; Hsieh, Shie-Liang; Leu, Chuen-Miin

    2012-06-15

    Decoy receptor 3 (DcR3) is a soluble protein in the TNFR superfamily. Its known ligands include Fas ligand, homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, TNF-like molecule 1A, and heparan sulfate proteoglycans. DcR3 has been reported to modulate the functions of T cells, dendritic cells, and macrophages; however, its role in regulating B cell activation is largely unknown. In this study, we found that the DcR3.Fc fusion protein bound to human and mouse B cells and suppressed the activation of B cells. DcR3.Fc attenuated Staphylococcus aureus, IgM-, Pam(3)CSK(4)-, and LPS-mediated B cell proliferation but did not affect cytokine-induced B cell growth. In the presence of these mitogens, DcR3.Fc did not induce B cell apoptosis, suggesting that DcR3 may inhibit the signal(s) important for B cell activation. Because the combination of Fas.Fc, LT-βR.Fc (homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes receptor), and DR3.Fc (TNF-like molecule 1A receptor) did not suppress B cell proliferation and because the biological effect of DcR3.Fc on B cells was not blocked by heparin, we hypothesize that a novel ligand(s) of DcR3 mediates its inhibitory activity on B cells. Moreover, we found that TLR2-stimulated NF-κB p65 activation and NF-κB-driven luciferase activity were attenuated by DcR3.Fc. The TLR2-induced cytokine production by B cells was consistently reduced by DcR3. These results imply that DcR3 may regulate B cell activation by suppressing the activation of NF-κB.

  5. Adenosine A2B and A3 receptor location at the mouse neuromuscular junction.

    PubMed

    Garcia, Neus; Priego, Mercedes; Hurtado, Erica; Obis, Teresa; Santafe, Manel M; Tomàs, Marta; Lanuza, Maria Angel; Tomàs, Josep

    2014-07-01

    To date, four subtypes of adenosine receptors have been cloned (A(1)R, A(2A)R, A(2B)R, and A(3)R). In a previous study we used confocal immunocytochemistry to identify A(1)R and A(2A)R receptors at mouse neuromuscular junctions (NMJs). The data shows that these receptors are localized differently in the three cells (muscle, nerve and glia) that configure the NMJs. A(1)R localizes in the terminal teloglial Schwann cell and nerve terminal, whereas A(2A)R localizes in the postsynaptic muscle and in the axon and nerve terminal. Here, we use Western blotting to investigate the presence of A(2B)R and A(3)R receptors in striated muscle and immunohistochemistry to localize them in the three cells of the adult neuromuscular synapse. The data show that A(2B)R and A(3)R receptors are present in the nerve terminal and muscle cells at the NMJs. Neither A(2B)R nor A(3)R receptors are localized in the Schwann cells. Thus, the four subtypes of adenosine receptors are present in the motor endings. The presence of these receptors in the neuromuscular synapse allows the receptors to be involved in the modulation of transmitter release. © 2014 Anatomical Society.

  6. [Study on the expression of bradykinin and its receptors B1R and B2R in the kidney immune injury in trichloroethylene-sensitized mouse].

    PubMed

    Wang, Hui; Zhang, Jiaxiang; Li, Shulong; Zha, Wansheng; Wang, Feng; Zhu, Qixing

    2015-07-01

    To study the expression of bradykinin and its receptors B1R and B2R in the kidney immune injury in trichloroethylene-sensitized mouse and discuss the pathogenesis of Dermatitis Medicamentosa-like of TCE (ODMLT). On the first days, intradermal injection by 50% TCE and the amount of FCA mixture 100 µl for initial sensitization; on 4, 7, 10 days, painted abdominal skin by 100 µl 50% TCE for three sensitization, on 17, 19 days, painted on the back skin by 100 µl 30% TCE for initial excitation and the last challenge; 24 h before each challenge, PKSI-527+TCE group received intraperitoneal injection by inhibitor PKSI-527 (50 mg/kg); solvent control group treat without TCE and sensitization and excitation reagent the same proportion of olive oil and acetone mixture, blank control group without any treatment. Before killing the mouse, renal weight and body weight were recorded. The renals and plasma were separated at 24 h, 48 h, 72 h and 7 d after the last challenge and observed pathological of the renals. Expression of B1R and B2R in renal were examined by immunofluorescence technique. Plasma were examined by ELISA for BK. The renal pathological examination revealed the apparent damage of TCE sensitized mice which compared to solvent control group showed obvious cellular infiltration, vacuolar degeneration of renal tubular epithelial cells. The renal damage of PKSI-527+TCE-sensitized groups which compared to the corresponding point of TCE-sensitized groups showed significantly reduced. The expression of BK in 24 h, 48 h and 72 h TCE-sensitized groups were significant higher than solvent control group and related TCE non-sensitized groups (P < 0.05) and 72 h point compared to the corresponding point of PKSI-527+TCE group was also increased, the difference was statistically significant (P < 0.05). The expression levels of B1R and B2R in the kidney in 24 h, 48 h, 72 h and 7 d TCE-sensitized groups were obviously higher than solvent control group and related TCE non

  7. H2O2 attenuates IGF-1R tyrosine phosphorylation and its survival signaling properties in neuronal cells via NR2B containing NMDA receptor.

    PubMed

    Zeng, Zhiwen; Wang, Dejun; Gaur, Uma; Rifang, Liao; Wang, Haitao; Zheng, Wenhua

    2017-09-12

    Impairment of insulin-like growth factor I (IGF-I) signaling plays an important role in the development of neurodegeneration. In the present study, we investigated the effect of H 2 O 2 on the survival signaling of IGF-1 and its underlying mechanisms in human neuronal cells SH-SY5Y. Our results showed that IGF-1 promoted cell survival and stimulated phosphorylation of IGF-1R as well as its downstream targets like AKT and ERK1/2 in these cells. Meanwhile, these effects of IGF-1 were abolished by H 2 O 2 at 200μM concentration which did not cause any significant toxicity to cells itself in our experiments. Moreover, studies using various glutamate receptor subtype antagonists displayed that N-methyl-D -aspartate (NMDA) receptor antagonist dizocilpine maleate (MK-801) blocked the effects of H 2 O 2 , whereas other glutamate receptor subtype antagonists, such as non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), metabolic glutamate receptor antagonists LY341495 and CPCCOEt, had no effect. Further studies revealed that NR2B-containing NMDARs are responsible for these effects as its effects were blocked by pharmacological inhibitor Ro25-698 or specific siRNA for NR2B, but not NR2A. Finally, our data also showed that Ca 2+ influx contributes to the effects of H 2 O 2 . Similar results were obtained in primary cultured cortical neurons. Taken together, the results from the present study suggested that H 2 O 2 attenuated IGF-1R tyrosine phosphorylation and its survival signaling properties via NR2B containing NMDA receptors and Ca 2+ influx in SH-SY5Y cells. Therefore, NMDAR antagonists, especially NR2B-selective ones, combined with IGF-1 may serve as an alternative therapeutic agent for oxidative stress related neurodegenerative disease.

  8. Expression of the ephrin receptor B2 in the embryonic chicken bursa of Fabricius

    USDA-ARS?s Scientific Manuscript database

    Chicken B-cells develop in a specific organ, the bursa of Fabricius. To understand the bursal microenvironment guiding B-cell development, previous studies identified ephrin (Eph) receptor B2 (EphB2) gene transcripts in the embryonic bursa. We hypothesize that the EphB2 receptors and their ligands r...

  9. Regulation of Fear Extinction in the Basolateral Amygdala by Dopamine D2 Receptors Accompanied by Altered GluR1, GluR1-Ser845 and NR2B Levels.

    PubMed

    Shi, Yan-Wei; Fan, Bu-Fang; Xue, Li; Wen, Jia-Ling; Zhao, Hu

    2017-01-01

    The amygdala, a critical structure for both Pavlovian fear conditioning and fear extinction, receives sparse but comprehensive dopamine innervation and contains dopamine D1 and D2 receptors. Fear extinction, which involves learning to suppress the expression of a previously learned fear, appears to require the dopaminergic system. The specific roles of D2 receptors in mediating associative learning underlying fear extinction require further study. Intra-basolateral amygdala (BLA) infusions of a D2 receptor agonist, quinpirole, and a D2 receptor antagonist, sulpiride, prior to fear extinction and extinction retention were tested 24 h after fear extinction training for long-term memory (LTM). LTM was facilitated by quinpirole and attenuated by sulpiride. In addition, A-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor glutamate receptor 1 (GluR1) subunit, GluR1 phospho-Ser845, and N -methyl-D-aspartic acid receptor NR2B subunit levels in the BLA were generally increased by quinpirole and down-regulated by sulpiride. The present study suggests that activation of D2 receptors facilitates fear extinction and that blockade of D2 receptors impairs fear extinction, accompanied by changes in GluR1, GluR1-Ser845 and NR2B levels in the amygdala.

  10. Glutamate receptor antibodies directed against AMPA receptors subunit 3 peptide B (GluR3B) can be produced in DBA/2J mice, lower seizure threshold and induce abnormal behavior.

    PubMed

    Ganor, Yonatan; Goldberg-Stern, Hadassa; Cohen, Ran; Teichberg, Vivian; Levite, Mia

    2014-04-01

    Anti-GluR3B antibodies (GluR3B Ab's), directed against peptide B/aa372-395 of GluR3 subunit of glutamate/AMPA receptors, are found in ∼35% of epilepsy patients, activate glutamate/AMPA receptors, evoke ion currents, kill neurons and damage the brain. We recently found that GluR3B Ab's also associate with neurological/psychiatric/behavioral abnormalities in epilepsy patients. Here we asked if GluR3B Ab's could be produced in DBA/2J mice, and also modulate seizure threshold and/or cause behavioral/motor impairments in these mice. DBA/2J mice were immunized with the GluR3B peptide in Complete Freund's Adjuvant (CFA), or with controls: ovalbumin (OVA), CFA, or phosphate-buffer saline (PBS). GluR3B Ab's and OVA Ab's were tested. Seizures were induced in all mice by the chemoconvulsant pentylenetetrazole (PTZ) at three time points, each time with less PTZ to avoid non-specific death. Behavior was examined in Open-Field, RotaRod and Grip tests. GluR3B Ab's were produced only in GluR3B-immunized mice, while OVA Ab's were produced only in OVA-immunized mice, showing high Ab's specificity. In GluR3B Ab's negative mice, seizure severity scores and percentages of animals developing generalized seizures declined in response to decreasing PTZ doses. In contrast, both parameters remained unchanged/high in the GluR3B Ab's positive mice, showing that these mice were more susceptible to seizures. The seizure scores associated significantly with the GluR3B Ab's levels. GluR3B Ab's positive mice were also more anxious in Open-Field test, fell faster in RotaRod test, and fell more in Grip test, compared to all the control mice. GluR3B Ab's are produced in DBA/2J mice, facilitate seizures and induce behavioral/motor impairments. This animal model can therefore serve for studying autoimmune epilepsy and abnormal behavior mediated by pathogenic anti-GluR3B Ab's. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. AMP and adenosine are both ligands for adenosine 2B receptor signaling.

    PubMed

    Holien, Jessica K; Seibt, Benjamin; Roberts, Veena; Salvaris, Evelyn; Parker, Michael W; Cowan, Peter J; Dwyer, Karen M

    2018-01-15

    Adenosine is considered the canonical ligand for the adenosine 2B receptor (A 2B R). A 2B R is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A 2B R signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A 2B R. The computational modeling suggested that AMP interacts with more favorable energy to A 2B R compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A 2B R, indicating preferential signaling via the G q pathway. Therefore, a putative AMP-A 2B R interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential G q activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Retinal plasma extravasation in streptozotocin-diabetic rats mediated by kinin B1 and B2 receptors

    PubMed Central

    Abdouh, M; Talbot, S; Couture, R; Hasséssian, H M

    2008-01-01

    Background and purpose: We investigated whether or not kinin receptors play a role in diabetic blood–retinal barrier breakdown, which is a leading cause of vision loss. Experimental approach: Blood–retinal barrier breakdown was quantified using Evans blue, and expression of kinin B1 receptor mRNA was measured using quantitative reverse transcrition-PCR. Diabetic rats (streptozotocin (STZ), 65 mg kg−1) received a single intraocular injection of bradykinin (BK) or des-Arg9-BK, alone, or in combination with antagonists for B1 (des-Arg10-Hoe140, R-715) and/or B2 (Hoe140) receptors, given intraocularly or intravenously (i.v.). Key results: In control rats, BK (0.1–10 nmol) dose-dependently increased plasma extravasation, which was inhibited by Hoe140 (0.2 nmol), whereas des-Arg9-BK (0.1 and 1 nmol) was without effect. B1 receptor mRNA was markedly increased in retinas of diabetic rats, and this was prevented by N-acetyl-L-cysteine (1 g kg−1 day−1 for 7 days). Plasma extravasation in retinas of STZ-diabetic rats was higher than in controls and enhanced by des-Arg9-BK. Response to des-Arg9-BK was inhibited by intraocular or i.v. injection of B1 receptor antagonists. Diabetes-induced plasma extravasation was inhibited only by a combination of des-Arg10-Hoe140 and Hoe 140 (100 nmol kg−1, i.v. 15 min earlier) or by R-715 (1 μmol kg−1, i.v.) injected daily for 7 days. Conclusions and implications: Kinin B1 receptors are upregulated in retinas of STZ-diabetic rats through a mechanism involving oxidative stress. Both kinin B1 and B2 receptors contribute to increased plasma extravasation in diabetic retinopathy. Chronic inhibition of both kinin receptors, possibly with antioxidant adjuvants, may be a novel therapeutic strategy for diabetic retinopathy. PMID:18311190

  13. Down-regulation of TGF-b1, TGF-b receptor 2, and TGF-b-associated microRNAs, miR-20a and miR-21, in skin lesions of sulfur mustard-exposed Iranian war veterans.

    PubMed

    Valizadeh, Mohadeseh; Mirzaei, Behnaz; Tavallaei, Mahmood; Noorani, Mohammad Reza; Amiri, Mojtaba; Soroush, Mohammad Reza; Mowla, Seyed Javad

    2015-01-01

    Sulfur mustard (SM) affects divergent cellular pathways including cell cycle, apoptosis, necrosis, and inflammatory responses. SM-induced lesions in skin include late-onset hyper-pigmentation, xerosis, and atrophy. It seems that TGF-b signaling pathway is a major player for SM pathogenesis. Here, we have employed a real-time polymerase chain reaction (PCR) approach to evaluate the expression alterations of all TGF-b variants and their receptors in skin biopsies obtained from 10 Iran-Iraq war veterans. Using specific LNA primers, the expression alteration of a TGF-bR2 regulator, miR-20a, and TGF-b downstream target, miR-21, was also assessed in the same samples Our real-time PCR data revealed a significant down-regulation of TGF-b1 and TGF-bR2, the major mediators of TGF-b signaling pathway, in skin biopsies of SM-exposed patients (p = 0.0015 and p = 0.0115, respectively). Down-regulation of TGF-b signaling pathway seems to contribute in severe inflammation observed in SM-exposed patients' tissues. MiR-20a and miR-21, as two important TGF-b associated microRNAs (miRNAs), were also down-regulated in SM-exposed skin lesions, compared to those of control group (p = 0.0003). Based on our findings, these miRNAs could be directly or indirectly involve in the pathogenesis of SM. Altogether, our data suggest the suitability of TGF-b1, TGF-bR2, as well as miR-20a and miR-21 as potential biomarkers for diagnosis and treatment of SM-exposed patients.

  14. The bradykinin B2 receptor in the early immune response against Listeria infection.

    PubMed

    Kaman, Wendy E; Wolterink, Arthur F W M; Bader, Michael; Boele, Linda C L; van der Kleij, Desiree

    2009-02-01

    The endogenous danger signal bradykinin was recently found implicated in the development of immunity against parasites via dendritic cells. We here report an essential role of the B(2) (B(2)R) bradykinin receptor in the early immune response against Listeria infection. Mice deficient in B(2)R (B(2)R(-/-) mice) were shown to suffer from increased hepatic bacterial burden and concomitant dramatic weight loss during infection with Listeria monocytogenes. Levels of cytokines known to play a pivotal role in the early phase immune response against L. monocytogenes, IL-12p70 and IFN-gamma, were reduced in B(2)R(-/-) mice. To extend these findings to the human system, we show that bradykinin potentiates the production of IL-12p70 in human monocyte-derived dendritic cells. Thus, we show that bradykinin and the B(2)R play a role in early innate immune functions during bacterial infection.

  15. The rat ErbB2 tyrosine kinase receptor produced in plants is immunogenic in mice and confers protective immunity against ErbB2(+) mammary cancer.

    PubMed

    Matić, Slavica; Quaglino, Elena; Arata, Lucia; Riccardo, Federica; Pegoraro, Mattia; Vallino, Marta; Cavallo, Federica; Noris, Emanuela

    2016-01-01

    The rat ErbB2 (rErbB2) protein is a 185-kDa glycoprotein belonging to the epidermal growth factor-related proteins (ErbB) of receptor tyrosine kinases. Overexpression and mutations of ErbB proteins lead to several malignancies including breast, lung, pancreatic, bladder and ovary carcinomas. ErbB2 is immunogenic and is an ideal candidate for cancer immunotherapy. We investigated the possibility of expressing the extracellular (EC) domain of rErbB2 (653 amino acids, aa) in Nicotiana benthamiana plants, testing the influence of the 23 aa transmembrane (TM) sequence on protein accumulation. Synthetic variants of the rErbB2 gene portion encoding the EC domain, optimized with a human codon usage and either linked to the full TM domain (rErbB2_TM, 676 aa), to a portion of it (rErbB2-pTM, 662 aa), or deprived of it (rErbB2_noTM, 653 aa) were cloned in the pEAQ-HT expression vector as 6X His tag fusions. All rErbB2 variants (72-74.5 kDa) were transiently expressed, but the TM was detrimental for rErbB2 EC accumulation. rERbB2_noTM was the most expressed protein; it was solubilized and purified with Nickel affinity resin. When crude soluble extracts expressing rErbB2_noTM were administered to BALB/c mice, specific rErbB2 immune responses were triggered. A potent antitumour activity was induced when vaccinated mice were challenged with syngeneic transplantable ErbB2(+) mammary carcinoma cells. To our knowledge, this is the first report of expression of rErbB2 in plants and of its efficacy in inducing a protective antitumour immune response, opening interesting perspectives for further immunological testing. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  16. COX-2 Elevates Oncogenic miR-526b in Breast Cancer by EP4 Activation.

    PubMed

    Majumder, Mousumi; Landman, Erin; Liu, Ling; Hess, David; Lala, Peeyush K

    2015-06-01

    MicroRNAs (miRs) are small regulatory molecules emerging as potential biomarkers in cancer. Previously, it was shown that COX-2 expression promotes breast cancer progression via multiple mechanisms, including induction of stem-like cells (SLC), owing to activation of the prostaglandin E2 receptor EP4 (PTGER4). COX-2 overexpression also upregulated microRNA-526b (miR-526b), in association with aggressive phenotype. Here, the functional roles of miR-526b in breast cancer and the mechanistic role of EP4 signaling in miR-526b upregulation were examined. A positive correlation was noted between miR-526b and COX-2 mRNA expression in COX-2 disparate breast cancer cell lines. Stable overexpression of miR-526b in poorly metastatic MCF7 and SKBR3 cell lines resulted in increased cellular migration, invasion, EMT phenotype and enhanced tumorsphere formation in vitro, and lung colony formation in vivo in immunodeficient mice. Conversely, knockdown of miR-526b in aggressive MCF7-COX-2 and SKBR3-COX-2 cells reduced oncogenic functions and reversed the EMT phenotype, in vitro. Furthermore, it was determined that miR-526b expression is dependent on EP4 receptor activity and downstream PI3K-AKT and cyclic AMP (cAMP) signaling pathways. PI3K-AKT inhibitors blocked EP4 agonist-mediated miR-526b upregulation and tumorsphere formation in MCF7 and SKBR3 cells. NF-κB inhibitor abrogates EP agonist-stimulated miRNA expression in MCF7 and T47D cells, indicating that the NF-κB pathway is also involved in miR-526b regulation. In addition, inhibition of COX-2, EP4, PI3K, and PKA in COX-2-overexpressing cells downregulated miR-526b and its functions in vitro. Finally, miR-526b expression was significantly higher in cancerous than in noncancerous breast tissues and associated with reduced patient survival. In conclusion, miR-526b promotes breast cancer progression, SLC-phenotype through EP4-mediated signaling, and correlates with breast cancer patient survival. This study presents novel

  17. Function of brain α2B-adrenergic receptor characterized with subtype-selective α2B antagonist and KO mice.

    PubMed

    Luhrs, Lauren; Manlapaz, Cynthia; Kedzie, Karen; Rao, Sandhya; Cabrera-Ghayouri, Sara; Donello, John; Gil, Daniel

    2016-12-17

    Noradrenergic signaling, through the α 2A and α 2C adrenergic receptors modulates the cognitive and behavioral symptoms of disorders such as schizophrenia, attention deficit hyperactivity disorder (ADHD), and addiction. However, it is unknown whether the α 2B receptor has any significant role in CNS function. The present study elucidates the potential role of the α 2B receptor in CNS function via the discovery and use of the first subtype-selective α 2B antagonist (AGN-209419), and behavioral analyses of α-receptor knockout (KO) mice. Using AGN-209419 as radioligand, α 2B receptor binding sites were identified within the olfactory bulb, cortex, thalamus, cerebellum, and striatum. Based on the observed expression patterns of α 2 subtypes in the brain, we compared α 2B KO, α 2A KO and α 2C KO mice behavioral phenotypes with their respective wild-type lines in anxiety (plus maze), compulsive (marble burying), and sensorimotor (prepulse inhibition) tasks. α 2B KO mice exhibited increased marble burying and α 2C KO mice exhibited an increased startle response to a pulse stimulus, but otherwise intact prepulse inhibition. To further explore compulsive behavior, we evaluated novelty-induced locomotor hyperactivity and found that α 2B KO and α 2C KO mice exhibited increased locomotion in the open field. Interestingly, when challenged with amphetamine, α 2C KO mice increased activity at lower doses relative to either α 2A KO or WT mice. However, α 2B KO mice exhibited stereotypy at doses of amphetamine that were only locomotor stimulatory to all other genotypes. Following co-administration of AGN-209419 with low-dose amphetamine in WT mice, stereotypy was observed, mimicking the α 2B KO phenotype. These findings suggest that the α 2B receptor is involved in CNS behaviors associated with sensorimotor gating and compulsivity, and may be therapeutically relevant for disorders such as schizophrenia, ADHD, post-traumatic stress disorder, addiction, and

  18. Bradykinin B2 receptors play a neuroprotective role in Hypoxia/reoxygenation injury related to pyroptosis pathway.

    PubMed

    Tang, Min; Li, Xia; Liu, Ping; Wang, Jianwen; He, Fangping; Zhu, Xiongchao

    2018-05-27

    Kinins are pro-inflammatory peptides that mediate numerous vascular and pain responses in tissue injury. Kinins exert their biological functions via two G-protein-coupled receptors: bradykinin 1 receptor (B1R) and bradykinin 2 receptor (B2R). We previously demonstrated the up-regulation of B2R after hypoxia/reoxygenation (H/R) injury in primary cultured cortical neurons. However, the role of B2R in inflammasome-induced pyroptosis remains unknown. We induced H/R neuronal injury in primary cultured cortical neurons harvested from embryonic day 17 brains. Next, we examined the neuroprotective function of B2R in H/R-induced neuronal apoptosis or necrosis using an annexin V FITC/propidium iodide (PI) double-staining technique. The pyroptosis signaling cascade, including caspase-1, IL-1β and IL-18 levels and cleaved gasdermin D (GSDMD) expression was examined by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting to explore the underlying molecular mechanism. H/R injury significantly increased B2R protein expression (P<0.05) as well as the percentage of early apoptotic and necrotic or late apoptotic neurons as verified by the annexin V FITC/PI flow cytometric analysis. Bradykinin (BK), a specific B2R agonist, caused a significant decrease in apoptotic neuronal death after H/R injury, while HOE140, a specific B2R antagonist, markedly reduced the neuoprotective effect of B2R. Following H/R injury, BK downregulated the caspase-1, IL-1β and IL-18 levels (P<0.01). In contrast, pretreatment with HOE140 significantly increased caspase-1, IL-1β, and IL-18 levels (P<0.01). Further analysis revealed that GSDMD, a key executioner of pyroptosis, is a target for B2R-mediated inhibition of neuronal pyroptosis. Cleaved GSDMD expression was significantly inhibited by BK pretreatment and significantly enhanced by HOE140 pretreatment (P<0.01). These results indicate that activation of B2R plays an important role in pyroptosis mediated

  19. Molecular and functional characterization of the first tick CAP2b (periviscerokinin) receptor from Rhipicephalus (Boophilus) microplus

    USDA-ARS?s Scientific Manuscript database

    The cDNA of the receptor for CAP2b/periviscerokinin (PVK) neuropeptides, designated Rhimi-CAP2b-R, was cloned from synganglia of tick Rhipicephalus (Boophilus) microplus. This receptor is the ortholog of the insect CAP2b/PVK receptor, as concluded from analyses of the predicted protein sequence, ph...

  20. Positive regulation of raphe serotonin neurons by serotonin 2B receptors.

    PubMed

    Belmer, Arnauld; Quentin, Emily; Diaz, Silvina L; Guiard, Bruno P; Fernandez, Sebastian P; Doly, Stéphane; Banas, Sophie M; Pitychoutis, Pothitos M; Moutkine, Imane; Muzerelle, Aude; Tchenio, Anna; Roumier, Anne; Mameli, Manuel; Maroteaux, Luc

    2018-06-01

    Serotonin is a neurotransmitter involved in many psychiatric diseases. In humans, a lack of 5-HT 2B receptors is associated with serotonin-dependent phenotypes, including impulsivity and suicidality. A lack of 5-HT 2B receptors in mice eliminates the effects of molecules that directly target serotonergic neurons including amphetamine derivative serotonin releasers, and selective serotonin reuptake inhibitor antidepressants. In this work, we tested the hypothesis that 5-HT 2B receptors directly and positively regulate raphe serotonin neuron activity. By ex vivo electrophysiological recordings, we report that stimulation by the 5-HT 2B receptor agonist, BW723C86, increased the firing frequency of serotonin Pet1-positive neurons. Viral overexpression of 5-HT 2B receptors in these neurons increased their excitability. Furthermore, in vivo 5-HT 2B -receptor stimulation by BW723C86 counteracted 5-HT 1A autoreceptor-dependent reduction in firing rate and hypothermic response in wild-type mice. By a conditional genetic ablation that eliminates 5-HT 2B receptor expression specifically and exclusively from Pet1-positive serotonin neurons (Htr2b 5-HTKO mice), we demonstrated that behavioral and sensitizing effects of MDMA (3,4-methylenedioxy-methamphetamine), as well as acute behavioral and chronic neurogenic effects of the antidepressant fluoxetine, require 5-HT 2B receptor expression in serotonergic neurons. In Htr2b 5-HTKO mice, dorsal raphe serotonin neurons displayed a lower firing frequency compared to control Htr2b lox/lox mice as assessed by in vivo extracellular recordings and a stronger hypothermic effect of 5-HT 1A -autoreceptor stimulation was observed. The increase in head-twitch response to DOI (2,5-dimethoxy-4-iodoamphetamine) further confirmed the lower serotonergic tone resulting from the absence of 5-HT 2B receptors in serotonin neurons. Together, these observations indicate that the 5-HT 2B receptor acts as a direct positive modulator of serotonin Pet1

  1. The Arrestin-selective Angiotensin AT1 Receptor Agonist [Sar1,Ile4,Ile8]-AngII Negatively Regulates Bradykinin B2 Receptor Signaling via AT1-B2 Receptor Heterodimers*

    PubMed Central

    Wilson, Parker C.; Lee, Mi-Hye; Appleton, Kathryn M.; El-Shewy, Hesham M.; Morinelli, Thomas A.; Peterson, Yuri K.; Luttrell, Louis M.; Jaffa, Ayad A.

    2013-01-01

    The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B1 and B2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT1-B2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT1 agonist, [Sar1,Ile4,Ile8]-AngII, but not the neutral AT1 antagonist, losartan, inhibits endogenous B2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar1,Ile4, Ile8]-AngII require the AT1 receptor and result from arrestin-dependent co-internalization of AT1-B2 heterodimers. BRET50 measurements indicate that AT1 and B2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar1,Ile4,Ile8]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar1,Ile4,Ile8]-AngII has no effect on B2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar1,Ile4,Ile8]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar1,Ile4,Ile8]-AngII exerts lateral allosteric modulation of B2 receptor signaling by binding to the orthosteric ligand binding site of the AT1 receptor and promoting co-sequestration of AT1-B2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT1 receptor ligands may translate into different pharmacologic actions. PMID:23661707

  2. The arrestin-selective angiotensin AT1 receptor agonist [Sar1,Ile4,Ile8]-AngII negatively regulates bradykinin B2 receptor signaling via AT1-B2 receptor heterodimers.

    PubMed

    Wilson, Parker C; Lee, Mi-Hye; Appleton, Kathryn M; El-Shewy, Hesham M; Morinelli, Thomas A; Peterson, Yuri K; Luttrell, Louis M; Jaffa, Ayad A

    2013-06-28

    The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B1 and B2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT1-B2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT1 agonist, [Sar(1),Ile(4),Ile(8)]-AngII, but not the neutral AT1 antagonist, losartan, inhibits endogenous B2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar(1),Ile(4), Ile(8)]-AngII require the AT1 receptor and result from arrestin-dependent co-internalization of AT1-B2 heterodimers. BRET50 measurements indicate that AT1 and B2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar(1),Ile(4),Ile(8)]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar(1),Ile(4),Ile(8)]-AngII has no effect on B2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar(1),Ile(4),Ile(8)]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar(1),Ile(4),Ile(8)]-AngII exerts lateral allosteric modulation of B2 receptor signaling by binding to the orthosteric ligand binding site of the AT1 receptor and promoting co-sequestration of AT1-B2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT1 receptor ligands may translate into different pharmacologic

  3. Molecular and functional characterization of the first tick CAP2b (periviscerokinin) receptor from Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

    PubMed

    Yang, Yunlong; Bajracharya, Prati; Castillo, Paula; Nachman, Ronald J; Pietrantonio, Patricia V

    2013-12-01

    The cDNA of the receptor for CAP(2b)/periviscerokinin (PVK) neuropeptides, designated Rhimi-CAP(2b)-R, was cloned from synganglia of tick Rhipicephalus (Boophilus) microplus. This receptor is the ortholog of the insect CAP(2b)/PVK receptor, as concluded from analyses of the predicted protein sequence, phylogenetics and functional expression. Expression analyses of synganglion, salivary gland, Malpighian tubule, and ovary revealed Rhimi-CAP(2b)-R transcripts. The expression in mammalian cells of the open reading frame of Rhimi-CAP(2b)-R cDNA fused with a hemagglutinin tag at the receptor N-terminus was confirmed by immunocytochemistry. In a calcium bioluminescence assay the recombinant receptor was activated by the tick Ixodes scapularis CAP(2b)/PVK and a PVK analog with EC₅₀s of 64 nM and 249 nM, respectively. Tick pyrokinins were not active. This is the first report on the functional characterization of the CAP(2b)/PVK receptor from any tick species which will now permit the discovery of the physiological roles of these neuropeptides in ticks, as neurohormones, neuromodulators and/or neurotransmitters. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Serotonin 2B Receptors in Mesoaccumbens Dopamine Pathway Regulate Cocaine Responses.

    PubMed

    Doly, Stéphane; Quentin, Emily; Eddine, Raphaël; Tolu, Stefania; Fernandez, Sebastian P; Bertran-Gonzalez, Jesus; Valjent, Emmanuel; Belmer, Arnauld; Viñals, Xavier; Callebert, Jacques; Faure, Philippe; Meye, Frank J; Hervé, Denis; Robledo, Patricia; Mameli, Manuel; Launay, Jean-Marie; Maldonado, Rafael; Maroteaux, Luc

    2017-10-25

    Addiction is a maladaptive pattern of behavior following repeated use of reinforcing drugs in predisposed individuals, leading to lifelong changes. Common among these changes are alterations of neurons releasing dopamine in the ventral and dorsal territories of the striatum. The serotonin 5-HT 2B receptor has been involved in various behaviors, including impulsivity, response to antidepressants, and response to psychostimulants, pointing toward putative interactions with the dopamine system. Despite these findings, it remains unknown whether 5-HT 2B receptors directly modulate dopaminergic activity and the possible mechanisms involved. To answer these questions, we investigated the contribution of 5-HT 2B receptors to cocaine-dependent behavioral responses. Male mice permanently lacking 5-HT 2B receptors, even restricted to dopamine neurons, developed heightened cocaine-induced locomotor responses. Retrograde tracing combined with single-cell mRNA amplification indicated that 5-HT 2B receptors are expressed by mesolimbic dopamine neurons. In vivo and ex vivo electrophysiological recordings showed that 5-HT 2B -receptor inactivation in dopamine neurons affects their neuronal activity and increases AMPA-mediated over NMDA-mediated excitatory synaptic currents. These changes are associated with lower ventral striatum dopamine activity and blunted cocaine self-administration. These data identify the 5-HT 2B receptor as a pharmacological intermediate and provide mechanistic insight into attenuated dopamine tone following exposure to drugs of abuse. SIGNIFICANCE STATEMENT Here we report that mice lacking 5-HT 2B receptors totally or exclusively in dopamine neurons exhibit heightened cocaine-induced locomotor responses. Despite the sensitized state of these mice, we found that associated changes include lower ventral striatum dopamine activity and lower cocaine operant self-administration. We described the selective expression of 5-HT 2B receptors in a subpopulation of

  5. Cloning and pharmacological characterization of the rabbit bradykinin B2 receptor.

    PubMed

    Bachvarov, D R; Saint-Jacques, E; Larrivée, J F; Levesque, L; Rioux, F; Drapeau, G; Marceau, F

    1995-12-01

    Degenerate primers, corresponding to consensus sequences of third and sixth transmembrane domains of G protein-coupled receptor superfamily, were used for the polymerase chain reaction amplification and consecutive characterization of G protein-coupled receptors present in cultured rabbit aortic smooth muscle cells. One of the isolated resulting fragments was highly homologous to the corresponding region of the bradykinin (BK) B2 receptor cloned in other species. The polymerase chain reaction fragment was used to screen a rabbit genomic library, which allowed the identification of an intronless 1101-nucleotide open reading frame which codes for a 367-amino acid receptor protein. The rabbit B2 receptor sequence is more than 80% identical to the ones determined in three other species and retain putative glycosylation, palmitoylation and phosphorylation sites. In the rabbit genomic sequence, an acceptor splice sequence was found 8 base pairs upstream of the start codon. Northern blot analysis showed a high expression of a major transcript (4.2 kilobases) in the rabbit kidney and duodenum, and a less abundant expression in other tissues. Southern blot experiments suggest that a single copy of this gene exists in the rabbit genome. The cloned rabbit B2 receptor expressed in COS-1 cells binds [3H]BK in a saturable manner (KD 2.1 nM) and this ligand competes with a series of kinin agonists and antagonist with a rank order consistent with the B2 receptor identity. The insurmountable character of the antagonism exerted by Hoe 140 against BK on the rabbit B2 receptor, previously shown in pharmacological experiments, was confirmed in binding experiments with the cloned receptor expressed in a controlled manner. By contrast, Hoe 140 competed with [3H]BK in a surmountable manner for the human B2 receptor expressed in COS-1 cells. The cloning of the rabbit B2 receptor will be useful notably for the study of the structural basis of antagonist binding and for studies on receptor

  6. Translational studies support a role for serotonin 2B receptor (HTR2B) gene in aggression-related cannabis response.

    PubMed

    Montalvo-Ortiz, Janitza L; Zhou, Hang; D'Andrea, Ivana; Maroteaux, Luc; Lori, Adriana; Smith, Alicia; Ressler, Kerry J; Nuñez, Yaira Z; Farrer, Lindsay A; Zhao, Hongyu; Kranzler, Henry R; Gelernter, Joel

    2018-06-06

    Cannabis use is increasing in the United States, as are its adverse effects. We investigated the genetics of an adverse consequence of cannabis use: cannabis-related aggression (CRA) using a genome-wide association study (GWAS) design. Our GWAS sample included 3269 African Americans (AAs) and 2546 European Americans (EAs). An additional 89 AA subjects from the Grady Trauma Project (GTP) were also examined using a proxy-phenotype replication approach. We identified genome-wide significant risk loci contributing to CRA in AAs at the serotonin receptor 2B receptor gene (HTR2B), and the lead SNP, HTR2B*rs17440378, showed nominal association to aggression in the GTP cohort of cannabis-exposed subjects. A priori evidence linked HTR2B to impulsivity/aggression but not to cannabis response. Human functional data regarding the HTR2B variant further supported our finding. Treating an Htr2b -/- knockout mouse with THC resulted in increased aggressive behavior, whereas wild-type mice following THC administration showed decreased aggression in the resident-intruder paradigm, demonstrating that HTR2B variation moderates the effects of cannabis on aggression. These concordant findings in mice and humans implicate HTR2B as a major locus associated with cannabis-induced aggression.

  7. Bifunctional fusion proteins containing the sequence of the bradykinin homologue maximakinin: activities at the rat bradykinin B2 receptor.

    PubMed

    Charest-Morin, Xavier; Lodge, Robert; Marceau, François

    2018-05-01

    To support bradykinin (BK) B 2 receptor (B 2 R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B 2 Rs (immunoblots, epifluorescence microscopy). APEX2-(NG) 15 -MK is a bona fide agonist of the rat, but not of the human B 2 R (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3',5,5'-tetramethylbenzidine (luminescence or colourimetric B 2 R detection, cell well plate format). APEX2-(NG) 15 -MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat B 2 R in the concentration range of 50-600 nmol/L. However, the non-secreted construction myc-HSA-MK is a B 2 R agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the B 2 R are stable but subjected to slow intracellular inactivation, strong species specificity, and possible steric hindrance between the receptor and large proteins.

  8. A cleavable signal peptide enhances cell surface delivery and heterodimerization of Cerulean-tagged angiotensin II AT1 and bradykinin B2 receptor.

    PubMed

    Quitterer, Ursula; Pohl, Armin; Langer, Andreas; Koller, Samuel; Abdalla, Said

    2011-06-10

    Heterodimerization of the angiotensin II AT1 receptor with the receptor for the vasodepressor bradykinin, B2R, is known to sensitize the AT1-stimulated response of hypertensive individuals in vivo. To analyze features of that prototypic receptor heterodimer in vitro, we established a new method that uses fluorescence resonance energy transfer (FRET) and applies for the first time AT1-Cerulean as a FRET donor. The Cerulean variant of the green fluorescent protein as donor fluorophore was fused to the C-terminus of AT1, and the enhanced yellow fluorescent protein (EYFP) as acceptor fluorophore was fused to B2R. In contrast to AT1-EGFP, the AT1-Cerulean fusion protein was retained intracellularly. To facilitate cell surface delivery of AT1-Cerulean, a cleavable signal sequence was fused to the receptor's amino terminus. The plasma membrane-localized AT1-Cerulean resembled the native AT1 receptor regarding ligand binding and receptor activation. A high FRET efficiency of 24.7% between membrane-localized AT1-Cerulean and B2R-EYFP was observed with intact, non-stimulated cells. Confocal FRET microscopy further revealed that the AT1/B2 receptor heterodimer was functionally coupled to receptor desensitization mechanisms because activation of the AT1-Cerulean/B2R-EYFP heterodimer with a single agonist triggered the co-internalization of AT1/B2R. Receptor co-internalization was sensitive to inhibition of G protein-coupled receptor kinases, GRKs, as evidenced by a GRK-specific peptide inhibitor. In agreement with efficient AT1/B2R heterodimerization, confocal FRET imaging of co-enriched receptor proteins immobilized on agarose beads also detected a high FRET efficiency of 24.0%. Taken together confocal FRET imaging revealed efficient heterodimerization of co-enriched and cellular AT1/B2R, and GRK-dependent co-internalization of the AT1/B2R heterodimer. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. SSR240612 [(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride], a new nonpeptide antagonist of the bradykinin B1 receptor: biochemical and pharmacological characterization.

    PubMed

    Gougat, Jean; Ferrari, Bernard; Sarran, Lionel; Planchenault, Claudine; Poncelet, Martine; Maruani, Jeanne; Alonso, Richard; Cudennec, Annie; Croci, Tiziano; Guagnini, Fabio; Urban-Szabo, Katalin; Martinolle, Jean-Pierre; Soubrié, Philippe; Finance, Olivier; Le Fur, Gérard

    2004-05-01

    The biochemical and pharmacological properties of a novel non-peptide antagonist of the bradykinin (BK) B(1) receptor, SSR240612 [(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride] were evaluated. SSR240612 inhibited the binding of [(3)H]Lys(0)-des-Arg(9)-BK to the B(1) receptor in human fibroblast MRC5 and to recombinant human B(1) receptor expressed in human embryonic kidney cells with inhibition constants (K(i)) of 0.48 and 0.73 nM, respectively. The compound selectivity for B(1) versus B(2) receptors was in the range of 500- to 1000-fold. SSR240612 inhibited Lys(0)-desAr(9)-BK (10 nM)-induced inositol monophosphate formation in human fibroblast MRC5, with an IC(50) of 1.9 nM. It also antagonized des-Arg(9)-BK-induced contractions of isolated rabbit aorta and mesenteric plexus of rat ileum with a pA(2) of 8.9 and 9.4, respectively. Antagonistic properties of SSR240612 were also demonstrated in vivo. SSR240612 inhibited des-Arg(9)-BK-induced paw edema in mice (3 and 10 mg/kg p.o. and 0.3 and 1 mg/kg i.p.). Moreover, SSR240612 reduced capsaicin-induced ear edema in mice (0.3, 3 and 30 mg/kg p.o.) and tissue destruction and neutrophil accumulation in the rat intestine following splanchnic artery occlusion/reperfusion (0.3 mg/kg i.v.). The compound also inhibited thermal hyperalgesia induced by UV irradiation (1 and 3 mg/kg p.o.) and the late phase of nociceptive response to formalin in rats (10 and 30 mg/kg p.o.). Finally, SSR240612 (20 and 30 mg/kg p.o.) prevented neuropathic thermal pain induced by sciatic nerve constriction in the rat. In conclusion, SSR240612 is a new, potent, and orally active specific non-peptide bradykinin B(1) receptor antagonist.

  10. SULT2B1b Sulfotransferase: Induction by Vitamin D Receptor and Reduced Expression in Prostate Cancer

    PubMed Central

    Seo, Young-Kyo; Mirkheshti, Nooshin; Song, Chung S.; Kim, Soyoung; Dodds, Sherry; Ahn, Soon C.; Christy, Barbara; Mendez-Meza, Rosario; Ittmann, Michael M.; Abboud-Werner, Sherry

    2013-01-01

    An elevated tumor tissue androgen level, which reactivates androgen receptor in recurrent prostate cancer, arises from the intratumor synthesis of 5α-dihydrotestosterone through use of the precursor steroid dehydroepiandrosterone (DHEA) and is fueled by the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD1), aldoketoreductase (AKR1C3), and steroid 5-alpha reductase, type 1 (SRD5A1) present in cancer tissue. Sulfotransferase 2B1b (SULT2B1b) (in short, SULT2B) is a prostate-expressed hydroxysteroid SULT that converts cholesterol, oxysterols, and DHEA to 3β-sulfates. DHEA metabolism involving sulfonation by SULT2B can potentially interfere with intraprostate androgen synthesis due to reduction of free DHEA pool and, thus, conversion of DHEA to androstenedione. Here we report that in prostatectomy specimens from treatment-naive patients, SULT2B expression is markedly reduced in malignant tissue (P < .001, Mann-Whitney U test) compared with robust expression in adjacent nonmalignant glands. SULT2B was detected in formalin-fixed specimens by immunohistochemistry on individual sections and tissue array. Immunoblotting of protein lysates of frozen cancer and matched benign tissue confirmed immunohistochemistry results. An in-house–developed rabbit polyclonal antibody against full-length human SULT2B was validated for specificity and used in the analyses. Ligand-activated vitamin D receptor induced the SULT2B1 promoter in vivo in mouse prostate and increased SULT2B mRNA and protein levels in vitro in prostate cancer cells. A vitamin D receptor/retinoid X receptor-α–bound DNA element (with a DR7 motif) mediated induction of the transfected SULT2B1 promoter in calcitriol-treated cells. SULT2B knockdown caused an increased proliferation rate of prostate cancer cells upon stimulation by DHEA. These results suggest that the tumor tissue SULT2B level may partly control prostate cancer growth, and its induction in a therapeutic setting may inhibit disease

  11. Synaptic GluN2A and GluN2B Containing NMDA Receptors within the Superficial Dorsal Horn Activated following Primary Afferent Stimulation

    PubMed Central

    MacDermott, Amy B.

    2014-01-01

    NMDA receptors are important elements in pain signaling in the spinal cord dorsal horn. They are heterotetramers, typically composed of two GluN1 and two of four GluN2 subunits: GluN2A-2D. Mice lacking some of the GluN2 subunits show deficits in pain transmission yet functional synaptic localization of these receptor subtypes in the dorsal horn has not been fully resolved. In this study, we have investigated the composition of synaptic NMDA receptors expressed in monosynaptic and polysynaptic pathways from peripheral sensory fibers to lamina I neurons in rats. We focused on substance P receptor-expressing (NK1R+) projection neurons, critical for expression of hyperalgesia and allodynia. EAB-318 and (R)-CPP, GluN2A/B antagonists, blocked both monosynaptic and polysynaptic NMDA EPSCs initiated by primary afferent activation by ∼90%. Physiological measurements exploiting the voltage dependence of monosynaptic EPSCs similarly indicated dominant expression of GluN2A/B types of synaptic NMDA receptors. In addition, at synapses between C fibers and NK1R+ neurons, NMDA receptor activation initiated a secondary, depolarizing current. Ifenprodil, a GluN2B antagonist, caused modest suppression of monosynaptic NMDA EPSC amplitudes, but had a widely variable, sometimes powerful, effect on polysynaptic responses following primary afferent stimulation when inhibitory inputs were blocked to mimic neuropathic pain. We conclude that GluN2B subunits are moderately expressed at primary afferent synapses on lamina I NK1R+ neurons, but play more important roles for polysynaptic NMDA EPSCs driven by primary afferents following disinhibition, supporting the view that the analgesic effect of the GluN2B antagonist on neuropathic pain is at least in part, within the spinal cord. PMID:25122884

  12. Contribution of B2 receptors for bradykinin in Arthus reaction-induced plasma extravasation in wild-type or B2 transgenic knockout mice

    PubMed Central

    Samadfam, R; Teixeira, C; Bkaily, G; Sirois, P; de Brum-Fernandes, A; D'Orleans-Juste, P

    2000-01-01

    The aim of the present study was to investigate the contribution of bradykinin (BK) B1 and B2 receptors in a model of type III hypersensitivity, the reverse passive Arthus reaction (RPA), in wild-type mice and transgenic B2 knockout littermates.BK (10 μg mouse−1) or bovine serum albumin (0.5 mg mouse−1) induced a sustained Evans blue extravasation for more than 80 min in naive or rabbit anti-bovine serum albumin-treated mice (RPA model), respectively. The response to the two stimuli was prevented by the B2 receptor antagonist, HOE-140, but not by [Leu8]desArg9-BK (B1 receptor antagonist).In contrast to the wild-type littermates, RPA and bradykinin were unable to trigger an increase in plasma extravasation in B2 knockout mice.Furthermore, endothelin-1 (5 μg mouse−1) and a selective NK-1 receptor agonist [Sar9,Met (O2)11]-SP (20 μg mouse−1), triggered a significant increase in peritoneal plasma extravasation in both wild-type and B2 knockout animals.A pretreatment with indomethacin (200 μg mouse−1) significantly reduced the RPA-induced but not the BK-induced increase in Evans blue extravasation. Furthermore, RPA, but not BK, triggered a significant indomethacin-sensitive increase in peritoneal prostaglandin E2 content.Our results suggest a pivotal role for B2 receptors in the mechanism of plasma extravasation which occurs during the reverse passive Arthus reaction in the mouse. Moreover, our results suggest an important contribution of prostanoids in the plasma leakage mechanisms triggered by RPA but not by bradykinin. PMID:10780980

  13. Pharmacological characterization of the bradykinin B2 receptor: inter-species variability and dissociation between binding and functional responses

    PubMed Central

    Paquet, J -L; Luccarini, J -M; Fouchet, C; Defrêne, E; Loillier, B; Robert, C; Bélichard, P; Cremers, B; Pruneau, D

    1999-01-01

    The present study addresses the differences in binding profiles and functional properties of the human and rat bradykinin (BK) B2 receptor using various kinin receptor peptide derivatives as well as the non-peptide receptor antagonists WIN 64338 (phosphonium, [[4-[[2-[[bis(cyclohexylamino)methylene]amino]-3-(2-naphtalenyl)1-oxopropyl]amino]-phenyl]-methyl]tributyl, chloride, monohydro-chloride), and FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[-N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]-phenyl]N-methylamino carbonyl methyl] acrylamide. [3H]-BK bound with a similar affinity to membranes of Chinese hamster ovary cells (CHO-K1) expressing the cloned human (hB2-CHO) or rat (rB2-CHO) B2 receptor, human embryonic intestine cells (INT407) expressing the native B2 receptor, human umbilical vein (HUV) and rat uterus (RU). WIN 64338 and FR173657 bound with a 3.8–6.6 fold and 7.0–16.3 fold higher affinity the rat than the human B2 receptor, respectively. The affinity values of BK derivatives as well as non-peptide antagonists were reduced by 6–23 fold in physiological HBSS compared to low ionic strength TES binding buffer. BK (0.01–3000 nM) increased inositol triphosphates (IP3) levels in hB2-CHO, rB2-CHO and INT407 cells. The B2 receptor antagonist, Hoe 140 (D-Arg0-[ Hyp3, Thi5, D-Tic7, Oic8]-BK) at 10−7 M, significantly shifted to the right the IP3 response curves to BK giving apparent pKB values of 8.56, 9.79 and 8.84 for hB2-CHO, rB2-CHO and INT407 cells, respectively. In human isolated umbilical vein, Hoe 140, D-Arg0-[Hyp3, D-Phe7, Leu8]-BK and NPC 567 had a lower potency in functional assays (pKB 8.18, 5.77 and 5.60, respectively) than expected from their affinity in binding studies (pKi 10.52, 8.64 and 8.27, respectively). FR173657 behaved as a high affinity ligand with pKi values of 8.59 and 9.81 and potent competitive antagonist with pKB values of 7.80 and 8.17 in HUV and RU, respectively. FR173657 bound with a similar affinity the cloned and

  14. 2-Methyltetrahydro-3-benzazepin-1-ols - The missing link in SAR of GluN2B selective NMDA receptor antagonists.

    PubMed

    Dey, Sougata; Schepmann, Dirk; Wünsch, Bernhard

    2018-01-15

    The NMDA receptor containing GluN2B subunits represents a promising target for the development of drugs for the treatment of various neurological disorders including neurodegenerative diseases. In order to study the role of CH 3 and OH moieties trisubstituted tetrahydro-3-benzazepines 4 were designed as missing link between tetra- and disubstituted 3-benzazepines 2 and 5. The synthesis of 4 comprises eight reaction steps starting from alanine. The intramolecular Friedel-Crafts acylation to obtain the ketone 12 and the base-catalyzed elimination of trifluoromethanesulfinate (CF 3 SO 2 - ) followed by NaBH 4 reduction represent the key steps. The GluN2B affinity of the cis-configured 3-benzazepin-1-ol cis-4a with a 4-phenylbutyl side chain (K i  = 252 nM) is considerably lower than the GluN2B affinity of (R,R)-2 (K i  = 17 nM) indicating the importance of the phenolic OH moiety for the interaction with the receptor protein. Introduction of an additional CH 3 moiety in 2-position led to a slight decrease of GluN2B affinity as can be seen by comparing the affinity data of cis-4a and 5. The homologous phenylpentyl derivative cis-4b shows the highest GluN2B affinity (K i  = 56 nM) of this series of compounds. According to docking studies cis-4a adopts the same binding mode as the cocrystallized ligand ifenprodil-keto 1A and 5 at the interface of the GluN2B and GluN1a subunits. The same crucial H-bonds are formed between the C(O)NH 2 moiety of Gln110 within the GluN2B subunit and the protonated amino moiety and the OH moiety of (R,R)-cis-4a. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Submicron R2Fe14B particles

    NASA Astrophysics Data System (ADS)

    Koylu-Alkan, O.; Barandiaran, J. M.; Salazar, D.; Hadjipanayis, G. C.

    2016-05-01

    Mechanochemical synthesis of submicron R2Fe14B particles with R = Dy, Nd, Pr has been performed successfully via high energy ball milling of rare-earth oxides, iron oxide and boron oxide in the presence of a reducing agent (Ca) and a dispersant material (CaO), followed by annealing at 800 - 900 °C. In the R = Nd system, we were able to fabricate particles embedded in a CaO matrix with coercivity (Hc) of 10.3 kOe after annealing at 900 °C for 5 min. After washing off the dispersant, the Hc was decreased to below 1 kOe because of hydrogen absorption that leads to the formation of the hydrated R2Fe14BHx phase that has a lower anisotropy. Upon removal of the hydrogen the coercivity was increased to 3.3 kOe. The average size of the Nd2Fe14B particles increases from 100 nm in a sample synthesized at 800 °C to 158 nm at 900 °C. The isotropic Dy2Fe14B particles showed a higher coercivity of 21 kOe in washed samples after annealing at 900 °C for 5 min. An average size of 71 nm is measured in samples synthesized at 800 °C and 107 nm at 900 °C. Fitting the high field M(H) measurements in Nd2Fe14B to the law of approach to saturation gave values for the magnetocrystalline anisotropy for the washed sample 2.23 × 107 erg/cm3 and for the vacuum annealed sample 4.15 × 107 erg/cm3, both of which are lower than the bulk values. This would explain the lower values of Hc observed in the particles.

  16. Existence of three subtypes of bradykinin B2 receptors in guinea pig.

    PubMed

    Seguin, L; Widdowson, P S; Giesen-Crouse, E

    1992-12-01

    We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Requirement of SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 for paired immunoglobulin-like receptor B (PIR-B)-mediated inhibitory signal.

    PubMed

    Maeda, A; Kurosaki, M; Ono, M; Takai, T; Kurosaki, T

    1998-04-20

    Paired immunoglobulin-like receptor B (PIR-B) (p91) molecule has been proposed to function as an inhibitory receptor in B cells and myeloid lineage cells. We demonstrate here that the cytoplasmic region of PIR-B is capable of inhibiting B cell activation. Mutational analysis of five cytoplasmic tyrosines indicate that tyrosine 771 in the motif VxYxxL plays the most crucial role in mediating the inhibitory signal. PIR-B-mediated inhibition was markedly reduced in the SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 double-deficient DT40 B cells, whereas this inhibition was unaffected in the inositol polyphosphate 5'-phosphatase SHIP-deficient cells. These data demonstrate that PIR-B can negatively regulate B cell receptor activation and that this PIR-B-mediated inhibition requires redundant functions of SHP-1 and SHP-2.

  18. CPG2 Recruits Endophilin B2 to the Cytoskeleton for Activity-Dependent Endocytosis of Synaptic Glutamate Receptors.

    PubMed

    Loebrich, Sven; Benoit, Marc Robert; Konopka, Jaclyn Aleksandra; Cottrell, Jeffrey Richard; Gibson, Joanne; Nedivi, Elly

    2016-02-08

    Internalization of glutamate receptors at the postsynaptic membrane via clathrin-mediated endocytosis (CME) is a key mechanism for regulating synaptic strength. A role for the F-actin cytoskeleton in CME is well established, and recently, PKA-dependent association of candidate plasticity gene 2 (CPG2) with the spine-cytoskeleton has been shown to mediate synaptic glutamate receptor internalization. Yet, how the endocytic machinery is physically coupled to the actin cytoskeleton to facilitate glutamate receptor internalization has not been demonstrated. Moreover, there has been no distinction of endocytic-machinery components that are specific to activity-dependent versus constitutive glutamate receptor internalization. Here, we show that CPG2, through a direct physical interaction, recruits endophilin B2 (EndoB2) to F-actin, thus anchoring the endocytic machinery to the spine cytoskeleton and facilitating glutamate receptor internalization. Regulation of CPG2 binding to the actin cytoskeleton by protein kinase A directly impacts recruitment of EndoB2 and clathrin. Specific disruption of EndoB2 or the CPG2-EndoB2 interaction impairs activity-dependent, but not constitutive, internalization of both NMDA- and AMPA-type glutamate receptors. These results demonstrate that, through direct interactions with F-actin and EndoB2, CPG2 physically bridges the spine cytoskeleton and the endocytic machinery, and this tripartite association is critical specifically for activity-dependent CME of synaptic glutamate receptors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Attenuation of Persistent Experimental Pancreatitis Pain by a Bradykinin B2 Receptor Antagonist

    PubMed Central

    Chen, Qingmin; Vera-Portocarrero, Louis P.; Ossipov, Michael H.; Vardanyan, Marina; Lai, Josephine; Porreca, Frank

    2017-01-01

    Objective The role of bradykinin (BK) receptors in activating and sensitizing peripheral nociceptors is well known. Recently, we showed that spinal dynorphin was pronociceptive through direct or indirect BK receptor activation. Here, we explored the potential role of BK receptors in pain associated with persistent pancreatitis in rats. Methods Experimental pancreatitis and abdominal hypersensitivity were induced by intravenous administrations of dibutyltin dichloride (DBTC). [des-Arg9-Leu8]BK (B1 antagonist) and HOE 140 (B2 antagonist) were given by intraperitoneal or intrathecal injection. Dynorphin antiserum was given intrathecally. Reverse transcription–polymerase chain reaction was used to detect spinal mRNA for BK receptors. Results Dibutyltin dichloride–induced pancreatitis upregulated B1 and B2 mRNA in the thoracic dorsal root ganglion and B2, but not B1, in the pancreas. No changes in spinal B1 or B2 mRNA were observed. Intraperitoneal or intrathecal administration of HOE 140 dose dependently abolished DBTC-induced abdominal hypersensitivity, whereas [des-Arg9-Leu8]BK was without effect by either route of administration. Antiserum to dynorphin (intrathecal) abolished DBTC-induced hypersensitivity. Conclusions These results suggest that blockade of peripheral or spinal BK B2 receptors may be an effective approach for diminishing pain associated with pancreatitis. Moreover, it is suggested that spinal dynorphin may maintain pancreatitis pain through direct or indirect activation of BK B2 receptors in the spinal cord. PMID:20531238

  20. Mutations in the PP2A regulatory subunit B family genes PPP2R5B, PPP2R5C and PPP2R5D cause human overgrowth.

    PubMed

    Loveday, Chey; Tatton-Brown, Katrina; Clarke, Matthew; Westwood, Isaac; Renwick, Anthony; Ramsay, Emma; Nemeth, Andrea; Campbell, Jennifer; Joss, Shelagh; Gardner, McKinlay; Zachariou, Anna; Elliott, Anna; Ruark, Elise; van Montfort, Rob; Rahman, Nazneen

    2015-09-01

    Overgrowth syndromes comprise a group of heterogeneous disorders characterised by excessive growth parameters, often in association with intellectual disability. To identify new causes of human overgrowth, we have been undertaking trio-based exome sequencing studies in overgrowth patients and their unaffected parents. Prioritisation of functionally relevant genes with multiple unique de novo mutations revealed four mutations in protein phosphatase 2A (PP2A) regulatory subunit B family genes protein phosphatase 2, regulatory Subunit B', beta (PPP2R5B); protein phosphatase 2, regulatory Subunit B', gamma (PPP2R5C); and protein phosphatase 2, regulatory Subunit B', delta (PPP2R5D). This observation in 3 related genes in 111 individuals with a similar phenotype is greatly in excess of the expected number, as determined from gene-specific de novo mutation rates (P = 1.43 × 10(-10)). Analysis of exome-sequencing data from a follow-up series of overgrowth probands identified a further pathogenic mutation, bringing the total number of affected individuals to 5. Heterozygotes shared similar phenotypic features including increased height, increased head circumference and intellectual disability. The mutations clustered within a region of nine amino acid residues in the aligned protein sequences (P = 1.6 × 10(-5)). We mapped the mutations onto the crystal structure of the PP2A holoenzyme complex to predict their molecular and functional consequences. These studies suggest that the mutations may affect substrate binding, thus perturbing the ability of PP2A to dephosphorylate particular protein substrates. PP2A is a major negative regulator of v-akt murine thymoma viral oncogene homolog 1 (AKT). Thus, our data further expand the list of genes encoding components of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signalling cascade that are disrupted in human overgrowth conditions. © The Author 2015. Published by Oxford University Press.

  1. Brain-derived neurotrophic factor (BDNF) in the rostral anterior cingulate cortex (rACC) contributes to neuropathic spontaneous pain-related aversion via NR2B receptors.

    PubMed

    Zhang, Le; Wang, Gongming; Ma, Jinben; Liu, Chengxiao; Liu, Xijiang; Zhan, Yufeng; Zhang, Mengyuan

    2016-10-01

    The rostral anterior cingulate cortex (rACC) plays an important role in pain affect. Previous investigations have reported that the rACC mediates the negative affective component of inflammatory pain and contributed to the aversive state of nerve injury-induced neuropathic pain. Brain-derived neurotrophic factor (BDNF), an activity-dependent neuromodulator in the adult brain, is believed to play a role in the development and maintenance of inflammatory and neuropathic pain in the spinal cord. However, whether and how BDNF in the rACC regulates pain-related aversion due to peripheral nerve injury is largely unknown. Behaviorally, using conditioned place preference (CPP) training in rats, which is thought to reveal spontaneous pain-related aversion, we found that CPP was acquired following spinal clonidine in rats with partial sciatic nerve transection. Importantly, BDNF was upregulated within the rACC in of rats with nerve injury and enhanced the CPP acquisition, while a local injection of a BDNF-tropomyosin receptor kinase B (TrkB) antagonist into the rACC completely blocked this process. Finally, we demonstrated that the BDNF/TrkB pathway exerted its function by activating the NR2B receptor, which is widely accepted to be a crucial factor contributing to pain affect. In conclusion, our results demonstrate that the BDNF/TrkB-mediated signaling pathway in the rACC is involved in the development of neuropathic spontaneous pain-related aversion and that this process is dependent upon activation of NR2B receptors. These findings suggest that suppression of the BDNF-related signaling pathway in the rACC may provide a novel strategy to overcome pain-related aversion. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Colonic motor dysfunctions in a mouse model of high-fat diet-induced obesity: an involvement of A2B adenosine receptors.

    PubMed

    Antonioli, Luca; Pellegrini, Carolina; Fornai, Matteo; Tirotta, Erika; Gentile, Daniela; Benvenuti, Laura; Giron, Maria Cecilia; Caputi, Valentina; Marsilio, Ilaria; Orso, Genny; Bernardini, Nunzia; Segnani, Cristina; Ippolito, Chiara; Csóka, Balázs; Németh, Zoltán H; Haskó, György; Scarpignato, Carmelo; Blandizzi, Corrado; Colucci, Rocchina

    2017-12-01

    Adenosine A 2B receptors (A 2B R) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of A 2B R in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic A 2B R localization was examined by immunofluorescence. The role of A 2B R in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, A 2B R were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the A 2B R antagonist MRS1754 enhanced electrically induced NK 1 -mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A 2B receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both A 2B R ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of A 2B R expression. A 2B R, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity.

  3. Relationship of angiotensin I-converting enzyme (ACE) and bradykinin B2 receptor (BDKRB2) polymorphism with diabetic nephropathy.

    PubMed

    Zou, Honghong; Wu, Guoqing; Lv, Jinlei; Xu, Gaosi

    2017-06-01

    To determine whether ACE 2 I/D and BDKRB2 3 +9/-9 polymorphism causatively affect diabetic nephropathy progression RESULTS: STZ-induced metabolic disorder, as well as inflammatory responses, was significantly aggravated in ACE II-B2R 4 +9bp, ACE DD-B2R+9bp, or ACE DD-B2R-9bp diabetic mice but not ACE II-B2R-9bp, indicating the genetic susceptibility of ACE DD or B2R+9bp to diabetic nephropathy. Furthermore, ACE II-B2R+9bp, ACE DD-B2R+9bp, or ACE DD-B2R-9bp rather than ACE II-B2R-9bp, worsened renal performance and enhanced pathological alterations induced by STZ. Markedly elevated monocyte chemoattractant protein-1(MCP-1), podocin, osteopontin (OPN), transforming growth factor-β1 (TGF-β1), and reduced nephrin, podocin were also detected both in diabetic mice and podocytes under hyperglycemic conditions in response to ACE II-B2R+9bp, ACE DD-B2R+9bp, or ACE DD-B2R-9bp, versus ACE II-B2R-9bp. In addition, high glucose-induced mitochondrial oxidative stress and cell apoptosis were observably increased in response to ACE II-B2R+9bp, ACE DD-B2R+9bp, or ACE DD-B2R-9bp but not ACE II-B2R-9bp. We provide first evidence indicating the causation between ACE DD or B2R+9bp genotype and the increased risk for diabetic nephropathy, broadening our horizon about the role of genetic modulators in this disease. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Grb2 regulates B-cell maturation, B-cell memory responses and inhibits B-cell Ca2+ signalling.

    PubMed

    Ackermann, Jochen A; Radtke, Daniel; Maurberger, Anna; Winkler, Thomas H; Nitschke, Lars

    2011-04-20

    Grb2 is a ubiquitously expressed adaptor protein, which activates Ras and MAP kinases in growth factor receptor signalling, while in B-cell receptor (BCR) signalling this role is controversial. In B cell lines it was shown that Grb2 can inhibit BCR-induced Ca(2+) signalling. Nonetheless, the physiological role of Grb2 in primary B cells is still unknown. We generated a B-cell-specific Grb2-deficient mouse line, which had a severe reduction of mature follicular B cells in the periphery due to a differentiation block and decreased B-cell survival. Moreover, we found several changes in important signalling pathways: enhanced BCR-induced Ca(2+) signalling, alterations in mitogen-activated protein kinase activation patterns and strongly impaired Akt activation, the latter pointing towards a defect in PI3K signalling. Interestingly, B-cell-specific Grb2-deficient mice showed impaired IgG and B-cell memory responses, and impaired germinal centre formation. Thus, Grb2-dependent signalling pathways are crucial for lymphocyte differentiation processes, as well as for control of secondary humoral immune responses.

  5. Uncoupling of the ITIM receptor G6b-B from the tyrosine phosphatases Shp1 and Shp2 disrupts platelet homeostasis in mice.

    PubMed

    Geer, Mitchell J; van Geffen, Johanna P; Gopalasingam, Piraveen; Vögtle, Timo; Smith, Christopher W; Heising, Silke; Kuijpers, Marijke J E; Tullemans, Bibian M E; Jarvis, Gavin E; Eble, Johannes A; Jeeves, Mark; Overduin, Michael; Heemskerk, Johan W M; Mazharian, Alexandra; Senis, Yotis A

    2018-06-11

    The immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B has emerged as a key regulator of platelet homeostasis. However, it remains unclear how it mediates its effects. Tyrosine phosphorylation of the ITIM and immunoreceptor tyrosine-based switch motif (ITSM) within the cytoplasmic tail of G6b-B provides a docking site for SH2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2, which are also critical regulators of platelet production and function. In this study, we investigate the physiological consequences of uncoupling G6b-B from Shp1 and Shp2. To address this, we generated a transgenic mouse model expressing a mutant form of G6b-B in which tyrosine (Y) residues 212 and 238 within the ITIM and ITSM were mutated to phenylalanine (F), respectively. Mice homozygous for the mutation (G6b-B diY/F) were macrothrombocytopenic, due to a reduction in platelet production, had large clusters of megakaryocytes and myelofibrosis at sites of hematopoiesis, similar to that observed in G6b knockout (G6b KO) mice. Platelets from G6b-B diY/F mice were hypo-responsive to collagen, due to a significant reduction in expression of the immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor complex GPVI-FcR γ-chain, and thrombin, that could be partially rescued by co-stimulating the platelets with ADP. In contrast, platelets from G6b-B diY/F, G6b KO and megakaryocyte-specific Shp2 KO mice were hyper-responsive to antibody-mediated cross-linking of the hemi-ITAM-containing podoplanin receptor CLEC-2, suggesting that G6b-B inhibits CLEC-2-mediated platelet activation through Shp2. Findings from this study demonstrate that G6b-B must engage with Shp1 and Shp2 in order to mediate its regulatory effects on platelet homeostasis. Copyright © 2018 American Society of Hematology.

  6. Thieno[3,2-b]- and thieno[2,3-b]pyrrole bioisosteric analogues of the hallucinogen and serotonin agonist N,N-dimethyltryptamine.

    PubMed

    Blair, J B; Marona-Lewicka, D; Kanthasamy, A; Lucaites, V L; Nelson, D L; Nichols, D E

    1999-03-25

    The synthesis and biological activity of 6-[2-(N, N-dimethylamino)ethyl]-4H-thieno[3,2-b]pyrrole (3a) and 4-[2-(N, N-dimethylamino)ethyl]-6H-thieno[2,3-b]pyrrole (3b), thienopyrroles as potential bioisosteres of N,N-dimethyltryptamine (1a), are reported. Hallucinogen-like activity was evaluated in the two-lever drug discrimination paradigm using LSD- and DOI-trained rats. Neither 3a nor 3b substituted for LSD or DOI up to doses of 50 micromol/kg. By comparison, 1a fully substituted in LSD-trained rats. However, 3a and 3b fully substituted for the 5-HT1A agonist LY293284 ((-)-(4R)-6-acetyl-4-(di-n-propylamino)-1,3,4, 5-tetrahydrobenz[c,d]indole). Both 3a and 3b induced a brief "serotonin syndrome" and salivation, an indication of 5-HT1A receptor activation. At the cloned human 5-HT2A receptor 3b had about twice the affinity of 3a. At the cloned human 5-HT2B and 5-HT2C receptors, however, 3a had about twice the affinity of 3b. Therefore, thiophene lacks equivalence as a replacement for the phenyl ring in the indole nucleus of tryptamines that bind to 5-HT2 receptor subtypes and possess LSD-like behavioral effects. Whereas both of the thienopyrroles had lower affinity than the corresponding 1a at 5-HT2 receptors, 3a and 3b had significantly greater affinity than 1a at the 5-HT1A receptor. Thus, thienopyrrole does appear to serve as a potent bioisostere for the indole nucleus in compounds that bind to the serotonin 5-HT1A receptor. These differences in biological activity suggest that serotonin receptor isoforms are very sensitive to subtle changes in the electronic character of the aromatic systems of indole compounds.

  7. Enhancement of bradykinin and resensitization of its B2 receptor.

    PubMed

    Marcic, B; Deddish, P A; Jackman, H L; Erdös, E G

    1999-03-01

    We studied the enhancement of the effects of bradykinin B2 receptor agonists by agents that react with active centers of angiotensin-converting enzyme (ACE) independent of enzymatic inactivation. The potentiation and the desensitization and resensitization of B2 receptor were assessed by measuring [3H]arachidonic acid release and [Ca2+]i mobilization in Chinese hamster ovary cells transfected to express human ACE and B2 receptor, or in endothelial cells with constitutively expressed ACE and receptor. Administration of bradykinin or its ACE-resistant analogue desensitized the receptor, but it was resensitized (arachidonic acid release or [Ca2+]i mobilization) by agents such as enalaprilat (1 micromol/L). Enalaprilat was inactive in the absence of ACE expression. La3+ (100 micromol/L) inhibited the apparent resensitization, probably by blocking the entry of extracellular calcium. Enalaprilat resensitized the receptor via ACE to release arachidonic acid by bradykinin at a lower concentration (5 nmol/L) than required to mobilize [Ca2+]i (1 micromol/L). Monoclonal antibodies inhibiting the ACE N-domain active center and polyclonal antiserum potentiated bradykinin. The snake venom peptide BPP5a and metabolites of angiotensin and bradykinin (angiotensin-[1-9], angiotensin-[1-7], bradykinin-[1-8]; 1 micromol/L) enhanced arachidonic acid release by bradykinin. Angiotensin-(1-9) and -(1-7) also resensitized the receptor. Enalaprilat potentiated the bradykinin effect in cells expressing a mutant ACE with a single N-domain active site. Agents that reacted with a single active site, on the N-domain or on the C-domain, potentiated bradykinin not by blocking its inactivation but by inducing crosstalk between ACE and the receptor. Enalaprilat enhanced signaling via ACE by Galphai in lower concentration than by Galphaq-coupled receptor.

  8. Old dance with a new partner: EGF receptor as the phenobarbital receptor mediating Cyp2B expression.

    PubMed

    Meyer, Sharon A; Jirtle, Randy L

    2013-05-07

    The decades-long quest for the phenobarbital (PhB) receptor that mediates activation of Cyp2B would appear fulfilled with the discovery by Mutoh et al., who found that PhB binds with pharmacological affinity to the epidermal growth factor receptor (EGFR). This finding provides a molecular basis for the suppression of hepatocyte EGFR signaling observed with PhB treatment, as previously noted in the context of tumor promotion. Although the PhB-mediated induction of Cyp2B expression through the association of a canonical nuclear receptor with the 5'-enhancer PBREM of Cyp2B is well known, direct binding of PhB to constitutive active androstane receptor (CAR, also known as NR1I3) typical of other xenobiotic-activated nuclear receptors has eluded detection. One EGF-activated pathway affected by the PhB-EGFR interaction is the loss of tyrosine phosphorylation of the scaffold protein RACK1. Dephosphorylated RACK1 provides the mechanistic link between the binding of PhB to EGFR and its effects on CAR by facilitating the interaction of serine/threonine phosphatase PP2A with inactive phosphorylated CAR. The dephosphorylation of CAR enables its translocation to the nucleus and activation of Cyp2B expression. Because EGFR and transducers RACK1, PP2A, and other partners are highly networked in numerous cellular pathways, this newly discovered partnership will surely reveal new fundamental roles for PhB beyond the regulation of drug metabolism.

  9. Structural and functional characterizations of activin type 2B receptor (acvr2b) ortholog from the marine fish, gilthead sea bream, Sparus aurata: evidence for gene duplication of acvr2b in fish.

    PubMed

    Funkenstein, Bruria; Krol, Ekaterina; Esterin, Elena; Kim, Yong-Soo

    2012-12-01

    Myostatin (MSTN), a negative regulator of muscle growth and a member of the transforming growth factor-β superfamily, can bind the two activin type 2 receptors (ACVR2). It has been previously shown that WT mice injected with ACVR2B extracellular domain (ACVR2B-ECD) had higher muscle mass. Likewise, fish larvae immersed in Pichia pastoris culture supernatant, containing goldfish Acvr2b-ECD, showed enhanced larval growth. However, it is not clear whether fish Mstn1 and Mstn2 signal through the same receptor and whether fish express more than one acvr2b gene. In the current study, three cDNAs encoding acvr2b (saacvr2b-1, saacvr2b-2a, and saacvr2b-2b) were cloned from gilthead sea bream. All three contain the short extracellular binding domain, a short transmembrane region, and a conserved catalytic domain of serine/threonine protein kinase. Bioinformatics analysis provided evidence for the existence of two acvr2b genes (acvr2b-1 and acvr2b-2) in several other fish species as well, probably as a result of gene or genome duplication. The two isoforms differ in their amino acid sequences. The direct inhibitory effect of Acvr2b-ECD on Mstn activity was tested in vitro. The saAcvr2b-1-ECD was expressed in the yeast P. pastoris. Evidence is provided for N-glycosylation of Acvr2b-1-ECD. The affinity-purified Acvr2b-1-ECD inhibited recombinant mouse/rat/human mature MSTN activity when determined in vitro using the CAGA-luciferase assay in A204 cells. A lower inhibitory activity was obtained when unprocessed purified, furin-digested, and activated saMstn1 was used. Results of this study demonstrate for the first time the existence of two acvr2b genes in fish. In addition, the study shows that bioactive fish Acvr2b-ECD can be produced from P. pastoris.

  10. Lung injury pathways: Adenosine receptor 2B signaling limits development of ischemic bronchiolitis obliterans organizing pneumonia.

    PubMed

    Densmore, John C; Schaid, Terry R; Jeziorczak, Paul M; Medhora, Meetha; Audi, Said; Nayak, Shraddha; Auchampach, John; Dwinell, Melinda R; Geurts, Aron M; Jacobs, Elizabeth R

    2017-02-01

    Purpose/Aim of the Study: Adenosine signaling was studied in bronchiolitis obliterans organizing pneumonia (BOOP) resulting from unilateral lung ischemia. Ischemia was achieved by either left main pulmonary artery or complete hilar ligation. Sprague-Dawley (SD) rats, Dahl salt sensitive (SS) rats and SS mutant rat strains containing a mutation in the A 2B adenosine receptor gene (Adora2b) were studied. Adenosine concentrations were measured in bronchoalveolar lavage (BAL) by HPLC. A 2A (A 2A AR) and A 2B adenosine receptor (A 2B AR) mRNA and protein were quantified. Twenty-four hours after unilateral PA ligation, BAL adenosine concentrations from ischemic lungs were increased relative to contralateral lungs in SD rats. A 2B AR mRNA and protein concentrations were increased after PA ligation while miR27a, a negatively regulating microRNA, was decreased in ischemic lungs. A 2A AR mRNA and protein concentrations remained unchanged following ischemia. A 2B AR protein was increased in PA ligated lungs of SS rats after 7 days, and 4 h after complete hilar ligation in SD rats. SS-Adora2b mutants showed a greater extent of BOOP relative to SS rats, and greater inflammatory changes. Increased A 2B AR and adenosine following unilateral lung ischemia as well as more BOOP in A 2B AR mutant rats implicate a protective role for A 2B AR signaling in countering ischemic lung injury.

  11. CD86 and beta2-adrenergic receptor signaling pathways, respectively, increase Oct-2 and OCA-B Expression and binding to the 3'-IgH enhancer in B cells.

    PubMed

    Podojil, Joseph R; Kin, Nicholas W; Sanders, Virginia M

    2004-05-28

    Stimulation of CD86 (formerly known as B7-2) and/or the beta2-adrenergic receptor on a CD40 ligand/interleukin-4-activated B cell increased the rate of mature IgG1 transcription. To identify the mechanism responsible for this effect, we determined whether CD86 and/or beta2-adrenergic receptor stimulation regulated transcription factor expression and binding to the 3'-IgH enhancer in vitro and in vivo. We showed that CD86 stimulation increased the nuclear localization of NF-kappaB1 (p50) and phosphorylated RelA (p65) and increased Oct-2 expression and binding to the 3'-IgH enhancer, in a protein kinase C-dependent manner. These effects were lost when CD86-deficient or NF-kappaB1-deficient B cells were used. CD86 stimulation also increased the level of IkappaB-alpha phosphorylation but in a protein kinase C-independent manner. Beta2-adrenergic receptor stimulation increased CREB phosphorylation, OCA-B expression, and OCA-B binding to the 3'-IgH enhancer in a protein kinase A-dependent manner, an effect lost when beta2-adrenergic receptor-deficient B cells were used. Also, the beta2-adrenergic receptor-induced increase in the level of mature IgG1 transcript was lost when OCA-B-deficient B cells were used. These data are the first to show that CD86 stimulation up-regulates the expression of the transcription factor Oct-2 in a protein kinase C- and NF-kappaB1-dependent manner, and that beta2-adrenergic receptor stimulation up-regulates the expression of the coactivator OCA-B in a protein kinase A-dependent manner to cooperate with Oct-2 binding to the 3'-IgH enhancer.

  12. Betacellulin-Induced Beta Cell Proliferation and Regeneration Is Mediated by Activation of ErbB-1 and ErbB-2 Receptors

    PubMed Central

    Oh, Yoon Sin; Shin, Seungjin; Lee, Youn-Jung; Kim, Eung Hwi; Jun, Hee-Sook

    2011-01-01

    Background Betacellulin (BTC), a member of the epidermal growth factor family, is known to play an important role in regulating growth and differentiation of pancreatic beta cells. Growth-promoting actions of BTC are mediated by epidermal growth factor receptors (ErbBs), namely ErbB-1, ErbB-2, ErbB-3 and ErbB-4; however, the exact mechanism for beta cell proliferation has not been elucidated. Therefore, we investigated which ErbBs are involved and some molecular mechanisms by which BTC regulates beta cell proliferation. Methodology/Principal Findings The expression of ErbB-1, ErbB-2, ErbB-3, and ErbB-4 mRNA was detected by RT-PCR in both a beta cell line (MIN-6 cells) and C57BL/6 mouse islets. Immunoprecipitation and western blotting analysis showed that BTC treatment of MIN-6 cells induced phosphorylation of only ErbB-1 and ErbB-2 among the four EGF receptors. BTC treatment resulted in DNA synthetic activity, cell cycle progression, and bromodeoxyuridine (BrdU)-positive staining. The proliferative effect was blocked by treatment with AG1478 or AG825, specific tyrosine kinase inhibitors of ErbB-1 and ErbB-2, respectively. BTC treatment increased mRNA and protein levels of insulin receptor substrate-2 (IRS-2), and this was blocked by the ErbB-1 and ErbB-2 inhibitors. Inhibition of IRS-2 by siRNA blocked cell cycle progression induced by BTC treatment. Streptozotocin-induced diabetic mice injected with a recombinant adenovirus expressing BTC and treated with AG1478 or AG825 showed reduced islet size, reduced numbers of BrdU-positive cells in the islets, and did not attain BTC-mediated remission of diabetes. Conclusions/Significance These results suggest that BTC exerts proliferative activity on beta cells through the activation of ErbB-1 and ErbB-2 receptors, which may increase IRS-2 expression, contributing to the regeneration of beta cells. PMID:21897861

  13. Role of kinin B1 and B2 receptors in memory consolidation during the aging process of mice.

    PubMed

    Lemos, Mayra Tolentino Resk; Amaral, Fabio Agostini; Dong, Karis Ester; Bittencourt, Maria Fernanda Queiroz Prado; Caetano, Ariadiny Lima; Pesquero, João Bosco; Viel, Tania Araujo; Buck, Hudson Sousa

    2010-04-01

    Under physiological conditions, elderly people present memory deficit associated with neuronal loss. This pattern is also associated with Alzheimer's disease but, in this case, in a dramatically intensified level. Kinin receptors have been involved in neurodegeneration and increase of amyloid-beta concentration, associated with Alzheimer's disease (AD). Considering these findings, this work evaluated the role of kinin receptors in memory consolidation during the aging process. Male C57Bl/6 (wt), knock-out B1 (koB1) or B2 (koB2) mice (3, 6, 12 and 18-month-old - mo; n=10 per group) were submitted to an acquisition session, reinforcement to learning (24h later: test 1) and final test (7days later: test 2), in an active avoidance apparatus, to evaluate memory. Conditioned avoidance responses (CAR, % of 50 trials) were registered. In acquisition sessions, similar CAR were obtained among age matched animals from all strains. However, a significant decrease in CAR was observed throughout the aging process (3mo: 8.8+/-2.3%; 6mo: 4.1+/-0.6%; 12mo: 2.2+/-0.6%, 18mo: 3.6+/-0.6%, P<0.01), indicating a reduction in the learning process. In test 1, as expected, memory retention increased significantly (P<0.05) in all 3- and 6-month-old animals as well as in 12-month-old-wt and 12-month-old-koB1 (P<0.01), compared to the training session. However, 12-month-old-koB2 and all 18-month-old animals did not show an increase in memory retention. In test 2, 3- and 6-month-old wt and koB1 mice of all ages showed a significant improvement in memory (P<0.05) compared to test 1. However, 12-month-old wt and koB2 mice of all ages showed no difference in memory retention. We suggest that, during the aging process, the B1 receptor could be involved in neurodegeneration and memory loss. Nevertheless, the B2 receptor is apparently acting as a neuroprotective factor. Copyright 2009 Elsevier Ltd. All rights reserved.

  14. R-Ras2 is required for germinal center formation to aid B cells during energetically demanding processes.

    PubMed

    Mendoza, Pilar; Martínez-Martín, Nuria; Bovolenta, Elena R; Reyes-Garau, Diana; Hernansanz-Agustín, Pablo; Delgado, Pilar; Diaz-Muñoz, Manuel D; Oeste, Clara L; Fernández-Pisonero, Isabel; Castellano, Ester; Martínez-Ruiz, Antonio; Alonso-Lopez, Diego; Santos, Eugenio; Bustelo, Xosé R; Kurosaki, Tomohiro; Alarcón, Balbino

    2018-05-29

    Upon antigen recognition within peripheral lymphoid organs, B cells interact with T cells and other immune cells to transiently form morphological structures called germinal centers (GCs), which are required for B cell clonal expansion, immunoglobulin class switching, and affinity maturation. This process, known as the GC response, is an energetically demanding process that requires the metabolic reprogramming of B cells. We showed that the Ras-related guanosine triphosphate hydrolase (GTPase) R-Ras2 (also known as TC21) plays an essential, nonredundant, and B cell-intrinsic role in the GC response. Both the conversion of B cells into GC B cells and their expansion were impaired in mice lacking R-Ras2, but not in those lacking a highly related R-Ras subfamily member or both the classic H-Ras and N-Ras GTPases. In the absence of R-Ras2, activated B cells did not exhibit increased oxidative phosphorylation or aerobic glycolysis. We showed that R-Ras2 was an effector of both the B cell receptor (BCR) and CD40 and that, in its absence, B cells exhibited impaired activation of the PI3K-Akt-mTORC1 pathway, reduced mitochondrial DNA replication, and decreased expression of genes involved in glucose metabolism. Because most human B cell lymphomas originate from GC B cells or B cells that have undergone the GC response, our data suggest that R-Ras2 may also regulate metabolism in B cell malignancies. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  15. Design and synthesis of novel sulfonamide-containing bradykinin hB2 receptor antagonists. 1. Synthesis and SAR of alpha,alpha-dimethylglycine sulfonamides.

    PubMed

    Fattori, Daniela; Rossi, Cristina; Fincham, Christopher I; Berettoni, Marco; Calvani, Federico; Catrambone, Fernando; Felicetti, Patrizia; Gensini, Martina; Terracciano, Rosa; Altamura, Maria; Bressan, Alessandro; Giuliani, Sandro; Maggi, Carlo A; Meini, Stefania; Valenti, Claudio; Quartara, Laura

    2006-06-15

    We recently published the extensive in vivo pharmacological characterization of MEN 16132 (J. Pharmacol. Exp. Ther. 2005, 616-623; Eur. J. Pharmacol. 2005, 528, 7), a member of the sulfonamide-containing human B(2) receptor (hB(2)R) antagonists. Here we report, in detail, how this family of compounds was designed, synthesized, and optimized to provide a group of products with subnanomolar affinity for the hB(2)R and high in vivo potency after topical administration to the respiratory tract. The series was designed on the basis of indications from the X-ray structures of the key structural motifs A and B present in known antagonists and is characterized by the presence of an alpha,alpha-dialkyl amino acid. The first lead (17) of the series was submitted to extensive chemical work to elucidate the structural requirements to increase hB(2) receptor affinity and antagonist potency in bioassays expressing the human B(2) receptor (hB(2)R). The following structural features were selected: a 2,4-dimethylquinoline moiety and a piperazine linker acylated with a basic amino acid. The representative lead compound 68 inhibited the specific binding of [(3)H]BK to hB(2)R with a pKi of 9.4 and antagonized the BK-induced inositolphosphate (IP) accumulation in recombinant cell systems expressing the hB(2)R with a pA(2) of 9.1. Moreover, compound 68 when administered (300 nmol/kg) intratracheally in the anesthetized guinea pig, was able to significantly inhibit BK-induced bronchoconstriction for up to 120 min after its administration, while having a lower and shorter lasting effect on hypotension.

  16. The relevance of kalikrein-kinin system via activation of B2 receptor in LPS-induced fever in rats.

    PubMed

    Soares, Denis de Melo; Santos, Danielle R; Rummel, Christoph; Ott, Daniela; Melo, Míriam C C; Roth, Joachim; Calixto, João B; Souza, Glória E P

    2017-11-01

    This study evaluated the involvement of endogenous kallikrein-kinin system and the bradykinin (BK) B 1 and B 2 receptors on LPS- induced fever and the POA cells involved in this response. Male Wistar rats received either i.v. (1 mg/kg), i.c.v. (20 nmol) or i.h. (2 nmol) injections of icatibant (B 2 receptor antagonist) 30 or 60 min, respectively, before the stimuli. DALBK (B 1 receptor antagonist) was given either 15min before BK (i.c.v.) or 30 min before LPS (i.v.). Captopril (5 mg/kg, sc.,) was given 1 h prior LPS or BK. Concentrations of BK and total kininogenon CSF, plasma and tissue kallikrein were evaluated. Rectal temperatures (rT) were assessed by telethermometry. Ca ++ signaling in POA cells was performed in rat pup brain tissue microcultures. Icatibant reduced LPS fever while, captopril exacerbated that response, an effect abolished by icatibant. Icatibant (i.h.) reduced fever to BK (i.h.) but not that induced by LPS (i.v.). BK increased intracellular calcium concentration in neurons and astrocytes. LPS increased levels of bradykinin, tissue kallikrein and total kininogen. BK (i.c.v.) increased rT and decreased tail skin temperature. Captopril potentiated BK-induced fever an effect abolished by icatibant. DALBK reduced the fever induced by BK. BK (i.c.v.) increased the CSF PGE 2 concentration. Effect abolished by indomethacin (i.p.). LPS activates endogenous kalikrein-kinin system leading to production of BK, which by acting on B 2 -receptors of POA cells causes prostaglandin synthesis that in turn produces fever. Thus, a kinin B 2 -receptor antagonist that enters into the brain could constitute a new and interesting strategy to treat fever. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Trophic Activity of Human P2X7 Receptor Isoforms A and B in Osteosarcoma

    PubMed Central

    Giuliani, Anna Lisa; Colognesi, Davide; Ricco, Tiziana; Roncato, Carlotta; Capece, Marina; Amoroso, Francesca; Wang, Qi Guang; De Marchi, Elena; Gartland, Allison; Di Virgilio, Francesco; Adinolfi, Elena

    2014-01-01

    The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tumours (81.4%), stained positive for both P2X7RA and B, thirty-one (57.4%) were positive using an anti-P2X7RA antibody, whereas fifteen of the total number (27.7%) expressed only P2X7RB. P2X7RB positive tumours showed increased cell density, at the expense of extracellular matrix. The human osteosarcoma cell line Te85, which lacks endogenous P2X7R expression, was stably transfected with either P2X7RA, P2X7RB, or both. Receptor expression was a powerful stimulus for cell growth, the most efficient growth-promoting isoform being P2X7RB alone. Growth stimulation was matched by increased Ca2+ mobilization and enhanced NFATc1 activity. Te85 P2X7RA+B cells presented pore formation as well as spontaneous extracellular ATP release. The ATP release was sustained in all clones by P2X7R agonist (BzATP) and reduced following P2X7R antagonist (A740003) application. BzATP also increased cell growth and activated NFATc1 levels. On the other hand cyclosporin A (CSA) affected both NFATc1 activation and cell growth, definitively linking P2X7R stimulation to NFATc1 and cell proliferation. All transfected clones also showed reduced RANK-L expression, and an overall decreased RANK-L/OPG ratio. Mineralization was increased in Te85 P2X7RA+B cells while it was significantly diminished in Te85 P2X7RB clones, in agreement with immunohistochemical results. In summary, our data show that the majority of human osteosarcomas express P2X7RA and B and suggest that expression of either isoform is differently

  18. Crosstalk between the equilibrative nucleoside transporter ENT2 and alveolar Adora2b adenosine receptors dampens acute lung injury

    PubMed Central

    Eckle, Tobias; Hughes, Kelly; Ehrentraut, Heidi; Brodsky, Kelley S.; Rosenberger, Peter; Choi, Doo-Sup; Ravid, Katya; Weng, Tingting; Xia, Yang; Blackburn, Michael R.; Eltzschig, Holger K.

    2013-01-01

    The signaling molecule adenosine has been implicated in attenuating acute lung injury (ALI). Adenosine signaling is terminated by its uptake through equilibrative nucleoside transporters (ENTs). We hypothesized that ENT-dependent adenosine uptake could be targeted to enhance adenosine-mediated lung protection. To address this hypothesis, we exposed mice to high-pressure mechanical ventilation to induce ALI. Initial studies demonstrated time-dependent repression of ENT1 and ENT2 transcript and protein levels during ALI. To examine the contention that ENT repression represents an endogenous adaptive response, we performed functional studies with the ENT inhibitor dipyridamole. Dipyridamole treatment (1 mg/kg; EC50=10 μM) was associated with significant increases in ALI survival time (277 vs. 395 min; P<0.05). Subsequent studies in gene-targeted mice for Ent1 or Ent2 revealed a selective phenotype in Ent2−/− mice, including attenuated pulmonary edema and improved gas exchange during ALI in conjunction with elevated adenosine levels in the bronchoalveolar fluid. Furthermore, studies in genetic models for adenosine receptors implicated the A2B adenosine receptor (Adora2b) in mediating ENT-dependent lung protection. Notably, dipyridamole-dependent attenuation of lung inflammation was abolished in mice with alveolar epithelial Adora2b gene deletion. Our newly identified crosstalk pathway between ENT2 and alveolar epithelial Adora2b in lung protection during ALI opens possibilities for combined therapies targeted to this protein set.—Eckle, T., Hughes, K., Ehrentraut, H., Brodsky, K. S., Rosenberger, P., Choi, D.-S., Ravid, K., Weng, T., Xia, Y., Blackburn, M. R., Eltzschig, H. K. Crosstalk between the equilibrative nucleoside transporter ENT2 and alveolar Adora2b adenosine receptors dampens acute lung injury. PMID:23603835

  19. MicroRNA-128b suppresses tumor growth and promotes apoptosis by targeting A2bR in gastric cancer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Ping; Guo, Xueyan; Zong, Wei

    2015-11-27

    MicroRNAs (miRNAs) play crucial roles in the development and progression of human cancers, including gastric cancer (GC). The discovery of miRNAs may provide a new and powerful tool for studying the mechanism, diagnosis, and treatment of GC. In this study, we aimed to investigate the role and mechanism of miR-128b in the development and progression of GC. Quantitative real-time PCR (qRT-PCR) was used to measure the expression level of miR-128b in GC tissues and cell lines. We found that miR-128b was significantly down-regulated in GC tissues and cell lines. In addition, over-expression of miR-128b inhibited GC cell proliferation, migration andmore » invasion of GC cells in vitro. Gain-of-function in vitro experiments further showed that the miR-128b mimic significantly promoted GC cell apoptosis. Subsequent dual-luciferase reporter assay identified one of the proto-oncogene A2bR as direct target of miR-128b. Therefore, our results indicate that miR-128b is a proto-oncogene miRNA that can suppresses GC proliferation and migration through down-regulation of the oncogene gene A2bR. Taken together, our results indicate that miR-128b could serve as a potential diagnostic biomarker and therapeutic option for human GC in the near future. - Highlights: • The expression of MiR-128b is significantly down-regulated in GC tissues and cell lines. • Ectopic expression of miR-128b directly affects cell proliferation, migration and invasion in vitro. • Overexpression of miR-128b increases apoptosis in GC cells. • A2bR is a candidate target gene of miR-128b. • MiR-128b represses cell proliferation, migration and invasion and promotes apoptosis by targeting A2bR in GC.« less

  20. [Immunoenzymatic assays of c-erbB-2 oncoprotein and epidermal growth factor receptor in breast cancer: correlation with clinical and biological parameters].

    PubMed

    Métayé, T; Bareille Saint-Gaudens, A; Millet, C; Ingrand, P; Daban, A; Bégon, F

    1996-01-01

    Two new immunoenzymatic assays for c-erbB-2 oncoprotein and epidermal growth factor receptor (EGF-R) (Oncogene Science) in human breast cancer were validated. Correlations between these assays and some clinical and biological parameters were also studied. The repeatability and reproducibility of standard curves for the two methods gave a coefficient of variation (CV) of less than 4% and about 10% respectively. The accuracy of c-erbB-2 oncoprotein and EGF-R assays was examined by using dilution and recovery tests throughout the standard curves. The linear relations between theoretical and measured values, for these tests, had slopes close to 1 and an intercept near 0. The median value for EGF-R, measured on solubilized membranes of 290 primary tumors, was 0.12 fmol/micrograms protein, the mean value was 0.37 (range 0 to 35.7). For c-erbB-2 oncoprotein, the median value, measured using the same population, was 2.75 human neu unit/micrograms protein, the mean value was 7.85 (range 1 to 125). There was an inverse relationship between EGF-R values and those for the estrogen receptor (ER), progesterone receptor and pS2 protein as well as menopausal status. C-erbB-2 oncoprotein concentrations were positively correlated with ER, pS2 protein and cathepsin D. Furthermore, a significant positive correlation was observed between EGF-R levels and c-erbB-2 oncoprotein levels. In conclusion, immunoenzymatic assays of EGF-R and c-erbB-2 oncoprotein are easy to use, sensitive and reliable. The accurate standardisation of immunoenzymatic assays could contribute to the clinical use of EGF-R and c-erbB-2 oncoprotein as prognostic factors in breast cancer.

  1. Molecular interactions between general anesthetics and the 5HT2B receptor.

    PubMed

    Matsunaga, Felipe; Gao, Lu; Huang, Xi-Ping; Saven, Jeffery G; Roth, Bryan L; Liu, Renyu

    2015-01-01

    Serotonin modulates many processes through a family of seven serotonin receptors. However, no studies have screened for interactions between general anesthetics currently in clinical use and serotonergic G-protein-coupled receptors (GPCRs). Given that both intravenous and inhalational anesthetics have been shown to target other classes of GPCRs, we hypothesized that general anesthetics might interact directly with some serotonin receptors and thus modify their function. Radioligand binding assays were performed to screen serotonin receptors for interactions with propofol and isoflurane as well as for affinity determinations. Docking calculations using the crystal structure of 5-HT2B were performed to computationally confirm the binding assay results and locate anesthetic binding sites. The 5-HT2B class of receptors interacted significantly with both propofol and isoflurane in the primary screen. The affinities for isoflurane and propofol were determined to be 7.78 and .95 μM, respectively, which were at or below the clinical concentrations for both anesthetics. The estimated free energy derived from docking calculations for propofol (-6.70 kcal/mol) and isoflurane (-5.10 kcal/mol) correlated with affinities from the binding assay. The anesthetics were predicted to dock at a pharmacologically relevant binding site of 5HT2B. The molecular interactions between propofol and isoflurane with the 5-HT2B class of receptors were discovered and characterized. This finding implicates the serotonergic GPCRs as potential anesthetic targets.

  2. Host Cell Invasion by TRYPANOSOMA cRUZI Is Potentiated by Activation of Bradykinin B2 Receptors

    PubMed Central

    Scharfstein, Julio; Schmitz, Veronica; Morandi, Veronica; Capella, Marcia M. A.; Lima, Ana Paula C. A.; Morrot, Alexandre; Juliano, Luiz; Müller-Esterl, Werner

    2000-01-01

    The parasitic protozoan Trypanosoma cruzi employs multiple molecular strategies to invade a broad range of nonphagocytic cells. Here we demonstrate that the invasion of human primary umbilical vein endothelial cells (HUVECs) or Chinese hamster ovary (CHO) cells overexpressing the B2 type of bradykinin receptor (CHO-B2R) by tissue culture trypomastigotes is subtly modulated by the combined activities of kininogens, kininogenases, and kinin-degrading peptidases. The presence of captopril, an inhibitor of bradykinin degradation by kininase II, drastically potentiated parasitic invasion of HUVECs and CHO-B2R, but not of mock-transfected CHO cells, whereas the B2R antagonist HOE 140 or monoclonal antibody MBK3 to bradykinin blocked these effects. Invasion competence correlated with the parasites' ability to liberate the short-lived kinins from cell-bound kininogen and to elicit vigorous intracellular free calcium ([Ca2+]i) transients through B2R. Invasion was impaired by membrane-permeable cysteine proteinase inhibitors such as Z-(SBz)Cys-Phe-CHN2 but not by the hydrophilic inhibitor 1-trans-epoxysuccinyl-l-leucyl-amido-(4-guanidino) butane or cystatin C, suggesting that kinin release is confined to secluded spaces formed by juxtaposition of host cell and parasite plasma membranes. Analysis of trypomastigote transfectants expressing various cysteine proteinase isoforms showed that invasion competence is linked to the kinin releasing activity of cruzipain, herein proposed as a factor of virulence in Chagas' disease. PMID:11067878

  3. NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin

    PubMed Central

    Alagarsamy, Sudar; Saugstad, Julie; Warren, Lee; Mansuy, Isabelle M.; Gereau, Robert W.; Conn, P. Jeffrey

    2010-01-01

    Previous reports have shown that activation of N-methyl-D-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor. 2005 Elsevier Ltd. All rights reserved. PMID:16005030

  4. Immunocytochemical localization of metabotropic (mGluR2/3 and mGluR4a) and ionotropic (GluR2/3) glutamate receptors in adrenal medullary ganglion cells.

    PubMed

    Sarría, R; Díez, J; Losada, J; Doñate-Oliver, F; Kuhn, R; Grandes, P

    2006-02-01

    The localization of metabotropic glutamate receptors of groups II (mGluR2/3) and III (mGluR4a) and the subunits 2 and 3 of alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) ionotropic glutamate receptors (GluR2/3) was investigated with immunocytochemical methods in the rat adrenal gland. MGluR2/3, mGluR4a and GluR2/3 immunoreactivities were observed in large-sized, centrally located type I adrenal medullary ganglion neurons. Furthermore, the small-sized type II adrenal ganglion neurons identified by their immunoreactivity to brain nitric oxide synthase (bNOS), also expressed mGluR2/3, mGluR4a and GluR2/3. These cells were disposed in the peripheral portion of the adrenal medulla. None of the type I neurons were positively labeled for bNOS. These morphological observations suggest that activation of glutamate receptors in ganglion neurons may be instrumental in the control of adrenal endocrine systems as well as blood regulation.

  5. Carboxypeptidase M Is a Positive Allosteric Modulator of the Kinin B1 Receptor*

    PubMed Central

    Zhang, Xianming; Tan, Fulong; Skidgel, Randal A.

    2013-01-01

    Ligand binding to extracellular domains of G protein-coupled receptors can result in novel and nuanced allosteric effects on receptor signaling. We previously showed that the protein-protein interaction of carboxypeptidase M (CPM) and kinin B1 receptor (B1R) enhances B1R signaling in two ways; 1) kinin binding to CPM causes a conformational activation of the B1R, and 2) CPM-generated des-Arg-kinin agonist is efficiently delivered to the B1R. Here, we show CPM is also a positive allosteric modulator of B1R signaling to its agonist, des-Arg10-kallidin (DAKD). In HEK cells stably transfected with B1R, co-expression of CPM enhanced DAKD-stimulated increases in intracellular Ca2+ or phosphoinositide turnover by a leftward shift of the dose-response curve without changing the maximum. CPM increased B1R affinity for DAKD by ∼5-fold but had no effect on basal B1R-dependent phosphoinositide turnover. Soluble, recombinant CPM bound to HEK cells expressing B1Rs without stimulating receptor signaling. CPM positive allosteric action was independent of enzyme activity but depended on interaction of its C-terminal domain with the B1R extracellular loop 2. Disruption of the CPM/B1R interaction or knockdown of CPM in cytokine-treated primary human endothelial cells inhibited the allosteric enhancement of CPM on B1R DAKD binding or ERK1/2 activation. CPM also enhanced the DAKD-induced B1R conformational change as detected by increased intramolecular fluorescence or bioluminescence resonance energy transfer. Thus, CPM binding to extracellular loop 2 of the B1R results in positive allosteric modulation of B1R signaling, and disruption of this interaction could provide a novel therapeutic approach to reduce pathological B1R signaling. PMID:24108126

  6. Mercury-Bridged Cobaltacarborane Complexes Containing B-Hg-B Three-Center Bonds. Synthesis and Structure of mu, mu’-((n5-C5R5)Co(CH3)2C2B3H4)Hg, mu-(n(5)-C5R5)Co(CH3)2C2B3H4)HgCl, (R=H, CH3) and Related Compounds.

    DTIC Science & Technology

    1980-11-01

    MERCURY-BRIDGED COBALTACARBORANE COMPLEXES CONTAINING B-HG-B TH--ETC(U) NOV 80 D C FINSTER . R N GRIMES N0 0 0 1 4-75-0305 UNCLASSXFIED TR󈧨 NL ILn...C5R5) Co 3)2C2B3 4 2 5 .- -C5R5 )Co(CH3)2C2B3H4 ]HgCl, (R=H, CH3 ) and Related Compounds, David C./ Finster -- Russell N./Grimes ( Department of Chemistry...Compounds 1 \\David C. Finster And Russell N. Grimes* Abstract. Reactions of the nid~p-cobaltacarborane anions 01CR )(C 3 )C BH and [n (H 1oC ihH~5n 5

  7. The structure, stability, and infrared spectrum of B 2N, B 2N +, B 2N -, BO, B 2O and B 2N 2.

    NASA Astrophysics Data System (ADS)

    Martin, J. M. L.; François, J. P.; Gijbels, R.

    1992-05-01

    The structure, infrared spectrum, and heat of formation of B 2N, B 2N -, BO, and B 2O have been studied ab initio. B 2N is very stable; B 2O even more so. B 2N, B 2N -, B 2O, and probably B 2N + have symmetric linear ground-state structures; for B 2O, an asymmetric linear structure lies about 12 kcal/mol above the ground state. B 2N +, B 2N - and B 2O have intense asymmetric stretching frequencies, predicted near 870, 1590 and 1400 cm -1, respectively. Our predicted harmonic frequencies and isotopic shifts for B 2O confirm the recent experimental identification by Andrews and Burkholder. Absorptions at 1889.5 and 1998.5 cm -1 in noble-gas trapped boron nitride vapor belong the BNB and BNBN ( 3Π), respectively; a tentative assignment of 882.5 cm -1 to BNB + is proposed. Total atomization energies Σ De (Σ D0) are computed (accuracy ±2 kcal/mol) as: BO 193.1 (190.4), B 2O 292.5 (288.7), B 2N 225.0 (250.3) kcal/mol. The ionization potential and electron affinity of B 2N are predicted to be 8.62±0.1 and 3.34±0.1 eV. The MP4-level additivity approximations involved in G1 theory results in errors on the order of 1 kcal/mol in the Σ De values.

  8. Comparative analysis of B7-1 and B7-2 costimulatory ligands: expression and function

    PubMed Central

    1994-01-01

    Antigen-specific T cell activation requires the engagement of the T cell receptor (TCR) with antigen as well as the engagement of appropriate costimulatory molecules. The most extensively characterized pathway of costimulation has been that involving the interaction of CD28 and CTLA4 on the T cell with B7 (now termed B7-1) on antigen presenting cells. Recently, B7-2 a second costimulatory ligand for CTLA4, was described, demonstrating the potential complexity of costimulatory interactions. This report examines and compares the expression and function of B7-1 and B7-2. Overall these results indicate that (a) B7-1 and B7-2 can be expressed by multiple cell types, including B cells, T cells, macrophages, and dendritic cells, all of which are therefore candidate populations for delivering costimulatory signals mediated by these molecules; (b) stimulating B cells with either LPS or anti-IgD-dextran induced expression of both B7- 1 and B7-2, and peak expression of both costimulatory molecules occurred after 18-42 h of culture. Expression of B7-2 on these B cell populations was significantly higher than expression of B7-1 at all times assayed after stimulation; (c) blocking of B7-2 costimulatory activity inhibited TCR-dependent T cell proliferation and cytokine production, without affecting early consequences of TCR signaling such as induction of CD69 or interleukin 2 receptor alpha (IL-2R alpha); and (d) expression of B7-1 and of B7-2 can be regulated by a variety of stimuli. Moreover, expression of B7-1 and B7-2 can be independently regulated by the same stimulus, providing an additional complexity in the mechanisms available for regulating costimulation and hence immune response. PMID:7519245

  9. Tumor Suppressor Activity of the EphB2 Receptor in Prostate Cancer

    DTIC Science & Technology

    2006-11-01

    should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of...information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE (DD-MM...identified in the EphB2 receptor gene in prostate cancer suggesting that EphB2, a member of the large Eph receptor tyrosine kinase family, is a tumor

  10. Bradykinin B1 and B2 receptors, tumour necrosis factor α and inflammatory hyperalgesia

    PubMed Central

    Poole, S; Lorenzetti, B B; Cunha, J M; Cunha, F Q; Ferreira, S H

    1999-01-01

    The effects of BK agonists and antagonists, and other hyperalgesic/antihyperalgesic drugs were measured (3 h after injection of hyperalgesic drugs) in a model of mechanical hyperalgesia (the end-point of which was indicated by a brief apnoea, the retraction of the head and forepaws, and muscular tremor). DALBK inhibited responses to carrageenin, bradykinin, DABK, and kallidin. Responses to kallidin and DABK were inhibited by indomethacin or atenolol and abolished by the combination of indomethacin+atenolol. DALBK or HOE 140, given 30 min before, but not 2 h after, carrageenin, BK, DABK and kallidin reduced hyperalgesic responses to these agents. A small dose of DABK+a small dose of BK evoked a response similar to the response to a much larger dose of DABK or BK, given alone. Responses to BK were antagonized by HOE 140 whereas DALBK antagonized only responses to larger doses of BK. The combination of a small dose of DALBK with a small dose of HOE 140 abolished the response to BK. The hyperalgesic response to LPS (1 μg) was inhibited by DALBK or HOE 140 and abolished by DALBK+HOE 140. The hyperalgesic response to LPS (5 μg) was not antagonized by DALBK+HOE 140. These data suggest: (a) a predominant role for B2 receptors in mediating hyperalgesic responses to BK and to drugs that stimulate BK release, and (b) activation of the hyperalgesic cytokine cascade independently of both B1 and B2 receptors if the hyperalgesic stimulus is of sufficient magnitude. PMID:10188975

  11. Design and synthesis of novel sulfonamide-containing bradykinin hB2 receptor antagonists. 2. Synthesis and structure-activity relationships of alpha,alpha-cycloalkylglycine sulfonamides.

    PubMed

    Fattori, Daniela; Rossi, Cristina; Fincham, Christopher I; Caciagli, Valerio; Catrambone, Fernando; D'Andrea, Piero; Felicetti, Patrizia; Gensini, Martina; Marastoni, Elena; Nannicini, Rossano; Paris, Marielle; Terracciano, Rosa; Bressan, Alessandro; Giuliani, Sandro; Maggi, Carlo A; Meini, Stefania; Valenti, Claudio; Quartara, Laura

    2007-02-08

    Recently we reported on the design and synthesis of a novel class of selective nonpeptide bradykinin (BK) B2 receptor antagonists (J. Med. Chem. 2006, 3602-3613). This work led to the discovery of MEN 15442, an antagonist with subnanomolar affinity for the human B2 receptor (hB2R), which also displayed significant and prolonged activity in vivo (for up to 210 min) against BK-induced bronchoconstriction in the guinea-pig at a dose of 300 nmol/kg (it), while demonstrating only a slight effect on BK-induced hypotension. Here we describe the further optimization of this series of compounds aimed at maximizing the effect on bronchoconstriction and minimizing the effect on hypotension, with a view to developing topically delivered drugs for airway diseases. The work led to the discovery of MEN 16132, a compound which, after intratracheal or aerosol administration, inhibited, in a dose-dependent manner, BK-induced bronchoconstricton in the airways, while showing minimal systemic activity. This compound was selected as a preclinical candidate for the topical treatment of airway diseases involving kinin B2 receptor stimulation.

  12. Cooperation of neurotrophin receptor TrkB and Her2 in breast cancer cells facilitates brain metastases.

    PubMed

    Choy, Cecilia; Ansari, Khairul I; Neman, Josh; Hsu, Sarah; Duenas, Matthew J; Li, Hubert; Vaidehi, Nagarajan; Jandial, Rahul

    2017-04-26

    Patients with primary breast cancer that is positive for human epidermal growth factor receptor 2 (Her2+) have a high risk of developing metastases in the brain. Despite gains with systemic control of Her2+ disease using molecular therapies, brain metastases remain recalcitrant to therapeutic discovery. The clinical predilection of Her2+ breast cancer cells to colonize the brain likely relies on paracrine mechanisms. The neural niche poses unique selection pressures, and neoplastic cells that utilize the brain microenvironment may have a survival advantage. Tropomyosin-related kinase B (TrkB), Her2, and downstream targets were analyzed in primary breast cancer, breast-to-brain metastasis (BBM) tissues, and tumor-derived cell lines using quantitative real-time PCR, western blot, and immunohistochemical assessment. TrkB function on BBM was confirmed with intracranial, intracardiac, or mammary fat pad xenografts in non-obese diabetic/severe combined immunodeficiency mice. The function of brain-derived neurotrophic factor (BDNF) on cell proliferation and TrkB/Her2 signaling and interactions were confirmed using selective shRNA knockdown and selective inhibitors. The physical interaction of Her2-TrkB was analyzed using electron microscopy, co-immunoprecipitation, and in silico analysis. Dual targeting of Her2 and TrkB was analyzed using clinically utilized treatments. We observed that patient tissues and cell lines derived from Her2+ human BBM displayed increased activation of TrkB, a neurotrophin receptor. BDNF, an extracellular neurotrophin, with roles in neuronal maturation and homeostasis, specifically binds to TrkB. TrkB knockdown in breast cancer cells led to decreased frequency and growth of brain metastasis in animal models, suggesting that circulating breast cancer cells entering the brain may take advantage of paracrine BDNF-TrkB signaling for colonization. In addition, we investigated a possible interaction between TrkB and Her2 receptors on brain metastatic

  13. Adenosine enhances sweet taste through A2B receptors in the taste bud

    PubMed Central

    Dando, Robin; Dvoryanchikov, Gennady; Pereira, Elizabeth; Chaudhari, Nirupa; Roper, Stephen D.

    2012-01-01

    Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (Type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. This ATP excites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Here we show that extracellular ATP is enzymatically degraded to adenosine within mouse vallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca2+ mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 µM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (Type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 µM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell RT-PCR on isolated vallate taste cells, we show that many Receptor cells express adenosine receptors, Adora2b, while Presynaptic (Type III) and Glial-like (Type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5′-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase (ACPP). Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet taste. PMID:22219293

  14. Adenosine enhances sweet taste through A2B receptors in the taste bud.

    PubMed

    Dando, Robin; Dvoryanchikov, Gennady; Pereira, Elizabeth; Chaudhari, Nirupa; Roper, Stephen D

    2012-01-04

    Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. This ATP excites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Here we show that extracellular ATP is enzymatically degraded to adenosine within mouse vallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca(2+) mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 μM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 μM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell reverse transcriptase (RT)-PCR on isolated vallate taste cells, we show that many Receptor cells express the adenosine receptor, Adora2b, while Presynaptic (type III) and Glial-like (type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5'-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase. Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry, and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet taste.

  15. Active G protein-coupled receptors (GPCR), matrix metalloproteinases 2/9 (MMP2/9), heparin-binding epidermal growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) are necessary for trenbolone acetate-induced alterations in protein turnover rate of fused bovine satellite cell cultures.

    PubMed

    Thornton, K J; Kamanga-Sollo, E; White, M E; Dayton, W R

    2016-06-01

    Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( < 0.05) protein synthesis rates and decreased ( < 0.05) protein degradation rates when compared to control cultures. Treatment of fused BSC cultures with 10 n TBA in the presence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( < 0.05) TBA-mediated increases in protein synthesis rate. Alternatively, inhibition of GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R in the presence of 10 n TBA each had no ( > 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( < 0.05) ability of TBA to decrease protein degradation rate. Additionally, fused BSC cultures treated with 10 n

  16. Submicron R{sub 2}Fe{sub 14}B particles

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Koylu-Alkan, O., E-mail: ozlem@udel.edu, E-mail: hadji@udel.edu; Hadjipanayis, G. C., E-mail: ozlem@udel.edu, E-mail: hadji@udel.edu; Barandiaran, J. M.

    Mechanochemical synthesis of submicron R{sub 2}Fe{sub 14}B particles with R = Dy, Nd, Pr has been performed successfully via high energy ball milling of rare-earth oxides, iron oxide and boron oxide in the presence of a reducing agent (Ca) and a dispersant material (CaO), followed by annealing at 800 - 900 °C. In the R = Nd system, we were able to fabricate particles embedded in a CaO matrix with coercivity (H{sub c}) of 10.3 kOe after annealing at 900 °C for 5 min. After washing off the dispersant, the H{sub c} was decreased to below 1 kOe because of hydrogen absorptionmore » that leads to the formation of the hydrated R{sub 2}Fe{sub 14}BH{sub x} phase that has a lower anisotropy. Upon removal of the hydrogen the coercivity was increased to 3.3 kOe. The average size of the Nd{sub 2}Fe{sub 14}B particles increases from 100 nm in a sample synthesized at 800 °C to 158 nm at 900 °C. The isotropic Dy{sub 2}Fe{sub 14}B particles showed a higher coercivity of 21 kOe in washed samples after annealing at 900 °C for 5 min. An average size of 71 nm is measured in samples synthesized at 800 °C and 107 nm at 900 °C. Fitting the high field M(H) measurements in Nd{sub 2}Fe{sub 14}B to the law of approach to saturation gave values for the magnetocrystalline anisotropy for the washed sample 2.23 × 10{sup 7} erg/cm{sup 3} and for the vacuum annealed sample 4.15 × 10{sup 7} erg/cm{sup 3}, both of which are lower than the bulk values. This would explain the lower values of H{sub c} observed in the particles.« less

  17. Functional and molecular characterization of kinin B1 and B 2 receptors in human bladder cancer: implication of the PI3Kγ pathway.

    PubMed

    Sgnaolin, V; Pereira, T C B; Bogo, M R; Zanin, R; Battastini, A M O; Morrone, F B; Campos, M M

    2013-08-01

    Kinins and their receptors have been recently implicated in cancer. Using functional and molecular approaches, we investigated the relevance of kinin B1 and B2 receptors in bladder cancer. Functional studies were conducted using bladder cancer cell lines, and human biopsies were employed for molecular studies. Both B1 des-Arg(9)-BK and B2 BK receptor agonists stimulated the proliferation of grade 3-derived T24 bladder cancer cells. Furthermore, treatment with B1 and B2 receptor antagonists (SSR240612 and HOE140) markedly inhibited the proliferation of T24 cells. Only higher concentrations of BK increased the proliferation of the grade 1 bladder cancer cell line RT4, while des-Arg(9)-BK completely failed to induce its proliferation. Real-time PCR revealed that the mRNA expression of kinin receptors, particularly B1 receptors, was increased in T24 cells relative to RT4 cells. Data from bladder cancer human biopsies revealed that B1 receptor expression was increased in all tumor samples and under conditions of chronic inflammation. We also show novel evidence demonstrating that the pharmacological inhibition of PI3Kγ (phosphatidylinositol 3-kinase) with AS252424, concentration-dependently reduced T24 cell proliferation induced by BK or des-Arg(9)-BK. Finally, the incubation of T24 cells with kinin agonists led to a marked activation of the PI3K/AKT and ERK 1/2 signaling pathways, whereas p38 MAP kinase remained unaffected. Kinin receptors, especially B1 receptors, appear to be implicated in bladder cancer progression. It is tempting to suggest that selective kinin antagonists might represent potential alternative therapies for bladder cancer.

  18. Dysregulated expression of miR-101b and miR-26b lead to age-associated increase in LPS-induced COX-2 expression in murine macrophage.

    PubMed

    Liu, Dan; Wang, Dongsheng; Xu, Zhenbiao; Gao, Jing; Liu, Min; Liu, Yanxin; Jiang, Minghong; Zheng, Dexian

    2015-10-01

    Aging is the natural process of decline in physiological structure and function of various molecules, cells, tissues, and organs. Growing evidence indicates that increased immune genetic diversity and dysfunction of immune system cause aging-related pathophysiological process with the growth of age. In the present study, we observed that LPS-induced higher activation of cyclooxygenase (COX)-2 promoter is associated with the upregulated binding activity of nuclear factor kappa B (NF-κB) in peritoneal macrophages of aged mice than young ones. Additionally, COX-2 is a direct target of miR-101b and miR-26b in the macrophages. Significant upregulation of miR-101b and miR-26b effectively prevented LPS-induced excessive expression of COX-2 in the young mice. Because these negative regulatory factors were unresponsive to LPS stimulation, the levels of COX-2 were markedly higher in the macrophages of aged mice. Further study showed that NF-κB activation contributed to the increase in the expression of miR-101b and miR-26b in the LPS-stimulated macrophages of young mice, but not aged ones. Moreover, histone deacetylase (HDAC) inhibitor trichostatin A (TSA) upregulated expression of miR-101b and miR-26b in the aged mouse macrophages only, but not the young cells. This demonstrated that HDAC suppressed the expression of miR-101b and miR-26b in the LPS-treated macrophages of aged mice and contributed to the aging process. TSA-induced increased expression of miR-101b and miR-26b could further suppress COX-2 expression. These findings provide novel evidence on the regulation of immune senescence and miR-101b and miR-26b, which might be promising targets in treating aged-related inflammatory diseases. Epigenetic regulation of the microRNAs (miRNAs) provides an important evidence for the treatment of innate inflammatory disease with HDAC inhibitors in elderly.

  19. Molecular recognition of human ephrinB2 cell surface receptor by an emergent African henipavirus

    PubMed Central

    Lee, Benhur; Pernet, Olivier; Ahmed, Asim A.; Zeltina, Antra; Beaty, Shannon M.; Bowden, Thomas A.

    2015-01-01

    The discovery of African henipaviruses (HNVs) related to pathogenic Hendra virus (HeV) and Nipah virus (NiV) from Southeast Asia and Australia presents an open-ended health risk. Cell receptor use by emerging African HNVs at the stage of host-cell entry is a key parameter when considering the potential for spillover and infection of human populations. The attachment glycoprotein from a Ghanaian bat isolate (GhV-G) exhibits <30% sequence identity with Asiatic NiV-G/HeV-G. Here, through functional and structural analysis of GhV-G, we show how this African HNV targets the same human cell-surface receptor (ephrinB2) as the Asiatic HNVs. We first characterized this virus−receptor interaction crystallographically. Compared with extant HNV-G–ephrinB2 structures, there was significant structural variation in the six-bladed β-propeller scaffold of the GhV-G receptor-binding domain, but not the Greek key fold of the bound ephrinB2. Analysis revealed a surprisingly conserved mode of ephrinB2 interaction that reflects an ongoing evolutionary constraint among geographically distal and phylogenetically divergent HNVs to maintain the functionality of ephrinB2 recognition during virus–host entry. Interestingly, unlike NiV-G/HeV-G, we could not detect binding of GhV-G to ephrinB3. Comparative structure–function analysis further revealed several distinguishing features of HNV-G function: a secondary ephrinB2 interaction site that contributes to more efficient ephrinB2-mediated entry in NiV-G relative to GhV-G and cognate residues at the very C terminus of GhV-G (absent in Asiatic HNV-Gs) that are vital for efficient receptor-induced fusion, but not receptor binding per se. These data provide molecular-level details for evaluating the likelihood of African HNVs to spill over into human populations. PMID:25825759

  20. Photochemical activation of MH3-B1/rGel: a HER2-targeted treatment approach for ovarian cancer

    PubMed Central

    Bull-Hansen, Bente; Berstad, Maria B.; Berg, Kristian; Cao, Yu; Skarpen, Ellen; Fremstedal, Ane Sofie; Rosenblum, Michael G.; Peng, Qian; Weyergang, Anette

    2015-01-01

    HER2-targeted therapy has been shown to have limited efficacy in ovarian cancer despite frequent overexpression of this receptor. Photochemical internalization (PCI) is a modality for cytosolic drug delivery, currently undergoing clinical evaluation. In the present project we studied the application of PCI in combination with the HER2-targeted recombinant fusion toxin, MH3-B1/rGel, for the treatment of ovarian cancer. The SKOV-3 cell line, resistant to trastuzumab- and MH3-B1/rGel- monotherapy, was shown to respond strongly to PCI of MH3-B1/rGel to a similar extent as observed for the treatment-sensitive SK-BR-3 breast cancer cells. Extensive hydrolytic degradation of MH3-B1/rGel in acidic endocytic vesicles was indicated as the mechanism of MH3-B1/rGel resistance in SKOV-3 cells. This was shown by the positive Pearson's correlation coefficient between Alexa488-labeled MH3-B1/rGel and Lysotracker in SKOV-3 cells in contrast to the negative Pearson's correlation coefficient in SK-BR-3 cells. The application of PCI to induce the release of MH3-B1/rGel was also demonstrated to be effective on SKOV-3 xenografts. Application of PCI with MH3-B1/rGel was further found highly effective in the HER2 expressing HOC-7 and NuTu-19 ovarian cancer cell lines. The presented results warrant future development of PCI in combination with MH3-B1/rGel as a novel therapeutic approach in preclinical models of ovarian cancer. PMID:26002552

  1. High-Resolution Synchrotron Infrared Spectroscopy of Thiophosgene: the νb{1}, νb{5}, 2νb{4}, and νb{2} + 2νb{6} bands

    NASA Astrophysics Data System (ADS)

    McKellar, Bob; Billinghurst, Brant E.

    2015-06-01

    Thiophosgene (Cl_2CS) is a favorite model system for studies of photophysics, vibrational dynamics, and intersystem interactions. But at high resolution its infrared spectrum is very congested due to hot bands and multiple isotopic species. Previously, we reported the first high resolution IR study of this molecule, analyzing the νb{2} (504 wn) and νb{4} (471 wn) fundamental bands. Here we continue, with analysis of the νb{1} (1139 wn) and νb{5} (820 wn) fundamentals for the two most abundant isotopologues, 35Cl2CS and 35Cl37ClCS, based on spectra with a resolution of about 0.001 wn obtained at the Canadian Light Source far-infrared beamline using synchrotron radiation and a Bruker IFS125 Fourier transform spectrometer. The νb{2} + νb{4} (942 wn) and νb{2} + 2νb{6} (1104 wn) bands are also studied here. But so far the νb{2} + νb{6} combination band (795 wn) resists analysis, as do the weak νb{3} (292.9 wn) and νb{6} (≈300? wn) fundamentals. A.R.W. McKellar, B.E.Billinghurst, J. Mol. Spectrosc. 260, 66 (2010).

  2. The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma.

    PubMed

    Gurd, J W; Bissoon, N

    1997-08-01

    The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-gamma was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-gamma and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.

  3. Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism

    PubMed Central

    Ding, Yan; Liu, Zixing; Desai, Shruti; Zhao, Yuhua; Liu, Hao; Pannell, Lewis K; Yi, Hong; Wright, Elizabeth R; Owen, Laurie B; Dean-Colomb, Windy; Fodstad, Oystein; Lu, Jianrong; LeDoux, Susan P; Wilson, Glenn L; Tan, Ming

    2012-01-01

    It is well known that ErbB2, a receptor tyrosine kinase, localizes on the plasma membrane. Here we describe a novel observation that ErbB2 also localizes in mitochondria of cancer cells and patient samples. We found that ErbB2 translocates into mitochondria through the association with mtHSP70. Additionally, mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complexes of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and the cellular ATP level also were decreased. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Interestingly, cancer cells with higher levels of mtErbB2 were more resistant to ErbB2 targeting antibody trastuzumab. Our study provides a novel perspective on the metabolic regulatory function of ErbB2 and reveals that mtErbB2 plays an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics. PMID:23232401

  4. HSD3B2, HSD17B1, HSD17B2, ESR1, ESR2 and AR expression in infertile women with endometriosis.

    PubMed

    Osiński, Maciej; Wirstlein, Przemysław; Wender-Ożegowska, Ewa; Mikołajczyk, Mateusz; Jagodziński, Paweł Piotr; Szczepańska, Małgorzata

    2018-01-01

    The development of endometriosis is associated with changes in the expression of genes encoding the 3β-hydroxysteroid dehydrogenase type II (HSD3B2) and 17β-hydroxysteroid dehydrogenase type II (HSD17B2), estrogen receptors 1 (ESR1) and 2 (ESR2) and the androgen receptor (AR). However, little is known about the expression of HSD3B2, HSD17B1, HSD17B2, ESR1 ESR2 and AR during the endometrial phases in eutopic endometrium from infertile women with endometriosis. Using RT-qPCR analysis, we assessed the expression of the studied genes in the follicular and luteal phases in eutopic endometrium from fertile women (n = 17) and infertile women (n = 35) with endometriosis. In the mid-follicular eutopic endometrium, we observed a significant increase in HSD3B2 transcript levels in all infertile women with endometriosis (p = 0.003), in infertile women with stage I/II endometriosis (p = 0.008) and in infertile women with stage III/IV endometriosis (p = 0.009) compared to all fertile women. There was a significant increase in ESR1 tran-scripts in all infertile women with endometriosis (p = 0.008) and in infertile women with stage I/II endometriosis (p = 0.019) and in infertile women with stage III/IV endometriosis (p = 0.023) compared to all fertile women. In the mid-luteal eutopic endometrium, we did not observe significant differences in HSD3B2, HSD17B1, HSD17B2, ESR1, ESR2 and AR transcripts between infertile women with endometriosis and fertile women. Observed significant increase in HSD3B2 and ESR1 transcripts in follicular eutopic endometrium from infer-tile women with endometriosis may be related to abnormal biological effect of E2 in endometrium, further affecting the development of human embryos.

  5. Lung Injury Pathways: Adenosine Receptor 2B Signaling Limits Development of Ischemic Bronchiolitis Obliterans Organizing Pneumonia

    PubMed Central

    Densmore, John C.; Schaid, Terry R.; Jeziorczak, Paul M.; Medhora, Meetha; Audi, Said; Nayak, Shraddha; Auchampach, John; Dwinell, Melinda R.; Geurts, Aron M.; Jacobs, Elizabeth R.

    2018-01-01

    Purpose/Aim of the study Adenosine signaling was studied in bronchiolitis obliterans organizing pneumonia (BOOP) resulting from unilateral lung ischemia. Materials and Methods Ischemia was achieved by either left main pulmonary artery or complete hilar ligation. Sprague Dawley (SD) rats, Dahl salt sensitive (SS) rats and SS mutant rat strain containing a mutation in the A2B adenosine receptor gene (Adora2b) were studied. Adenosine concentrations were measured in bronchoalveolar lavage (BAL) by HPLC. A2A (A2AAR) and A2B adenosine receptor (A2BAR) mRNA and protein were quantified. Results 24h after unilateral PA ligation, BAL adenosine concentrations from ischemic lungs were increased relative to contralateral lungs in SD rats. A2BAR mRNA and protein concentrations were increased after PA ligation while miR27a, a negatively regulating microRNA, was decreased in ischemic lungs. A2AAR mRNA and protein concentrations remained unchanged following ischemia. A2BAR protein was increased in PA ligated lungs of SS rats after 7d, and 4h after complete hilar ligation in SD rats. SS-Adora2b mutants showed a greater extent of BOOP relative to SS rats, and greater inflammatory changes. Conclusions Increased A2BAR and adenosine following unilateral lung ischemia as well as more BOOP in A2BAR mutant rats implicate a protective role for A2BAR signaling in countering ischemic lung injury. PMID:28266889

  6. Site-specific magnetic anisotropies in R2Fe14B systems

    NASA Astrophysics Data System (ADS)

    Yoshioka, T.; Tsuchiura, H.

    2018-04-01

    The local magnetic anisotropy of R ions in R2Fe14B (R = Dy, Ho) systems is studied based on a microscopic effective spin model constructed from the information obtained by using first-principles calculations. By taking into account up to 6-th order crystal electric field parameters, the model satisfactory describes the observed magnetization curves and the temperature dependence of anisotropy constants. We found that at low temperatures, the noncollinear structure appears in the Ho2Fe14B system reflecting the local magnetic anisotropy.

  7. Src family kinases mediate the inhibition of substance P release in the rat spinal cord by μ-opioid receptors and GABA(B) receptors, but not α2 adrenergic receptors.

    PubMed

    Zhang, Guohua; Chen, Wenling; Marvizón, Juan Carlos G

    2010-09-01

    GABA(B) , μ-opioid and adrenergic α(2) receptors inhibit substance P release from primary afferent terminals in the dorsal horn. Studies in cell expression systems suggest that μ-opioid and GABA(B) receptors inhibit transmitter release from primary afferents by activating Src family kinases (SFKs), which then phosphorylate and inhibit voltage-gated calcium channels. This study investigated whether SFKs mediate the inhibition of substance P release by these three receptors. Substance P release was measured as neurokinin 1 receptor (NK1R) internalization in spinal cord slices and in vivo. In slices, NK1R internalization induced by high-frequency dorsal root stimulation was inhibited by the μ-opioid agonist DAMGO and the GABA(B) agonist baclofen. This inhibition was reversed by the SFK inhibitor PP1. NK1R internalization induced by low-frequency stimulation was also inhibited by DAMGO, but PP1 did not reverse this effect. In vivo, NK1R internalization induced by noxious mechanical stimulation of the hind paw was inhibited by intrathecal DAMGO and baclofen. This inhibition was reversed by intrathecal PP1, but not by the inactive PP1 analog PP3. PP1 produced no effect by itself. The α(2) adrenergic agonists medetomidine and guanfacine produced a small but statistically significant inhibition of NK1R internalization induced by low-frequency dorsal root stimulation. PP1 did not reverse the inhibition by guanfacine. These results show that SFKs mediate the inhibition of substance P release by μ-opioid and GABA(B) receptors, but not by α(2) receptors, which is probably mediated by the binding of G protein βγ subunits to calcium channels. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd. No claim to original US government works.

  8. Evaluation of 11C-Me-NB1 as a Potential PET Radioligand for Measuring GluN2B-Containing NMDA Receptors, Drug Occupancy, and Receptor Cross Talk.

    PubMed

    Krämer, Stefanie D; Betzel, Thomas; Mu, Linjing; Haider, Ahmed; Herde, Adrienne Müller; Boninsegni, Anna K; Keller, Claudia; Szermerski, Marina; Schibli, Roger; Wünsch, Bernhard; Ametamey, Simon M

    2018-04-01

    Clinical and preclinical research with modulators at the N -methyl-d-aspartate (NMDA) receptor GluN2B N-terminal domain (NTD) aims for the treatment of various neurologic diseases. The interpretation of the results is hampered by the lack of a suitable NMDA PET tracer for assessing the receptor occupancy of potential drugs. We have developed 11 C-Me-NB1 as a PET tracer for imaging GluN1/GluN2B-containing NMDA receptors and used it to investigate in rats the dose-dependent receptor occupancy of eliprodil, a GluN2B NTD modulator. Methods: 11 C-Me-NB1 was synthesized and characterized by in vitro displacement binding experiments with rat brain membranes, in vitro autoradiography, and blocking and displacement experiments by PET and PET kinetic modeling. Receptor occupancy by eliprodil was studied by PET with 11 C-Me-NB1. Results: 11 C-Me-NB1 was synthesized at 290 ± 90 GBq/μmol molar activity, 7.4 ± 1.9 GBq total activity at the end of synthesis ( n = 17), and more than 99% radiochemical purity. 11 C-Me-NB1 binding in rat brain was blocked in vitro and in vivo by the NTD modulators Ro-25-6981 and eliprodil. Half-maximal receptor occupancy by eliprodil occurred at 1.5 μg/kg. At 1 mg/kg of eliprodil, a dose with reported neuroprotective effects, more than 99.5% of binding sites were occupied. In vitro, 11 C-Me-NB1 binding was independent of the σ-1 receptor (Sigma1R), and the Sigma1R agonist (+)-pentazocine did not compete for high-affinity binding. In vivo, a 2.5 mg/kg dose of (+)-pentazocine abolished 11 C-Me-NB1-specific binding, indicating an indirect effect of Sigma1R on 11 C-Me-NB1 binding. Conclusion: 11 C-Me-NB1 is suitable for the in vivo imaging of NMDA GluN1/GluN2B receptors and the assessment of receptor occupancy by NTD modulators. GluN1/GluN2B NMDA receptors are fully occupied at neuroprotective doses of eliprodil. Furthermore, 11 C-Me-NB1 enables imaging of GluN1/GluN2B NMDA receptor cross talk. © 2018 by the Society of Nuclear Medicine and

  9. Targeted Segment Transfer from Rye Chromosome 2R to Wheat Chromosomes 2A, 2B, and 7B.

    PubMed

    Ren, Tianheng; Li, Zhi; Yan, Benju; Tan, Feiquan; Tang, Zongxiang; Fu, Shulan; Yang, Manyu; Ren, Zhenglong

    2017-01-01

    Increased chromosome instability was induced by a rye (Secale cereale L.) monosomic 2R chromosome into wheat (Triticum aestivum L.). Centromere breakage and telomere dysfunction result in high rates of chromosome aberrations, including breakages, fissions, fusions, deletions, and translocations. Plants with target traits were sequentially selected to produce a breeding population, from which 3 translocation lines with target traits have been selected. In these lines, wheat chromosomes 2A, 2B, and 7B recombined with segments of the rye chromosome arm 2RL. This was detected by FISH analysis using repeat sequences pSc119.2, pAs1 and genomic DNA of rye together as probes. The translocation chromosomes in these lines were named as 2ASMR, 2BSMR, and 7BSMR. The small segments that were transferred into wheat consisted of pSc119.2 repeats and other chromatin regions that conferred resistance to stripe rust and expressed target traits. These translocation lines were highly resistant to stripe rust, and expressed several typical traits that were associated with chromosome arm 2RL, which are better than those of its wheat parent, disomic addition, and substitution lines that show agronomic characteristics. The integration of molecular methods and conventional techniques to improve wheat breeding schemes are discussed. © 2017 S. Karger AG, Basel.

  10. Human umbilical vein: involvement of cyclooxygenase-2 pathway in bradykinin B1 receptor-sensitized responses.

    PubMed

    Errasti, A E; Rey-Ares, V; Daray, F M; Rogines-Velo, M P; Sardi, S P; Paz, C; Podestá, E J; Rothlin, R P

    2001-08-01

    In isolated human umbilical vein (HUV), the contractile response to des-Arg9-bradykinin (des-Arg9-BK), selective BK B1 receptor agonist, increases as a function of the incubation time. Here, we evaluated whether cyclooxygenase (COX) pathway is involved in BK B1-sensitized response obtained in 5-h incubated HUV rings. The effect of different concentrations of indomethacin, sodium salicylate, ibuprofen, meloxicam, lysine clonixinate or NS-398 administrated 30 min before concentration-response curves (CRC) was studied. All treatments produced a significant rightward shift of the CRC to des-Arg9-BK in a concentration-dependent manner, which provides pharmacological evidence that COX pathway is involved in the BK B1 responses. Moreover, in this tissue, the NS-398 pKb (5.2) observed suggests that COX-2 pathway is the most relevant. The strong correlation between published pIC50 for COX-2 and the NSAIDs' pKbs estimated further supports the hypothesis that COX-2 metabolites are involved in BK B1 receptor-mediated responses. In other rings, indomethacin (30, 100 micromol/l) or NS-398 (10, 30 micromol/l) produced a significant rightward shift of the CRC to BK, selective BK B2 agonist, and its pKbs were similar to the values to inhibit BK B1 receptor responses, suggesting that COX-2 pathway also is involved in BK B2 receptor responses. Western blot analysis shows that COX-1 and COX-2 isoenzymes are present before and after 5-h in vitro incubation and apparently COX-2 does not suffer additional induction.

  11. Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia

    NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with boundmore » ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.« less

  12. Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

    PubMed Central

    Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia; Pomper, Martin G.; De Micheli, Carlo; Conti, Paola; Pinto, Andrea

    2017-01-01

    NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity. PMID:28760974

  13. MiR-344b-1-3p targets TLR2 and negatively regulates TLR2 signaling pathway

    PubMed Central

    Xu, Hong; Wu, Yuting; Li, Li; Yuan, Weifeng; Zhang, Deming; Yan, Qitao; Guo, Zhenhui; Huang, Wenjie

    2017-01-01

    Objectives COPD is an abnormal inflammatory response characterized by decreased expression of TLR2 in patients, which is suggested to induce invasive pulmonary aspergillosis (IPA). MicroRNAs (miRNAs) have been shown to play important roles in the pathogenesis of human respiratory system disorders. Therefore, the aim of this study was to identify the miRNAs involved in the regulation of TLR2 signaling in COPD. Materials and methods miRNA microarray analysis was performed to screen for the dysregulated miRNAs in alveolar macrophages (AMs) isolated from COPD rats. The interaction between these miRNAs and TLR2 gene was predicted using miRBase and validated using dual luciferase assay. Based on the analysis, a novel miR-344b-1-3p was identified as a novel modulator of TLR2 gene. Then, the mechanism through which miR-344b-1-3p regulated TLR2 expression was explored using cigarette smoke extract (CSE)-pretreated NR8383 cells. Moreover, by subjecting CSE-pretreated NR8383 cells to Pam3CSK4, the effect of miR-344b-1-3p on NF-κB activity and other important mediators of COPD, including IRAK-1, ERK, TNF-α, IL-1β, and MIP-2, was also assessed. Results COPD rat model was successfully induced by smoke inhalation. Among the 11 upregulated miRNAs in AMs from COPD rats, miR-344b-1-3p was predicted to be a novel miRNA targeting TLR2 gene. In the CSE pretreated NR8383 cells exposed to Pam3CSK4, miR-344b-1-3p inhibition increased the expression levels of TLR2, TNF-α, and IL-1β and decreased the expression levels of MIP-2. In addition, the phosphorylation of IRAK-1, IκBα, and IRK was augmented by miR-344b-1-3p inhibition. Conclusion Findings outlined in this study suggest that miR-344b-1-3p was an effective modulator of TLR2 gene, which can be employed as a promising therapeutic and preventive target of IPA in COPD patients. PMID:28243080

  14. Upregulated miR-29b promotes neuronal cell death by inhibiting Bcl2L2 after ischemic brain injury.

    PubMed

    Shi, Guodong; Liu, Yang; Liu, Tielong; Yan, Wangjun; Liu, Xiaowei; Wang, Yuan; Shi, Jiangang; Jia, Lianshun

    2012-01-01

    It is increasingly clear that microRNAs (miRNAs) play an important role in controlling cell survival. However, the functional significance of miRNAs in ischemic brain injury remains poorly understood. In the present study, we assayed the expression levels of miR-29b after ischemic brain injury, and defined the target genes and biological functions of miR-29b. We found that the miR-29b levels were significantly increased in rat brain after transient middle cerebral artery occlusion and neurons after oxygen-glucose deprivation. Moreover, ectopic expression of miR-29b promoted neuronal cell death, whereas its repression decreased cell death. Furthermore, we verified that miR-29b directly targeted and inhibited Bcl2L2 gene expression, and then increased neuronal cell death. Importantly, Bcl2L2 overexpression rescued neuronal cell death induced by miR-29b. These results suggest an important role of miR-29b in regulating neuronal cell death, thus offering a new target for the development of therapeutic agents against ischemic brain injury.

  15. Diadenosine polyphosphates as antagonists of the endogenous P2Y1 receptor in rat brain capillary endothelial cells of the B7 and B10 clones

    PubMed Central

    Vigne, Paul; Breittmayer, Jean Philippe; Frelin, Christian

    2000-01-01

    Diadenosine polyphosphates (ApnAs, n=2–7) are considered as stress mediators in the cardiovascular system. They act both via identified P2 purinoceptors and via yet to be characterized receptors. This study analyses the actions of ApnAs in clones of rat brain capillary endothelial cells that express P2Y1 receptors (B10 cells) or both P2Y1 and P2Y2 receptors (B7 cells).B10 cells responded to Ap3A with rises in intracellular Ca2+ concentration ([Ca2+]i). This response was prevented by adenosine-3′-phosphate-5′-phosphate, an antagonist of P2Y1 receptors. It was largely suppressed by a treatment with apyrase VII or with creatine phosphokinase/creatine phosphate to degrade contaminating ADP.ApnAs inhibited ADP induced increases in [Ca2+]i mediated by P2Y1 receptors by shifting ADP concentration-response curves to larger concentrations. Apparent Ki values were estimated to be 6 μM for Ap4A, 10 μM for Ap5A and 47 μM for Ap6A. Ap2A and Ap3A were much less active.ApnAs were neither agonists nor antagonists of the endogenous P2Y2 receptor in B7 cells.ApnAs are neither agonists nor antagonists of the Gi-coupled, ADP receptor in B10 cells.The results suggest that most actions of ApnAs in B7 and B10 cells can be accounted for by endogenous P2Y1 receptors. Ap4A, Ap5A and Ap6A are specific antagonists of endogenous Ca2+-coupled P2Y1 receptors. PMID:10742308

  16. Activation of bradykinin B2 receptor induced the inflammatory responses of cytosolic phospholipase A2 after the early traumatic brain injury.

    PubMed

    Chao, Honglu; Liu, Yinlong; Lin, Chao; Xu, Xiupeng; Li, Zheng; Bao, Zhongyuan; Fan, Liang; Tao, Chao; Zhao, Lin; Liu, Yan; Wang, Xiaoming; You, Yongping; Liu, Ning; Ji, Jing

    2018-06-09

    Phospholipase A 2 is a known aggravator of inflammation and deteriorates neurological outcomes after traumatic brain injury (TBI), however the exact inflammatory mechanisms remain unknown. This study investigated the role of bradykinin and its receptor, which are known initial mediators within inflammation activation, as well as the mechanisms of the cytosolic phospholipase A 2 (cPLA 2 )-related inflammatory responses after TBI. We found that cPLA 2 and bradykinin B2 receptor were upregulated after a TBI. Rats treated with the bradykinin B2 receptor inhibitor LF 16-0687 exhibited significantly less cPLA 2 expression and related inflammatory responses in the brain cortex after sustaining a controlled cortical impact (CCI) injury. Both the cPLA 2 inhibitor and the LF16-0687 improved CCI rat outcomes by decreasing neuron death and reducing brain edema. The following TBI model utilized both primary astrocytes and primary neurons in order to gain further understanding of the inflammation mechanisms of the B2 bradykinin receptor and the cPLA 2 in the central nervous system. There was a stronger reaction from the astrocytes as well as a protective effect of LF16-0687 after the stretch injury and bradykinin treatment. The protein kinase C pathway was thought to be involved in the B2 bradykinin receptor as well as the cPLA 2 -related inflammatory responses. Rottlerin, a Protein Kinase C (PKC) δ inhibitor, decreased the activity of the cPLA 2 activity post-injury, and LF16-0687 suppressed both the PKC pathway and the cPLA 2 activity within the astrocytes. These results indicated that the bradykinin B2 receptor-mediated pathway is involved in the cPLA 2 -related inflammatory response from the PKC pathway. Copyright © 2018. Published by Elsevier B.V.

  17. Ibrutinib (ImbruvicaTM) potently inhibits ErbB receptor phosphorylation and cell viability of ErbB2-positive breast cancer cells.

    PubMed

    Grabinski, Nicole; Ewald, Florian

    2014-12-01

    Ibrutinib (formerly PCI-32765) is a specific, irreversible, and potent inhibitor of Burton's tyrosine kinase (BTK) developed for the treatment of several forms of blood cancer. It is now an FDA-approved drug marketed under the name Imbruvica(TM) (Pharmacyclics, Inc.) and successfully used as an orally administered second-line drug in the treatment of mantle cell lymphoma. Since BTK is predominantly expressed in hematopoietic cells, the sensitivity of solid tumor cells to Ibrutinib has not been analyzed. In this study, we determined the effect of Ibrutinib on breast cancer cells. We demonstrate that Ibrutinib efficiently reduces the phosphorylation of the receptor tyrosine kinases ErbB1, ErbB2 and ErbB3, thereby suppressing AKT and MAPK signaling in ErbB2-positive (ErbB2+) breast cancer cell lines. Treatment with Ibrutinib significantly reduced the viability of ErbB2+ cell lines with IC50 values at nanomolar concentrations, suggesting therapeutic potential of Ibrutinib in breast cancer. Combined treatment with Ibrutinib and the dual PI3K/mTOR inhibitor BEZ235 synergistically reduces cell viability of ErbB2+ breast cancer cells. Combination indices below 0.25 at 50% inhibition of cell viability were determined by the Chou-Talalay method. Therefore, the combination of Ibrutinib and canonical PI3K pathway inhibitors could be a new and effective approach in the treatment of breast cancer with activated ErbB receptors. Ibrutinib could thus become a valuable component of targeted therapy in aggressive ErbB2+ breast cancer.

  18. Clofibric acid induces hepatic CYP 2B1/2 via constitutive androstane receptor not via peroxisome proliferator activated receptor alpha in rat.

    PubMed

    Ibrahim, Zein Shaban; Ahmed, Mohamed Mohamed; El-Shazly, Samir Ahmed; Ishizuka, Mayumi; Fujita, Shoichi

    2014-01-01

    Peroxisome proliferator activated receptor α (PPARα) ligands, fibrates used to control hyperlipidemia. We demonstrated CYP2B induction by clofibric acid (CFA) however, the mechanism was not clear. In this study, HepG2 cells transfected with expression plasmid of mouse constitutive androstane receptor (CAR) or PPARα were treated with CFA, phenobarbital (PB) or TCPOBOP. Luciferase assays showed that CFA increased CYP2B1 transcription to the same level as PB, or TCPOBOP in HepG2 transfected with mouse CAR But failed to induce it in PPARα transfected cells. CYP2B expressions were increased with PB or CFA in Wistar female rats (having normal levels of CAR) but not in Wistar Kyoto female rats (having low levels of CAR). The induction of CYP2B by PB or CFA was comparable to nuclear CAR levels. CAR nuclear translocation was induced by CFA in both rat strains. This indicates that fibrates can activate CAR and that fibrates-insulin sensitization effect may occur through CAR, while hypolipidemic effect may operate through PPARα.

  19. Structure and functional interaction of the extracellular domain of human GABA[subscript B] receptor GBR2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Geng, Yong; Xiong, Dazhi; Mosyak, Lidia

    2012-10-24

    Inhibitory neurotransmission is mediated primarily by GABA. The metabotropic GABA{sub B} receptor is a G protein-coupled receptor central to mammalian brain function. Malfunction of GABA{sub B} receptor has been implicated in several neurological disorders. GABA{sub B} receptor functions as a heterodimeric assembly of GBR1 and GBR2 subunits, where GBR1 is responsible for ligand-binding and GBR2 is responsible for G protein coupling. Here we demonstrate that the GBR2 ectodomain directly interacts with the GBR1 ectodomain to increase agonist affinity by selectively stabilizing the agonist-bound conformation of GBR1. We present the crystal structure of the GBR2 ectodomain, which reveals a polar heterodimericmore » interface. We also identify specific heterodimer contacts from both subunits, and GBR1 residues involved in ligand recognition. Lastly, our structural and functional data indicate that the GBR2 ectodomain adopts a constitutively open conformation, suggesting a structural asymmetry in the active state of GABA{sub B} receptor that is unique to the GABAergic system.« less

  20. miR-26b enhances radiosensitivity of hepatocellular carcinoma cells by targeting EphA2

    PubMed Central

    Jin, Qiao; Li, Xiang Jun; Cao, Pei Guo

    2016-01-01

    Objective(s): Although low-dose radiotherapy (RT) that involves low collateral damage is more suitable for hepatocellular carcinoma (HCC) than traditional high-dose RT, but to achieve satisfactory therapeutic effect with low-dose RT, it is necessary to sensitize HCC cells to irradiation. This study was aimed to determine whether radiosensitivity of HCC cells can be enhanced using miR-26b by targeting erythropoietin producing human hepatocelluar A2 (EphA2). Materials and Methods: The levels of miR-26b and EphA2 expression in multiple HCC cell lines were assessed by qPCR and western blotting, respectively, and compared with those in a hepatic cell line. HCC 97H cells were transfected with miR-26b mimics, EphA2-ShRNA or EphA2 over-expression vector before exposure to low-dose irradiation. Results: Different degrees of miR-26b down-regulation and EphA2 up-regulation were observed in all HCC cell lines, among which the HCC 97H cell line expressed the lowest level of miR-26b and highest level of EphA2. EphA2 was verified as the target of miR-26b by dual luciferase reporter assay. HCC 97H cells transfected with miR-26b mimics or EphA2-ShRNA reduced the expression of EphA2 protein, with significantly lower cell proliferation rate and cell invasion ability and higher apoptosis rate in response to low-dose irradiation than those in the non-transfected cells. These results were reversed after EphA2 was overexpressed by transfection with the EphA2 overexpression vector. Co-transfection with miR-26b mimics and EphA2 overexpression vector barely altered EphA2 expression level and cell response to low-dose irradiation. Conclusion: These data suggest that miR-26b enhances radiosensitivity of HCC 97H cells by targeting EphA2 protein. PMID:27746866

  1. miR-26b enhances radiosensitivity of hepatocellular carcinoma cells by targeting EphA2.

    PubMed

    Jin, Qiao; Li, Xiang Jun; Cao, Pei Guo

    2016-08-01

    Although low-dose radiotherapy (RT) that involves low collateral damage is more suitable for hepatocellular carcinoma (HCC) than traditional high-dose RT, but to achieve satisfactory therapeutic effect with low-dose RT, it is necessary to sensitize HCC cells to irradiation. This study was aimed to determine whether radiosensitivity of HCC cells can be enhanced using miR-26b by targeting erythropoietin producing human hepatocelluar A2 (EphA2). The levels of miR-26b and EphA2 expression in multiple HCC cell lines were assessed by qPCR and western blotting, respectively, and compared with those in a hepatic cell line. HCC 97H cells were transfected with miR-26b mimics, EphA2-ShRNA or EphA2 over-expression vector before exposure to low-dose irradiation. Different degrees of miR-26b down-regulation and EphA2 up-regulation were observed in all HCC cell lines, among which the HCC 97H cell line expressed the lowest level of miR-26b and highest level of EphA2. EphA2 was verified as the target of miR-26b by dual luciferase reporter assay. HCC 97H cells transfected with miR-26b mimics or EphA2-ShRNA reduced the expression of EphA2 protein, with significantly lower cell proliferation rate and cell invasion ability and higher apoptosis rate in response to low-dose irradiation than those in the non-transfected cells. These results were reversed after EphA2 was overexpressed by transfection with the EphA2 overexpression vector. Co-transfection with miR-26b mimics and EphA2 overexpression vector barely altered EphA2 expression level and cell response to low-dose irradiation. These data suggest that miR-26b enhances radiosensitivity of HCC 97H cells by targeting EphA2 protein.

  2. Adenosine A2B receptor: from cell biology to human diseases

    NASA Astrophysics Data System (ADS)

    Sun, Ying; Huang, Pingbo

    2016-08-01

    Extracellular adenosine is a ubiquitous signaling molecule that modulates a wide array of biological processes. Recently, significant advances have been made in our understanding of A2B adenosine receptor (A2BAR). In this review, we first summarize some of the general characteristics of A2BAR, and then we describe the multiple binding partners of the receptor, such as newly identified α-actinin-1 and p105, and discuss how these associated proteins could modulate A2BAR’s functions, including certain seemingly paradoxical functions of the receptor. Growing evidence indicates a critical role of A2BAR in cancer, renal disease, and diabetes, in addition to its importance in the regulation of vascular diseases and lung disease. Here, we also discuss the role of A2BAR in cancer, renal disease, and diabetes and the potential of the receptor as a target for treating these three diseases.

  3. Gender-related associations of genetic polymorphisms of α-adrenergic receptors, endothelial nitric oxide synthase and bradykinin B2 receptor with treadmill exercise test responses.

    PubMed

    Nunes, Rafael Amorim Belo; Barroso, Lúcia Pereira; Pereira, Alexandre da Costa; Krieger, José Eduardo; Mansur, Alfredo José

    2014-01-01

    Treadmill exercise test responses have been associated with cardiovascular prognosis in individuals without overt heart disease. Neurohumoral and nitric oxide responses may influence cardiovascular performance during exercise testing. Therefore, we evaluated associations between functional genetic polymorphisms of α-adrenergic receptors, endothelial nitric oxide synthase, bradykinin receptor B2 and treadmill exercise test responses in men and women without overt heart disease. We enrolled 766 (417 women; 349 men) individuals without established heart disease from a check-up programme at the Heart Institute, University of São Paulo Medical School. Exercise capacity, chronotropic reserve, maximum heart-rate achieved, heart-rate recovery, exercise systolic blood pressure (SBP), exercise diastolic blood pressure (DBP) and SBP recovery were assessed during exercise testing. Genotypes for the α-adrenergic receptors ADRA1A Arg347Cys (rs1048101), ADRA2A 1780 C>T (rs553668), ADRA2B Del 301-303 (rs28365031), endothelial nitric synthase (eNOS) 786 T>C (rs2070744), eNOS Glu298Asp (rs1799983) and BK2R (rs5810761) polymorphisms were assessed by PCR and high-resolution melting analysis. Maximum SBP was associated with ADRA1A rs1048101 (p=0.008) and BK2R rs5810761 (p=0.008) polymorphisms in men and ADRA2A rs553668 (p=0.008) and ADRA2B rs28365031 (p=0.022) in women. Maximum DBP pressure was associated with ADRA2A rs553668 (p=0.002) and eNOS rs1799983 (p=0.015) polymorphisms in women. Exercise capacity was associated with eNOS rs2070744 polymorphisms in women (p=0.01) and with eNOS rs1799983 in men and women (p=0.038 and p=0.024). The findings suggest that genetic variants of α-adrenergic receptors and bradykinin B2 receptor may be involved with blood pressure responses during exercise tests. Genetic variants of endothelial nitric oxide synthase may be involved with exercise capacity and blood pressure responses during exercise tests. These responses may be gender-related.

  4. Gender-related associations of genetic polymorphisms of α-adrenergic receptors, endothelial nitric oxide synthase and bradykinin B2 receptor with treadmill exercise test responses

    PubMed Central

    Nunes, Rafael Amorim Belo; Barroso, Lúcia Pereira; Pereira, Alexandre da Costa; Krieger, José Eduardo; Mansur, Alfredo José

    2014-01-01

    Background Treadmill exercise test responses have been associated with cardiovascular prognosis in individuals without overt heart disease. Neurohumoral and nitric oxide responses may influence cardiovascular performance during exercise testing. Therefore, we evaluated associations between functional genetic polymorphisms of α-adrenergic receptors, endothelial nitric oxide synthase, bradykinin receptor B2 and treadmill exercise test responses in men and women without overt heart disease. Methods We enrolled 766 (417 women; 349 men) individuals without established heart disease from a check-up programme at the Heart Institute, University of São Paulo Medical School. Exercise capacity, chronotropic reserve, maximum heart-rate achieved, heart-rate recovery, exercise systolic blood pressure (SBP), exercise diastolic blood pressure (DBP) and SBP recovery were assessed during exercise testing. Genotypes for the α-adrenergic receptors ADRA1A Arg347Cys (rs1048101), ADRA2A 1780 C>T (rs553668), ADRA2B Del 301–303 (rs28365031), endothelial nitric synthase (eNOS) 786 T>C (rs2070744), eNOS Glu298Asp (rs1799983) and BK2R (rs5810761) polymorphisms were assessed by PCR and high-resolution melting analysis. Results Maximum SBP was associated with ADRA1A rs1048101 (p=0.008) and BK2R rs5810761 (p=0.008) polymorphisms in men and ADRA2A rs553668 (p=0.008) and ADRA2B rs28365031 (p=0.022) in women. Maximum DBP pressure was associated with ADRA2A rs553668 (p=0.002) and eNOS rs1799983 (p=0.015) polymorphisms in women. Exercise capacity was associated with eNOS rs2070744 polymorphisms in women (p=0.01) and with eNOS rs1799983 in men and women (p=0.038 and p=0.024). Conclusions The findings suggest that genetic variants of α-adrenergic receptors and bradykinin B2 receptor may be involved with blood pressure responses during exercise tests. Genetic variants of endothelial nitric oxide synthase may be involved with exercise capacity and blood pressure responses during exercise tests

  5. Diversity in peptide recognition by the SH2 domain of SH2B1.

    PubMed

    McKercher, Marissa A; Guan, Xiaoyang; Tan, Zhongping; Wuttke, Deborah S

    2018-02-01

    SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein-protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate-1 and -2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C-terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high-resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand-binding interface of SH2B1 with high specificity. © 2017 Wiley Periodicals, Inc.

  6. Jak2 and Ca2+/calmodulin are key intermediates for bradykinin B2 receptor-mediated activation of Na+/H+ exchange in KNRK and CHO cells.

    PubMed

    Lefler, David; Mukhin, Yurii V; Pettus, Tobiah; Leeb-Lundberg, L M Fredrik; Garnovskaya, Maria N; Raymond, John R

    2003-04-01

    Na(+)/H(+) exchangers are ubiquitous in mammalian cells, carrying out key functions, such as cell volume defense, acid-base homeostasis, and regulation of the cytoskeleton. We used two screening technologies (FLIPR and microphysiometry) to characterize the signal transduction pathway used by the bradykinin B(2) receptor to activate Na(+)/H(+) exchange in two cell lines, KNRK and CHO. In both cell types, B(2) receptor activation resulted in rapid increases in the rate of proton extrusion that were sodium-dependent and could be blocked by the Na(+)/H(+) exchange inhibitors EIPA and MIA or by replacing extracellular sodium with TMA. Activation of Na(+)/H(+) exchange by bradykinin was concentration-dependent and could be blocked by the selective B(2) receptor antagonist HOE140, but not by the B(1) receptor antagonist des-Arg10-HOE140. Inhibitors of Jak2 tyrosine kinase (genistein and AG490) and of CAM (W-7 and calmidazolium) attenuated bradykinin-induced activation of Na(+)/H(+) exchange. Bradykinin induced formation of a complex between CAM and Jak2, supporting a regulatory role for Jak2 and CAM in the activation of Na(+)/H(+) exchange in KNRK and CHO cells. We propose that this pathway (B(2) receptor --> Jak2 --> CAM --> Na(+)/H(+) exchanger) is a fundamental regulator of Na(+)/H(+) exchange activity.

  7. A2B Adenosine Receptor Agonist Improves Erectile Function in Diabetic Rats.

    PubMed

    Wen, Jiaming; Wang, Bohan; Du, Chuanjun; Xu, Gang; Zhang, Zhewei; Li, Yi; Zhang, Nan

    2015-10-01

    Diabetes is an important risk factor for erectile dysfunction (ED). Recent studies have indicated that A2B adenosine receptor (ADORA2B) signaling is essential for penile erection. Thus, we hypothesize that diabetic ED may be attributed to impaired A2B adenosine signaling. To test this hypothesis, we generated diabetic rats by injecting streptozocin as animal model. After 12 weeks, immunohistochemistry staining was used to localize the expression of ADORA2B. Western Blot and quantitative PCR were employed to determine ADORA2B expression level. Intracavernosal pressure (ICP) measurement was used to evaluate erectile function. Diabetic rats received a single intravenous injection of BAY 60-6583, an ADORA2B agonist, or vehicle solution, at 60 min before the ICP measurement. The results showed that ADORA2B expressed in the nerve bundle, smooth muscle, and endothelium in penile tissue of control mice. Western Blot and quantitative PCR results indicated that the expression levels of ADORA2B protein and mRNA were significantly reduced in penile tissues of diabetic rats. Functional studies showed that the erectile response induced by electrical stimulation was remarkably decreased in diabetic rats, compared with age-matched control rats. However, at 60 min after BAY 60-6583 treatment, the erectile function was improved in diabetic rats, suggesting that enhancement of ADORA2B signaling may improve erectile function in diabetic ED. This preclinical study has revealed a previously unrecognized therapeutic possibility of BAY 60-6583 as an effective and mechanism-based drug to treat diabetic ED. In conclusion, we propose that impaired A2B adenosine signaling is one of the pathological mechanisms of diabetic ED.

  8. Dm5-HT2B: Pharmacological Characterization of the Fifth Serotonin Receptor Subtype of Drosophila melanogaster.

    PubMed

    Blenau, Wolfgang; Daniel, Stöppler; Balfanz, Sabine; Thamm, Markus; Baumann, Arnd

    2017-01-01

    Serotonin (5-hydroxytryptamine, 5-HT) is an important regulator of physiological and behavioral processes in both protostomes (e.g., insects) and deuterostomes (e.g., mammals). In insects, serotonin has been found to modulate the heart rate and to control secretory processes, development, circadian rhythms, aggressive behavior, as well as to contribute to learning and memory. Serotonin exerts its activity by binding to and activating specific membrane receptors. The clear majority of these receptors belong to the superfamily of G-protein-coupled receptors. In Drosophila melanogaster , a total of five genes have been identified coding for 5-HT receptors. From this family of proteins, four have been pharmacologically examined in greater detail, so far. While Dm5-HT 1A , Dm5-HT 1B , and Dm5-HT 7 couple to cAMP signaling cascades, the Dm5-HT 2A receptor leads to Ca 2+ signaling in an inositol-1,4,5-trisphosphate-dependent manner. Based on sequence similarity to homologous genes in other insects, a fifth D. melanogaster gene was uncovered coding for a Dm5-HT 2B receptor. Knowledge about this receptor's pharmacological properties is very limited. This is quite surprising because Dm5-HT 2B has been attributed to distinct physiological functions based on genetic interference with its gene expression. Mutations were described reducing the response of the larval heart to 5-HT, and specific knockdown of Dm5-HT 2B mRNA in hemocytes resulted in a higher susceptibility of the flies to bacterial infection. To gain deeper understanding of Dm5-HT 2B 's pharmacology, we evaluated the receptor's response to a series of established 5-HT receptor agonists and antagonists in a functional cell-based assay. Metoclopramide and mianserin were identified as two potent antagonists that may allow pharmacological interference with Dm5-HT 2B signaling in vitro and in vivo .

  9. Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling*♦

    PubMed Central

    Walliser, Claudia; Tron, Kyrylo; Clauss, Karen; Gutman, Orit; Kobitski, Andrei Yu.; Retlich, Michael; Schade, Anja; Röcker, Carlheinz; Henis, Yoav I.; Nienhaus, G. Ulrich; Gierschik, Peter

    2015-01-01

    The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca2+. The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca2+ and regulation of Ca2+-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca2+ release from intracellular stores; (iii) Ca2+ entry from the extracellular compartment; and (iv) nuclear translocation of the Ca2+-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca2+ signaling. PMID:25903139

  10. The trophoblast cell surface antigen 2 and miR-125b axis in urothelial bladder cancer

    PubMed Central

    Lazzarini, Raffaella; Gesuita, Rosaria; Guerra, Emanuela; Tossetta, Giovanni; Castellucci, Clara; Giannubilo, Stefano Raffaele; Procopio, Antonio; Alberti, Saverio; Mazzucchelli, Roberta

    2017-01-01

    Human trophoblast cell surface antigen 2 (Trop-2) is a 40-kDa transmembrane glycoprotein that was first identified as a marker of human trophoblast cells. Trop-2 acts on cell proliferation, adhesion, and migration by activating a number of intracellular signalling pathways. Elevated Trop-2 expression has been demonstrated in several types of cancer and correlated with aggressiveness and poor prognosis. Since no data are available on Trop-2 in bladder cancer (BC), the purpose of the study was to determine its levels in tissue specimens from normal individuals and patients with BC at different stages. Moreover, since according to recent evidence Trop-2 is a miR-125b target, miR-125b expression was also assessed in tissue specimens. Finally, the effect of the Trop-2/miR-125b axis on the proliferation and migration of BC cells was evaluated in vitro. The Trop-2/miR-125b axis was seen to be differentially expressed in normal urothelium, non-invasive BC and invasive BC tissue. Significant miR-125b down-regulation was associated with a significant increase in Trop-2 protein levels in BC tissue and correlated with disease severity. In vitro analysis confirmed the role of miR-125b in down-modulation of Trop-2 protein levels and showed that Trop-2/miR-125b axis affects cellular proliferation in bladder tissue. In conclusion, our findings highlight a role for the Trop-2/miR-125b axis in BC progression and suggest Trop-2 and miR-125b as diagnostic/prognostic marker candidates as well as druggable targets for innovative therapeutic approaches. PMID:28938585

  11. The selectivity of conantokin-G for ion channel inhibition of NR2B subunit-containing NMDA receptors is regulated by amino acid residues in the S2 region of NR2B

    PubMed Central

    Sheng, Zhenyu; Liang, Zhong; Geiger, James H.; Prorok, Mary; Castellino, Francis J.

    2009-01-01

    The conantokins are short, naturally-occurring peptides that inhibit ion flow through N-methyl-D-aspartate receptor (NMDAR) channels. One member of this peptide family, conantokin-G (con-G), specifically antagonizes NR2B-containing NMDAR channels, whereas other known conantokins are less selective inhibitors with regard to the nature of the NR2 subunit of the NMDAR complex. In order to define the molecular determinants of NR2B that govern con-G selectivity, we evaluated the ability of con-G to inhibit NMDAR ion channels expressed in human embryonic kidney (HEK)293 cells transfected with NR1, in combination with various NR2A/2B chimeras and point mutants, by electrophysiology using cells voltage-clamped in the whole cell configuration. We found that a variant of the con-G-insensitive subunit, NR2A, in which the 158 residues comprising the S2 peptide segment (E657-I814) were replaced by the corresponding S2 region of NR2B (E658-I815), results in receptors that are highly sensitive to inhibition by con-G. Of the 22 amino acids that are different between the NR2A-S2 and the NR2B-S2 regions, exchange of one of these, M739 of NR2B for the equivalent K738 of NR2A, was sufficient to completely import the inhibitory activity of con-G into NR1b/NR2A-containing NMDARs. Some reinforcement of this effect was found by substitution of a second amino acid, K755 of NR2B for Y754 of NR2A. The discovery of the molecular determinants of NR2B selectivity with con-G has implications for the design of subunit-selective neurobiological probes and drug therapies, in addition to advancing our understanding of NR2B- versus NR2A-mediated neurological processes. PMID:19427876

  12. MicroRNA-34b/c inhibits aldosterone-induced vascular smooth muscle cell calcification via a SATB2/Runx2 pathway.

    PubMed

    Hao, Jianbing; Zhang, Lei; Cong, Guangting; Ren, Liansheng; Hao, Lirong

    2016-12-01

    Increasing evidence shows that aldosterone and specific microRNAs (miRs) contribute to vascular smooth muscle cell (VSMC) calcification. In this study, we aim to explore the mechanistic links between miR-34b/c and aldosterone in VSMC calcification. VSMC calcification models were established both in vitro and in vivo. First, the levels of aldosterone, miR-34b/c and special AT-rich sequence-binding protein 2 (SATB2) were measured. Then, miR-34b/c mimics or inhibitors were transfected into VSMCs to evaluate the function of miR-34b/c. Luciferase reporter assays were used to demonstrate whether SATB2 was a direct target of miR-34b/c. Aldosterone and SATB2 were found to be markedly upregulated during VSMC calcification, whereas miR-34b/c expression was downregulated. Treatment with the mineralocorticoid receptor (MR) antagonist eplerenone inhibited VSMC calcification. In aldosterone-induced VSMC calcification, miR-34b/c levels were downregulated and SATB2 protein was upregulated. Furthermore, miR-34b/c overexpression alleviated aldosterone-induced VSMC calcification as well as inhibited the expression of SATB2 protein, whereas miR-34b/c inhibition markedly enhanced VSMC calcification and upregulated SATB2 protein. In addition, luciferase reporter assays showed that SATB2 is a direct target of miR-34b/c in VSMCs. Overexpression of SATB2 induced Runx2 overproduction and VSMC calcification. Therefore, miR-34b/c participates in aldosterone-induced VSMC calcification via a SATB2/Runx2 pathway. As miR-34b/c appears to be a negative regulator, it has potential as a therapeutic target of VSMC calcification.

  13. Co-Targeting HER2 and EphB4 Pathways

    DTIC Science & Technology

    2013-07-01

    progress has been made toward the clinic. An IND has been obtained for sEphbB4- HSA, an albumin stabilized soluble EphB4 decoy receptor that efficiently...for activated HER2/HER family receptors , angiogenic markers (EphrinB2, EphB1,2,3,4,6, VEGF, VEGFR-1, 2, 3 and PDGFR), vessel density, signal...ABOVE ADDRESS. 1. REPORT DATE 2. REPORT TYPE 3 . DATES COVERED 4. TITLE AND SUBTITLE Co-Targeting HER2 and EphB4 Pathways 5a. CONTRACT NUMBER 5b

  14. Structural insights into FRS2α PTB domain recognition by neurotrophin receptor TrkB.

    PubMed

    Zeng, Lei; Kuti, Miklos; Mujtaba, Shiraz; Zhou, Ming-Ming

    2014-07-01

    The fibroblast growth factor receptor (FGFR) substrate 2 (FRS2) family proteins function as scaffolding adapters for receptor tyrosine kinases (RTKs). The FRS2α proteins interact with RTKs through the phosphotyrosine-binding (PTB) domain and transfer signals from the activated receptors to downstream effector proteins. Here, we report the nuclear magnetic resonance structure of the FRS2α PTB domain bound to phosphorylated TrkB. The structure reveals that the FRS2α-PTB domain is comprised of two distinct but adjacent pockets for its mutually exclusive interaction with either nonphosphorylated juxtamembrane region of the FGFR, or tyrosine phosphorylated peptides TrkA and TrkB. The new structural insights suggest rational design of selective small molecules through targeting of the two conjunct pockets in the FRS2α PTB domain. © 2014 Wiley Periodicals, Inc.

  15. Radioligand binding characterization of the bradykinin B(2) receptor in the rabbit and pig ileal smooth muscle.

    PubMed

    Meini, Stefania; Cucchi, Paola; Catalani, Claudio; Bellucci, Francesca; Santicioli, Paolo; Giuliani, Sandro; Maggi, Carlo Alberto

    2010-06-10

    Several species-related differences have been reported in kinin B(2) receptor pharmacology. The present study aimed to evaluate the affinity of the bradykinin B(2) receptor antagonist MEN16132 for the rabbit and pig B(2) receptor, and radioligand binding experiments using [(3)H]bradykinin and membranes of rabbit and pig ileum smooth muscle were conducted. The [(3)H]bradykinin binding was characterized by homologous displacement curves indicating K(d) values of 0.65 and 0.33nM in rabbit and pig, respectively. The B(2) receptor specificity of [(3)H]bradykinin binding was shown by the low affinity (>microM) displayed by agonists ([desArg(9)]bradykinin and Lys[desArg(9)]bradykinin) and antagonists [Leu(8),desArg(9)]bradykinin and Lys[Leu(8),desArg(9)]bradykinin) selective for the B(1) receptor. The affinity of MEN16132 and other antagonists was determined by inhibition curves (pK(i) values in the rabbit and pig assay, respectively): MEN16132 (10.4 and 10.3) and peptide compounds such as icatibant (10.1 and 9.9) and MEN11270 (10.3 and 10.1) displayed subnanomolar potency in both assays; the nonpeptide LF16-0687 (8.4 and 8.5) and FR173657 (8.2 and 9.1) exhibited a different affinity pattern, whereas WIN64338 displayed low affinity (5.7 and B(2) receptor. An attempt to highlight differences which can undertake ligands selectivity across the species is presented. In conclusion, the present study indicates MEN16132 as the only nonpeptidic compound which displays an even subnanomolar affinity for the rabbit and pig B(2) receptor.

  16. Adenosine A2A receptors are required for glutamate mGluR5- and dopamine D1 receptor-evoked ERK1/2 phosphorylation in rat hippocampus: involvement of NMDA receptor.

    PubMed

    Krania, Paraskevi; Dimou, Eleni; Bantouna, Maria; Kouvaros, Stylianos; Tsiamaki, Eirini; Papatheodoropoulos, Costas; Sarantis, Konstantinos; Angelatou, Fevronia

    2018-05-01

    Interaction between mGluR5 and NMDA receptors (NMDAR) is vital for synaptic plasticity and cognition. We recently demonstrated that stimulation of mGluR5 enhances NMDAR responses in hippocampus by phosphorylating NR2B(Tyr1472) subunit, and this reaction was enabled by adenosine A 2A receptors (A 2A R) (J Neurochem, 135, 2015, 714). In this study, by using in vitro phosphorylation and western blot analysis in hippocampal slices of male Wistar rats, we show that mGluR5 stimulation or mGluR5/NMDARs co-stimulation synergistically activate ERK1/2 signaling leading to c-Fos expression. Interestingly, both reactions are under the permissive control of endogenous adenosine acting through A 2A Rs. Moreover, mGluR5-mediated ERK1/2 phosphorylation depends on NMDAR, which however exhibits a metabotropic way of function, since no ion influx through its ion channel is required. Furthermore, our results demonstrate that mGluR5 and mGluR5/NMDAR-evoked ERK1/2 activation correlates well with the mGluR5/NMDAR-evoked NR2B(Tyr1472) phosphorylation, since both phenomena coincide temporally, are Src dependent, and are both enabled by A 2A Rs. This indicates a functional involvement of NR2B(Tyr1472) phosphorylation in the ERK1/2 activation. Our biochemical results are supported by electrophysiological data showing that in CA1 region of hippocampus, the theta burst stimulation (TBS)-induced long-term potentiation coincides temporally with an increase in ERK1/2 activation and both phenomena are dependent on the tripartite A 2A , mGlu5, and NMDARs. Furthermore, we show that the dopamine D1 receptors evoked ERK1/2 activation as well as the NR2B(Tyr1472) phosphorylation are also regulated by endogenous adenosine and A 2A Rs. In conclusion, our results highlight the A 2A Rs as a crucial regulator not only for NMDAR responses, but also for regulating ERK1/2 signaling and its downstream pathways, leading to gene expression, synaptic plasticity, and memory consolidation. © 2017 International

  17. Conversion of (η(5)-C2B9H10R)TaX3 (X = Me, NMe2) to (η(6)-C2B9H10R)TaX' (X' = NMe2, azaallyl) in the absence of a reducing agent: synthesis and structure of tantallacarboranes incorporating an arachno-η(6)-C2B9(4-) ligand.

    PubMed

    Xiang, Li; Xie, Zuowei

    2014-08-04

    Heating a benzene solution of [η(5)-(Me2NCH2CH2)C2B9H10] Ta(NMe2)3 (1) in the presence of pyridine gave an unprecedented complex [η(1):η(6)-(Me2NCH2CH2)C2B9H10]Ta (NMe2)(NC5H5) (2). On the other hand, reaction of (η(5)-C2B9H11)TaMe3 with adamantly isonitrile (AdNC) in dimethoxyethane (DME) at room temperature afforded another unexpected complex (η(6)-C2B9H11)Ta[η(3)-C,C,N-CH2C(CH3)NAd](DME) (4). These results show that pyridine and DME are essential for the formation of 2 and 4, respectively. It is suggested that the nido-η(5)-C2B9H10R(2−) ligand in tantallacarboranes takes up two electrons released by reductive elimination to form an arachno-η(6)-C2B9H10R(4−) fragment via the cage C–C bond cleavage.

  18. Magnetic and superconducting phase diagrams of single-crystal Er0.8R0.2Ni2B2C (R=Tb,Lu) and ErNi1.9Co0.1B2C: Identification of pair-breaking mechanisms

    NASA Astrophysics Data System (ADS)

    Takeya, H.; El Massalami, M.

    2004-01-01

    We investigated the magnetism, superconductivity and their interplay in single crystals Er0.8R0.2Ni2B2C (R=Tb,Lu) and ErNi1.9Co0.1B2C. In contrast to Co substitution, R substitutions induce considerable modifications in the magnetism of Er sublattice: e.g., Tb (Lu) substitution enhances (reduces) TN and critical fields. Both R substitutions introduce size effects and pinning centers; the former modifies the magnon specific heat while the latter hinders the formation of a weak ferromagnetism. The superconductivity, on the other hand, is strongly (weakly) influenced by Tb and Co (Lu) substitution. Taking LuNi2B2C as a nonmagnetic superconducting limit, we analyzed their superconductivities, as well as that of ErNi2B2C, in terms of multiple pair breaking theory on dirty superconductors. Based on this analysis, many of their superconducting features can be explained: The breakdown of de Gennes scaling is due to the presence of multiple pair breakers, the anisotropy of Hc2(T) is related to the magnetic anisotropy, the absence of a structure in Hc2(T) at TN of Lu substitution (TN2(T) of Tb and Co substitutions is in part due to a saturation of antiferromagnetic correlations. For Lu substitution, the strength of magnon mediated pair breaking process(es) is substantially reduced.

  19. Epithelial-specific A2B adenosine receptor signaling protects the colonic epithelial barrier during acute colitis

    PubMed Central

    Aherne, CM; Saeedi, B; Collins, CB; Masterson, JC; McNamee, EN; Perrenoud, L; Rapp, CR; Curtis, VF; Bayless, A; Fletcher, A; Glover, LE; Evans, CM; Jedlicka, P; Furuta, GT; de Zoeten, EF; Colgan, SP; Eltzschig, HK

    2015-01-01

    Central to inflammatory bowel disease (IBD) pathogenesis is loss of mucosal barrier function. Emerging evidence implicates extracellular adenosine signaling in attenuating mucosal inflammation. We hypothesized that adenosine-mediated protection from intestinal barrier dysfunction involves tissue-specific signaling through the A2B adenosine receptor (Adora2b) at the intestinal mucosal surface. To address this hypothesis, we combined pharmacologic studies and studies in mice with global or tissue-specific deletion of the Adora2b receptor. Adora2b−/− mice experienced a significantly heightened severity of colitis, associated with a more acute onset of disease and loss of intestinal epithelial barrier function. Comparison of mice with Adora2b deletion on vascular endothelial cells (Adora2bfl/flVeCadCre+) or intestinal epithelia (Adora2bfl/flVillinCre+) revealed a selective role for epithelial Adora2b signaling in attenuating colonic inflammation. In vitro studies with Adora2b knockdown in intestinal epithelial cultures or pharmacologic studies highlighted Adora2b-driven phosphorylation of vasodilator-stimulated phosphoprotein (VASP) as a specific barrier repair response. Similarly, in vivo studies in genetic mouse models or treatment studies with an Adora2b agonist (BAY 60-6583) recapitulate these findings. Taken together, our results suggest that intestinal epithelial Adora2b signaling provides protection during intestinal inflammation via enhancing mucosal barrier responses. PMID:25850656

  20. MiR-26b inhibits hepatocellular carcinoma cell proliferation, migration, and invasion by targeting EphA2.

    PubMed

    Li, Hesheng; Sun, Qinglei; Han, Bing; Yu, Xingquan; Hu, Baoguang; Hu, Sanyuan

    2015-01-01

    Deregulated microRNAs (miRNAs) have been shown to play important roles in cancer progression as a result of changes in expression of their target genes. In this study, we investigated the expression of miR-16b in eight hepatocellular carcinoma (HCC) cell lines, revealed the roles of miR-26b on hepatocellular carcinoma (HCC) cell proliferation, migration, and invasion, and confirmed that EphA2 is a direct target of miR-26b. The miR-26b expression was decreased and EphA2 expression was evaluated in HCC cell lines. Luciferase assays revealed that miR-26b inhibited EphA2 expression by targeting the 3'-untranslated region of EphA2 mRNA. Overexpression of miR-26b dramatically inhibited the proliferation, invasion, and migration of HCC cells by targeting EphA2. Moreover, miR-26b down-regulated c-Myc and CyclinD1 expression, which was reversed by overexpressed EphA2. Taken together, our data demonstrated the mechanism of miR-26b contributed to HCC progression and implicated that miR-26b's potential in HCC therapy.

  1. MiR-26b Mimic Inhibits Glioma Proliferation In Vitro and In Vivo Suppressing COX-2 Expression.

    PubMed

    Chen, Zheng-Gang; Zheng, Chuan-Yi; Cai, Wang-Qing; Li, Da-Wei; Ye, Fu-Yue; Zhou, Jian; Wu, Ran; Yang, Kun

    2017-08-11

    Glioma is the most common malignant tumor of the nervous system. Studies have shown the microRNA (miR)-26b/cyclooxygenase (COX)-2 axis in the development and progression in many tumor cells. Our study aims to investigate the effect and mechanism of miR-26b/COX-2 axis in glioma. Decreased expression of miR-26b with increased level of COX-2 was found in glioma tissues compared with matched normal tissues. A strong negative correlation was observed between the level of miR-26b and COX-2 in 30 glioma tissues. The miR-26b was then overexpressed by transfecting miR-26b mimic into U-373 cells. The invasive cell number and wounld closing rate were reduced in U-373 cells transfected with miR-26b mimic. Besides, COX2 siRNA enhanced the effect of miR-26b mimic in suppressing the expression of p-ERK1 and p-JNK. Finally, the in vivo experiment revealed that miR-26b mimic transfection strongly reduced the tumor growth, tumor volume and the expression of matrix metalloproteinase (MMP)-2, MMP-9). Taken together, our research indicated a miR-26b/COX-2/ERK/JNK axis in regulating the motility of glioma in vitro and in vivo, providing a new sight for treatment of glioma.

  2. Kinin-B2 receptor expression and activity during differentiation of embryonic rat neurospheres.

    PubMed

    Martins, Antonio H; Alves, Janaína M; Trujillo, Cleber A; Schwindt, Telma T; Barnabé, Gabriela F; Motta, Fabiana L T; Guimaraes, Alessander O; Casarini, Dulce E; Mello, Luiz E; Pesquero, João B; Ulrich, Henning

    2008-04-01

    Neural progenitor cells were isolated from rat fetal telencephalon and proliferate as neurospheres in the presence of EGF, FGF-2, and heparin. In the absence of these growth factors, neurospheres differentiate into neurons, astrocytes, and oligodendrocytes. Using an embryonal carcinoma cell line as in vitro differentiation model, we have already demonstrated the presence of an autocrine loop system between kinin-B2 receptor activity and secretion of its ligand bradykinin (BK) as prerequisites for final neuronal differentiation (Martins et al., J Biol Chem 2005; 280: 19576-19586). The aim of this study was to verify the activity of the kallikrein-kinin system (KKS) during neural progenitor cell differentiation. Immunofluorescence studies and flow cytometry analysis revealed increases in glial fibrillary acidic protein and beta-3 tubulin expression and decrease in the number of nestin-positive cells along neurospheres differentiation, indicating the transition of neural progenitor cells to astrocytes and neurons. Kinin-B2 receptor expression and activity, secretion of BK into the medium, and presence of high-molecular weight kininogen suggest the participation of the KKS in neurosphere differentiation. Functional kinin-B2 receptors and BK secretion indicate an autocrine loop during neurosphere differentiation to neurons, astrocytes, and oligodendrocytes, reflecting events occurring during early brain development. (c) 2008 International Society for Analytical Cytology.

  3. MicroRNA-26b Enhances the Radiosensitivity of Hepatocellular Carcinoma Cells by Targeting EphA2.

    PubMed

    Jin, Qiao; Li, Xiang Jun; Cao, Pei Guo

    2016-02-01

    Sensitizing hepatocellular carcinoma (HCC) cells to irradiation is important to achieve satisfactory therapeutic effect with low-dose radiotherapy. Erythropoietin-producing hepatocellular carcinoma A2 (EphA2) is a member of the Eph receptor family that constitutes the largest family of tyrosine kinase receptors. EphA2 overexpression is one of the poor prognostic factors in many progressive cancers. Importantly, EphA2 is a potential target of microRNA-26b (miR-26b), and miR-26b expression is down-regulated in several types of cancer. In this study, we measured the expression levels of miR-26b and EphA2 protein in seven human HCC cell lines by quantitative PCR and western blot analysis, respectively. Overall, lower miR-26b expression levels tended to be associated with higher EphA2 levels in HCC cell lines. Among the cell lines examined, 97H HCC cells expressed the lowest level of miR-26b and highest level of EphA2 protein. Thus, using 97H HCC cells, EphA2 mRNA was verified as the target of miR-26b by the luciferase reporter assay. Accordingly, a synthetic miR-26b, miR-26b mimics, was used to mimic the function of endogenous miR-26b. In 97H HCC cells transfected with miR-26b mimics or short-hairpin RNA targeting EphA2 mRNA, expression of EphA2 protein was reduced, which was associated with significantly lower proliferation rate and invasion ability and with higher apoptosis rate in response to low-dose irradiation, compared to control cells. In contrast, 97H HCC cells over-expressing EphA2 showed higher proliferation rate and invasion ability and lower apoptosis rate upon irradiation. These data suggest that miR-26b enhances the radiosensitivity of 97H HCC cells by targeting EphA2 protein.

  4. SETDB1 HISTONE METHYLTRANSFERASE REGULATES MOOD-RELATED BEHAVIORS AND EXPRESSION OF THE NMDA RECEPTOR SUBUNIT NR2B

    PubMed Central

    Jiang, Yan; Jakovcevski, Mira; Bharadwaj, Rahul; Connor, Caroline; Schroeder, Frederick A.; Lin, Cong L.; Straubhaar, Juerg; Martin, Gilles; Akbarian, Schahram

    2010-01-01

    Histone methyltransferases specific for the histone H3-lysine 9 (H3K9) residue, including Setdb1 (Set domain, bifurcated 1)/Eset/Kmt1e are associated with repressive chromatin remodeling and expressed in adult brain, but potential effects on neuronal function and behavior remain unexplored. Here, we report that transgenic mice with increased Setdb1 expression in adult forebrain neurons show antidepressant-like phenotypes in behavioral paradigms for anhedonia, despair and learned helplessness. Chromatin immunoprecipitation in conjunction with DNA tiling arrays (ChIP-chip) revealed that genomic occupancies of neuronal Setdb1 are limited to less than 1% of annotated genes, which include the NMDA receptor subunit NR2B/Grin2B and other ionotropic glutamate receptor genes. Chromatin conformation capture (“3C”) and Setdb1-ChIP revealed a loop formation tethering the NR2B/Grin2b promoter to the Setdb1 target site positioned 30Kb downstream of the transcription start site. In hippocampus and ventral striatum, two key structures in the neuronal circuitry regulating mood-related behaviors, Setdb1-mediated repressive histone methylation at NR2B/Grin2b was associated with decreased NR2B expression and EPSP insensitivity to pharmacological blockade of NR2B, and accelerated NMDA receptor desensitization consistent with a shift in NR2A/B subunit ratios. In wildtype mice, systemic treatment with the NR2B antagonist, Ro-256981, and hippocampal siRNA-mediated NR2B/Grin2b knockdown, resulted in behavioral changes similar to those elicited by the Setdb1 transgene. Together, these findings point to a role for neuronal Setdb1 in the regulation of affective and motivational behaviors through repressive chromatin remodeling at a select set of target genes, resulting in altered NMDA receptor subunit composition and other molecular adaptations. PMID:20505083

  5. Enhancement of B-cell receptor signaling by a point mutation of adaptor protein 3BP2 identified in human inherited disease cherubism.

    PubMed

    Ogi, Kazuhiro; Nakashima, Kenji; Chihara, Kazuyasu; Takeuchi, Kenji; Horiguchi, Tomoko; Fujieda, Shigeharu; Sada, Kiyonao

    2011-09-01

    Tyrosine phosphorylation of adaptor protein c-Abl-Src homology 3 (SH3) domain-binding protein-2 (3BP2, also referred to SH3BP2) positively regulates the B-cell antigen receptor (BCR)-mediated signal transduction, leading to the activation of nuclear factor of activated T cells (NFAT). Here we showed the effect of the proline to arginine substitution of 3BP2 in which is the most common mutation in patients with cherubism (P418R) on B-cell receptor signaling. Comparing to the wild type, overexpression of the mutant form of 3BP2 (3BP2-P416R, corresponding to P418R in human protein) enhanced BCR-mediated activation of NFAT. 3BP2-P416R increased the signaling complex formation with Syk, phospholipase C-γ2 (PLC-γ2), and Vav1. In contrast, 3BP2-P416R could not change the association with the negative regulator 14-3-3. Loss of the association mutant that was incapable to associate with 14-3-3 could not mimic BCR-mediated NFAT activation in Syk-deficient cells. Moreover, BCR-mediated phosphorylation of extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was not affected by P416R mutation. These results showed that P416R mutation of 3BP2 causes the gain of function in B cells by increasing the interaction with specific signaling molecules. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  6. Nogo-B Receptor stabilizes Niemann-Pick Type C2 protein and regulates intracellular cholesterol trafficking

    PubMed Central

    Harrison, Kenneth D.; Miao, Robert Qing; Fernandez-Hernándo, Carlos; Suárez, Yajaira; Dávalos, Alberto; Sessa, William C.

    2009-01-01

    Summary The Nogo-B Receptor (NgBR) is a recently identified receptor for the N-terminus of Reticulon 4B/Nogo-B. Other than its role in binding Nogo-B, little is known about the biology of NgBR. To elucidate a basic cellular role for NgBR, we performed a yeast-2-hybrid screen for interacting proteins using the C-terminal domain as bait and identified Niemann-Pick Type C2 protein (NPC2) as an NgBR-interacting protein. NPC2 protein levels are increased in the presence of NgBR and NgBR enhances NPC2 protein stability. NgBR localizes primarily to the endoplasmic reticulum (ER), and regulates the stability of nascent NPC2. RNAi-mediated disruption of NgBR or genetic deficiency in NgBR leads to a decrease in NPC2 levels, increased intracellular cholesterol accumulation and a loss of sterol sensing, all hallmarks of an NPC2 mutation. These data identify NgBR as an NPC2-interacting protein and provide evidence of a role for NgBR in intracellular cholesterol trafficking. PMID:19723497

  7. New insights into the stereochemical requirements of the bradykinin B2 receptor antagonists binding

    NASA Astrophysics Data System (ADS)

    Lupala, Cecylia S.; Gomez-Gutierrez, Patricia; Perez, Juan J.

    2016-01-01

    Bradykinin (BK) is a member of the kinin family, released in response to inflammation, trauma, burns, shock, allergy and some cardiovascular diseases, provoking vasodilatation and increased vascular permeability among other effects. Their actions are mediated through at least two G-protein coupled receptors, B1 a receptor up-regulated during inflammation episodes or tissue trauma and B2 that is constitutively expressed in a variety of cell types. The goal of the present work is to carry out a structure-activity study of BK B2 antagonism, taking into account the stereochemical features of diverse non-peptide antagonists and the way these features translate into ligand anchoring points to complementary regions of the receptor, through the analysis of the respective ligand-receptor complex. For this purpose an atomistic model of the BK B2 receptor was built by homology modeling and subsequently refined embedded in a lipid bilayer by means of a 600 ns molecular dynamics trajectory. The average structure from the last hundred nanoseconds of the molecular dynamics trajectory was energy minimized and used as model of the receptor for docking studies. For this purpose, a set of compounds with antagonistic profile, covering maximal diversity were selected from the literature. Specifically, the set of compounds include Fasitibant, FR173657, Anatibant, WIN64338, Bradyzide, CHEMBL442294, and JSM10292. Molecules were docked into the BK B2 receptor model and the corresponding complexes analyzed to understand ligand-receptor interactions. The outcome of this study is summarized in a 3D pharmacophore that explains the observed structure-activity results and provides insight into the design of novel molecules with antagonistic profile. To prove the validity of the pharmacophore hypothesized a virtual screening process was also carried out. The pharmacophore was used as query to identify new hits using diverse databases of molecules. The results of this study revealed a set of new

  8. Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.

    PubMed

    Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

    2003-01-01

    Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.

  9. AHR-16303B, a novel antagonist of 5-HT2 receptors and voltage-sensitive calcium channels

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Barrett, R.J.; Appell, K.C.; Kilpatrick, B.F.

    1991-01-01

    In vivo and in vitro methods were used to characterize AHR-16303B, a novel compound with antagonistic action at 5-HT2 receptors and voltage-sensitive calcium channels. The 5-HT2 receptor-antagonistic properties of AHR-16303B were demonstrated by inhibition of (a) (3H)ketanserin binding to rat cerebral cortical membranes (IC50 = 165 nM); (b) 5-hydroxytryptamine (5-HT)-induced foot edema in rats (minimum effective dose, (MED) = 0.32 mg/kg orally, p.o.); (c) 5-HT-induced vasopressor responses in spontaneously hypertensive rats (SHR) (ID50 = 0.18 mg/kg intravenously (i.v.), 1.8 mg/kg p.o.), (d) 5-HT-induced antidiuresis in rats (MED = 1 mg/kg p.o.), and (e) platelet aggregation induced by 5-HT + ADPmore » (IC50 = 1.5 mM). The calcium antagonist properties of AHR-16303B were demonstrated by inhibition of (a) (3H)nimodipine binding to voltage-sensitive calcium channels on rabbit skeletal muscle membranes (IC50 = 15 nM), (b) KCl-stimulated calcium flux into cultured PC12 cells (IC50 = 81 nM), and (c) CaCl2-induced contractions of rabbit thoracic aortic strips (pA2 = 8.84). AHR-16303B had little or no effect on binding of radioligands to dopamine2 (DA2) alpha 1, alpha 2, H1, 5-HT1 alpha, beta 2, muscarinic M1, or sigma opioid receptors; had no effect on 5-HT3 receptor-mediated vagal bradycardia; and had only minor negative inotropic, chronotropic, and dromotropic effects on isolated guinea pig atria. In conscious SHR, 30 mg/kg p.o. AHR-16303B completely prevented the vasopressor responses to i.v. 5-HT, and decreased blood pressure (BP) by 24% 3 h after dosing.« less

  10. Cloning and expression of the recombinant crustacean hyperglycemic hormone isoform B2 (rCHH-B2) and its effects on the metabolism and osmoregulation of the Pacific white shrimp Litopenaeus vannamei.

    PubMed

    Camacho-Jiménez, Laura; Sánchez-Castrejón, Edna; Díaz, Fernando; Aguilar, Manuel B; Muñoz-Márquez, Ma Enriqueta; Ponce-Rivas, Elizabeth

    2017-11-01

    Crustacean hyperglycemic hormones (CHHs) are multifunctional neuropeptides ubiquitous in crustaceans. In Litopenaeus vannamei, CHH-B2 is a CHH eyestalk isoform whose expression has been shown to vary with enviromental conditions, suggesting its relevance for ecophysiological performance of shrimp, controlling processes related to metabolism and osmo-ionic regulation. To study the involvement of CHH-B2 in these processes, we cloned and expressed a recombinant version with a free C-terminal glycine (rCHH-B2-Gly) in the methylotrophic yeast Pichia pastoris. The rCHH-B2-Gly peptide secreted to the culture medium was purified by RP-HPLC and used for in vivo glucose, triglyceride, and osmoregulation dose-response analyses with juvenile shrimp. The peptide was also amidated at the C-terminus using an α-amidating enzyme to produce rCHH-B2-amide. The shrimp showed a dose-dependent effect of rCHH-B2-Gly to hemolymph glucose and triglyceride levels, inducing maximal increases by injecting 500 and 1000pmol of hormone, respectively. Additionally, 10pmol of hormone was sufficient to reduce the hypo-osmoregulatory capacity of shrimp at 35‰. These findings suggest that CHH-B2 has regulatory roles in carbohydrate and lipid metabolism, and a potential involvement in osmoregulation of L. vannamei. Injection of 100pmol of rCHH-B2-amide increased glucose and triglyceride levels by 15 and 28%, respectively in comparison with rCHH-B2-Gly, suggesting an important role for the C-terminal amidation. Additionally, an in silico structural analysis done with the CHH-B1 and rCHH-B2-Gly peptides suggests that the C-terminal region may be relevant for the activity of the L. vannamei isoforms and explain the functional divergence from other crustacean CHH/CHH-like peptides. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Requirement for ErbB2/ErbB signaling in developing cartilage and bone.

    PubMed

    Fisher, Melanie C; Clinton, Gail M; Maihle, Nita J; Dealy, Caroline N

    2007-08-01

    During endochondral ossification, the skeletal elements of vertebrate limbs form and elongate via coordinated control of chondrocyte and osteoblast differentiation and proliferation. The role of signaling by the ErbB family of receptor tyrosine kinases, which consists of ErbB1 (epidermal growth factor receptor or EGFR), ErbB2, ErbB3 and ErbB4, has been little studied during cartilage and bone development. Signaling by the ErbB network generates a diverse array of cellular responses via formation of ErbB dimers activated by distinct ligands that produce distinct signal outputs. Herstatin is a soluble ErbB2 receptor that acts in a dominant negative fashion to inhibit ErbB signaling by binding to endogenous ErbB receptors, preventing functional dimer formation. Here, we examine the effects of Herstatin on limb skeletal element development in transgenic mice, achieved via Prx1 promoter-driven expression in limb cartilage and bone. The limb skeletal elements of Prx1-Herstatin embryos are shortened, and chondrocyte maturation and osteoblast differentiation are delayed. In addition, proliferation by chondrocytes and periosteal cells of Prx1-Herstatin limb skeletal elements is markedly reduced. Our study identifies requirements for ErbB signaling in the maintenance of chondrocyte and osteoblast proliferation involved in the timely progression of chondrocyte maturation and periosteal osteoblast differentiation.

  12. Comprehensive Binary Interaction Mapping of SH2 Domains via Fluorescence Polarization Reveals Novel Functional Diversification of ErbB Receptors

    PubMed Central

    Ciaccio, Mark F.; Chuu, Chih-pin; Jones, Richard B.

    2012-01-01

    First-generation interaction maps of Src homology 2 (SH2) domains with receptor tyrosine kinase (RTK) phosphosites have previously been generated using protein microarray (PM) technologies. Here, we developed a large-scale fluorescence polarization (FP) methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB) domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry procedures. This

  13. Transcriptional regulation of miR-146b by C/EBPβ LAP2 in esophageal cancer cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Junxia; Shan, Fabo; Xiong, Gang

    2014-03-28

    Highlights: • MiR-146b promotes esophageal cancer cell proliferation. • MiR-146b inhibits esophageal cancer cell apoptosis. • C/EBPβ directly binds to miR-146b promoter conserved region. • MiR-146b is up-regulated by C/EBPβ LAP2 transcriptional activation. - Abstract: Recent clinical study indicated that up-regulation of miR-146b was associated with poor overall survival of patients in esophageal squamous cell carcinoma. However, the underlying mechanism of miR-146b dysregulation remains to be explored. Here we report that miR-146b promotes cell proliferation and inhibits cell apoptosis in esophageal cancer cell lines. Mechanismly, two C/EBPβ binding motifs are located in the miR-146b promoter conserved region. Among the threemore » isoforms of C/EBPβ, C/EBPβ LAP2 positively regulated miR-146b expression and increases miR-146b levels in a dose-dependent manner through transcription activation of miR-146b gene. Together, these results suggest a miR-146b regulatory mechanism involving C/EBPβ, which may contribute to the up-regulation of miR-146b in esophageal squamous cell carcinoma.« less

  14. Dibenzo[b,f][1,4]oxazepines and dibenzo[b,e]oxepines: Influence of the chlorine substitution pattern on the pharmacology at the H1R, H4R, 5-HT2AR and other selected GPCRs.

    PubMed

    Naporra, Franziska; Gobleder, Susanne; Wittmann, Hans-Joachim; Spindler, Julia; Bodensteiner, Michael; Bernhardt, Günther; Hübner, Harald; Gmeiner, Peter; Elz, Sigurd; Strasser, Andrea

    2016-11-01

    Inspired by VUF6884 (7-Chloro-11-(4-methylpiperazin-1-yl)dibenzo[b,f][1,4]oxazepine), reported as a dual H 1 /H 4 receptor ligand (pK i : 8.11 (human H 1 R (hH 1 R)), 7.55 (human H 4 R (hH 4 R))), four known and 28 new oxazepine and related oxepine derivatives were synthesised and pharmacologically characterized at histamine receptors and selected aminergic GPCRs. In contrast to the oxazepine series, within the oxepine series, the new compounds showed high affinity to the hH 1 R (pK i : 6.8-8.7), but no or moderate affinity to the hH 4 R (pK i :≤5.3). For one oxepine derivative (1-(2-Chloro-6,11-dihydrodibenzo[b,e]oxepin-11-yl)-4-methylpiperazine), the enantiomers were separated and the R-enantiomer was identified as the eutomer at the hH 1 R (pK i : 8.83 (R), 7.63 (S)) and the guinea-pig H 1 R (gpH 1 R) (pK i : 8.82 (R), 7.41 (S)). Molecular dynamic studies suggest that the tricyclic core of the compounds is bound in a similar mode into the binding pocket, as described for doxepine in the hH 1 R crystal structure. Moreover, docking studies of all oxepine derivatives at the hH 1 R indicate that the oxygen and the position of the chlorine in the tricyclic core determines, if the R- or the S-enantiomer is the eutomer. For some of the oxazepines and oxepines the affinity to other aminergic GPCRs is in the same range as to hH 1 R or hH 4 R, thus, those compounds have to be classified as dirty drugs. However, one oxazepine derivative (3,7-Dichloro-11-(4-methylpiperazin-1-yl)dibenzo[b,f][1,4]oxazepine was identified as dual hH 1 /h5-HT 2A receptor ligand (pK i : 9.23 (hH 1 R), 8.74 (h5-HT 2A R), ≤7 at other analysed GPCRs), whereas one oxepine derivative (1-(3,8-Dichloro-6,11-dihydrodibenzo[b,e]oxepin-11-yl)-4-methylpiperazine) was identified as selective hH 1 R antagonist (pK i : 8.44 (hH 1 R), ≤6.7 at other analyzed GPCRs). Thus, the pharmacological results suggest that the oxazepine/oxepine moiety and additionally the chlorine substitution pattern toggles

  15. Protease-activated Receptor-2 (PAR-2)-mediated Nf-κB Activation Suppresses Inflammation-associated Tumor Suppressor MicroRNAs in Oral Squamous Cell Carcinoma*

    PubMed Central

    Johnson, Jeff J.; Miller, Daniel L.; Jiang, Rong; Liu, Yueying; Shi, Zonggao; Tarwater, Laura; Williams, Russell; Balsara, Rashna; Sauter, Edward R.; Stack, M. Sharon

    2016-01-01

    Oral cancer is the sixth most common cause of death from cancer with an estimated 400,000 deaths worldwide and a low (50%) 5-year survival rate. The most common form of oral cancer is oral squamous cell carcinoma (OSCC). OSCC is highly inflammatory and invasive, and the degree of inflammation correlates with tumor aggressiveness. The G protein-coupled receptor protease-activated receptor-2 (PAR-2) plays a key role in inflammation. PAR-2 is activated via proteolytic cleavage by trypsin-like serine proteases, including kallikrein-5 (KLK5), or by treatment with activating peptides. PAR-2 activation induces G protein-α-mediated signaling, mobilizing intracellular calcium and Nf-κB signaling, leading to the increased expression of pro-inflammatory mRNAs. Little is known, however, about PAR-2 regulation of inflammation-related microRNAs. Here, we assess PAR-2 expression and function in OSCC cell lines and tissues. Stimulation of PAR-2 activates Nf-κB signaling, resulting in RelA nuclear translocation and enhanced expression of pro-inflammatory mRNAs. Concomitantly, suppression of the anti-inflammatory tumor suppressor microRNAs let-7d, miR-23b, and miR-200c was observed following PAR-2 stimulation. Analysis of orthotopic oral tumors generated by cells with reduced KLK5 expression showed smaller, less aggressive lesions with reduced inflammatory infiltrate relative to tumors generated by KLK5-expressing control cells. Together, these data support a model wherein KLK5-mediated PAR-2 activation regulates the expression of inflammation-associated mRNAs and microRNAs, thereby modulating progression of oral tumors. PMID:26839311

  16. Protease-activated Receptor-2 (PAR-2)-mediated Nf-κB Activation Suppresses Inflammation-associated Tumor Suppressor MicroRNAs in Oral Squamous Cell Carcinoma.

    PubMed

    Johnson, Jeff J; Miller, Daniel L; Jiang, Rong; Liu, Yueying; Shi, Zonggao; Tarwater, Laura; Williams, Russell; Balsara, Rashna; Sauter, Edward R; Stack, M Sharon

    2016-03-25

    Oral cancer is the sixth most common cause of death from cancer with an estimated 400,000 deaths worldwide and a low (50%) 5-year survival rate. The most common form of oral cancer is oral squamous cell carcinoma (OSCC). OSCC is highly inflammatory and invasive, and the degree of inflammation correlates with tumor aggressiveness. The G protein-coupled receptor protease-activated receptor-2 (PAR-2) plays a key role in inflammation. PAR-2 is activated via proteolytic cleavage by trypsin-like serine proteases, including kallikrein-5 (KLK5), or by treatment with activating peptides. PAR-2 activation induces G protein-α-mediated signaling, mobilizing intracellular calcium and Nf-κB signaling, leading to the increased expression of pro-inflammatory mRNAs. Little is known, however, about PAR-2 regulation of inflammation-related microRNAs. Here, we assess PAR-2 expression and function in OSCC cell lines and tissues. Stimulation of PAR-2 activates Nf-κB signaling, resulting in RelA nuclear translocation and enhanced expression of pro-inflammatory mRNAs. Concomitantly, suppression of the anti-inflammatory tumor suppressor microRNAs let-7d, miR-23b, and miR-200c was observed following PAR-2 stimulation. Analysis of orthotopic oral tumors generated by cells with reduced KLK5 expression showed smaller, less aggressive lesions with reduced inflammatory infiltrate relative to tumors generated by KLK5-expressing control cells. Together, these data support a model wherein KLK5-mediated PAR-2 activation regulates the expression of inflammation-associated mRNAs and microRNAs, thereby modulating progression of oral tumors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Structural insights into the extracellular recognition of the human serotonin 2B receptor by an antibody

    PubMed Central

    Wacker, Daniel; Kapoor, Mili; Zhang, Ai; Han, Gye Won; Basu, Shibom; Patel, Nilkanth; Messerschmidt, Marc; Weierstall, Uwe; Liu, Wei; Katritch, Vsevolod; Roth, Bryan L.; Stevens, Raymond C.

    2017-01-01

    Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases. Given the importance of G protein-coupled receptors (GPCRs) as pharmaceutical targets, there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs. Here we present the 3.0-Å resolution structure of a complex between the human 5-hydroxytryptamine 2B (5-HT2B) receptor and an antibody Fab fragment bound to the extracellular side of the receptor, determined by serial femtosecond crystallography with an X-ray free-electron laser. The antibody binds to a 3D epitope of the receptor that includes all three extracellular loops. The 5-HT2B receptor is captured in a well-defined active-like state, most likely stabilized by the crystal lattice. The structure of the complex sheds light on the mechanism of selectivity in extracellular recognition of GPCRs by monoclonal antibodies. PMID:28716900

  18. A Significant Role of the Truncated Ghrelin Receptor GHS-R1b in Ghrelin-induced Signaling in Neurons*

    PubMed Central

    Navarro, Gemma; Aguinaga, David; Angelats, Edgar; Medrano, Mireia; Moreno, Estefanía; Mallol, Josefa; Cortés, Antonio; Canela, Enric I.; Casadó, Vicent; McCormick, Peter J.; Lluís, Carme; Ferré, Sergi

    2016-01-01

    The truncated non-signaling ghrelin receptor growth hormone secretagogue R1b (GHS-R1b) has been suggested to simply exert a dominant negative role in the trafficking and signaling of the full and functional ghrelin receptor GHS-R1a. Here we reveal a more complex modulatory role of GHS-R1b. Differential co-expression of GHS-R1a and GHS-R1b, both in HEK-293T cells and in striatal and hippocampal neurons in culture, demonstrates that GHS-R1b acts as a dual modulator of GHS-R1a function: low relative GHS-R1b expression potentiates and high relative GHS-R1b expression inhibits GHS-R1a function by facilitating GHS-R1a trafficking to the plasma membrane and by exerting a negative allosteric effect on GHS-R1a signaling, respectively. We found a preferential Gi/o coupling of the GHS-R1a-GHS-R1b complex in HEK-293T cells and, unexpectedly, a preferential Gs/olf coupling in both striatal and hippocampal neurons in culture. A dopamine D1 receptor (D1R) antagonist blocked ghrelin-induced cAMP accumulation in striatal but not hippocampal neurons, indicating the involvement of D1R in the striatal GHS-R1a-Gs/olf coupling. Experiments in HEK-293T cells demonstrated that D1R co-expression promotes a switch in GHS-R1a-G protein coupling from Gi/o to Gs/olf, but only upon co-expression of GHS-R1b. Furthermore, resonance energy transfer experiments showed that D1R interacts with GHS-R1a, but only in the presence of GHS-R1b. Therefore, GHS-R1b not only determines the efficacy of ghrelin-induced GHS-R1a-mediated signaling but also determines the ability of GHS-R1a to form oligomeric complexes with other receptors, promoting profound qualitative changes in ghrelin-induced signaling. PMID:27129257

  19. Role of RBP2-Induced ER and IGF1R-ErbB Signaling in Tamoxifen Resistance in Breast Cancer.

    PubMed

    Choi, Hee-Joo; Joo, Hyeong-Seok; Won, Hee-Young; Min, Kyueng-Whan; Kim, Hyung-Yong; Son, Taekwon; Oh, Young-Ha; Lee, Jeong-Yeon; Kong, Gu

    2018-04-01

    Despite the benefit of endocrine therapy, acquired resistance during or after treatment still remains a major challenge in estrogen receptor (ER)-positive breast cancer. We investigated the potential role of histone demethylase retinoblastoma-binding protein 2 (RBP2) in endocrine therapy resistance of breast cancer. Survival of breast cancer patients according to RBP2 expression was analyzed in three different breast cancer cohorts including METABRIC (n = 1980) and KM plotter (n = 1764). RBP2-mediated tamoxifen resistance was confirmed by invitro sulforhodamine B (SRB) colorimetric, colony-forming assays, and invivo xenograft models (n = 8 per group). RNA-seq analysis and receptor tyrosine kinase assay were performed to identify the tamoxifen resistance mechanism by RBP2. All statistical tests were two-sided. RBP2 was associated with poor prognosis to tamoxifen therapy in ER-positive breast cancer (P = .04 in HYU cohort, P = .02 in KM plotter, P = .007 in METABRIC, log-rank test). Furthermore, RBP2 expression was elevated in patients with tamoxifen-resistant breast cancer (P = .04, chi-square test). Knockdown of RBP2 conferred tamoxifen sensitivity, whereas overexpression of RBP2 induced tamoxifen resistance invitro and invivo (MCF7 xenograft: tamoxifen-treated control, mean [SD] tumor volume = 70.8 [27.9] mm3, vs tamoxifen-treated RBP2, mean [SD] tumor volume = 387.9 [85.1] mm3, P < .001). Mechanistically, RBP2 cooperated with ER co-activators and corepressors and regulated several tamoxifen resistance-associated genes, including NRIP1, CCND1, and IGFBP4 and IGFBP5. Furthermore, epigenetic silencing of IGFBP4/5 by RBP2-ER-NRIP1-HDAC1 complex led to insulin-like growth factor-1 receptor (IGF1R) activation. RBP2 also increased IGF1R-ErbB crosstalk and subsequent PI3K-AKT activation via demethylase activity-independent ErbB protein stabilization. Combinational treatment with tamoxifen and PI3K inhibitor could overcome RBP2-mediated tamoxifen

  20. Immunocytochemical localization of the NMDA-R2A receptor subunit in the cat retina.

    PubMed

    Goebel, D J; Aurelia, J L; Tai, Q; Jojich, L; Poosch, M S

    1998-10-19

    Immunocytochemical studies were performed to determine the distribution and cellular localization of the NMDA-R2A receptor subunit (R2A) in the cat retina. R2A-immunoreactivity (R2A-IR) was noted in all layers of the retina, with specific localizations in the outer segments of red/green and blue cone photoreceptors, B-type horizontal cells, several types of amacrine cells, Müller cells and the majority of cells in the ganglion cell layer. In the inner nuclear layer, 48% of all cells residing in the amacrine cell layer were R2A-IR including a cell resembling the GABAergic A17 amacrine cell. Interestingly, the AII rod amacrine cell was devoid of R2A-IR. Although the localization of the R2A subunit was anticipated in ganglion cells, amacrines and Müller cells, the presence of this receptor subunit to the cells in the outer retina was not expected. Here, both the R2A and the R2B subunits were found to be present in the outer segments of cone photoreceptors and to the tips of rod outer segments. Although the function of these receptor subunits in rod and cone photoreceptors remains to be determined, the fact that both R2A and R2B receptor subunits are localized to cone outer segments suggests a possible alternative pathway for calcium entry into a region where this cation plays such a crucial role in the process of phototransduction. To further classify the cells that display NR2A-IR, we performed dual labeling experiments showing the relationship between R2A-labeled cells with GABA. Results showed that all GABAergic-amacrines and displaced amacrines express the R2A-subunit protein. In addition, approximately 11% of the NR2A-labeled amacrines, did not stain for GABA. These findings support pharmacological data showing that NMDA directly facilitates GABA release in retina and retinal cultures [I.L. Ferreira, C.B. Duarte, P.F. Santos, C.M. Carvalho, A.P. Carvalho, Release of [3H]GABA evoked by glutamate receptor agonist in cultured chick retinal cells: effect of Ca2

  1. Ionizing Radiation–Inducible miR-27b Suppresses Leukemia Proliferation via Targeting Cyclin A2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Bo; Li, Dongping; Kovalchuk, Anna

    2014-09-01

    Purpose: Ionizing radiation is a common carcinogen that is important for the development of leukemia. However, the underlying epigenetic mechanisms remain largely unknown. The goal of the study was to explore microRNAome alterations induced by ionizing radiation (IR) in murine thymus, and to determine the role of IR-inducible microRNA (miRNA/miR) in the development of leukemia. Methods and Materials: We used the well-established C57BL/6 mouse model and miRNA microarray profiling to identify miRNAs that are differentially expressed in murine thymus in response to irradiation. TIB152 human leukemia cell line was used to determine the role of estrogen receptor–α (ERα) in miR-27bmore » transcription. The biological effects of ectopic miR-27b on leukemogenesis were measured by western immunoblotting, cell viability, apoptosis, and cell cycle analyses. Results: Here, we have shown that IR triggers the differential expression of miR-27b in murine thymus tissue in a dose-, time- and sex-dependent manner. miR-27b was significantly down-regulated in leukemia cell lines CCL119 and TIB152. Interestingly, ERα was overexpressed in those 2 cell lines, and it was inversely correlated with miR-27b expression. Therefore, we used TIB152 as a model system to determine the role of ERα in miR-27b expression and the contribution of miR-27b to leukemogenesis. β-Estradiol caused a rapid and transient reduction in miR-27b expression reversed by either ERα-neutralizing antibody or ERK1/2 inhibitor. Ectopic expression of miR-27b remarkably suppressed TIB152 cell proliferation, at least in part, by inducing S-phase arrest. In addition, it attenuated the expression of cyclin A2, although it had no effect on the levels of PCNA, PPARγ, CDK2, p21, p27, p-p53, and cleaved caspase-3. Conclusion: Our data reveal that β-estradiol/ERα signaling may contribute to the down-regulation of miR-27b in acute leukemia cell lines through the ERK1/2 pathway, and that miR-27b may function as a

  2. The Vaporization of B2O3(l) to B2O3(g) and B2O2(g)

    NASA Technical Reports Server (NTRS)

    Jacobson, Nathan S.; Myers, Dwight L.

    2011-01-01

    The vaporization of B2O3 in a reducing environment leads to formation of both B2O3(g) and B2O2(g). While formation of B2O3(g) is well understood, many questions about the formation of B2O2(g) remain. Previous studies using B(s) + B2O3(l) have led to inconsistent thermodynamic data. In this study, it was found that after heating, B(s) and B2O3(l) appear to separate and variations in contact area likely led to the inconsistent vapor pressures of B2O2(g). To circumvent this problem, an activity of boron is fixed with a two-phase mixture of FeB and Fe2B. Both second and third law enthalpies of formation were measured for B2O2(g) and B2O3(g). From these the enthalpies of formation at 298.15 K are calculated to be -479.9 +/- 41.5 kJ/mol for B2O2(g) and -833.4 +/- 13.1 kJ/mol for B2O3(g). Ab initio calculations to determine the enthalpies of formation of B2O2(g) and B2O3(g) were conducted using the W1BD composite method and show good agreement with the experimental values.

  3. Dm5-HT2B: Pharmacological Characterization of the Fifth Serotonin Receptor Subtype of Drosophila melanogaster

    PubMed Central

    Blenau, Wolfgang; Daniel, Stöppler; Balfanz, Sabine; Thamm, Markus; Baumann, Arnd

    2017-01-01

    Serotonin (5-hydroxytryptamine, 5-HT) is an important regulator of physiological and behavioral processes in both protostomes (e.g., insects) and deuterostomes (e.g., mammals). In insects, serotonin has been found to modulate the heart rate and to control secretory processes, development, circadian rhythms, aggressive behavior, as well as to contribute to learning and memory. Serotonin exerts its activity by binding to and activating specific membrane receptors. The clear majority of these receptors belong to the superfamily of G-protein-coupled receptors. In Drosophila melanogaster, a total of five genes have been identified coding for 5-HT receptors. From this family of proteins, four have been pharmacologically examined in greater detail, so far. While Dm5-HT1A, Dm5-HT1B, and Dm5-HT7 couple to cAMP signaling cascades, the Dm5-HT2A receptor leads to Ca2+ signaling in an inositol-1,4,5-trisphosphate-dependent manner. Based on sequence similarity to homologous genes in other insects, a fifth D. melanogaster gene was uncovered coding for a Dm5-HT2B receptor. Knowledge about this receptor’s pharmacological properties is very limited. This is quite surprising because Dm5-HT2B has been attributed to distinct physiological functions based on genetic interference with its gene expression. Mutations were described reducing the response of the larval heart to 5-HT, and specific knockdown of Dm5-HT2B mRNA in hemocytes resulted in a higher susceptibility of the flies to bacterial infection. To gain deeper understanding of Dm5-HT2B’s pharmacology, we evaluated the receptor’s response to a series of established 5-HT receptor agonists and antagonists in a functional cell-based assay. Metoclopramide and mianserin were identified as two potent antagonists that may allow pharmacological interference with Dm5-HT2B signaling in vitro and in vivo. PMID:28553207

  4. A soluble form of IL-13 receptor alpha 1 promotes IgG2a and IgG2b production by murine germinal center B cells.

    PubMed

    Poudrier, J; Graber, P; Herren, S; Gretener, D; Elson, G; Berney, C; Gauchat, J F; Kosco-Vilbois, M H

    1999-08-01

    A functional IL-13R involves at least two cell surface proteins, the IL-13R alpha 1 and IL-4R alpha. Using a soluble form of the murine IL-13R alpha 1 (sIL-13R), we reveal several novel features of this system. The sIL-13R promotes proliferation and augmentation of Ag-specific IgM, IgG2a, and IgG2b production by murine germinal center (GC) B cells in vitro. These effects were enhanced by CD40 signaling and were not inhibited by an anti-IL4R alpha mAb, a result suggesting other ligands. In GC cell cultures, sIL-13R also promoted IL-6 production, and interestingly, sIL-13R-induced IgG2a and IgG2b augmentation was absent in GC cells isolated from IL-6-deficient mice. Furthermore, the effects of the sIL-13R molecule were inhibited in the presence of an anti-IL-13 mAb, and preincubation of GC cells with IL-13 enhanced the sIL-13R-mediated effects. When sIL-13R was injected into mice, it served as an adjuvant-promoting production to varying degrees of IgM and IgG isotypes. We thus propose that IL-13R alpha 1 is a molecule involved in B cell differentiation, using a mechanism that may involve regulation of IL-6-responsive elements. Taken together, our data reveal previously unknown activities as well as suggest that the ligand for the sIL-13R might be a component of the IL-13R complex or a counterstructure yet to be defined.

  5. Circular RNA circMAN2B2 facilitates lung cancer cell proliferation and invasion via miR-1275/FOXK1 axis.

    PubMed

    Ma, Xuemei; Yang, Xiaodong; Bao, Wenhua; Li, Shumin; Liang, Shanshan; Sun, Yunhui; Zhao, Yunwei; Wang, Jing; Zhao, Chenxu

    2018-04-15

    Lung cancer remains a leading cause of cancer-related deaths worldwide. In the past years, increasing reports indicate that circular RNAs (circRNAs) exert a great important role in human cancers, including lung cancer. However, the knowledge about circRNA in lung cancer remains very little so far. In the present study, we screened out a highly expressed novel circRNA named circMAN2B2 in lung cancer tissues. We investigated the function of circMAN2B2 and found that circMAN2B2 knockdown significantly inhibited cell proliferation and invasion in both H1299 and A549 lung cancer cells. Mechanistically, we found that circMAN2B2 could sponge miR-1275 to inhibit its level. Through a series of functional experiments, we dissected the role of miR-1275 in lung cancer and proved the anti-tumor role of miR-1275. Furthermore, we found that miR-1275 exerted its role in lung cancer by regulating FOXK1 expression. In addition, we demonstrated that restoration of FOXK1 could rescue circMAN2B2 knockdown-induced repression of cell proliferation and invasion. Taken together, our study demonstrated that circMAN2B2 acts as an oncogenic role in lung cancer through promoting FOXK1 expression by sponging miR-1275. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Inhibition of the Activin Receptor Type-2B Pathway Restores Regenerative Capacity in Satellite Cell-Depleted Skeletal Muscle.

    PubMed

    Formicola, Luigi; Pannérec, Alice; Correra, Rosa Maria; Gayraud-Morel, Barbara; Ollitrault, David; Besson, Vanessa; Tajbakhsh, Shahragim; Lachey, Jennifer; Seehra, Jasbir S; Marazzi, Giovanna; Sassoon, David A

    2018-01-01

    Degenerative myopathies typically display a decline in satellite cells coupled with a replacement of muscle fibers by fat and fibrosis. During this pathological remodeling, satellite cells are present at lower numbers and do not display a proper regenerative function. Whether a decline in satellite cells directly contributes to disease progression or is a secondary result is unknown. In order to dissect these processes, we used a genetic model to reduce the satellite cell population by ~70-80% which leads to a nearly complete loss of regenerative potential. We observe that while no overt tissue damage is observed following satellite cell depletion, muscle fibers atrophy accompanied by changes in the stem cell niche cellular composition. Treatment of these mice with an Activin receptor type-2B (AcvR2B) pathway blocker reverses muscle fiber atrophy as expected, but also restores regenerative potential of the remaining satellite cells. These findings demonstrate that in addition to controlling fiber size, the AcvR2B pathway acts to regulate the muscle stem cell niche providing a more favorable environment for muscle regeneration.

  7. Inhibition of the Activin Receptor Type-2B Pathway Restores Regenerative Capacity in Satellite Cell-Depleted Skeletal Muscle

    PubMed Central

    Formicola, Luigi; Pannérec, Alice; Correra, Rosa Maria; Gayraud-Morel, Barbara; Ollitrault, David; Besson, Vanessa; Tajbakhsh, Shahragim; Lachey, Jennifer; Seehra, Jasbir S.; Marazzi, Giovanna; Sassoon, David A.

    2018-01-01

    Degenerative myopathies typically display a decline in satellite cells coupled with a replacement of muscle fibers by fat and fibrosis. During this pathological remodeling, satellite cells are present at lower numbers and do not display a proper regenerative function. Whether a decline in satellite cells directly contributes to disease progression or is a secondary result is unknown. In order to dissect these processes, we used a genetic model to reduce the satellite cell population by ~70–80% which leads to a nearly complete loss of regenerative potential. We observe that while no overt tissue damage is observed following satellite cell depletion, muscle fibers atrophy accompanied by changes in the stem cell niche cellular composition. Treatment of these mice with an Activin receptor type-2B (AcvR2B) pathway blocker reverses muscle fiber atrophy as expected, but also restores regenerative potential of the remaining satellite cells. These findings demonstrate that in addition to controlling fiber size, the AcvR2B pathway acts to regulate the muscle stem cell niche providing a more favorable environment for muscle regeneration. PMID:29881353

  8. Modulation by Cocaine of Dopamine Receptors through miRNA-133b in Zebrafish Embryos

    PubMed Central

    Barreto-Valer, Katherine; López-Bellido, Roger; Macho Sánchez-Simón, Fátima; Rodríguez, Raquel E.

    2012-01-01

    The use of cocaine during pregnancy can affect the mother and indirectly might alter the development of the embryo/foetus. Accordingly, in the present work our aim was to study in vivo (in zebrafish embryos) the effects of cocaine on the expression of dopamine receptors and on miR-133b. These embryos were exposed to cocaine hydrochloride (HCl) at 5 hours post-fertilization (hpf) and were then collected at 8, 16, 24, 48 and 72 hpf to study the expression of dopamine receptors, drd1, drd2a, drd2b and drd3, by quantitative real time PCR (qPCR) and in situ hybridization (ISH, only at 24 hpf). Our results indicate that cocaine alters the expression of the genes studied, depending on the stage of the developing embryo and the type of dopamine receptor. We found that cocaine reduced the expression of miR-133b at 24 and 48 hpf in the central nervous system (CNS) and at the periphery by qPCR and also that the spatial distribution of miR-133b was mainly seen in somites, a finding that suggests the involvement of miR-133b in the development of the skeletal muscle. In contrast, at the level of the CNS miR-133b had a weak and moderate expression at 24 and 48 hpf. We also analysed the interaction of miR-133b with the Pitx3 and Pitx3 target genes drd2a and drd2b, tyrosine hydroxylase (th) and dopamine transporter (dat) by microinjection of the Pitx3-3'UTR sequence. Microinjection of Pitx3-3'UTR affected the expression of pitx3, drd2a, drd2b, th and dat. In conclusion, in the present work we describe a possible mechanism to account for cocaine activity by controlling miR-133b transcription in zebrafish. Via miR-133b cocaine would modulate the expression of pitx3 and subsequently of dopamine receptors, dat and th. These results indicate that miRNAs can play an important role during embryogenesis and in drug addiction. PMID:23285158

  9. Magnetic behavior of R 2Co 14B hydrides (R = La, Pr, Sm, Gd, Tb and Y)

    NASA Astrophysics Data System (ADS)

    Zhang, L. Y.; Pourarian, F.; Wallace, W. E.

    1988-08-01

    The structure and magnetic properties of R 2Co 14B sysstems(R = La, Pr, Nd, Sm, Gd, Tb and Y) and their hydrides were studied by means of bulk magnetometry. All R 2Co 14B hydrides presently studied occur in the tetragonal Nd 2Fe 14B-type crystal structure. The composition-temperature isotherms measured fro selected Gd- and Nd- containing systems exhibit some indication of a platuau pressure at higher hydrogen concentrations. Hydrogenation expands the unit volume, Vc, by 1.5 to 3.0%, depending on the nature of R and the content of hydrogen. It was found that introduction of hydrogen into the lattice decreases Ms of the Co sublittice. This is attributed to the effect of electron charge transfer from Hto Co-3d sublittice. Hydrogennation significantly decreases the anistropy fields, HA, and the spin-reorientation transition temperatur, TSR, for Prand Tb-based intermetallics. The results indicates that the hydrogen makes the compounds magnetically softer, which is attributed to the influence of hydrogen on both the 3d and R sublittices. Two types of spin-reorientation transition for the Nd 2Co 14B system were observed. Hydrogenaration reduces both the low transition temperature, TSR 1, and the high transition temperature, TSR 2, which is explained using the Boltich-Wallace mechanism.

  10. Gentiopicroside attenuates morphine rewarding effect through downregulation of GluN2B receptors in nucleus accumbens.

    PubMed

    Liu, Shui-Bing; Ma, Lan; Guo, Hong-Ju; Feng, Bin; Guo, Yan-Yan; Li, Xiao-Qiang; Sun, Wen-Ji; Zheng, Lian-He; Zhao, Ming-Gao

    2012-08-01

    Gentiopicroside (Gent) is one of the secoiridoid compound isolated from Gentiana lutea. This compound exhibits analgesic activities and inhibits the expression of GluN2B-containing N-methyl-D-aspartate (NMDA) receptors in the anterior cingulate cortex in mice. Nucleus accumbens (NAc) is a forebrain structure known for its role in drug addiction. However, little is known about the role of Gent on morphine dependence and synaptic transmission changes in the NAc. Conditioned place preference (CPP) test and behavioral sensitization of locomotor activity were used to investigate drug-seeking related behaviors. Brain slices containing NAc were prepared, and whole-cell patch-clamp recordings were performed to record the excitatory postsynaptic currents (EPSCs). Expression of proteins was detected by Western blot analysis. Systemic administration of Gent attenuated the CPP effect induced by morphine, but had no effect on morphine-induced behavioral sensitization. Gent significantly reversed overexpression of GluN2B-containing NMDA receptors and dopamine D2 receptors in NAc during the first week of morphine withdrawal. However, the compound did not affect the overexpression of GluN2A-containing NMDA receptors, GluA1, and dopamine D1 receptors. Lastly, Gent significantly reduced NMDA receptors-mediated EPSCs in the NAc. Our study provides strong evidence that Gent inhibits morphine dependence through downregulation of GluN2B-containing NMDA receptors in the NAc. © 2012 Blackwell Publishing Ltd.

  11. The role of serotonin-2 (5-HT2) and dopamine receptors in the behavioral actions of the 5-HT2A/2C agonist, DOI, and putative 5-HT2C inverse agonist, SR46349B.

    PubMed

    Scarlota, Laura C; Harvey, John A; Aloyo, Vincent J

    2011-02-01

    Atypical antipsychotic efficacy is often attributed to actions at serotonin-2 (5-HT(2)) and dopamine receptors, indicating a potential benefit of understanding the interplay between these systems. Currently, it is known that 5-HT(2) receptors modulate dopamine release, although the role of specific dopamine receptors in 5-HT(2)-mediated behavior is not well understood. We examined the role of 5-HT(2A), 5-HT(2C), and dopamine (D1 and D2) receptors in the behavioral response to a 5-HT(2A/2C) agonist (DOI) and 5-HT(2A/2C) antagonist (SR46349B). Effects were assessed by measuring rabbit head bobs (previously characterized as 5-HT(2A) receptor-mediated) and body shakes (5-HT(2C)-mediated). As expected, DOI produced head bobs and body shakes, and these DOI-elicited behaviors were attenuated by the SR46349B pretreatment. Unexpectedly, SR46349B also induced head bobs when administered alone. However, SR46349B-elicited head bobs are distinguishable from those produced by DOI since the 5-HT(2A) antagonist, ketanserin, only attenuated DOI-elicited head bobs. Conversely, 5-HT(2C) ligands (SB242084 and SB206553) inhibited SR46349B but not DOI-induced head bobs. Furthermore, when administered alone, SB206553 (a 5-HT(2C) inverse agonist) produced head bobs, indicating the behavior can be either 5-HT(2A) or 5-HT(2C) mediated. Next, it was revealed that D1 and D2 receptors play a role in DOI-elicited head bobs, but only D1 receptors are required for SR46349B-elicited head bobs. 5-HT(2A) receptor agonism and 5-HT(2C) inverse agonism produce the same behavior, likely due to similar downstream actions at D1 receptors. Consequently, 5-HT(2C) agonism or D1 agonism may be effective therapies for disorders, such as schizophrenia, currently being treated with 5-HT(2A) antagonists.

  12. Immunostimulatory CpG-oligonucleotides induce functional high affinity IL-2 receptors on B-CLL cells: costimulation with IL-2 results in a highly immunogenic phenotype.

    PubMed

    Decker, T; Schneller, F; Kronschnabl, M; Dechow, T; Lipford, G B; Wagner, H; Peschel, C

    2000-05-01

    CpG-oligodeoxynucleotides (CpG-ODN) have been shown to induce proliferation, cytokine production, and surface molecule regulation in normal and malignant human B cells. In the present study, we investigated the potential of CpG-ODN to induce functional high-affinity receptors in leukemic and normal B cells and the effects of costimulation with IL-2 on proliferation, cytokine secretion, and surface molecule regulation. Highly purified B cells from B-CLL patients and normal controls were stimulated with CpG-ODN with or without IL-2. Expression of CD25 was determined using FACS, and the presence of high-affinity IL-2 receptors was determined by scatchard analysis. Costimulatory effects of IL-2 and CpG-ODN were investigated using proliferation assays, ELISA (IL-6, TNF-alpha), and FACS analysis (CD80, CD86 expression). Reactivity of autologous and allogeneic T cells toward activated B-CLL cells was determined in mixed lymphocyte reactions and Interferon-gamma Elispot assays. The CpG-ODN DSP30 caused a significantly stronger induction of the IL-2 receptor alpha chain in malignant as compared with normal B cells (p = 0.03). This resulted in the expression of functional high-affinity IL-2 receptors in B-CLL cells, but fewer numbers of receptors with less affinity were expressed in normal B cells. Although addition of IL-2 to CpG-ODN-stimulated cells augmented proliferation in both normal B cells and B-CLL cells, no costimulatory effect on cytokine production or surface molecule expression could be observed in normal B cells. In contrast, TNF-alpha and IL-6 production was increased in B-CLL cells, and the expression of CD80 and CD86 was further enhanced when IL-2 was used as a costimulus. Autologous and allogeneic immune recognition of B-CLL cells stimulated with CpG-ODN and IL-2 was increased compared with B-CLL cells stimulated with CpG-ODN alone. Stimulation of B-CLL cells with CpG-ODN and IL-2 might be an attractive strategy for potential immunotherapies for B

  13. Phosphatidylserine as an anchor for plasminogen and its plasminogen receptor, Histone H2B, to the macrophage surface

    PubMed Central

    DAS, R.; PLOW, E. F.

    2013-01-01

    Summary Background Plasminogen (Plg) binding to cell surface Plg receptors (Plg-Rs) on the surface of macrophages facilitates Plg activation and migration of these cells. Histone H2B (H2B) acts as a Plg-R and its cell surface expression is upregulated when monocytes are differentiated to macrophages via a pathway dependent on L-type Ca2+ channels and intracellular Ca2+. Objectives We sought to investigate the mechanism by which H2B, a protein without a transmembrane domain, is retained on themacrophage surface. Methods THP-1 monocytoid cells were induced to differentiate with interferon gamma + Vitamin D3 or to undergo apoptosis by treatment with camptothecin. Flow cytometry and cell surface biotinylation followed by Western blotting were used to measure the interrelationship between Plg binding, cell surface expression of H2B and outermembrane exposure of phosphatidylserine (PS). Results H2B interacted directly with PS via an electrostatic interaction. Anti-PS or PS binding proteins, annexin V and protein S, diminished H2B interaction with PS on the surface of differentiated or apoptotic cells and these same reagents inhibited Plg binding to these cells. L-type Ca2+ channels played a significant role in PS exposure, H2B surface expression and Plg binding induced either by differentiation or apoptosis. Conclusions These data suggest that H2B tethers to the surface of cells by interacting with PS on differentiated or apoptotic monocytoid cells. L-type Ca2+ channels regulate PS exposure on the surface of these cells. The exposed PS interacts directly with H2B and hence provides sites for Plg to bind to. PMID:21040449

  14. A Significant Role of the Truncated Ghrelin Receptor GHS-R1b in Ghrelin-induced Signaling in Neurons.

    PubMed

    Navarro, Gemma; Aguinaga, David; Angelats, Edgar; Medrano, Mireia; Moreno, Estefanía; Mallol, Josefa; Cortés, Antonio; Canela, Enric I; Casadó, Vicent; McCormick, Peter J; Lluís, Carme; Ferré, Sergi

    2016-06-17

    The truncated non-signaling ghrelin receptor growth hormone secretagogue R1b (GHS-R1b) has been suggested to simply exert a dominant negative role in the trafficking and signaling of the full and functional ghrelin receptor GHS-R1a. Here we reveal a more complex modulatory role of GHS-R1b. Differential co-expression of GHS-R1a and GHS-R1b, both in HEK-293T cells and in striatal and hippocampal neurons in culture, demonstrates that GHS-R1b acts as a dual modulator of GHS-R1a function: low relative GHS-R1b expression potentiates and high relative GHS-R1b expression inhibits GHS-R1a function by facilitating GHS-R1a trafficking to the plasma membrane and by exerting a negative allosteric effect on GHS-R1a signaling, respectively. We found a preferential Gi/o coupling of the GHS-R1a-GHS-R1b complex in HEK-293T cells and, unexpectedly, a preferential Gs/olf coupling in both striatal and hippocampal neurons in culture. A dopamine D1 receptor (D1R) antagonist blocked ghrelin-induced cAMP accumulation in striatal but not hippocampal neurons, indicating the involvement of D1R in the striatal GHS-R1a-Gs/olf coupling. Experiments in HEK-293T cells demonstrated that D1R co-expression promotes a switch in GHS-R1a-G protein coupling from Gi/o to Gs/olf, but only upon co-expression of GHS-R1b. Furthermore, resonance energy transfer experiments showed that D1R interacts with GHS-R1a, but only in the presence of GHS-R1b. Therefore, GHS-R1b not only determines the efficacy of ghrelin-induced GHS-R1a-mediated signaling but also determines the ability of GHS-R1a to form oligomeric complexes with other receptors, promoting profound qualitative changes in ghrelin-induced signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Anti-Ephrin Type-B Receptor 2 (EphB2) and Anti-Three Prime Histone mRNA EXonuclease 1 (THEX1) Autoantibodies in Scleroderma and Lupus

    PubMed Central

    Azzouz, Doua F.; Martin, Gabriel V.; Arnoux, Fanny; Balandraud, Nathalie; Martin, Thierry; Dubucquoi, Sylvain; Hachulla, Eric; Farge-Bancel, Dominique; Tiev, Kiet; Cabane, Jean; Bardin, Nathalie; Chiche, Laurent; Martin, Marielle; Caillet, Eléonore C.; Kanaan, Sami B.; Harlé, Jean Robert; Granel, Brigitte; Diot, Elisabeth; Roudier, Jean; Auger, Isabelle; Lambert, Nathalie C.

    2016-01-01

    In a pilot ProtoArray analysis, we identified 6 proteins out of 9483 recognized by autoantibodies (AAb) from patients with systemic sclerosis (SSc). We further investigated the 6 candidates by ELISA on hundreds of controls and patients, including patients with Systemic Lupus Erythematosus (SLE), known for high sera reactivity and overlapping AAb with SSc. Only 2 of the 6 candidates, Ephrin type-B receptor 2 (EphB2) and Three prime Histone mRNA EXonuclease 1 (THEX1), remained significantly recognized by sera samples from SSc compared to controls (healthy or with rheumatic diseases) with, respectively, 34% versus 14% (P = 2.10−4) and 60% versus 28% (P = 3.10−8). Above all, EphB2 and THEX1 revealed to be mainly recognized by SLE sera samples with respectively 56%, (P = 2.10−10) and 82% (P = 5.10−13). As anti-EphB2 and anti-THEX1 AAb were found in both diseases, an epitope mapping was realized on each protein to refine SSc and SLE diagnosis. A 15-mer peptide from EphB2 allowed to identify 35% of SLE sera samples (N = 48) versus only 5% of any other sera samples (N = 157), including SSc sera samples. AAb titers were significantly higher in SLE sera (P<0.0001) and correlated with disease activity (p<0.02). We could not find an epitope on EphB2 protein for SSc neither on THEX1 for SSc or SLE. We showed that patients with SSc or SLE have AAb against EphB2, a protein involved in angiogenesis, and THEX1, a 3’-5’ exoribonuclease involved in histone mRNA degradation. We have further identified a peptide from EphB2 as a specific and sensitive tool for SLE diagnosis. PMID:27617966

  16. A Novel Aminotetralin-Type Serotonin (5-HT) 2C Receptor-Specific Agonist and 5-HT2A Competitive Antagonist/5-HT2B Inverse Agonist with Preclinical Efficacy for Psychoses

    PubMed Central

    Morgan, Drake; Felsing, Daniel; Kondabolu, Krishnakanth; Rowland, Neil E.; Robertson, Kimberly L.; Sakhuja, Rajeev; Booth, Raymond G.

    2014-01-01

    Development of 5-HT2C agonists for treatment of neuropsychiatric disorders, including psychoses, substance abuse, and obesity, has been fraught with difficulties, because the vast majority of reported 5-HT2C selective agonists also activate 5-HT2A and/or 5-HT2B receptors, potentially causing hallucinations and/or cardiac valvulopathy. Herein is described a novel, potent, and efficacious human 5-HT2C receptor agonist, (−)-trans-(2S,4R)-4-(3′[meta]-bromophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (−)-MBP), that is a competitive antagonist and inverse agonist at human 5-HT2A and 5-HT2B receptors, respectively. (−)-MBP has efficacy comparable to the prototypical second-generation antipsychotic drug clozapine in three C57Bl/6 mouse models of drug-induced psychoses: the head-twitch response elicited by [2,5]-dimethoxy-4-iodoamphetamine; hyperlocomotion induced by MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (dizocilpine maleate)]; and hyperlocomotion induced by amphetamine. (−)-MBP, however, does not alter locomotion when administered alone, distinguishing it from clozapine, which suppresses locomotion. Finally, consumption of highly palatable food by mice was not increased by (−)-MBP at a dose that produced at least 50% maximal efficacy in the psychoses models. Compared with (−)-MBP, the enantiomer (+)-MBP was much less active across in vitro affinity and functional assays using mouse and human receptors and also translated in vivo with comparably lower potency and efficacy. Results indicate a 5-HT2C receptor-specific agonist, such as (−)-MBP, may be pharmacotherapeutic for psychoses, without liability for obesity, hallucinations, heart disease, sedation, or motoric disorders. PMID:24563531

  17. Transport properties of RCo_2B_2C with R = Dy, Ho, and Pr single

    NASA Astrophysics Data System (ADS)

    Duran, Alejandro; Escudero, Roberto

    2002-03-01

    Single crystals of (Dy, Ho, Pr)Co_2B_2C have been grown by a cold copper crucible method. Metallurgical and structural studies indicate that this borocarbide family melts incongruently and crystallizes as a derivative structure of the ThCr_2Si_2. The family accepts rare earth atoms depending on the type of transition metals used to form the compound. For instance with Ni atoms, all lanthanides ranging from the large lanthanum to lutetium ions are reported to form RNi_2B_2C single crystals, so far no single crystals have been obtained when changing Ni by Cobalt. A comparison of the structural parameters of the RCo_2B_2C with the RNiHo, Pr) compounds indicate that the atomic distance between transition metal atoms contracts with the insertion of the Co ion, resulting in an increasing of the c parameter and decreasing volume. Several recent reports published in the current literature related on the physical properties of RCo_2B_2C (R = rare earth metals and Y) have been only performed on polycrystalline samples, they commonly contain small amounts of second phases. High quality single crystals are necessaries in order to better understand the physical properties, such as anisotropy in the transport and in the magnetic properties. In this report we show magnetic susceptibility and resistivity measurements performed in single crystals in the ab-plane and c direction for 2 - 320 K temperature range for the three single crystals of (Dy, Ho, Pr)Co_2B_2C.

  18. Non-selectivity of new bradykinin antagonists for B1 receptors.

    PubMed

    Rhaleb, N E; Gobeil, F; Regoli, D

    1992-01-01

    Two new B1 receptor antagonists, [Hyp3,Thi5,DTic7,Oic8]desArg9-BK and DArg[Hyp3,Thi5,DTic7,Oic8]desArg9-BK were tested in vitro on the rabbit jugular vein and the guinea pig ileum (preparations containing B2 receptors) and on the rabbit aorta (preparation containing B1 receptors) for pharmacological characterization. The results indicate that both compounds are antagonists on both B1 and B2 receptors, are competitive and discriminate between B2A and B2B receptor subtypes.

  19. Dependence of purinergic P2X receptor activity on ectodomain structure.

    PubMed

    He, Mu-Lan; Zemkova, Hana; Stojilkovic, Stanko S

    2003-03-21

    Purinergic receptors (P2XRs) activate and desensitize in response to the binding of extracellular nucleotides in a receptor- and ligand-specific manner, but the structural bases of their ligand preferences and channel kinetics have been incompletely characterized. Here we tested the hypothesis that affinity of agonists for binding domain accounts for a ligand-specific desensitization pattern. We generated chimeras using receptors with variable sensitivity to ATP in order: P2X(4)R > P2X(2a)R = P2X(2b)R P2X(7)R. Chimeras having the ectodomain Ile(66)-Tyr(310) sequence of P2X(2)R and Val(61)-Phe(313) sequence of P2X(7)R in the backbone of P2X(4)R were expressed but were non-functioning channels. P2X(2a) + X(4)R and P2X(2b) + X(4)R chimeras having the Val(66)-Tyr(315) ectodomain sequence of P2X(4)R in the backbones of P2X(2a)R and P2X(2b)R were functional and exhibited increased sensitivity to ligands as compared with both parental receptors. These chimeras also desensitized faster than parental receptors and in a ligand-nonspecific manner. However, like parental P2X(2b)R and P2X(2a)R, chimeric P2X(2b) + X(4)R desensitized more rapidly than P2X(2a) + X(4)R, and the rate of desensitization of P2X(2a)+X(4)R increased by substituting its Arg(371)-Pro(376) intracellular C-terminal sequence with the Glu(376)-Gly(381) sequence of P2X(4)R. These results indicate the relevance of interaction between the ectodomain and flanking regions around the transmembrane domains on ligand potency and receptor activation. Furthermore, the ligand potency positively correlates with the rate of receptor desensitization but does not affect the C-terminal-specific pattern of desensitization.

  20. The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

    PubMed Central

    Zhong, Shumei; Sun, Shihua; Teng, Ba-Bie

    2004-01-01

    Background In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. Methods We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. Results The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene

  1. The role of GluN2B-containing NMDA receptors in short- and long-term fear recall.

    PubMed

    Mikics, Eva; Toth, Mate; Biro, Laszlo; Bruzsik, Biborka; Nagy, Boglarka; Haller, Jozsef

    2017-08-01

    N-methyl-d-aspartate (NMDA) receptors are crucial synaptic elements in long-term memory formation, including the associative learning of fearful events. Although NMDA blockers were consistently shown to inhibit fear memory acquisition and recall, the clinical use of general NMDA blockers is hampered by their side effects. Recent studies revealed significant heterogeneity in the distribution and neurophysiological characteristics of NMDA receptors with different GluN2 (NR2) subunit composition, which may have differential role in fear learning and recall. To investigate the specific role of NMDA receptor subpopulations with different GluN2 subunit compositions in the formation of lasting traumatic memories, we contrasted the effects of general NMDA receptor blockade with GluN2A-, GluN2B-, and GluN2C/D subunit selective antagonists (MK-801, PEAQX, Ro25-6981, PPDA, respectively). To investigate acute and lasting consequences, behavioral responses were investigated 1 and 28days after fear conditioning. We found that MK-801 (0.05 and 0.1mg/kg) decreased fear recall at both time points. GluN2B receptor subunit blockade produced highly similar effects, albeit efficacy was somewhat smaller 28days after fear conditioning. Unlike MK-801, Ro25-6981 (3 and 10mg/kg) did not affect locomotor activity in the open-field. In contrast, GluN2A and GluN2C/D blockers (6 and 20mg/kg PEAQX; 3 and 10mg/kg PPDA, respectively) had no effect on conditioned fear recall at any time point and dose. This sharp contrast between GluN2B- and other subunit-containing NMDA receptor function indicates that GluN2B receptor subunits are intimately involved in fear memory formation, and may provide a novel pharmacological target in post-traumatic stress disorder or other fear-related disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. An ErbB2-Muc4 complex in rat ocular surface epithelia.

    PubMed

    Swan, Jeremy S; Arango, Maria E; Carothers Carraway, Coralie A; Carraway, Kermit L

    2002-05-01

    To show the presence and localization of type 1 growth factor receptors (ErbB2, ErbB3 and ErbB4) in rat corneal and conjunctival epithelia and investigate the association of ErbB2 with its intramembrane ligand Muc4. Methacarn-fixed, paraffin-embedded sections of corneas and eyelids from female adult rats were immunocytochemically stained using antibodies against the ErbB receptors and Muc4. Sequential immunoprecipitation and immunoblot analyses were performed on epithelial lysates to investigate the presence of a complex of Muc4 and ErbB2 in corneal and conjunctival epithelia. Immunocytochemical staining demonstrated the presence of ErbB2, ErbB3 and ErbB4 growth factor receptors throughout the rat corneal and conjunctival epithelia. Co-immunoprecipitation of the epithelial lysates demonstrated that Muc4 and ErbB2 are present as a complex. The three type 1 growth factor receptors (ErbB2, ErbB3 and ErbB4) are present in the rat corneal and conjunctival epithelia, and ErbB2 is at least partly associated with Muc4. This demonstration of the presence and localization of these three type 1 growth factor receptors may help in understanding how these receptors contribute to ocular epithelial behavior and functions.

  3. Co-Targeting HER2 and EphB4 Pathways

    DTIC Science & Technology

    2013-09-01

    soluble EphB4 decoy receptor that efficiently blocks EphB4/EphB2 signaling. A phase I study for solid tumors using this agent given intravenously...June 2013 3 . DATES COVERED 4. TITLE AND SUBTITLE Co-Targeting HER2 and EphB4 Pathways 5a. CONTRACT NUMBER W81XWH-11-1-0471 5b. GRANT...13. SUPPLEMENTARY NOTES 14. ABSTRACT Multiple receptor pathways allow for redundancy in growth pathways that are dysregulated in cancer and lead to

  4. Long noncoding RNA FTX regulates cardiomyocyte apoptosis by targeting miR-29b-1-5p and Bcl2l2.

    PubMed

    Long, Bo; Li, Na; Xu, Xi-Xia; Li, Xiao-Xin; Xu, Xin-Jie; Guo, Dan; Zhang, Dong; Wu, Zhi-Hong; Zhang, Shu-Yang

    2018-01-01

    Cardiomyocyte apoptosis correlates with the pathogenesis of heart disease. Long noncoding RNA (LncRNA) emerges as a class of noncoding RNAs that regulate gene expression and participate in various cellular processes. However, the role of lncRNAs in cardiomyocyte apoptosis remains to be elucidated. In our study, we found that lncRNA FTX is significantly down-regulated upon ischemia/reperfusion injury and hydrogen peroxide treatment. Enhanced expression of FTX inhibits cardiomyocyte apoptosis induced by hydrogen peroxide. miR-29b-1-5p was found to interact with FTX and regulate the expression of Bcl2l2. Inhibition of miR-29b-1-5p attenuated cardiomyocyte apoptosis upon hydrogen peroxide treatment. We then found that FTX functions as endogenous sponge for miR-29b-1-5p and regulates the activity of miR-29b-1-5p. The results demonstrate that FTX regulates cardiomyocyte apoptosis through modulating the expression of Bcl2l2 which is mediated by miR-29b-1-5p. Our findings reveal a novel regulatory model which is composed of FTX, miR-29b-1-5p and Bcl2l2. Manipulating of their levels may become a new approach to tackling cardiomyocyte apoptosis related heart diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Ephrin-B2 prevents N-methyl-D-aspartate receptor antibody effects on memory and neuroplasticity.

    PubMed

    Planagumà, Jesús; Haselmann, Holger; Mannara, Francesco; Petit-Pedrol, Mar; Grünewald, Benedikt; Aguilar, Esther; Röpke, Luise; Martín-García, Elena; Titulaer, Maarten J; Jercog, Pablo; Graus, Francesc; Maldonado, Rafael; Geis, Christian; Dalmau, Josep

    2016-09-01

    To demonstrate that ephrin-B2 (the ligand of EphB2 receptor) antagonizes the pathogenic effects of patients' N-methyl-D-aspartate receptor (NMDAR) antibodies on memory and synaptic plasticity. One hundred twenty-two C57BL/6J mice infused with cerebrospinal fluid (CSF) from patients with anti-NMDAR encephalitis or controls, with or without ephrin-B2, were investigated. CSF was infused through ventricular catheters connected to subcutaneous osmotic pumps over 14 days. Memory, behavioral tasks, locomotor activity, presence of human antibodies specifically bound to hippocampal NMDAR, and antibody effects on the density of cell-surface and synaptic NMDAR and EphB2 were examined at different time points using reported techniques. Short- and long-term synaptic plasticity were determined in acute brain sections; the Schaffer collateral pathway was stimulated and the field excitatory postsynaptic potentials were recorded in the CA1 region of the hippocampus. Mice infused with patients' CSF, but not control CSF, developed progressive memory deficit and depressive-like behavior along with deposits of NMDAR antibodies in the hippocampus. These findings were associated with a decrease of the density of cell-surface and synaptic NMDAR and EphB2, and marked impairment of long-term synaptic plasticity without altering short-term plasticity. Administration of ephrin-B2 prevented the pathogenic effects of the antibodies in all the investigated paradigms assessing memory, depressive-like behavior, density of cell-surface and synaptic NMDAR and EphB2, and long-term synaptic plasticity. Administration of ephrin-B2 prevents the pathogenic effects of anti-NMDAR encephalitis antibodies on memory and behavior, levels of cell-surface NMDAR, and synaptic plasticity. These findings reveal a strategy beyond immunotherapy to antagonize patients' antibody effects. Ann Neurol 2016;80:388-400. © 2016 American Neurological Association.

  6. Ephrin-B3 regulates glutamate receptor signaling at hippocampal synapses

    PubMed Central

    Antion, Marcia D.; Christie, Louisa A.; Bond, Allison M.; Dalva, Matthew B.; Contractor, Anis

    2010-01-01

    B-ephrin - EphB receptor signaling modulates NMDA receptors by inducing tyrosine phosphorylation of NR2 subunits. Ephrins and EphB RTKs are localized to postsynaptic compartments in the CA1, and therefore potentially interact in a non-canonical cis-configuration. However, it is not known whether cis- configured receptor-ligand signaling is utilized by this class of RTKs, and whether this might influence excitatory synapses. We found that ablation of ephrin-B3 results in an enhancement of the NMDA receptor component of synaptic transmission relative to the AMPA receptor component in CA1 synapses. Synaptic AMPA receptor expression is reduced in ephrin-B3 knockout mice, and there is a marked enhancement of tyrosine phosphorylation of the NR2B receptor subunit. In a reduced system co-expression of ephrin-B3 attenuated EphB2-mediated NR2B tyrosine phosphorylation. Moreover, phosphorylation of EphB2 was elevated in the hippocampus of ephrin-B3 knockout mice, suggesting that regulation of EphB2 activity is lost in these mice. Direct activation of EphB RTKs resulted in phosphorylation of NR2B and a potential signaling partner, the non-receptor tyrosine kinase Pyk2. Our data suggests that ephrin-B3 limits EphB RTK-mediated phosphorylation of the NR2B subunit through an inhibitory cis- interaction which is required for the correct function of glutamatergic CA1 synapses. PMID:20678574

  7. Adaptor protein SH2-B linking receptor-tyrosine kinase and Akt promotes adipocyte differentiation by regulating peroxisome proliferator-activated receptor gamma messenger ribonucleic acid levels.

    PubMed

    Yoshiga, Daigo; Sato, Naoichi; Torisu, Takehiro; Mori, Hiroyuki; Yoshida, Ryoko; Nakamura, Seiji; Takaesu, Giichi; Kobayashi, Takashi; Yoshimura, Akihiko

    2007-05-01

    Adipocyte differentiation is regulated by insulin and IGF-I, which transmit signals by activating their receptor tyrosine kinase. SH2-B is an adaptor protein containing pleckstrin homology and Src homology 2 (SH2) domains that have been implicated in insulin and IGF-I receptor signaling. In this study, we found a strong link between SH2-B levels and adipogenesis. The fat mass and expression of adipogenic genes including peroxisome proliferator-activated receptor gamma (PPARgamma) were reduced in white adipose tissue of SH2-B-/- mice. Reduced adipocyte differentiation of SH2-B-deficient mouse embryonic fibroblasts (MEFs) was observed in response to insulin and dexamethasone, whereas retroviral SH2-B overexpression enhanced differentiation of 3T3-L1 preadipocytes to adipocytes. SH2-B overexpression enhanced mRNA level of PPARgamma in 3T3-L1 cells, whereas PPARgamma levels were reduced in SH2-B-deficient MEFs in response to insulin. SH2-B-mediated up-regulation of PPARgamma mRNA was blocked by a phosphatidylinositol 3-kinase inhibitor, but not by a MAPK kinase inhibitor. Insulin-induced Akt activation and the phosphorylation of forkhead transcription factor (FKHR/Foxo1), a negative regulator of PPARgamma transcription, were up-regulated by SH2-B overexpression, but reduced in SH2-B-deficient MEFs. These data indicate that SH2-B is a key regulator of adipogenesis both in vivo and in vitro by regulating the insulin/IGF-I receptor-Akt-Foxo1-PPARgamma pathway.

  8. Kinin B1 receptor antagonists containing alpha-methyl-L-phenylalanine: in vitro and in vivo antagonistic activities.

    PubMed

    Gobeil, F; Charland, S; Filteau, C; Perron, S I; Neugebauer, W; Regoli, D

    1999-03-01

    -To protect from metabolism and to improve potency of the AcLys-[D-betaNal7,Ile8]desArg9-bradykinin (BK) (R 715), we prepared and tested 3 analogues containing alpha-methyl-L-Phe ([alphaMe]Phe) in position 5: these are the AcLys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 892), Lys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 913), and AcLys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 914). The new compounds were tested against the contractile effect induced by desArg9BK on 2 B1 receptor bioassays, the human umbilical vein, and the rabbit aorta. Their antagonistic activities were compared with those of the early prototypes (Lys-[Leu8]desArg9BK and [Leu8]desArg9BK) and with other recently described peptide antagonists. The 3 (alphaMe)Phe analogues showed high antagonistic potencies (pA2) at both the human (8.8, 7.7, and 8. 7, respectively) and rabbit (8.6, 7.8, and 8.6, respectively) B1 receptors. No antagonistic effects (pA2<5) were observed on the B2 receptors that mediate the contractile effects of BK on the human umbilical vein, the rabbit jugular vein, and the guinea pig ileum. Moreover, these new B1 antagonists were found to be resistant to in vitro degradation by purified angiotensin-converting enzyme from rabbit lung. The Nalpha-acetylated forms, R 892 and R 914, were resistant to aminopeptidases from human plasma. In vivo antagonistic potencies (ID50) of B1 receptor antagonists were evaluated in anesthetized lipopolysaccharide-treated (for B1 receptor) and nontreated (for B2 receptor) rabbits against the hypotensive effects of exogenous desArg9BK and BK. R 892 efficiently inhibited (ID50 2.8 nmol/kg IV) hypotension induced by desArg9BK without affecting that evoked by BK (ID50 >600 nmol/kg IV). Conversely, the peptide antagonists Lys-Lys-[Hyp3,Igl5,D-Igl7,Oic8]desArg9BK (B 9858) and DArg-[Hyp3,Thi5,D-Tic7,Oic8] desArg9BK (S 0765) showed dual B1/B2 receptor antagonism in vitro and in vivo. It is concluded that R 892 and congeners provide selective

  9. CD22 ligation inhibits downstream B cell receptor signaling and Ca(2+) flux upon activation.

    PubMed

    Sieger, N; Fleischer, S J; Mei, H E; Reiter, K; Shock, A; Burmester, G R; Daridon, C; Dörner, T

    2013-03-01

    CD22 is a surface molecule exclusively expressed on B cells that regulates adhesion and B cell receptor (BCR) signaling as an inhibitory coreceptor of the BCR. Central downstream signaling molecules that are activated upon BCR engagement include spleen tyrosine kinase (Syk) and, subsequently, phospholipase Cγ2 (PLCγ2), which results in calcium (Ca(2+)) mobilization. The humanized anti-CD22 monoclonal antibody epratuzumab is currently being tested in clinical trials. This study was undertaken to determine the potential mechanism by which this drug regulates B cell activation. Purified B cells were preincubated with epratuzumab, and the colocalization of CD22 and CD79α, without BCR engagement, was assessed by confocal microscopy. The phosphorylation of Syk (Y348, Y352) and PLCγ2 (Y759) as well as the Ca(2+) flux in the cells were analyzed by flow cytometry upon stimulation of the BCR and/or Toll-like receptor 9 (TLR-9). The influence of CD22 ligation on BCR signaling was assessed by pretreating the cells with epratuzumab or F(ab')(2) fragment of epratuzumab, in comparison with control cells (medium alone or isotype-matched IgG1). Epratuzumab induced colocalization of CD22 and components of the BCR independent of BCR engagement, and also reduced intracellular Ca(2+) mobilization and diminished the phosphorylation of Syk and PLCγ2 after BCR stimulation in vitro. Inhibition of kinase phosphorylation was demonstrated in both CD27- and CD27+ B cells, and this appeared to be independent of Fc receptor signaling. Preactivation of the cells via the stimulation of TLR-9 did not circumvent the inhibitory effect of epratuzumab on BCR signaling. These findings are consistent with the concept of targeting CD22 to raise the threshold of BCR activation, which could offer therapeutic benefit in patients with autoimmune diseases. Copyright © 2013 by the American College of Rheumatology.

  10. Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses

    PubMed Central

    Ferreira, Joana S; Papouin, Thomas; Ladépêche, Laurent; Yao, Andrea; Langlais, Valentin C; Bouchet, Delphine; Dulong, Jérôme; Mothet, Jean-Pierre; Sacchi, Silvia; Pollegioni, Loredano; Paoletti, Pierre; Oliet, Stéphane Henri Richard; Groc, Laurent

    2017-01-01

    The subunit composition of synaptic NMDA receptors (NMDAR), such as the relative content of GluN2A- and GluN2B-containing receptors, greatly influences the glutamate synaptic transmission. Receptor co-agonists, glycine and D-serine, have intriguingly emerged as potential regulators of the receptor trafficking in addition to their requirement for its activation. Using a combination of single-molecule imaging, biochemistry and electrophysiology, we show that glycine and D-serine relative availability at rat hippocampal glutamatergic synapses regulate the trafficking and synaptic content of NMDAR subtypes. Acute manipulations of co-agonist levels, both ex vivo and in vitro, unveil that D-serine alter the membrane dynamics and content of GluN2B-NMDAR, but not GluN2A-NMDAR, at synapses through a process requiring PDZ binding scaffold partners. In addition, using FRET-based FLIM approach, we demonstrate that D-serine rapidly induces a conformational change of the GluN1 subunit intracellular C-terminus domain. Together our data fuels the view that the extracellular microenvironment regulates synaptic NMDAR signaling. DOI: http://dx.doi.org/10.7554/eLife.25492.001 PMID:28598327

  11. Tiam–Rac signaling mediates trans-endocytosis of ephrin receptor EphB2 and is important for cell repulsion

    PubMed Central

    2016-01-01

    Ephrin receptors interact with membrane-bound ephrin ligands to regulate contact-mediated attraction or repulsion between opposing cells, thereby influencing tissue morphogenesis. Cell repulsion requires bidirectional trans-endocytosis of clustered Eph–ephrin complexes at cell interfaces, but the mechanisms underlying this process are poorly understood. Here, we identified an actin-regulating pathway allowing ephrinB+ cells to trans-endocytose EphB receptors from opposing cells. Live imaging revealed Rac-dependent F-actin enrichment at sites of EphB2 internalization, but not during vesicle trafficking. Systematic depletion of Rho family GTPases and their regulatory proteins identified the Rac subfamily and the Rac-specific guanine nucleotide exchange factor Tiam2 as key components of EphB2 trans-endocytosis, a pathway previously implicated in Eph forward signaling, in which ephrins act as in trans ligands of Eph receptors. However, unlike in Eph signaling, this pathway is not required for uptake of soluble ligands in ephrinB+ cells. We also show that this pathway is required for EphB2-stimulated contact repulsion. These results support the existence of a conserved pathway for EphB trans-endocytosis that removes the physical tether between cells, thereby enabling cell repulsion. PMID:27597758

  12. Solid-solution stability and preferential site-occupancy in (R-R′){sub 2}Fe{sub 14}B compounds

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Colin, C. V.; Dempsey, N. M.; Univ. Grenoble Alpes, Inst NEEL, F-38000 Grenoble

    The rare-earth (R) uniaxial anisotropy of R{sub 2}Fe{sub 14}B compounds with magnetic R atoms (e.g., Nd or Pr) is at the origin of the exceptional hard magnetic properties achieved in magnets based on these compounds. The uniaxial anisotropy found in Ce{sub 2}Fe{sub 14}B is attributed mainly to the magnetism of Fe. Ce is the most abundant R element and there has been much recent effort to fabricate magnets in which Ce is partially substituted for Nd. In the present neutron study of (R{sub 1−x}Ce{sub x}){sub 2}Fe{sub 14}B (R = La or Nd), Ce is found to enter the R{sub 2}Fe{submore » 14}B phase over the entire composition range. The crystallographic parameters decrease with increasing Ce content and the Ce atoms preferentially occupy the smaller 4f sites. It is concluded that Ce in these (RR′){sub 2}Fe{sub 14}B compounds essentially maintains the intermediate valence character found in Ce{sub 2}Fe{sub 14}B. It is proposed that, in this intermediate valence state, Ce weakly contributes to uniaxial anisotropy, thus making a link with the fact that significant coercivity is preserved in Ce-substituted NdFeB magnets.« less

  13. The downregulation of miR-3173 in B-cell acute lymphoblastic leukaemia promotes cell invasion via PTK2.

    PubMed

    Tian, Lijun; Cao, Junhua; Ji, Qiang; Zhang, Chuanling; Qian, Tong; Song, Xianchuan; Huang, Baoshan; Tian, Xinyi

    2017-12-16

    MicroRNAs are important regulators of the pathogenesis of B-cell acute lymphoblastic leukaemia (B-ALL). In this study, we identified miR-3173 and its predicted target gene PTK2 were correspondingly differentially expressed in B-ALL patients. In B-ALL cell lines, CCK-8 proliferation assay revealed that miR-3173 could inhibit the cell proliferation. Moreover, transwell assay revealed that miR-3173 could also inhibit cell migration and invasion in B-ALL cell lines. Luciferase assays revealed that miR-3173 directly bound to the 3'untranslated region of PTK2, and western blotting showed that miR-3173 suppressed the expression of PTK2 at the protein level. Generally, this study indicates that miR-3173 negatively regulates PTK2 and inhibits proliferation and invasion of B-ALL cell lines. Thus, miR-3173 may represent a potential therapeutic molecule for B-ALL intervention. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. The potential role of myocardial serotonin receptor 2B expression in canine dilated cardiomyopathy.

    PubMed

    Fonfara, Sonja; Hetzel, Udo; Oyama, Mark A; Kipar, Anja

    2014-03-01

    Serotonin signalling in the heart is mediated by receptor subtype 2B (5-HTR2B). A contribution of serotonin to valvular disease has been reported, but myocardial expression of 5-HTR2B and its role in canine dilated cardiomyopathy (DCM) is not known. The aim of the present study was to investigate myocardial 5-HTR2B mRNA expression in dogs with DCM and to correlate results with expression of markers for inflammation and remodelling. Myocardial samples from eight healthy dogs, four dogs with DCM, five with cardiac diseases other than DCM and six with systemic non-cardiac diseases were investigated for 5-HTR2B mRNA expression using quantitative PCR (qPCR). The results were compared to mRNA expression of selected cytokines, matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinase (TIMP). Laser microdissection with subsequent qPCR and immunohistochemistry were employed to identify the cells expressing 5-HTR2B. The myocardium of control dogs showed constitutive 5-HTR2B mRNA expression. In dogs with DCM, 5-HTR2B mRNA values were significantly greater than in all other groups, with highest levels of expression in the left ventricle and right atrium. Myocytes were identified as the source of 5-HTR2B mRNA and protein. A significant positive correlation of 5-HTR2B mRNA with expression of several cytokines, MMPs and TIMPs was observed. The findings suggest that serotonin might play a role in normal cardiac structure and function and could contribute to myocardial remodelling and functional impairment in dogs with DCM. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Targeting Sulfotransferase (SULT) 2B1b as a regulator of Cholesterol Metabolism in Prostate Cancer

    DTIC Science & Technology

    2016-10-01

    Approved for Public Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author(s) and...and PCa cell lines and that genetic knock down suppresses LNCaP growth and diminishes androgen receptor ( AR ) activity. It is hypothesized that SULT2B1b...knock down suppresses LNCaP growth and diminishes androgen receptor ( AR ) activity. It is hypothesized that SULT2B1b modulates PCa growth and

  16. Biotransformation of the mineralocorticoid receptor antagonists spironolactone and canrenone by human CYP11B1 and CYP11B2: Characterization of the products and their influence on mineralocorticoid receptor transactivation.

    PubMed

    Schiffer, Lina; Müller, Anne-Rose; Hobler, Anna; Brixius-Anderko, Simone; Zapp, Josef; Hannemann, Frank; Bernhardt, Rita

    2016-10-01

    Spironolactone and its major metabolite canrenone are potent mineralocorticoid receptor antagonists and are, therefore, applied as drugs for the treatment of primary aldosteronism and essential hypertension. We report that both compounds can be converted by the purified adrenocortical cytochromes P450 CYP11B1 and CYP11B2, while no conversion of the selective mineralocorticoid receptor antagonist eplerenone was observed. As their natural function, CYP11B1 and CYP11B2 carry out the final steps in the biosynthesis of gluco- and mineralocorticoids. Dissociation constants for the new exogenous substrates were determined by a spectroscopic binding assay and demonstrated to be comparable to those of the natural substrates, 11-deoxycortisol and 11-deoxycorticosterone. Metabolites were produced at preparative scale with a CYP11B2-dependent Escherichia coli whole-cell system and purified by HPLC. Using NMR spectroscopy, the metabolites of spironolactone were identified as 11β-OH-spironolactone, 18-OH-spironolactone and 19-OH-spironolactone. Canrenone was converted to 11β-OH-canrenone, 18-OH-canrenone as well as to the CYP11B2-specific product 11β,18-diOH-canrenone. Therefore, a contribution of CYP11B1 and CYP11B2 to the biotransformation of drugs should be taken into account and the metabolites should be tested for their potential toxic and pharmacological effects. A mineralocorticoid receptor transactivation assay in antagonist mode revealed 11β-OH-spironolactone as pharmaceutically active metabolite, whereas all other hydroxylation products negate the antagonist properties of spironolactone and canrenone. Thus, human CYP11B1 and CYP11B2 turned out to metabolize steroid-based drugs additionally to the liver-dependent biotransformation of drugs. Compared with the action of the parental drug, changed properties of the metabolites at the target site have been observed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Laminar shear stress regulates endothelial kinin B1 receptor expression and function: potential implication in atherogenesis

    PubMed Central

    Duchene, Johan; Cayla, Cécile; Vessillier, Sandrine; Scotland, Ramona; Yamashiro, Kazuo; Lecomte, Florence; Syed, Irfan; Vo, Phuong; Marrelli, Alessandra; Pitzalis, Costantino; Cipollone, Francesco; Schanstra, Joost; Bascands, Jean-Loup; Hobbs, Adrian J; Perretti, Mauro; Ahluwalia, Amrita

    2009-01-01

    OBJECTIVE The pro-inflammatory phenotype induced by low laminar shear stress (LSS) is implicated in atherogenesis. The kinin B1 receptor (B1R), known to be induced by inflammatory stimuli, exerts many pro-inflammatory effects including vasodilatation and leukocyte recruitment. We investigated whether low LSS is a stimulus for endothelial B1R expression and function. METHODS AND RESULTS Human and mouse atherosclerotic plaques expressed high level of B1R mRNA and protein. In addition, B1R expression was upregulated in the aortic arch (low LSS region) of ApoE-/- mice fed a high fat diet compared to vascular regions of high LSS and animals fed normal chow. Of interest, a greater expression of B1R was noticed in endothelial cells from regions of low LSS in aortic arch of ApoE-/- mice. B1R was also upregulated in human umbilical vein endothelial cells (HUVEC) exposed to low LSS (0-2dyn/cm2) compared to physiological LSS (6-10dyn/cm2): an effect similarly evident in murine vascular tissue perfused ex vivo. Functionally, B1R activation increased prostaglandin and CXCL5 expression in cells exposed to low, but not physiological, LSS. IL-1β and ox-LDL induced B1R expression and function in HUVECs, a response substantially enhanced under low LSS conditions and inhibited by blockade of NFκB activation. CONCLUSION Herein, we show that LSS is a major determinant of functional B1R expression in endothelium. Furthermore, whilst physiological high LSS is a powerful repressor of this inflammatory receptor, low LSS at sites of atheroma are associated with substantial upregulation, identifying this receptor as a potential therapeutic target. CONDENSED ABSTRACT Low laminar shear stress (LSS) underlies the pro-inflammatory processes in atherogenesis. Herein, we demonstrate that whilst physiological LSS represses inflammatory kinin B1 receptor (B1R) expression/function, low atherogenic LSS is associated with profound upregulation of both in atherosclerosis in both humans and animal

  18. The E3 ubiquitin ligase mind bomb-2 (MIB2) protein controls B-cell CLL/lymphoma 10 (BCL10)-dependent NF-κB activation.

    PubMed

    Stempin, Cinthia C; Chi, Liying; Giraldo-Vela, Juan P; High, Anthony A; Häcker, Hans; Redecke, Vanessa

    2011-10-28

    B-cell CLL/lymphoma 10 (BCL10) is crucial for the activation of NF-κB in numerous immune receptor signaling pathways, including the T-cell receptor (TCR) and B-cell receptor signaling pathways. However, the molecular mechanisms that lead to signal transduction from BCL10 to downstream NF-κB effector kinases, such as TAK1 and components of the IKK complex, are not entirely understood. Here we used a proteomic approach and identified the E3 ligase MIB2 as a novel component of the activated BCL10 complex. In vitro translation and pulldown assays suggest direct interaction between BCL10 and MIB2. Overexpression experiments show that MIB2 controls BCL10-mediated activation of NF-κB by promoting autoubiquitination and ubiquitination of IKKγ/NEMO, as well as recruitment and activation of TAK1. Knockdown of MIB2 inhibited BCL10-dependent NF-κB activation. Together, our results identify MIB2 as a novel component of the activated BCL10 signaling complex and a missing link in the BCL10-dependent NF-κB signaling pathway.

  19. Presynaptic muscarinic acetylcholine autoreceptors (M1, M2 and M4 subtypes), adenosine receptors (A1 and A2A) and tropomyosin-related kinase B receptor (TrkB) modulate the developmental synapse elimination process at the neuromuscular junction.

    PubMed

    Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A; Santafé, Manel; Tomàs, Josep

    2016-06-23

    The development of the nervous system involves an initially exuberant production of neurons that make an excessive number of synaptic contacts. The initial overproduction of synapses promotes connectivity. Hebbian competition between axons with different activities (the least active are punished) leads to the loss of roughly half of the overproduced elements and this refines connectivity and increases specificity. The neuromuscular junction is innervated by a single axon at the end of the synapse elimination process and, because of its relative simplicity, has long been used as a model for studying the general principles of synapse development. The involvement of the presynaptic muscarinic ACh autoreceptors may allow for the direct competitive interaction between nerve endings through differential activity-dependent acetylcholine release in the synaptic cleft. Then, the most active ending may directly punish the less active ones. Our previous results indicate the existence in the weakest axons on the polyinnervated neonatal NMJ of an ACh release inhibition mechanism based on mAChR coupled to protein kinase C and voltage-dependent calcium channels. We suggest that this mechanism plays a role in the elimination of redundant neonatal synapses. Here we used confocal microscopy and quantitative morphological analysis to count the number of brightly fluorescent axons per endplate in P7, P9 and P15 transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice. We investigate the involvement of individual mAChR M1-, M2- and M4-subtypes in the control of axonal elimination after the Levator auris longus muscle had been exposed to agonist and antagonist in vivo. We also analysed the role of adenosine receptor subtypes (A1 and A2A) and the tropomyosin-related kinase B receptor. The data show that postnatal axonal elimination is a regulated multireceptor mechanism that guaranteed the monoinnervation of the neuromuscular synapses. The three receptor sets considered (mAChR, AR and TrkB receptors

  20. The NF-κB family member RelB regulates microRNA miR-146a to suppress cigarette smoke-induced COX-2 protein expression in lung fibroblasts.

    PubMed

    Zago, Michela; Rico de Souza, Angela; Hecht, Emelia; Rousseau, Simon; Hamid, Qutayba; Eidelman, David H; Baglole, Carolyn J

    2014-04-21

    Diseases due to cigarette smoke exposure, including chronic obstructive pulmonary disease (COPD) and lung cancer, are associated with chronic inflammation typified by the increased expression of cyclooxygenase-2 (COX-2) protein. RelB is an NF-κB family member that suppresses cigarette smoke induction of COX-2 through an unknown mechanism. The ability of RelB to regulate COX-2 expression may be via miR-146a, a miRNA that attenuates COX-2 in lung fibroblasts. In this study we tested whether RelB attenuation of cigarette smoke-induced COX-2 protein is due to miR-146a. Utilizing pulmonary fibroblasts deficient in RelB expression, together with siRNA knock-down of RelB, we show the essential role of RelB in diminishing smoke-induced COX-2 protein expression despite robust activation of the canonical NF-κB pathway and subsequent induction of Cox-2 mRNA. RelB did not regulate COX-2 protein expression at the level of mRNA stability. Basal levels of miR-146a were significantly lower in Relb-deficient cells and cigarette smoke increased miR-146a expression only in Relb-expressing cells. Inhibition of miR-146a had no effects on Relb expression or induction of Cox-2 mRNA by cigarette smoke but significantly increased COX-2 protein. These data highlight the potential of a RelB-miR-146a axis as a novel regulatory pathway that attenuates inflammation in response to respiratory toxicants. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  1. Targeting Anti-Insulin B Cell Receptors Improves Receptor Editing in Type 1 Diabetes-Prone Mice1, 2, 3

    PubMed Central

    Bonami, Rachel H.; Thomas, James W.

    2015-01-01

    Autoreactive B lymphocytes that commonly arise in the developing repertoire can be salvaged by receptor editing, a central tolerance mechanism that alters BCR specificity through continued L chain rearrangement. It is unknown whether autoantigens with weak cross-linking potential, such as insulin, elicit receptor editing, or if this process is dysregulated in related autoimmunity. To resolve these issues, an editing-competent model was developed in which anti-insulin Vκ125 was targeted to the Igκ locus and paired with anti-insulin VH125Tg. Physiologic, circulating insulin increased RAG-2 expression and was associated with BCR replacement that eliminated autoantigen recognition in a proportion of developing anti-insulin B lymphocytes. The proportion of anti-insulin B cells that underwent receptor editing was reduced in the type 1 diabetes-prone NOD strain relative to a non-autoimmune strain. Resistance to editing was associated with increased surface IgM expression on immature (but not transitional or mature) anti-insulin B cells in the NOD strain. The actions of mAb123 on central tolerance were also investigated, as selective targeting of insulin-occupied BCR by mAb123 eliminates anti-insulin B lymphocytes and prevents type 1 diabetes. Autoantigen-targeting by mAb123 increased RAG-2 expression and dramatically enhanced BCR replacement in newly developed B lymphocytes. Administering F(ab’)2123 induced IgM downregulation and reduced the frequency of anti-insulin B lymphocytes within the polyclonal repertoire of VH125Tg/NOD mice, suggesting enhanced central tolerance by direct BCR interaction. These findings indicate that weak or faulty checkpoints for central tolerance can be overcome by autoantigen-specific immunomodulatory therapy. PMID:26432895

  2. Polycomb Repressive Complex 2 Regulates MiR-200b in Retinal Endothelial Cells: Potential Relevance in Diabetic Retinopathy

    PubMed Central

    Ruiz, Michael Anthony; Feng, Biao; Chakrabarti, Subrata

    2015-01-01

    Glucose-induced augmented vascular endothelial growth factor (VEGF) production is a key event in diabetic retinopathy. We have previously demonstrated that downregulation of miR-200b increases VEGF, mediating structural and functional changes in the retina in diabetes. However, mechanisms regulating miR-200b in diabetes are not known. Histone methyltransferase complex, Polycomb Repressive Complex 2 (PRC2), has been shown to repress miRNAs in neoplastic process. We hypothesized that, in diabetes, PRC2 represses miR-200b through its histone H3 lysine-27 trimethylation mark. We show that human retinal microvascular endothelial cells exposed to high levels of glucose regulate miR-200b repression through histone methylation and that inhibition of PRC2 increases miR-200b while reducing VEGF. Furthermore, retinal tissue from animal models of diabetes showed increased expression of major PRC2 components, demonstrating in vivo relevance. This research established a repressive relationship between PRC2 and miR-200b, providing evidence of a novel mechanism of miRNA regulation through histone methylation. PMID:25884496

  3. Phenformin inhibits growth and epithelial-mesenchymal transition of ErbB2-overexpressing breast cancer cells through targeting the IGF1R pathway.

    PubMed

    Guo, Zhiying; Zhao, Ming; Howard, Erin W; Zhao, Qingxia; Parris, Amanda B; Ma, Zhikun; Yang, Xiaohe

    2017-09-01

    Reports suggest that metformin, a popular anti-diabetes drug, prevents breast cancer through various systemic effects, including insulin-like growth factor receptor (IGFR) regulation. Although the anti-cancer properties of metformin have been well-studied, reports on a more bioavailable/potent biguanide, phenformin, remain sparse. Phenformin exerts similar functional activity to metformin and has been reported to impede mammary carcinogenesis in rats. Since the effects of phenformin on specific breast cancer subtypes have not been fully explored, we used ErbB2-overexpressing breast cancer cell and animal models to test the anti-cancer potential of phenformin. We report that phenformin (25-75 μM) decreased cell proliferation and impaired cell cycle progression in SKBR3 and 78617 breast cancer cells. Reduced tumor size after phenformin treatment (30 mg/kg/day) was demonstrated in an MMTV-ErbB2 transgenic mouse syngeneic tumor model. Phenformin also blocked epithelial-mesenchymal transition, decreased the invasive phenotype, and suppressed receptor tyrosine kinase signaling, including insulin receptor substrate 1 and IGF1R, in ErbB2-overexpressing breast cancer cells and mouse mammary tumor-derived tissues. Moreover, phenformin suppressed IGF1-stimulated proliferation, receptor tyrosine kinase signaling, and epithelial-mesenchymal transition markers in vitro . Together, our study implicates phenformin-mediated IGF1/IGF1R regulation as a potential anti-cancer mechanism and supports the development of phenformin and other biguanides as breast cancer therapeutics.

  4. Phenformin inhibits growth and epithelial-mesenchymal transition of ErbB2-overexpressing breast cancer cells through targeting the IGF1R pathway

    PubMed Central

    Guo, Zhiying; Zhao, Ming; Howard, Erin W.; Zhao, Qingxia; Parris, Amanda B.; Ma, Zhikun; Yang, Xiaohe

    2017-01-01

    Reports suggest that metformin, a popular anti-diabetes drug, prevents breast cancer through various systemic effects, including insulin-like growth factor receptor (IGFR) regulation. Although the anti-cancer properties of metformin have been well-studied, reports on a more bioavailable/potent biguanide, phenformin, remain sparse. Phenformin exerts similar functional activity to metformin and has been reported to impede mammary carcinogenesis in rats. Since the effects of phenformin on specific breast cancer subtypes have not been fully explored, we used ErbB2-overexpressing breast cancer cell and animal models to test the anti-cancer potential of phenformin. We report that phenformin (25–75 μM) decreased cell proliferation and impaired cell cycle progression in SKBR3 and 78617 breast cancer cells. Reduced tumor size after phenformin treatment (30 mg/kg/day) was demonstrated in an MMTV-ErbB2 transgenic mouse syngeneic tumor model. Phenformin also blocked epithelial-mesenchymal transition, decreased the invasive phenotype, and suppressed receptor tyrosine kinase signaling, including insulin receptor substrate 1 and IGF1R, in ErbB2-overexpressing breast cancer cells and mouse mammary tumor-derived tissues. Moreover, phenformin suppressed IGF1-stimulated proliferation, receptor tyrosine kinase signaling, and epithelial-mesenchymal transition markers in vitro. Together, our study implicates phenformin-mediated IGF1/IGF1R regulation as a potential anti-cancer mechanism and supports the development of phenformin and other biguanides as breast cancer therapeutics. PMID:28947975

  5. Memory in aged mice is rescued by enhanced expression of the GluN2B subunit of the NMDA receptor

    PubMed Central

    Brim, B. L.; Haskell, R.; Awedikian, R.; Ellinwood, N.M.; Jin, L.; Kumar, A.; Foster, T.C.; Magnusson, K.

    2012-01-01

    The GluN2B subunit of the N-methyl-D-aspartate (NMDA) receptor shows age-related declines in expression across the frontal cortex and hippocampus. This decline is strongly correlated to age-related memory declines. This study was designed to determine if increasing GluN2B subunit expression in the frontal lobe or hippocampus would improve memory in aged mice. Mice were injected bilaterally with either the GluN2B vector, containing cDNA specific for the GluN2B subunit and enhanced Green Fluorescent Protein (eGFP); a control vector or vehicle. Spatial memory, cognitive flexibility, and associative memory were assessed using the Morris water maze. Aged mice, with increased GluN2B subunit expression, exhibited improved long-term spatial memory, comparable to young mice. However, memory was rescued on different days in the Morris water maze; early for hippocampal GluN2B subunit enrichment and later for the frontal lobe. A higher concentration of the GluN2B antagonist, Ro 25-6981, was required to impair long-term spatial memory in aged mice with enhanced GluN2B expression, as compared to aged controls, suggesting there was an increase in the number of GluN2B-containing NMDA receptors. In addition, hippocampal slices from aged mice with increased GluN2B subunit expression exhibited enhanced NMDA receptor-mediated excitatory post-synaptic potentials (EPSP). Treatment with Ro 25-6981 showed that a greater proportion of the NMDA receptor-mediated EPSP was due to the GluN2B subunit in these animals, as compared to aged controls. These results suggest that increasing the production of the GluN2B subunit in aged animals enhances memory and synaptic transmission. Therapies that enhance GluN2B subunit expression within the aged brain may be useful for ameliorating age-related memory declines. PMID:23103326

  6. Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

    PubMed

    Cai, Yingying; Liu, Yuting; Culhane, Kelly J; DeVree, Brian T; Yang, Yang; Sunahara, Roger K; Yan, Elsa C Y

    2017-01-01

    Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

  7. Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor

    PubMed Central

    Cai, Yingying; Liu, Yuting; Culhane, Kelly J.; DeVree, Brian T.; Yang, Yang; Sunahara, Roger K.; Yan, Elsa C. Y.

    2017-01-01

    Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms. PMID:28609478

  8. Endothelins activate Ca(2+)-gated K(+) channels via endothelin B receptors in CD-1 mouse erythrocytes.

    PubMed

    Rivera, A; Rotter, M A; Brugnara, C

    1999-10-01

    Cell dehydration mediated by Ca(2+)-activated K(+) channels plays an important role in the pathogenesis of sickle cell disease. CD-1 mouse erythrocytes possess a Ca(2+)-activated K(+) channel (Gardos channel) with maximal velocity (V(max)) of 0.154 +/- 0.02 mmol. l cells(-1). min(-1) and an affinity constant (K(0.5)) for Ca(2+) of 286 +/- 83 nM in the presence of A-23187. Cells pretreated with 500 nM endothelin-1 (ET-1) increased their V(max) by 88 +/- 9% (n = 8) and decreased their K(0.5) for Ca(2+) to 139 +/- 63 nM (P < 0.05; n = 4). Activation of the Gardos channel resulted in an EC(50) of 75 +/- 20 nM for ET-1 and 374 +/- 97 nM for ET-3. Analysis of the affinity of unlabeled ET-1 for its receptor showed two classes of binding sites with apparent dissociation constants of 167 +/- 51 and 785 +/- 143 nM and with capacity of binding sites of 298 +/- 38 and 1,568 +/- 211 sites/cell, respectively. The Gardos channel was activated by the endothelin B (ET(B)) receptor agonist IRL 1620 and inhibited by BQ-788, demonstrating the involvement of ET(B) receptors. Calphostin C inhibited 73% of ET-1-induced Gardos activation and 84% of the ET-1-induced membrane protein kinase C activity. Thus endothelins regulate erythrocyte Gardos channels via ET(B) receptors and a calphostin-sensitive mechanism.

  9. MEN16132, a novel potent and selective nonpeptide antagonist for the human bradykinin B2 receptor. In vitro pharmacology and molecular characterization.

    PubMed

    Cucchi, Paola; Meini, Stefania; Bressan, Alessandro; Catalani, Claudio; Bellucci, Francesca; Santicioli, Paolo; Lecci, Alessandro; Faiella, Angela; Rotondaro, Luigi; Giuliani, Sandro; Giolitti, Alessandro; Quartara, Laura; Maggi, Carlo Alberto

    2005-12-28

    The pharmacological characterization of the novel nonpeptide antagonist for the B2 receptor, namely MEN16132 (4-(S)-Amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2H-4-pyranylcarbonyl}piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride) is presented. The affinity of MEN16132 for the bradykinin B2 receptor has been investigated by means of competition studies at [3H]bradykinin binding to membranes prepared from Chinese Hamster Ovary (CHO) cells expressing the human bradykinin B2 receptor (pKi 10.5), human lung fibroblasts (pKi 10.5), guinea pig airways (pKi 10.0), guinea pig ileum longitudinal smooth muscle (pKi 10.2), or guinea pig cultured colonic myocytes (pKi 10.3). In all assays MEN16132 was as potent as the peptide antagonist Icatibant, and from 3- to 100-fold more potent than the reference nonpeptide antagonists FR173657 or LF16-0687. The selectivity for the bradykinin B2 receptor was checked at the human bradykinin B1 receptor (pKi<5), and at a panel of 26 different receptors and channels. The antagonist potency was measured in functional assays, i.e., in blocking the bradykinin induced inositolphosphates (IP) accumulation at the human (CHO: pKB 10.3) and guinea pig (colonic myocytes: pKB 10.3) B2 receptor, or in antagonizing the bradykinin induced contractile responses in human (detrusor smooth muscle: pKB 9.9) and guinea pig (ileum longitudinal smooth muscle: pKB 10.1) tissues. In both functional assay types MEN16132 exerted a different antagonist pattern, i.e., surmountable at the human and insurmountable at the guinea pig bradykinin B2 receptors. Moreover, the receptor determinants important for the high affinity interaction of MEN16132 with the human bradykinin B2 receptor were investigated by means of radioligand binding studies performed at 24 point-mutated receptors. The results obtained revealed that residues in transmembrane segment 2 (W86A), 3 (I110A), 6 (W256A), and 7 (Y295A, Y295F but

  10. Toll-Like Receptor 2 Mediates Cellular Activation by the B Subunits of Type II Heat-Labile Enterotoxins

    PubMed Central

    Hajishengallis, George; Tapping, Richard I.; Martin, Michael H.; Nawar, Hesham; Lyle, Elizabeth A.; Russell, Michael W.; Connell, Terry D.

    2005-01-01

    The type II heat-labile enterotoxins (LT-IIa and LT-IIb) of Escherichia coli have an AB5 subunit structure similar to that of cholera toxin (CT) and other type I enterotoxins, despite significant differences in the amino acid sequences of their B subunits and different ganglioside receptor specificities. LT-II holotoxins and their nontoxic B subunits display unique properties as immunological adjuvants distinct from those of CT and its B subunits. In contrast to type II holotoxins, the corresponding pentameric B subunits, LT-IIaB and LT-IIbB, stimulated cytokine release in both human and mouse cells dependent upon Toll-like receptor 2 (TLR2). Induction of interleukin-1β (IL-1β), IL-6, IL-8, or tumor necrosis factor alpha in human THP-1 cells by LT-IIaB or LT-IIbB was inhibited by anti-TLR2 but not by anti-TLR4 antibody. Furthermore, transient expression of TLR1 and TLR2 in human embryonic kidney 293 cells resulted in activation of a nuclear factor-κB-dependent luciferase gene in response to LT-IIaB or LT-IIbB. Moreover, peritoneal macrophages from TLR2-deficient mice failed to respond to LT-IIaB or LT-IIbB, in contrast to wild-type or TLR4-deficient cells. These results demonstrate that besides their established binding to gangliosides, the B subunits of type II enterotoxins also interact with TLR2. Although a ganglioside-nonbinding mutant (T34I) of LT-IIaB effectively induced cytokine release, a phenotypically similar point mutation (T13I) in LT-IIbB abrogated cytokine induction, suggesting a variable requirement for gangliosides as coreceptors in TLR2 agonist activity. TLR2-dependent activation of mononuclear cells by type II enterotoxin B subunits appears to be a novel mechanism whereby these molecules may exert their immunomodulatory and adjuvant activities. PMID:15731031

  11. Targeted mutagenesis of aryl hydrocarbon receptor 2a and 2b genes in Atlantic killifish (Fundulus heteroclitus)

    PubMed Central

    Aluru, Neelakanteswar; Karchner, Sibel I.; Franks, Diana G.; Nacci, Diane; Champlin, Denise; Hahn, Mark E.

    2014-01-01

    Understanding molecular mechanisms of toxicity is facilitated by experimental manipulations, such as disruption of function by gene targeting, that are especially challenging in non-standard model species with limited genomic resources. While loss-of-function approaches have included gene knock-down using morpholino-modified oligonucleotides and random mutagenesis using mutagens or retroviruses, more recent approaches include targeted mutagenesis using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. These latter methods provide more accessible opportunities to explore gene function in non-traditional model species. To facilitate evaluations of toxic mechanisms for important categories of aryl hydrocarbon pollutants, whose actions are known to be receptor mediated, we used ZFN and CRISPR-Cas9 approaches to generate aryl hydrocarbon receptor 2a (AHR2a) and AHR2b gene mutations in Atlantic killifish (Fundulus heteroclitus) embryos. This killifish is a particularly valuble non-traditional model for this study, with multiple paralogs of AHR whose functions are not well characterized. In addition, some populations of this species have evolved resistance to toxicants such as halogenated aromatic hydrocarbons. AHR-null killifish will be valuable for characterizing the role of the individual AHR paralogs in evolved resistance, as well as in normal development. We first used five-finger ZFNs targeting exons 1 and 3 of AHR2a. Subsequently, CRISPR-Cas9 guide RNAs were designed to target regions in exon 2 and 3 of AHR2a and AHR2b. We successfully induced frameshift mutations in AHR2a exon 3 with ZFN and CRISPR-Cas9 guide RNAs, with mutation frequencies of 10% and 16%, respectively. In AHR2b, mutations were induced using CRISPR-Cas9 guide RNAs targeting sites in both exon 2 (17%) and exon 3 (63%). We screened AHR2b exon 2 CRISPR-Cas9-injected embryos for

  12. Homeobox NKX2-3 promotes marginal-zone lymphomagenesis by activating B-cell receptor signalling and shaping lymphocyte dynamics

    PubMed Central

    Robles, Eloy F.; Mena-Varas, Maria; Barrio, Laura; Merino-Cortes, Sara V.; Balogh, Péter; Du, Ming-Qing; Akasaka, Takashi; Parker, Anton; Roa, Sergio; Panizo, Carlos; Martin-Guerrero, Idoia; Siebert, Reiner; Segura, Victor; Agirre, Xabier; Macri-Pellizeri, Laura; Aldaz, Beatriz; Vilas-Zornoza, Amaia; Zhang, Shaowei; Moody, Sarah; Calasanz, Maria Jose; Tousseyn, Thomas; Broccardo, Cyril; Brousset, Pierre; Campos-Sanchez, Elena; Cobaleda, Cesar; Sanchez-Garcia, Isidro; Fernandez-Luna, Jose Luis; Garcia-Muñoz, Ricardo; Pena, Esther; Bellosillo, Beatriz; Salar, Antonio; Baptista, Maria Joao; Hernandez-Rivas, Jesús Maria; Gonzalez, Marcos; Terol, Maria Jose; Climent, Joan; Ferrandez, Antonio; Sagaert, Xavier; Melnick, Ari M.; Prosper, Felipe; Oscier, David G.; Carrasco, Yolanda R.; Dyer, Martin J. S.; Martinez-Climent, Jose A.

    2016-01-01

    NKX2 homeobox family proteins have a role in cancer development. Here we show that NKX2-3 is overexpressed in tumour cells from a subset of patients with marginal-zone lymphomas, but not with other B-cell malignancies. While Nkx2-3-deficient mice exhibit the absence of marginal-zone B cells, transgenic mice with expression of NKX2-3 in B cells show marginal-zone expansion that leads to the development of tumours, faithfully recapitulating the principal clinical and biological features of human marginal-zone lymphomas. NKX2-3 induces B-cell receptor signalling by phosphorylating Lyn/Syk kinases, which in turn activate multiple integrins (LFA-1, VLA-4), adhesion molecules (ICAM-1, MadCAM-1) and the chemokine receptor CXCR4. These molecules enhance migration, polarization and homing of B cells to splenic and extranodal tissues, eventually driving malignant transformation through triggering NF-κB and PI3K-AKT pathways. This study implicates oncogenic NKX2-3 in lymphomagenesis, and provides a valid experimental mouse model for studying the biology and therapy of human marginal-zone B-cell lymphomas. PMID:27297662

  13. Synthesis of t-Butyl (2R)-Hydroxyisovalerate, A Precursor of Aureobasidin B

    NASA Astrophysics Data System (ADS)

    Maharani, R.; Puspitasari, D.; Taufiqqurahman; Huspa, D. H. P.; Hidayat, A. T.; Sumiarsa, D.; Hidayat, I. W.

    2017-02-01

    Aureobasidins are a family of cyclodepsipeptides have antifungal properties. They were isolated from the black yeast Aureobasidium pullulans R106 and over 30 derivatives have been successfully characterized. There are few publications reporting the total synthesis of aureobasidins. The limited reports of the synthesis of the aureobasidin derivatives are due to the difficult access to the preparations of precursors. The aim of this research is to synthesise a precursor of aureobasidin B, t-butyl (2R)-hydroxyisovalerate (t-Bu-Hiv), that is prepared for the total synthesis of aureobasidin B. The synthesis of AbB is planned to be undertaken by using a solid phase method, so the ester formation between t-Bu-Hiv and the Fmoc-β-hydroxymethylvaline will be carried out in solution phase to form depsidipeptide. The t-butyl group was used as protecting agent that is due to the straightforward elimination of the protecting group from the Fmoc-depsidipeptide. The t-Bu-Hiv acid was prepared from D-valine through diazotisation to form (2R)-acetyloxyisovaleric acid in 62.7% yield. Product of the first step was then protected by t-butyl group by using Boc-anhydride in t-butanol to give t-butil (2R)-acetyloxyisovalerate in 44% yield. In the last step, the acetyloxy group was eliminated by using potassium carbonate in methanol/water to give the desired product, t-Bu-Hiv in 33.5% yield. The t-Bu-Hiv is ready to be combined with Fmoc-β-hydroxymethylvaline to result in depsidipeptide that will be attached to the resin in the total synthesis of AbB. Each stage of this synthesis was controlled by thin layer chromatography and all products were purified by open column chromatography. All the synthesized products were characterized by various spectroscopic techniques, including infrared spectrophotometer, mass spectroscopy (ESI-MS), 1H-NMR and 13C-NMR.

  14. A Gammaherpesvirus Bcl-2 Ortholog Blocks B Cell Receptor-Mediated Apoptosis and Promotes the Survival of Developing B Cells In Vivo

    PubMed Central

    Coleman, Carrie B.; McGraw, Jennifer E.; Feldman, Emily R.; Roth, Alexa N.; Keyes, Lisa R.; Grau, Katrina R.; Cochran, Stephanie L.; Waldschmidt, Thomas J.; Liang, Chengyu; Forrest, J. Craig; Tibbetts, Scott A.

    2014-01-01

    Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) establish lifelong latency in their hosts and are associated with the development of several types of malignancies, including a subset of B cell lymphomas. These viruses are thought to co-opt the process of B cell differentiation to latently infect a fraction of circulating memory B cells, resulting in the establishment of a stable latency setpoint. However, little is known about how this infected memory B cell compartment is maintained throughout the life of the host. We have previously demonstrated that immature and transitional B cells are long-term latency reservoirs for murine gammaherpesvirus 68 (MHV68), suggesting that infection of developing B cells contributes to the maintenance of lifelong latency. During hematopoiesis, immature and transitional B cells are subject to B cell receptor (BCR)-mediated negative selection, which results in the clonal deletion of autoreactive B cells. Interestingly, numerous gammaherpesviruses encode homologs of the anti-apoptotic protein Bcl-2, suggesting that virus inhibition of apoptosis could subvert clonal deletion. To test this, we quantified latency establishment in mice inoculated with MHV68 vBcl-2 mutants. vBcl-2 mutant viruses displayed a marked decrease in the frequency of immature and transitional B cells harboring viral genome, but this attenuation could be rescued by increased host Bcl-2 expression. Conversely, vBcl-2 mutant virus latency in early B cells and mature B cells, which are not targets of negative selection, was remarkably similar to wild-type virus. Finally, in vivo depletion of developing B cells during chronic infection resulted in decreased mature B cell latency, demonstrating a key role for developing B cells in the maintenance of lifelong latency. Collectively, these findings support a model in which gammaherpesvirus latency in circulating mature B cells is sustained in part through the

  15. Valsartan ameliorates KIR2.1 in rats with myocardial infarction via the NF-κB-miR-16 pathway.

    PubMed

    Li, Xinran; Hu, Hesheng; Wang, Ye; Xue, Mei; Li, Xiaolu; Cheng, Wenjuan; Xuan, Yongli; Yin, Jie; Yang, Na; Yan, Suhua

    2016-09-30

    MicroRNAs have an important role in regulating arrhythmogenesis. MicroRNA-16 (miR-16) is predicted to target KCNJ2. The regulation of miR-16 is primarily due to NF-κB. Whether valsartan could downregulate miR-16 via the inhibition of NF-κB after MI and whether miR-16 targets KCNJ2 remain unclear. MI rats received valsartan or saline for 7days. The protein levels of NF-κB p65, inhibitor κBα (IκBα), and Kir2.1 were detected by Western blot analysis. The mRNA levels of Kir2.1 and miR-16 were examined by quantitative real-time PCR. Whole cell patch-clamp techniques were applied to record IK1. MiR-16 expression was higher in the infarct border, and was accompanied by a depressed IK1/KIR2.1 level. Additionally, miR-16 overexpression suppressed KCNJ2/KIR2.1 expression. In contrast, miR-16 inhibition or binding-site mutation enhanced KCNJ2/KIR2.1 expression, establishing KCNJ2 as a miR-16 target. In the MI rats, compared to saline treatment, valsartan reduced NF-κB p65 and miR-16 expression and increased IκBα and Kir2.1 expression. In vitro, angiotensin II increased miR-16 expression and valsartan inhibited it. Overexpressing miR-16 in cells treated with valsartan abrogated its beneficial effect on KCNJ2/Kir2.1. NF-κB activation directly upregulates miR-16 expression. miR-16 controls KCNJ2 expression, and valsartan ameliorates Kir2.1 after MI partly depending on the NF-κB-miR-16 pathway. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Neuron-to-glia signaling mediated by excitatory amino acid receptors regulates ErbB receptor function in astroglial cells of the neuroendocrine brain.

    PubMed

    Dziedzic, Barbara; Prevot, Vincent; Lomniczi, Alejandro; Jung, Heike; Cornea, Anda; Ojeda, Sergio R

    2003-02-01

    Hypothalamic astroglial erbB tyrosine kinase receptors are required for the timely initiation of mammalian puberty. Ligand-dependent activation of these receptors sets in motion a glia-to-neuron signaling pathway that prompts the secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual development, from hypothalamic neuroendocrine neurons. The neuronal systems that may regulate this growth factor-mediated back signaling to neuroendocrine neurons have not been identified. Here we demonstrate that hypothalamic astrocytes contain metabotropic receptors of the metabotropic glutamate receptor 5 subtype and the AMPA receptor subunits glutamate receptor 2 (GluR2) and GluR3. As in excitatory synapses, these receptors are in physical association with their respective interacting/clustering proteins Homer and PICK1. In addition, they are associated with erbB-1 and erbB-4 receptors. Concomitant activation of astroglial metabotropic and AMPA receptors results in the recruitment of erbB tyrosine kinase receptors and their respective ligands to the glial cell membrane, transactivation of erbB receptors via a mechanism requiring metalloproteinase activity, and increased erbB receptor gene expression. By facilitating erbB-dependent signaling and promoting erbB receptor gene expression in astrocytes, a neuron-to-glia glutamatergic pathway may represent a basic cell-cell communication mechanism used by the neuroendocrine brain to coordinate the facilitatory transsynaptic and astroglial input to LHRH neurons during sexual development.

  17. Site-preference and valency for rare-earth sites in (R-Ce)2Fe14B magnets

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alam, Aftab; Khan, Mahmud; McCallum, R. W.

    2013-01-28

    Rare-earth (R) permanent magnets of R2Fe14B have technological importance due to their high energy products, and they have two R-sites (Wyckoff 4f and 4g, with four-fold multiplicity) that affect chemistry and valence. Designing magnetic behavior and stability via alloying is technologically relevant to reduce critical (expensive) R-content while retaining key properties; cerium, an abundant (cheap) R-element, offers this potential. We calculate magnetic properties and Ce site preference in (R1-xCex)2Fe14B [R=La,Nd] using density functional theory (DFT) methods—including a DFT+U scheme to treat localized 4f-electrons. Fe moments compare well with neutron data—almost unaffected by Hubbard U, and weakly affected by spin-orbit coupling.more » In La2Fe14B, Ce alloys for 0 ≤ x ≤ 1 and prefers smaller R(4f) sites, as observed, a trend we find unaffected by valence. Whereas, in Nd2Fe14B, Ce is predicted to have limited alloying (x ≤ 0.3) with a preference for larger R(4g) sites, resulting in weak partial ordering and segregation. The Curie temperatures versus x for (Nd,Ce) were predicted for a typical sample processing and verified experimentally.« less

  18. miR-125b controls apoptosis and temozolomide resistance by targeting TNFAIP3 and NKIRAS2 in glioblastomas

    PubMed Central

    Haemmig, S; Baumgartner, U; Glück, A; Zbinden, S; Tschan, M P; Kappeler, A; Mariani, L; Vajtai, I; Vassella, E

    2014-01-01

    Diffusely infiltrating gliomas are among the most prognostically discouraging neoplasia in human. Temozolomide (TMZ) in combination with radiotherapy is currently used for the treatment of glioblastoma (GBM) patients, but less than half of the patients respond to therapy and chemoresistance develops rapidly. Epigenetic silencing of the O6-methylguanine-DNA methyltransferase (MGMT) has been associated with longer survival in GBM patients treated with TMZ, but nuclear factor κB (NF-κB)-mediated survival signaling and TP53 mutations contribute significantly to TMZ resistance. Enhanced NF-κB is in part owing to downregulation of negative regulators of NF-κB activity, including Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and NF-κB inhibitor interacting RAS-like 2 (NKIRAS2). Here we provide a novel mechanism independent of TP53 and MGMT by which oncogenic miR-125b confers TMZ resistance by targeting TNFAIP3 and NKIRAS2. GBM cells overexpressing miR-125b showed increased NF-κB activity and upregulation of anti-apoptotic and cell cycle genes. This was significantly associated with resistance of GBM cells to TNFα- and TNF-related inducing ligand-induced apoptosis as well as resistance to TMZ. Conversely, overexpression of anti-miR-125b resulted in cell cycle arrest, increased apoptosis and increased sensitivity to TMZ, indicating that endogenous miR-125b is sufficient to control these processes. GBM cells overexpressing TNFAIP3 and NKIRAS2 were refractory to miR-125b-induced apoptosis resistance as well as TMZ resistance, indicating that both genes are relevant targets of miR-125b. In GBM tissues, high miR-125b expression was significantly correlated with nuclear NF-κB confirming that miR-125b is implicated in NF-κB signaling. Most remarkably, miR-125b overexpression was clearly associated with shorter overall survival of patients treated with TMZ, suggesting that this microRNA is an important predictor of response to therapy. PMID:24901050

  19. Differential Conductance Measurements of MgB2/I/Pb Heterojunctions and all-MgB2 Junctions

    NASA Astrophysics Data System (ADS)

    Cusick, David; Eckhardt, Matthew; Dai, Wenqing; Li, Qi; Chen, Ke; Cunnane, Daniel; Zhuang, C. G.; Xi, X. X.; Naito, Michio; Ramos, Roberto

    2015-03-01

    We present our work characterizing several types of Magnesium Diboride Josephson junctions, including MgB2/I/Pb heterojunctions and all-MgB2 junctions. We will report on the I-V and dI/dV-V data collected at various temperatures using both a cryocooler-based experimental platform between 2 and 20 Kelvin and using a 3He probe platform between 0.3 and 1.0 Kelvin. These were both developed by undergraduates in a liberal arts university. Using high-sampling rates with a 24-bit data acquisition card and access to a broad of range of temperatures, we track and report energy gap distributions and temperature-dependent features of dI/dV peaks of MgB2, comparing these with theoretical predictions. R.C.R. acknowledges support from National Science Foundation Grant # DMR-1206561.

  20. MiR-181b targets Six2 and inhibits the proliferation of metanephric mesenchymal cells in vitro

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lyu, Zhongshi; Mao, Zhaomin; Wang, Honglian

    2013-11-01

    Highlights: •We do bio-informatics websites to analysis of Six2 3′UTR. •MiR181b is a putative miRNA which can targets Six2 3′UTR. •MiR-181b binding site in the 3′UTR of Six2 is functional. •MiR-181b suppresses MK3 cells cell proliferation by targeting Six2. -- Abstract: MicroRNAs (miRNAs) are small non-coding RNAs that down-regulate gene expression by binding to target mRNA for cleavage or translational repression, and play important regulatory roles in renal development. Despite increasing genes have been predicted to be miRNA targets by bioinformatic analysis during kidney development, few of them have been verified by experiment. The objective of our study is tomore » identify the miRNAs targeting Six2, a critical transcription factor that maintains the mesenchymal progenitor pool via self-renewal (proliferation) during renal development. We initially analyzed the 3′UTR of Six2 and found 37 binding sites targeted by 50 putative miRNAs in the 3′UTR of Six2. Among the 50 miRNAs, miR-181b is the miRNAs predicted by the three used websites. In our study, the results of luciferase reporter assay, realtime-PCR and Western blot demonstrated that miR-181b directly targeted on the 3′UTR of Six2 and down-regulate the expression of Six2 at mRNA and protein levels. Furthermore, EdU proliferation assay along with the Six2 rescue strategy showed that miR-181b suppresses the proliferation of metanephric mesenchymal by targeting Six2 in part. In our research, we concluded that by targeting the transcription factor gene Six2, miR-181b inhibits the proliferation of metanephric mesenchymal cells in vitro and might play an important role in the formation of nephrons.« less

  1. Effects of nucleotides on [3H]bradykinin binding in guinea pig: further evidence for multiple B2 receptor subtypes.

    PubMed

    Seguin, L; Widdowson, P S

    1993-02-01

    We have suggested recently the existence of three subtypes of B2 bradykinin receptors in tissues of guinea pigs. We have classified these B2 bradykinin receptors into B2a, B2b, and B2c subtypes depending on their affinity for various bradykinin antagonists. Because the actions of bradykinin in different cell systems appear to be both dependent on and independent of G proteins, we sought to determine whether the binding of [3H]bradykinin to the B2 subtypes is sensitive to guanine nucleotides and, therefore, possibly coupled to G proteins. In the ileum, where we have demonstrated B2a and B2b subtypes, specific [3H]bradykinin binding was reduced with GDP (100 microM) and the nonmetabolized analogue of GTP, guanyl-5'-yl-imidodiphosphate (GppNHp; 100 microM). Competition studies with bradykinin and with [Hyp3]bradykinin, which shows approximately 20-fold greater selectivity for the B2a subtype than bradykinin, were performed in the presence or absence of GppNHp (100 microM). The competition experiments demonstrated that binding to the B2a subtype, which has higher affinity for [Hyp3]bradykinin and bradykinin than the B2b subtype, was lost in the presence of GppNHp, whereas binding to the B2b subtype was unaffected. In contrast, GppNHp (100 microM) and GDP (100 microM) failed to alter specific [3H]bradykinin binding to B2b and B2c subtypes in lung. [3H]Bradykinin binding was unaffected by AMP, ADP, ATP, and GMP (100 microM each). Based on this evidence, we suggest that the B2a bradykinin subtype is coupled to G proteins. The B2b and B2c subtypes are either not coupled to G proteins, or may be coupled to the Go-type GTP binding proteins, which have been suggested to be less sensitive to guanine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Positive feedback of NR2B-containing NMDA receptor activity is the initial step toward visual imprinting: a model for juvenile learning.

    PubMed

    Nakamori, Tomoharu; Sato, Katsushige; Kinoshita, Masae; Kanamatsu, Tomoyuki; Sakagami, Hiroyuki; Tanaka, Kohichi; Ohki-Hamazaki, Hiroko

    2015-01-01

    Imprinting in chicks is a good model for elucidating the processes underlying neural plasticity changes during juvenile learning. We recently reported that neural activation of a telencephalic region, the core region of the hyperpallium densocellulare (HDCo), was critical for success of visual imprinting, and that N-Methyl-D-aspartic (NMDA) receptors containing the NR2B subunit (NR2B/NR1) in this region were essential for imprinting. Using electrophysiological and multiple-site optical imaging techniques with acute brain slices, we found that long-term potentiation (LTP) and enhancement of NR2B/NR1 currents in HDCo neurons were induced in imprinted chicks. Enhancement of NR2B/NR1 currents as well as an increase in surface NR2B expression occurred even following a brief training that was too weak to induce LTP or imprinting behavior. This means that NR2B/NR1 activation is the initial step of learning, well before the activation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors which induces LTP. We also showed that knockdown of NR2B/NR1 inhibited imprinting, and inversely, increasing the surface NR2B expression by treatment with a casein kinase 2 inhibitor successfully reduced training time required for imprinting. These results suggest that imprinting stimuli activate post-synaptic NR2B/NR1 in HDCo cells, increase NR2B/NR1 signaling through up-regulation of its expression, and induce LTP and memory acquisition. The study investigated the neural mechanism underlying juvenile learning. In the initial stage of chick imprinting, NMDA receptors containing the NMDA receptor subunit 2B (NR2B) are activated, surface expression of NR2B/NR1 (NMDA receptor subunit 1) is up-regulated, and consequently long-term potentiation is induced in the telencephalic neurons. We suggest that the positive feedback in the NR2B/NR1 activation is a unique process of juvenile learning, exhibiting rapid memory acquisition. © 2014 International Society for Neurochemistry.

  3. miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis in osteosarcoma cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Zhi; Li, Youjun, E-mail: liyoujunn@126.com; Wang, Nan

    miR-130b was significantly up-regulated in osteosarcoma (OS) cells. Naked cuticle homolog 2 (NKD2) inhibited tumor growth and metastasis in OS by suppressing Wnt signaling. We used three miRNA target analysis tools to identify potential targets of miR-130b, and found that NKD2 is a potential target of miR-130b. Based on these findings, we hypothesize that miR-130b might target NKD2 and regulate the Wnt signaling to promote OS growth. We detected the expression of miR-130b and NKD2 mRNA and protein by quantitative Real-Time PCR (qRT-PCR) and western blot assays, respectively, and found up-regulation of miR-130b and down-regulation of NKD2 mRNA and proteinmore » exist in OS cell lines. MTT and flow cytometry assays showed that miR-130b inhibitors inhibit proliferation and promote apoptosis in OS cells. Furthermore, we showed that NKD2 is a direct target of miR-130b, and miR-130b regulated proliferation and apoptosis of OS cells by targeting NKD2. We further investigated whether miR-130b and NKD2 regulate OS cell proliferation and apoptosis by inhibiting Wnt signaling, and the results confirmed our speculation that miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis of OS cells. These findings will offer new clues for OS development and progression, and novel potential therapeutic targets for OS. - Highlights: • miR-130b is up-regulated and NKD2 is down-regulated in osteosarcoma cell lines. • Down-regulation of miR-130b inhibits proliferation of osteosarcoma cells. • Down-regulation of miR-130b promotes apoptosis of osteosarcoma cells. • miR-130b directly targets NKD2. • NKD2 regulates OS cell proliferation and apoptosis by inhibiting the Wnt signaling.« less

  4. Plexin-B2 promotes invasive growth of malignant glioma

    PubMed Central

    Pingle, Sandeep C.; Kesari, Santosh; Wang, Huaien; Yong, Raymund L.; Zou, Hongyan; Friedel, Roland H.

    2015-01-01

    Invasive growth is a major determinant of the high lethality of malignant gliomas. Plexin-B2, an axon guidance receptor important for mediating neural progenitor cell migration during development, is upregulated in gliomas, but its function therein remains poorly understood. Combining bioinformatic analyses, immunoblotting and immunohistochemistry of patient samples, we demonstrate that Plexin-B2 is consistently upregulated in all types of human gliomas and that its expression levels correlate with glioma grade and poor survival. Activation of Plexin-B2 by Sema4C ligand in glioblastoma cells induced actin-based cytoskeletal dynamics and invasive migration in vitro. This proinvasive effect was associated with activation of the cell motility mediators RhoA and Rac1. Furthermore, costimulation of Plexin-B2 and the receptor tyrosine kinase Met led to synergistic Met phosphorylation. In intracranial glioblastoma transplants, Plexin-B2 knockdown hindered invasive growth and perivascular spreading, and resulted in decreased tumor vascularity. Our results demonstrate that Plexin-B2 promotes glioma invasion and vascularization, and they identify Plexin-B2 as a potential novel prognostic marker for glioma malignancy. Targeting the Plexin-B2 pathway may represent a novel therapeutic approach to curtail invasive growth of glioblastoma. PMID:25762646

  5. CARDIO-i2b2: integrating arrhythmogenic disease data in i2b2.

    PubMed

    Segagni, Daniele; Tibollo, Valentina; Dagliati, Arianna; Napolitano, Carlo; G Priori, Silvia; Bellazzi, Riccardo

    2012-01-01

    The CARDIO-i2b2 project is an initiative to customize the i2b2 bioinformatics tool with the aim to integrate clinical and research data in order to support translational research in cardiology. In this work we describe the implementation and the customization of i2b2 to manage the data of arrhytmogenic disease patients collected at the Fondazione Salvatore Maugeri of Pavia in a joint project with the NYU Langone Medical Center (New York, USA). The i2b2 clinical research chart data warehouse is populated with the data obtained by the research database called TRIAD. The research infrastructure is extended by the development of new plug-ins for the i2b2 web client application able to properly select and export phenotypic data and to perform data analysis.

  6. Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Casabar, Richard C.T.; Das, Parikshit C.; DeKrey, Gregory K.

    Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold)more » and CYP3A4 (11-fold) promoter activities over control at 10 {mu}M. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 {mu}M. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 {mu}M, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 {mu}M and 10 {mu}M, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5 mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.« less

  7. Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor.

    PubMed

    Casabar, Richard C T; Das, Parikshit C; Dekrey, Gregory K; Gardiner, Catherine S; Cao, Yan; Rose, Randy L; Wallace, Andrew D

    2010-06-15

    Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 microM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 microM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 microM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 microM and 10 microM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates. Copyright 2010 Elsevier Inc. All rights reserved.

  8. GABA/sub B/ receptor activation inhibits Ca/sup 2 +/-activated potassium channels in synaptosomes: involvement of G-proteins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ticku, M.K.; Delgado, A.

    1989-01-01

    /sup 86/Rb-efflux assay from preloaded synaptosomes of rat cerebral cortex was developed to study the effect of GABA/sub B/ receptor agonist baclofen on Ca/sup 2 +/-activated K/sup +/-channels. Depolarization of /sup 86/Rb-loaded synaptosomes in physiological buffer increased Ca/sup 2 +/-activated /sup 86/Rb-efflux by 400%. The /sup 86/Rb-efflux was blocked by quinine sulfate, tetraethylammonium, and La/sup 3 +/ indicating the involvement of Ca/sup 2 +/-activated K/sup +/-channels. (-)Baclofen inhibited Ca/sup 2 +/-activated /sup 86/Rb-efflux in a stereospecific manner. The inhibitory effect of (-)baclofen was mediated by GABA/sub B/ receptor activation, since it was blocked by GABA/sub B/ antagonist phaclofen, but notmore » by bicuculline. Further, pertussis toxin also blocked the ability of baclofen or depolarizing action to affect Ca/sup 2 +/-activated K/sup +/-channels. These results suggest that baclofen inhibits Ca/sup 2 +/-activated K/sup +/-channels in synaptosomes and these channels are regulated by G-proteins. This assay may provide an ideal in vitro model to study GABA/sub B/ receptor pharmacology.« less

  9. Site-preference and valency for rare-earth sites in (R-Ce)2Fe14B [R =La,Nd] magnets

    NASA Astrophysics Data System (ADS)

    Alam, Aftab; Khan, Mahmud; McCallum, R. W.; Johnson, D. D.

    2013-03-01

    Rare-earth (R) permanent magnets of R2Fe14B have technological importance due to their high energy products, and they have two symmetry distinct R-sites (Wyckoff 4f and 4g) that affect chemistry and valence. Designing magnetic behavior and stability via alloying is technologically relevant to reduce critical (expensive) R-content while retaining key properties; cerium, an abundant (cheap) R-element, offers this potential. We calculate magnetic properties and Ce site preference in (R1-xCex)Fe14B [R=La,Nd] using density functional theory (DFT) methods. The Fe moments compare well with neutron scattering data - remain weakly affected by Hubbard U, but improved with spin-orbit coupling. In (La,Ce)2Fe14B, Ce alloys for 0 < x < 1 with a preference for smaller R(4f) sites, as observed, a trend we find unaffected by valence. Whereas in (Nd,Ce)2Fe14B, Ce is predicted to have limited alloying (x < 0.3) with a preference for larger R(4g) sites, resulting in weak partial ordering and segregation. Curie temperatures versus x were predicted for a typical sample processing and verified experimentally. We shall also present some initial results on the critical mixed valency of Ce in related compounds. Work at Ames Laboratory was supported by the U.S. Department of Energy, ARPA-E under the REACT program (0472-1526)

  10. Subtype specific internalization of P2Y1 and P2Y2 receptors induced by novel adenosine 5′-O-(1-boranotriphosphate) derivatives

    PubMed Central

    Tulapurkar, M E; Laubinger, W; Nahum, V; Fischer, B; Reiser, G

    2004-01-01

    P2Y-nucleotide receptors represent important targets for drug development. The lack of stable and receptor specific agonists, however, has prevented successful therapeutic applications. A novel series of P-boronated ATP derivatives (ATP-α-B) were synthesized by substitution of a nonbridging O at Pα with a BH3 group. This introduces a chiral center, thus resulting in diastereoisomers. In addition, at C2 of the adenine ring a further substitution was made (Cl- or methylthio-). The pairs of diastereoisomers were denoted here as A and B isomers. Here, we tested the receptor subtype specificity of these analogs on HEK 293 cells stably expressing rat P2Y1 and rat P2Y2 receptors, respectively, both attached to the fluorescent marker protein GFP (rP2Y1-GFP, rP2Y2-GFP). We investigated agonist-induced receptor endocytosis, [Ca2+]i rise and arachidonic acid (AA) release. Agonist-induced endocytosis of rP2Y1-GFP was more pronounced for the A isomers than the corresponding B counterparts for all ATP-α-B analogs. Both 2-MeS-substituted diastereoisomers induced a greater degree of agonist-induced receptor endocytosis as compared to the 2-Cl-substituted derivatives. Endocytosis results are in accordance with the potency to induce Ca2+ release by these compounds in HEK 293 cells stably transfected with rP2Y1. In case of rP2Y2-GFP, the borano-nucleotides were very weak agonists in comparison to UTP and ATP in terms of Ca2+ release, AA release and in inducing receptor endocytosis. The different ATP-α-B derivatives and also the diastereoisomers were equally ineffective. Thus, the new agonists may be considered as potent and highly specific agonist drug candidates for P2Y1 receptors. The difference in activity of the ATP analogs at P2Y receptors could be used as a tool to investigate structural differences between P2Y receptor subtypes. PMID:15197109

  11. Down-regulation of NR2B receptors partially contributes to analgesic effects of Gentiopicroside in persistent inflammatory pain.

    PubMed

    Chen, Lei; Liu, Jin-cheng; Zhang, Xiao-nan; Guo, Yan-yan; Xu, Zhao-hui; Cao, Wei; Sun, Xiao-li; Sun, Wen-ji; Zhao, Ming-Gao

    2008-06-01

    Gentiopicroside is one of the secoiridoid compound isolated from Gentiana lutea. It exhibits analgesic activities in the mice. The anterior cingulate cortex (ACC) is a forebrain structure known for its roles in pain transmission and modulation. Painful stimuli potentiate the prefrontal synaptic transmission and induce glutamate NMDA NR2B receptor expression in the ACC. But little is known about Gentiopicroside on the persistent inflammatory pain and chronic pain-induced synaptic transmission changes in the ACC. The present study was undertaken to investigate its analgesic activities and central synaptic modulation to the peripheral painful inflammation. Gentiopicroside produced significant analgesic effects against persistent inflammatory pain stimuli in mice. Systemic administration of Gentiopicroside significantly reversed NR2B over-expression during the chronic phases of persistent inflammation caused by hind-paw administration of complete Freunds adjuvant (CFA) in mice. Whole-cell patch clamp recordings revealed that Gentiopicroside significantly reduced NR2B receptors mediated postsynaptic currents in the ACC. Our findings provide strong evidence that analgesic effects of Gentiopicroside involve down-regulation of NR2B receptors in the ACC to persistent inflammatory pain.

  12. Role of G protein-coupled receptors (GPCR), matrix metalloproteinases 2 and 9 (MMP2 and MMP9), heparin-binding epidermal growth factor-like growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) in trenbolone acetate-stimulated bovine satellite cell proliferation.

    PubMed

    Thornton, K J; Kamange-Sollo, E; White, M E; Dayton, W R

    2015-09-01

    Implanting cattle with steroids significantly enhances feed efficiency, rate of gain, and muscle growth. However, the mechanisms responsible for these improvements in muscle growth have not been fully elucidated. Trenbolone acetate (TBA), a testosterone analog, has been shown to increase proliferation rate in bovine satellite cell (BSC) cultures. The classical genomic actions of testosterone have been well characterized; however, our results indicate that TBA may also initiate a quicker, nongenomic response that involves activation of G protein-coupled receptors (GPCR) resulting in activation of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) that release membrane-bound heparin-binding epidermal growth factor-like growth factor (hbEGF), which then binds to and activates the epidermal growth factor receptor (EGFR) and/or erbB2. Furthermore, the EGFR has been shown to regulate expression of the IGF-1 receptor (IGF-1R), which is well known for its role in modulating muscle growth. To determine whether this nongenomic pathway is potentially involved in TBA-stimulated BSC proliferation, we analyzed the effects of treating BSC with guanosine 5'-O-2-thiodiphosphate (GDPβS), an inhibitor of all GPCR; a MMP2 and MMP9 inhibitor (MMPI); CRM19, a specific inhibitor of hbEGF; AG1478, a specific EGFR tyrosine kinase inhibitor; AG879, a specific erbB2 kinase inhibitor; and AG1024, an IGF-1R tyrosine kinase inhibitor on TBA-stimulated proliferation rate (H-thymidine incorporation). Assays were replicated at least 9 times for each inhibitor experiment using BSC cultures obtained from at least 3 different animals. Bovine satellite cell cultures were obtained from yearling steers that had no previous exposure to androgenic or estrogenic compounds. As expected, BSC cultures treated with 10 n TBA showed ( < 0.05) increased proliferation rate when compared with control cultures. Additionally, treatment with 5 ng hbEGF/mL stimulated proliferation in BSC cultures ( < 0.05). Treatment

  13. Critical Role of the Src Homology 2 (SH2) Domain of Neuronal SH2B1 in the Regulation of Body Weight and Glucose Homeostasis in Mice

    PubMed Central

    Morris, David L.; Cho, Kae Won; Rui, Liangyou

    2010-01-01

    SH2B1 is an SH2 domain-containing adaptor protein that plays a key role in the regulation of energy and glucose metabolism in both rodents and humans. Genetic deletion of SH2B1 in mice results in obesity and type 2 diabetes. Single-nucleotide polymorphisms in the SH2B1 loci and chromosomal deletions of the SH2B1 loci associate with obesity and insulin resistance in humans. In cultured cells, SH2B1 promotes leptin and insulin signaling by binding via its SH2 domain to phosphorylated tyrosines in Janus kinase 2 and the insulin receptor, respectively. Here we generated three lines of mice to analyze the role of the SH2 domain of SH2B1 in the central nervous system. Transgenic mice expressing wild-type, SH2 domain-defective (R555E), or SH2 domain-alone (ΔN503) forms of SH2B1 specifically in neurons were crossed with SH2B1 knockout mice to generate KO/SH2B1, KO/R555E, or KO/ΔN503 compound mutant mice. R555E had a replacement of Arg555 with Glu within the SH2 domain. ΔN503 contained an intact SH2 domain but lacked amino acids 1-503. Neuron-specific expression of recombinant SH2B1, but not R555E or ΔN503, corrected hyperphagia, obesity, glucose intolerance, and insulin resistance in SH2B1 null mice. Neuron-specific expression of R555E in wild-type mice promoted obesity and insulin resistance. These results indicate that in addition to the SH2 domain, N-terminal regions of neuronal SH2B1 are also required for the maintenance of normal body weight and glucose metabolism. Additionally, mutations in the SH2 domain of SH2B1 may increase the susceptibility to obesity and type 2 diabetes in a dominant-negative manner. PMID:20484460

  14. Critical role of the Src homology 2 (SH2) domain of neuronal SH2B1 in the regulation of body weight and glucose homeostasis in mice.

    PubMed

    Morris, David L; Cho, Kae Won; Rui, Liangyou

    2010-08-01

    SH2B1 is an SH2 domain-containing adaptor protein that plays a key role in the regulation of energy and glucose metabolism in both rodents and humans. Genetic deletion of SH2B1 in mice results in obesity and type 2 diabetes. Single-nucleotide polymorphisms in the SH2B1 loci and chromosomal deletions of the SH2B1 loci associate with obesity and insulin resistance in humans. In cultured cells, SH2B1 promotes leptin and insulin signaling by binding via its SH2 domain to phosphorylated tyrosines in Janus kinase 2 and the insulin receptor, respectively. Here we generated three lines of mice to analyze the role of the SH2 domain of SH2B1 in the central nervous system. Transgenic mice expressing wild-type, SH2 domain-defective (R555E), or SH2 domain-alone (DeltaN503) forms of SH2B1 specifically in neurons were crossed with SH2B1 knockout mice to generate KO/SH2B1, KO/R555E, or KO/DeltaN503 compound mutant mice. R555E had a replacement of Arg(555) with Glu within the SH2 domain. DeltaN503 contained an intact SH2 domain but lacked amino acids 1-503. Neuron-specific expression of recombinant SH2B1, but not R555E or DeltaN503, corrected hyperphagia, obesity, glucose intolerance, and insulin resistance in SH2B1 null mice. Neuron-specific expression of R555E in wild-type mice promoted obesity and insulin resistance. These results indicate that in addition to the SH2 domain, N-terminal regions of neuronal SH2B1 are also required for the maintenance of normal body weight and glucose metabolism. Additionally, mutations in the SH2 domain of SH2B1 may increase the susceptibility to obesity and type 2 diabetes in a dominant-negative manner.

  15. Bisphenol-A rapidly promotes dynamic changes in hippocampal dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDA receptor subunit NR2B

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xu Xiaohong, E-mail: xuxh63@zjnu.cn; Ye Yinping; Li Tao

    Bisphenol-A (BPA) is known to be a potent endocrine disrupter. Evidence is emerging that estrogen exerts a rapid influence on hippocampal synaptic plasticity and the dendritic spine density, which requires activation of NMDA receptors. In the present study, we investigated the effects of BPA (ranging from 1 to 1000 nM), focusing on the rapid dynamic changes in dendritic filopodia and the expressions of estrogen receptor (ER) {beta} and NMDA receptor, as well as the phosphorylation of NMDA receptor subunit NR2B in the cultured hippocampal neurons. A specific ER antagonist ICI 182,780 was used to examine the potential involvement of ERs.more » The results demonstrated that exposure to BPA (ranging from 10 to 1000 nM) for 30 min rapidly enhanced the motility and the density of dendritic filopodia in the cultured hippocampal neurons, as well as the phosphorylation of NR2B (pNR2B), though the expressions of NMDA receptor subunits NR1, NR2B, and ER{beta} were not changed. The antagonist of ERs completely inhibited the BPA-induced increases in the filopodial motility and the number of filopodia extending from dendrites. The increased pNR2B induced by BPA (100 nM) was also completely eliminated. Furthermore, BPA attenuated the effects of 17{beta}-estradiol (17{beta}-E{sub 2}) on the dendritic filopodia outgrowth and the expression of pNR2B when BPA was co-treated with 17{beta}-E{sub 2}. The present results suggest that BPA, like 17{beta}-E{sub 2}, rapidly results in the enhanced motility and density of dendritic filopodia in the cultured hippocampal neurons with the concomitant activation of NMDA receptor subunit NR2B via an ER-mediated signaling pathway. Meanwhile, BPA suppressed the enhancement effects of 17{beta}-E{sub 2} when it coexists with 17{beta}-E{sub 2}. These results provided important evidence suggesting the neurotoxicity of the low levels of BPA during the early postnatal development of the brain.« less

  16. B11 NMR in the layered diborides OsB2 and RuB2

    NASA Astrophysics Data System (ADS)

    Suh, B. J.; Zong, X.; Singh, Y.; Niazi, A.; Johnston, D. C.

    2007-10-01

    B11 nuclear magnetic resonance (NMR) measurements have been performed on B11 enriched OsB2 and RuB2 polycrystalline powder samples in an external field of 4.7T and in the temperature range, 4.2KB2 and RuB2 , respectively. The experimental results indicate that a p character dominates the conduction electron wave function at the B site with a negligibly small s character in both compounds.

  17. Site-preference and valency for rare-earth sites in (R-Ce)(2)Fe14B magnets

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alam, A; Khan, M; McCallum, RW

    2013-01-28

    Rare-earth (R) permanent magnets of R2Fe14B have technological importance due to their high energy products, and they have two R-sites (Wyckoff 4f and 4g, with four-fold multiplicity) that affect chemistry and valence. Designing magnetic behavior and stability via alloying is technologically relevant to reduce critical (expensive) R-content while retaining key properties; cerium, an abundant (cheap) R-element, offers this potential. We calculate magnetic properties and Ce site preference in (R1-xCex)(2)Fe14B [R = La, Nd] using density functional theory (DFT) methods-including a DFT+U scheme to treat localized 4f-electrons. Fe moments compare well with neutron data-almost unaffected by Hubbard U, and weakly affectedmore » by spin-orbit coupling. In La2Fe14B, Ce alloys for 0 <= x <= 1 and prefers smaller R(4f) sites, as observed, a trend we find unaffected by valence. Whereas, in Nd2Fe14B, Ce is predicted to have limited alloying (x <= 0.3) with a preference for larger R(4g) sites, resulting in weak partial ordering and segregation. The Curie temperatures versus x for (Nd, Ce) were predicted for a typical sample processing and verified experimentally. (C) 2013 American Institute of Physics. [http://dx.doi.org/10.1063/1.4789527]« less

  18. The AhR and NF-κB/Rel Proteins Mediate the Inhibitory Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the 3′ Immunoglobulin Heavy Chain Regulatory Region

    PubMed Central

    Salisbury, Richard L.; Sulentic, Courtney E. W.

    2015-01-01

    Transcriptional regulation of the murine immunoglobulin (Ig) heavy chain gene (Igh) involves several regulatory elements including the 3′Igh regulatory region (3′IghRR), which is composed of at least 4 enhancers (hs3A, hs1.2, hs3B, and hs4). The hs1.2 and hs4 enhancers exhibit the greatest transcriptional activity and contain binding sites for several transcription factors including nuclear factor kappaB/Rel (NF-κB/Rel) proteins and the aryl hydrocarbon receptor (AhR). Interestingly, the environmental immunosuppressant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which potently inhibits antibody secretion, also profoundly inhibits 3′IghRR and hs1.2 enhancer activation induced by the B-lymphocyte activator lipopolysaccharide (LPS), but enhances LPS-induced activation of the hs4 enhancer. Within the hs1.2 and hs4 enhancers, the AhR binding site is in close proximity or overlaps an NF-κB/Rel binding site suggesting a potential reciprocal modulation of the 3′IghRR by AhR and NF-κB/Rel. The objective of the current study was to evaluate the role of NF-κB/Rel and the AhR on the 3′IghRR and its enhancers using the AhR ligand TCDD, the AhR antagonist CH223191, and toll-like receptor agonists LPS, Resiquimod (R848), or cytosine-phosphate-guanine-oligodeoxynucleotides (CpG). Utilizing the CH12.LX B-lymphocyte cell line and variants expressing either a 3′IghRR-regulated transgene reporter or an inducible IκBα (inhibitor kappa B-alpha protein) superrepressor (IκBαAA), we demonstrate an AhR- and NF-κB/Rel-dependent modulation of 3′IghRR and hs4 activity. Additionally, in mouse splenocytes or CH12.LX cells, binding within the hs1.2 and hs4 enhancer of the AhR and the NF-κB/Rel proteins RelA and RelB was differentially altered by the cotreatment of LPS and TCDD. These results suggest that the AhR and NF-κB/Rel protein binding profile within the 3′IghRR mediates the inhibitory effects of TCDD on Ig expression and therefore antibody levels. PMID:26377645

  19. 15 CFR Appendix B to Subpart R of... - Minor Projects for Purposes of § 922.193(a)(2)(iii)

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ....193(a)(2)(iii) B Appendix B to Subpart R of Part 922 Commerce and Foreign Trade Regulations Relating... Thunder Bay National Marine Sanctuary and Underwater Preserve Pt. 922, Subpt. R, App. B Appendix B to Subpart R of Part 922—Minor Projects for Purposes of § 922.193(a)(2)(iii) Pursuant to Michigan State...

  20. 15 CFR Appendix B to Subpart R of... - Minor Projects for Purposes of § 922.193(a)(2)(iii)

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ....193(a)(2)(iii) B Appendix B to Subpart R of Part 922 Commerce and Foreign Trade Regulations Relating... Thunder Bay National Marine Sanctuary and Underwater Preserve Pt. 922, Subpt. R, App. B Appendix B to Subpart R of Part 922—Minor Projects for Purposes of § 922.193(a)(2)(iii) Pursuant to Michigan State...

  1. 15 CFR Appendix B to Subpart R of... - Minor Projects for Purposes of § 922.193(a)(2)(iii)

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ....193(a)(2)(iii) B Appendix B to Subpart R of Part 922 Commerce and Foreign Trade Regulations Relating... Thunder Bay National Marine Sanctuary and Underwater Preserve Pt. 922, Subpt. R, App. B Appendix B to Subpart R of Part 922—Minor Projects for Purposes of § 922.193(a)(2)(iii) Pursuant to Michigan State...

  2. Panicolytic-like action of bradykinin in the dorsal periaqueductal gray through μ-opioid and B2-kinin receptors.

    PubMed

    Sestile, Caio César; Maraschin, Jhonatan Christian; Gama, Vanessa Scalco; Zangrossi, Hélio; Graeff, Frederico Guilherme; Audi, Elisabeth Aparecida

    2017-09-01

    A wealth of evidence has shown that opioid and kinin systems may control proximal defense in the dorsal periaqueductal gray matter (dPAG), a critical panic-associated area. Studies with drugs that interfere with serotonin-mediated neurotransmission suggest that the μ-opioid receptor (MOR) synergistically interacts with the 5-HT 1A receptor in the dPAG to inhibit escape, a panic-related behavior. A similar inhibitory effect has also been reported after local administration of bradykinin (BK), which is blocked by the non-selective opioid receptor antagonist naloxone. The latter evidence, points to an interaction between BK and opioids in the dPAG. We further explored the existence of this interaction through the dPAG electrical stimulation model of panic. We also investigated whether intra-dPAG injection of captopril, an inhibitor of the angiotensin-converting enzyme (ACE) that also degrades BK, causes a panicolytic-like effect. Our results showed that intra-dPAG injection of BK inhibited escape performance in a dose-dependent way, and this panicolytic-like effect was blocked by the BK type 2 receptor (B2R) antagonist HOE-140, and by the selective MOR antagonist CTOP. Conversely, the panicolytic-like effect caused by local administration of the selective MOR agonist DAMGO was antagonized by pre-treatment with either CTOP or HOE-140, indicating cross-antagonism between MOR and B2R. Finally, intra-dPAG injection of captopril also impaired escape in a dose-dependent way, and this panicolytic-like effect was blocked by pretreatment with HOE-140, suggesting mediation by endogenous BK. The panicolytic-like effect of captopril indicates that the use of ACE inhibitors in the clinical management of panic disorder may be worth exploring. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ma, Zhiyuan; Sun, Xiaorui; Xu, Dequan

    Myogenesis is a complex process including myoblast proliferation, differentiation and myotube formation and is controlled by myogenic regulatory factors (MRFs), MyoD, MyoG, Myf5 and Myf6 (also known as MRF4). MicroRNA is a kind of ∼22 nt-long non-coding small RNAs, and act as key transcriptional or post-transcriptional regulators of gene expression. Identification of miRNAs involved in the regulation of muscle genes could improve our understanding of myogenesis process. In this study, we investigated the regulation of Myf6 gene by miRNAs. We showed that miR-374b specifically bound to the 3'untranslated region (UTR) of Myf6 and down-regulated the expression of Myf6 gene at bothmore » mRNA and protein level. Furthermore, miR-374b is ubiquitously expressed in the tissues of adult C57BL6 mouse, and the mRNA abundance increases first and then decreases during C2C12 myoblasts differentiation. Over-expression of miR-374b impaired C2C12 cell differentiation, while inhibiting miR-374b expression by 2′-O-methyl antisense oligonucleotides promoted C2C12 cell differentiation. Taken together, our findings identified miR-374b directly targets Myf6 and negatively regulates myogenesis. - Highlights: • MiR-374b directly targets 3′UTR of Myf6. • MiR-374b negatively regulates Myf6 in C2C12 cells. • MiR-374b abundance significiently changes during C2C12 cells differentiation. • MiR-374b negatively regulates C2C12 cells differentiation.« less

  4. Investigation the influences of B2O3 and R2O on the structure and crystallization behaviors of CaO-Al2O3 based F-free mold flux

    NASA Astrophysics Data System (ADS)

    Li, Jiangling; Kong, Bowen; Gao, Xiangyu; Liu, Qingcai; Shu, Qifeng; Chou, Kuochih

    2018-04-01

    The influences of B2O3 and R2O on the structure and crystallization of CaO-Al2O3 based F-free mold flux were investigated by Raman Spectroscopy and Differential Scanning Calorimetry Technique, respectively, for developing a new type of F-free mold flux. The results of structural investigations showed that B3+ is mainly in the form of [BO3]. And [BO3] appears to form BIII-O-Al linkage which will produce a positive effect on forming [AlO4] network. The number of bridging oxygen and the degree of polymerization of [AlO4] network structure for CaO-Al2O3 system were also increased with the increasing of B2O3. On the contrary, with the addition of R2O into CaO-Al2O3-B2O3 system, the number of bridging oxygen and the degree of polymerization of [AlO4] network were decreased. DSC results showed that the crystallization process became more sluggish with the increase of B2O3, which indicated that the crystallization ability was weakened. While the quenched mold fluxes crystallized more rapidly when introducing R2O. In other word, the crystallization rates of CaO-Al2O3 based slags were accelerated by the introduction of R2O. The liquidus temperature and crystallization temperature were decreased with the increasing amount of B2O3 or by addition of R2O into CaO-Al2O3 system. Only one kind of crystal was precipitated in 8% B2O3 and %R2O-containing samples, which was CaAl2O4 identified by SEM-EDS. When the content of B2O3 increased from 8% to 16%, Ca3B2O6 is clearly observed, demonstrating that the crystallization ability of Ca3B2O6 is enhanced by the increasing concentration of B2O3 in mold flux. The Ca/Al ratio of the generated calcium aluminate has been altered from 1:2 to 1:4 with the increasing of B2O3. The size of CaAl2O4 crystal is gradually increased with the addition of R2O. The crystallization ability of CaAl2O4 is promoted by R2O.

  5. Conservation, expression, and knockdown of zebrafish plxnb2a and plxnb2b.

    PubMed

    Perälä, Nina; Peitsaro, Nina; Sundvik, Maria; Koivula, Henri; Sainio, Kirsi; Sariola, Hannu; Panula, Pertti; Immonen, Tiina

    2010-10-01

    In mice lacking Plexin B2, a receptor of the axon guidance molecules Semaphorin 4C and Semaphorin 4D, the closure of the neural tube and structural organization of the cerebellum are severely impaired. We cloned two Plexin B2 orthologs, plxnb2a and plxnb2b, in zebrafish, which is a widely used model for the development of the vertebrate central nervous system (CNS). The predicted proteins, Plexin B2a and Plexin B2b, contain all the conserved and functional domains of the plexin B-subfamily. During embryonic development, plxnb2a is expressed, e.g., in pharyngeal arches while plxnb2b expression is more confined to neuronal structures like the cerebellum. However, both plxnb2a and plxnb2b are expressed at the midbrain-hindbrain boundary, in the otic vesicles, facial ganglia, and pectoral fins. Knockdown of both plxnb2a and plxnb2b simultaneously (>95% and 45%, respectively) resulted in normal CNS structure, axon guidance and swimming performance of the morphants.

  6. Class I odorant receptors, TAS1R and TAS2R taste receptors, are markers for subpopulations of circulating leukocytes

    PubMed Central

    Malki, Agne; Fiedler, Julia; Fricke, Kristina; Ballweg, Ines; Pfaffl, Michael W.; Krautwurst, Dietmar

    2015-01-01

    Our cellular immune system has to cope constantly with foodborne substances that enter the bloodstream postprandially. Here, they may activate leukocytes via specific but yet mostly unknown receptors. Ectopic RNA expression out of gene families of chemosensory receptors, i.e., the ∼400 ORs, ∼25 TAS2R bitter-taste receptors, and the TAS1R umami- and sweet-taste receptor dimers by which we typically detect foodborne substances, has been reported in a variety of peripheral tissues unrelated to olfaction or taste. In the present study, we have now discovered, by gene-specific RT-PCR experiments, the mRNA expression of most of the Class I ORs (TAS1R) and TAS2R in 5 different types of blood leukocytes. Surprisingly, we did not detect Class II OR mRNA. By RT-qPCR, we show the mRNA expression of human chemosensory receptors and their cow orthologs in PMN, thus suggesting an evolutionary concept. By immunocytochemistry, we demonstrate that some olfactory and taste receptors are expressed, on average, in 40–60% of PMN and T or B cells and largely coexpress in the same subpopulation of PMN. The mRNA expression and the size of subpopulations expressing certain chemosensory receptors varied largely among individual blood samples, suggesting a regulated expression of olfactory and taste receptors in these cells. Moreover, we show mRNA expression of their downstream signaling molecules and demonstrate that PTX abolishes saccharin- or 2-PEA-induced PMN chemotactic migration, indicating a role for Gi-type proteins. In summary, our data suggest "chemosensory"-type subpopulations of circulating leukocytes. PMID:25624459

  7. Fibroblast Growth Factor 10-Fibroblast Growth Factor Receptor 2b Mediated Signaling Is Not Required for Adult Glandular Stomach Homeostasis

    PubMed Central

    Sala, Frederic G.; Ford, Henri R.; Bellusci, Saverio; Grikscheit, Tracy C.

    2012-01-01

    The signaling pathways that are essential for gastric organogenesis have been studied in some detail; however, those that regulate the maintenance of the gastric epithelium during adult homeostasis remain unclear. In this study, we investigated the role of Fibroblast growth factor 10 (FGF10) and its main receptor, Fibroblast growth factor receptor 2b (FGFR2b), in adult glandular stomach homeostasis. We first showed that mouse adult glandular stomach expressed Fgf10, its receptors, Fgfr1b and Fgfr2b, and most of the other FGFR2b ligands (Fgf1, Fgf7, Fgf22) except for Fgf3 and Fgf20. Fgf10 expression was mesenchymal whereas FGFR1 and FGFR2 expression were mostly epithelial. Studying double transgenic mice that allow inducible overexpression of Fgf10 in adult mice, we showed that Fgf10 overexpression in normal adult glandular stomach increased epithelial proliferation, drove mucous neck cell differentiation, and reduced parietal and chief cell differentiation. Although a similar phenotype can be associated with the development of metaplasia, we found that Fgf10 overexpression for a short duration does not cause metaplasia. Finally, investigating double transgenic mice that allow the expression of a soluble form of Fgfr2b, FGF10's main receptor, which acts as a dominant negative, we found no significant changes in gastric epithelial proliferation or differentiation in the mutants. Our work provides evidence, for the first time, that the FGF10-FGFR2b signaling pathway is not required for epithelial proliferation and differentiation during adult glandular stomach homeostasis. PMID:23133671

  8. The α2B adrenergic receptor is mutant in cortical myoclonus and epilepsy

    PubMed Central

    De Fusco, Maurizio; Vago, Riccardo; Striano, Pasquale; Di Bonaventura, Carlo; Zara, Federico; Mei, Davide; Kim, Min Seuk; Muallem, Shmuel; Chen, Yunjia; Wang, Qin; Guerrini, Renzo; Casari, Giorgio

    2013-01-01

    Objective Autosomal dominant cortical myoclonus and epilepsy (ADCME) is characterized by distal, fairly rhythmic myoclonus and epilepsy with variable severity. We have previously mapped the disease locus on chromosome 2p11.1-q12.2 by genome-wide linkage analysis. Additional pedigrees affected by similar forms of epilepsy have been associated to chromosome 8q, 5p and 3q, but none of the causing genes has been identified. We aim at identifying the mutant gene responsible for this epileptic form. Methods Genes included in the ADCME critical region were prioritized and directly sequenced. Co-immunoprecipitation, immunofluorescence and electrophysiology approaches on transfected human cells have been utilized for testing the functional significance of the identified mutation. Results Here we show that mutation in the α2-adrenergic receptor subtype B2B-AR) associates to ADCME by identifying a novel in-frame insertion/deletion in two Italian families. The mutation alters several conserved residues of the third intracellular (3i) loop, neither hampering the α2B-AR plasma membrane localization nor the arrestin-mediated internalization capacity, but altering the binding with the scaffolding protein spinophilin upon neurotransmitter activation. Spinophilin, in turn, regulates interaction of GPCRs with Regulators of G proteins Signaling proteins. Accordingly, the mutant α2B-AR increases the epinephrine-stimulated calcium signaling. Interpretation The identified mutation is responsible for ADCME, as the loss of α2B-AR/spinophilin interaction causes a gain of function effect. This work implicates for the first time the α-adrenergic system in human epilepsy and opens new ways for understanding the molecular pathway of epileptogenesis, widening the spectrum of possible therapeutic targets. PMID:24114805

  9. Adenosine A2B receptor modulates intestinal barrier function under hypoxic and ischemia/reperfusion conditions.

    PubMed

    Yang, Yang; Qiu, Yuan; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Zhang, Chaojun; Yang, Hanwenbo; Teitelbaum, Daniel H; Sun, Li-Hua; Yang, Hua

    2014-01-01

    Intestinal barrier function failure from ischemia/reperfusion (I/R) and acute hypoxia has been implicated as a critical determinant in the predisposition to intestinal inflammation and a number of inflammatory disorders. Here, we identified the role of Adenosine A2B receptor (A2BAR) in the regulation of intestinal barrier function under I/R and acute hypoxic conditions. C57BL/6J mice were used, and were randomized into three groups: Sham, I/R, IR+PSB1115 (a specific A2BAR antagonist) groups. After surgery, the small bowel was harvested for immunohistochemical staining, RNA and protein content, and intestinal permeability analyses. Using an epithelial cell culture model, we investigated the influence of hypoxia on the epithelial function, and the role of A2BAR in the expressions of tight junction and epithelial permeability. The expressions of Claudin-1, occludin and ZO-1 were detected by RT-PCR and Western-Blot. Epithelial barrier function was assessed with transepithelial resistance (TER). The A2BAR antagonist, PSB1115, significantly increased tight junction protein expression after intestinal I/R or acute hypoxia conditions. PSB1115 also attenuated the disrupted distribution of TJ proteins. Furthermore, inhibition of A2BAR attenuated the decrease in TER induced by I/R or acute hypoxic conditions, and maintained intestinal barrier function. Antagonism of A2BAR activity improves intestinal epithelial structure and barrier function in a mouse model of intestinal I/R and a cell model of acute hypoxia. These findings support a potentially destructive role for A2BAR under intestinal I/R and acute hypoxic conditions.

  10. Transcriptional regulation by retinoic acid of interleukin-2 alpha receptors in human B cells.

    PubMed Central

    Bhatti, L; Sidell, N

    1994-01-01

    In this study, we demonstrated that retinoic acid (RA) up-regulated interleukin-2 receptor-alpha (IL-2R alpha) expression on two human B-cell lines, IE8.6 and SKW6.4. Deleted forms of the human IL-2R alpha promoter linked to the bacterial chloramphenicol acetyltransferase reporter gene were transfected into IE8.6 cells in order to define RA-responsive regulatory domains. Experiments using the -1.6 kb construct, which contains all known regulatory regions in the IL-2R alpha promoter, indicated that RA could induce IL-2R alpha promoter activity. The basal activity of the -471 construct was initially low, but was markedly enhanced by the addition of RA. Deletion of promoter sequences between -471 and -317 resulted in a significant augmentation of basal promoter activity and abolished promoter induction by RA. This finding revealed a requirement for sequences 5' of base -317 for RA-induced promoter activation, raising the possibility of the presence of both a RA response element and a negative regulatory element (NRE) upstream of base -317. Transfection studies with internal deletion mutants with the putative NRE removed resulted in increases in basal promoter activity and unresponsiveness to RA similar to the -317 construct. In contrast, an internal deletion mutant with the NRE intact had low basal activity and was inducible by RA similar to the -471 construct. Taken together, our results suggested that RA-induced activation of the IL-2R alpha promoter was through changes in the function of a NRE present between bases -400 and -368. This 31-base pair element may interact with an adjacent RA-responsive regulatory site as well as being responsible for down-regulation of basal IL-2R alpha expression under certain conditions. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8157276

  11. Identification of a second binding site on the TRIM25 B30.2 domain.

    PubMed

    D'Cruz, Akshay A; Kershaw, Nadia J; Hayman, Thomas J; Linossi, Edmond M; Chiang, Jessica J; Wang, May K; Dagley, Laura F; Kolesnik, Tatiana B; Zhang, Jian-Guo; Masters, Seth L; Griffin, Michael D W; Gack, Michaela U; Murphy, James M; Nicola, Nicos A; Babon, Jeffrey J; Nicholson, Sandra E

    2018-01-23

    The r etinoic acid- i nducible g ene- I (RIG-I) receptor recognizes short 5'-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tri partite m otif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 ( c aspase a ctivation and r ecruitment d omains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  12. Natural Infection of a Homozygous Δ24 CCR5 Red-capped Mangabey with an R2b-Tropic Simian Immunodeficiency Virus

    PubMed Central

    Chen, Zhiwei; Kwon, Douglas; Jin, Zhanqun; Monard, Simon; Telfer, Paul; Jones, Morris S.; Lu, Chang Y.; Aguilar, Roberto F.; Ho, David D.; Marx, Preston A.

    1998-01-01

    A homozygous 24-bp deletion (Δ24) was found in the CC chemokine receptor 5 (CCR5) of 11 out of 15 red-capped mangabeys (RCMs), Cercocebus torquatus torquatus, both in Africa and in an American zoo. The CCR5 Δ24 defect encompassed eight amino acids in frame in the fourth transmembrane region. Unexpectedly, RCM-009, one of 11 homozygotes (Δ24CCR5/ Δ24CCR5), was found to be naturally infected with a divergent simian immunodeficiency virus (SIV) strain, which was not R5-tropic, but used CCR2b (R2b) as its major coreceptor. SIVrcmGab1 was the only R2b-tropic SIV among other divergent SIVs tested. Cells transfected with the Δ24 CCR5 did not support entry of R5-tropic SIVmac, SIVcpz, SIVmne, HIV-2, or HIV-1, and were also inactive in signal transduction mediated by β-chemokines. At 86.6%, the Δ24 allelic frequency was significantly higher than that of the 32-bp deletion found in humans. The Δ24 frequency was 4.1% in 34 sooty mangabeys (SMs), a geographically isolated subspecies that was naturally infected with R5-tropic SIV. Finding identical deletions in two mangabey subspecies separated for 10,000 years or more dates the Δ24 CCR5 deletion as ancient. However, the source of the selective pressure for the high rate of CCR5 deletion in RCMs remains to be determined. The high allelic frequency of the Δ24 CCR5 in RCMs, in comparison to that of SMs, suggests that R2b-tropism may have been acquired by SIVrcm, as an adaptation to CCR5 genetic defects appeared in its host. PMID:9841919

  13. Alterations in GluR2 AMPA receptor phosphorylation at serine 880 following group I metabotropic glutamate receptor stimulation in the rat dorsal striatum.

    PubMed

    Ahn, Sung Min; Choe, Eun Sang

    2010-04-01

    Phosphorylation of ionotropic glutamate receptors in the brain plays a crucial role in the regulation of synaptic plasticity. In this study, we investigated the regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor phosphorylation by the stimulation of group I metabotropic glutamate receptors (mGluRs) in the dorsal striatum in vivo. The results showed that intrastriatal infusion of the group I mGluR agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG, 250 nmol), enhanced the sensitivity of GluR2 subunit in its phosphorylation at serine 880 (S880) in the dorsal striatum. This enhancement of the sensitivity of GluR2-S880 phosphorylation was reduced by blocking group I mGluRs and N-methyl-D-aspartate (NMDA) receptors. Similar reduction of the enhancement was also induced by inhibiting phospholipase C (PLC), calcium/calmodulin-dependent protein kinase (CaMK), c-Jun N-terminal kinase (JNK), and protein kinase C (PKC). Inhibition of protein phosphatase (PP) 1/2A and calcineurin (PP2B) alone enhanced GluR2-S880 phosphorylation in the dorsal striatum, whereas inhibition of these phosphatases did not further enhance the S880 phosphorylation by DHPG stimulation. In addition, inhibition of PP1/2A or PP2B also enhanced the phosphorylation of CaMKII, JNK and PKC. These data suggest that the phosphorylation of AMPA receptor GluR2 subunit at S880 is subject to the upregulation by the stimulation of group I mGluRs. Interactions among glutamate receptors, protein kinases, and PPs participate in this upregulation. (c) 2009 Wiley-Liss, Inc.

  14. Celecoxib offsets the negative renal influences of cyclosporine via modulation of the TGF-β1/IL-2/COX-2/endothelin ET(B) receptor cascade.

    PubMed

    El-Gowelli, Hanan M; Helmy, Maged W; Ali, Rabab M; El-Mas, Mahmoud M

    2014-03-01

    Endothelin (ET) signaling provokes nephrotoxicity induced by the immunosuppressant drug cyclosporine A (CSA). We tested the hypotheses that (i): celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, counterbalances renal derangements caused by CSA in rats and (ii) the COX-2/endothelin ET(B) receptor signaling mediates the CSA-celecoxib interaction. Ten-day treatment with CSA (20 mg/kg/day) significantly increased biochemical indices of renal function (serum urea, creatinine), inflammation (interleukin-2, IL-2) and fibrosis (transforming growth factor-β₁, TGF-β₁). Histologically, CSA caused renal tubular atrophy along with interstitial fibrosis. These detrimental renal effects of CSA were largely reduced in rats treated concurrently with celecoxib (10 mg/kg/day). We also report that cortical glomerular and medullary tubular protein expressions of COX-2 and ET(B) receptors were reduced by CSA and restored to near-control values in rats treated simultaneously with celecoxib. The importance of ET(B) receptors in renal control and in the CSA-celecoxib interaction was further verified by the findings (i) most of the adverse biochemical, inflammatory, and histopathological profiles of CSA were replicated in rats treated with the endothelin ETB receptor antagonist BQ788 (0.1 mg/kg/day, 10 days), and (ii) the BQ788 effects, like those of CSA, were alleviated in rats treated concurrently with celecoxib. Together, the data suggest that the facilitation of the interplay between the TGF-β1/IL-2/COX-2 pathway and the endothelin ET(B) receptors constitutes the cellular mechanism by which celecoxib ameliorates the nephrotoxic manifestations of CSA in rats. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Impact and benefit of A(2B)-adenosine receptor agonists for the respiratory tract: mucociliary clearance, ciliary beat frequency, trachea muscle tonus and cytokine release.

    PubMed

    Walaschewski, Robin; Begrow, Frank; Verspohl, Eugen J

    2013-01-01

    Adenosine is known to induce a bronchospasm in asthma- and COPD patients. The role of A(2B) receptors was investigated with respect to several parameters of the respiratory tract: tonus of smooth muscle, ciliary beat frequency as measured by high-speed video camera connected to a microscope (both in rats) and mucociliary clearance (MCC; transport of a fluorescent dye using a microdialysis procedure) in mice.  NECA (5'-N-ethylcarboxamidoadenosine) (a non-selective adenosine receptor agonist) was able to acutely induce a contraction, which was reversed to a relaxation after repeated dosing. This relaxation was completely abolished by PSB-1115, an A(2B) receptor antagonist. IL-13 (cytokine) was not involved mediating acute contractility effects. MCC was increased by BAY 60-6583 (A(2B) receptor agonist) and NECA (counteracted by the A(2B) receptor antagonist PSB-1115). Activation of A(2B) adenosine receptors by BAY 60-6583 induced an increase of the ciliary beat frequency, which could be reduced by administration of PSB-1115. Several cytokines were increased by NECA although only some are relevant because they are not blocked by A(2B) receptor antagonism. The A(2B) receptors are involved in airway relaxation, MCC improvement and ciliary beat frequency. A(2B) receptor agonists may be of therapeutic value and should be developed. © 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society.

  16. Cryo-EM structure of the serotonin 5-HT1B receptor coupled to heterotrimeric Go.

    PubMed

    García-Nafría, Javier; Nehmé, Rony; Edwards, Patricia C; Tate, Christopher G

    2018-06-20

    G-protein-coupled receptors (GPCRs) form the largest family of receptors encoded by the human genome (around 800 genes). They transduce signals by coupling to a small number of heterotrimeric G proteins (16 genes encoding different α-subunits). Each human cell contains several GPCRs and G proteins. The structural determinants of coupling of G s to four different GPCRs have been elucidated 1-4 , but the molecular details of how the other G-protein classes couple to GPCRs are unknown. Here we present the cryo-electron microscopy structure of the serotonin 5-HT 1B receptor (5-HT 1B R) bound to the agonist donitriptan and coupled to an engineered G o heterotrimer. In this complex, 5-HT 1B R is in an active state; the intracellular domain of the receptor is in a similar conformation to that observed for the β 2 -adrenoceptor (β 2 AR) 3 or the adenosine A 2A receptor (A 2A R) 1 in complex with G s . In contrast to the complexes with G s , the gap between the receptor and the Gβ-subunit in the G o -5-HT 1B R complex precludes molecular contacts, and the interface between the Gα-subunit of G o and the receptor is considerably smaller. These differences are likely to be caused by the differences in the interactions with the C terminus of the G o α-subunit. The molecular variations between the interfaces of G o and G s in complex with GPCRs may contribute substantially to both the specificity of coupling and the kinetics of signalling.

  17. Adenosine A2B Receptor Deficiency Promotes Host Defenses against Gram-Negative Bacterial Pneumonia

    PubMed Central

    Barletta, Kathryn E.; Cagnina, R. Elaine; Burdick, Marie D.; Linden, Joel

    2012-01-01

    Rationale: Activation of the adenosine A2B receptor (A2BR) promotes antiinflammatory effects in diverse biological settings, but the role of this receptor in antimicrobial host defense in the lung has not been established. Gram-negative bacillary pneumonia is a common and serious illness associated with high morbidity and mortality, the treatment of which is complicated by increasing rates of antibiotic resistance. Objectives: To test the hypothesis that absence of adenosine A2B receptor signaling promotes host defense against bacterial pneumonia. Methods: We used a model of Klebsiella pneumoniae pneumonia in wild-type mice and mice with targeted deletion of the A2BR. Host responses were compared in vivo and leukocyte responses to the bacteria were examined in vitro. Measurements and Main Results: A2BR–/– mice demonstrated enhanced bacterial clearance from the lung and improved survival after infection with K. pneumoniae compared with wild-type controls, an effect that was mediated by bone marrow–derived cells. Leukocyte recruitment to the lungs and expression of inflammatory cytokines did not differ between A2BR–/– and wild-type mice, but A2BR–/– neutrophils exhibited sixfold greater bactericidal activity and enhanced production of neutrophil extracellular traps compared with wild-type neutrophils when incubated with K. pneumoniae. Consistent with this finding, bronchoalveolar lavage fluid from A2BR–/– mice with Klebsiella pneumonia contained more extracellular DNA compared with wild-type mice with pneumonia. Conclusions: These data suggest that the absence of A2BR signaling enhances antimicrobial activity in gram-negative bacterial pneumonia. PMID:22997203

  18. Cross-talk between Carboxypeptidase M and the Kinin B1 Receptor Mediates a New Mode of G Protein-coupled Receptor Signaling*

    PubMed Central

    Zhang, Xianming; Tan, Fulong; Brovkovych, Viktor; Zhang, Yongkang; Skidgel, Randal A.

    2011-01-01

    G protein-coupled receptor (GPCR) signaling is affected by formation of GPCR homo- or heterodimers, but GPCR regulation by other cell surface proteins is not well understood. We reported that the kinin B1 receptor (B1R) heterodimerizes with membrane carboxypeptidase M (CPM), facilitating receptor signaling via CPM-mediated conversion of bradykinin or kallidin to des-Arg kinin B1R agonists. Here, we found that a catalytically inactive CPM mutant that still binds substrate (CPM-E264Q) also facilitates efficient B1R signaling by B2 receptor agonists bradykinin or kallidin. This response required co-expression of B1R and CPM-E264Q in the same cell, was disrupted by antibody that dissociates CPM from B1R, and was not found with a CPM-E264Q-B1R fusion protein. An additional mutation that reduced the affinity of CPM for C-terminal Arg and increased the affinity for C-terminal Lys inhibited the B1R response to bradykinin (with C-terminal Arg) but generated a response to Lys9-bradykinin. CPM-E264Q-mediated activation of B1Rs by bradykinin resulted in increased intramolecular fluorescence resonance energy transfer (FRET) in a B1R FRET construct, similar to that generated directly by a B1R agonist. In cytokine-treated human lung microvascular endothelial cells, disruption of B1R-CPM heterodimers inhibited B1R-dependent NO production stimulated by bradykinin and blocked the increased endothelial permeability caused by treatment with bradykinin and pyrogallol (a superoxide generator). Thus, CPM and B1Rs on cell membranes form a critical complex that potentiates B1R signaling. Kinin peptide binding to CPM causes a conformational change in the B1R leading to intracellular signaling and reveals a new mode of GPCR activation by a cell surface peptidase. PMID:21454694

  19. Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain.

    PubMed

    Abe, Tetsuya; Matsumura, Shinji; Katano, Tayo; Mabuchi, Tamaki; Takagi, Kunio; Xu, Li; Yamamoto, Akitsugu; Hattori, Kotaro; Yagi, Takeshi; Watanabe, Masahiko; Nakazawa, Takanobu; Yamamoto, Tadashi; Mishina, Masayoshi; Nakai, Yoshihide; Ito, Seiji

    2005-09-01

    Despite abundant evidence implicating the importance of N-methyl-D-aspartate (NMDA) receptors in the spinal cord for pain transmission, the signal transduction coupled to NMDA receptor activation is largely unknown for the neuropathic pain state that lasts over periods of weeks. To address this, we prepared mice with neuropathic pain by transection of spinal nerve L5. Wild-type, NR2A-deficient, and NR2D-deficient mice developed neuropathic pain; in addition, phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 was observed in the superficial dorsal horn of the spinal cord 1 week after nerve injury. Neuropathic pain and NR2B phosphorylation at Tyr1472 were attenuated by the NR2B-selective antagonist CP-101,606 and disappeared in mice lacking Fyn kinase, a Src-family tyrosine kinase. Concomitant with the NR2B phosphorylation, an increase in neuronal nitric oxide synthase activity was visualized in the superficial dorsal horn of neuropathic pain mice by NADPH diaphorase histochemistry. Electron microscopy showed that the phosphorylated NR2B was localized at the postsynaptic density in the spinal cord of mice with neuropathic pain. Indomethacin, an inhibitor of prostaglandin (PG) synthesis, and PGE receptor subtype EP1-selective antagonist reduced the NR2B phosphorylation in these mice. Conversely, EP1-selective agonist stimulated Fyn kinase-dependent nitric oxide formation in the spinal cord. The present study demonstrates that Tyr1472 phosphorylation of NR2B subunits by Fyn kinase may have dual roles in the retention of NMDA receptors in the postsynaptic density and in activation of nitric oxide synthase, and suggests that PGE2 is involved in the maintenance of neuropathic pain via the EP1 subtype.

  20. Coexistence of Eph receptor B1 and ephrin B2 in port-wine stain endothelial progenitor cells contributes to clinicopathological vasculature dilatation.

    PubMed

    Tan, W; Wang, J; Zhou, F; Gao, L; Yin, R; Liu, H; Sukanthanag, A; Wang, G; Mihm, M C; Chen, D-B; Nelson, J S

    2017-12-01

    Port-wine stain (PWS) is a vascular malformation characterized by progressive dilatation of postcapillary venules, but the molecular pathogenesis remains obscure. To illustrate that PWS endothelial cells (ECs) present a unique molecular phenotype that leads to pathoanatomical PWS vasculatures. Immunohistochemistry and transmission electron microscopy were used to characterize the ultrastructure and molecular phenotypes of PWS blood vessels. Primary culture of human dermal microvascular endothelial cells and in vitro tube formation assay were used for confirmative functional studies. Multiple clinicopathological features of PWS blood vessels during the development and progression of the disease were shown. There were no normal arterioles and venules observed phenotypically and morphologically in PWS skin; arterioles and venules both showed differentiation impairments, resulting in a reduction of arteriole-like vasculatures and defects in capillary loop formation in PWS lesions. PWS ECs showed stemness properties with expression of endothelial progenitor cell markers CD133 and CD166 in non-nodular lesions. They also expressed dual venous/arterial identities, Eph receptor B1 (EphB1) and ephrin B2 (EfnB2). Co-expression of EphB1 and EfnB2 in normal human dermal microvascular ECs led to the formation of PWS-like vasculatures in vitro, for example larger-diameter and thick-walled capillaries. PWS ECs are differentiation-impaired, late-stage endothelial progenitor cells with a specific phenotype of CD133 + /CD166 + /EphB1 + /EfnB2 + , which form immature venule-like pathoanatomical vasculatures. The disruption of normal EC-EC interactions by coexistence of EphB1 and EfnB2 contributes to progressive dilatation of PWS vasculatures. © 2017 British Association of Dermatologists.

  1. RNi2B2C (R = Ho, Dy, Tb and Pr) single crystals grown by the cold copper crucible method

    NASA Astrophysics Data System (ADS)

    Durán, A.; Munoz, E.; Bernès, S.; Escudero, R.

    2000-08-01

    Single crystals of RNi2B2C (R = Ho, Dy, Tb, Pr) have been grown on cold copper crucibles in a high-frequency induction furnace. As a result, shiny metallic and brittle platelike single crystals were obtained. They were examined by x-ray and scanning electron microscopy with WDX/EDX for local composition analysis and show a very good crystallographic structure and compositions. Resistivity and dc magnetic measurements were performed to study superconducting and magnetic properties. Besides known electronic properties of the RNi2B2C family, we report for the first time results for PrNi2B2C single crystals successfully obtained by this technique.

  2. Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways.

    PubMed

    Guillermet-Guibert, J; Saint-Laurent, N; Davenne, L; Rochaix, P; Cuvillier, O; Culler, M D; Pradayrol, L; Buscail, L; Susini, C; Bousquet, C

    2007-02-01

    Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.

  3. Activation-Induced Killer Cell Immunoglobulin-like Receptor 3DL2 Binding to HLA-B27 Licenses Pathogenic T Cell Differentiation in Spondyloarthritis.

    PubMed

    Ridley, Anna; Hatano, Hiroko; Wong-Baeza, Isabel; Shaw, Jacqueline; Matthews, Katherine K; Al-Mossawi, Hussein; Ladell, Kristin; Price, David A; Bowness, Paul; Kollnberger, Simon

    2016-04-01

    In the spondyloarthritides (SpA), increased numbers of CD4+ T cells express killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). The aim of this study was to determine the factors that induce KIR-3DL2 expression, and to characterize the relationship between HLA-B27 and the phenotype and function of KIR-3DL2-expressing CD4+ T cells in SpA. In total, 34 B27+ patients with SpA, 28 age- and sex-matched healthy controls (20 B27- and 8 B27+), and 9 patients with rheumatoid arthritis were studied. KIR-3DL2 expression and other phenotypic characteristics of peripheral blood and synovial fluid CD4+ T cells were studied by flow cytometry, quantitative polymerase chain reaction, and Western blotting. T cell receptor clonality was determined by template-switch anchored reverse transcription-polymerase chain reaction and sequencing analysis. Cytokines were measured by enzyme-linked immunosorbent assay. Cellular activation induced KIR-3DL2 expression on both naive and effector CD4+ T cells. KIR-3DL2 binding to B27+ cells promoted expression of KIR-3DL2, the Th17-specific transcription factor retinoic acid receptor-related orphan nuclear receptor γt, and the antiapoptotic factor B cell lymphoma 2. KIR-3DL2+CD4+ T cells in patients with ankylosing spondylitis were oligoclonal and enriched for markers of T cell activation and for the gut homing receptor CCR9. In the presence of B27+ antigen-presenting cells, KIR-3DL2+CD4+ T cells produced less interleukin-2 (IL-2) but more IL-17. This effect was blocked by HC10, an antibody that inhibits the binding of KIR-3DL2 to B27 heavy chains. KIR-3DL2 binding to HLA-B27 licenses Th17 cell differentiation in SpA. These findings raise the therapeutic potential of targeting HLA-B27-KIR-3DL2 interactions for the treatment of B27+ patients with SpA. © 2016 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.

  4. Ryanodine Receptor Calcium Leak in Circulating B-Lymphocytes as a Biomarker in Heart Failure.

    PubMed

    Kushnir, Alexander; Santulli, Gaetano; Reiken, Steven R; Coromilas, Ellie; Godfrey, Sarah J; Brunjes, Danielle L; Colombo, Paolo C; Yuzefpolskaya, Melana; Sokol, Seth I; Kitsis, Richard N; Marks, Andrew R

    2018-03-28

    Background -Advances in congestive heart failure (CHF) management depend on biomarkers for monitoring disease progression and therapeutic response. During systole, intracellular Ca2 + is released from the sarcoplasmic reticulum (SR) into the cytoplasm through type 2 ryanodine receptor/Ca2 + release channels (RyR2). In CHF, chronically elevated circulating catecholamine levels cause pathologic remodeling of RyR2 resulting in diastolic SR Ca2 + leak, and decreased myocardial contractility. Similarly, skeletal muscle contraction requires SR Ca2 + release through type-1 ryanodine receptors (RyR1), and chronically elevated catecholamine levels in CHF cause RyR1 mediated SR Ca2 + leak, contributing to myopathy and weakness. Circulating B-lymphocytes express RyR1 and catecholamine responsive signaling cascades, making them a potential surrogate for defects in intracellular Ca2 + handling due to leaky RyR channels in CHF. Methods -Whole blood was collected from patients with CHF, CHF status-post left-ventricular assist devices (LVAD), and controls. Blood was also collected from mice with ischemic CHF, ischemic CHF + S107 (a drug that specifically reduces RyR channel Ca2 + leak), and WT controls. Channel macromolecular complex was assessed by immunostaining RyR1 immunoprecipitated from lymphocyte enriched preparations. RyR1 Ca2 + leak was assessed using flow cytometry to measure Ca2 + fluorescence in B-lymphocytes, in the absence and presence of RyR1 agonists that empty RyR1 Ca2 + stores within the endoplasmic reticulum (ER). Results -Circulating B-lymphocytes from humans and mice with CHF exhibited remodeled RyR1 and decreased ER Ca2 + stores, consistent with chronic intracellular Ca2 + leak. This Ca2 + leak correlated with circulating catecholamine levels. The intracellular Ca2 + leak was significantly reduced in mice treated with the Rycal S107. CHF patients treated with LVAD exhibited a heterogeneous response. Conclusions -In CHF, B-lymphocytes exhibit remodeled leaky

  5. Activation of farnesoid X receptor promotes triglycerides lowering by suppressing phospholipase A2 G12B expression.

    PubMed

    Liu, Qingli; Yang, Meng; Fu, Xuekun; Liu, Renzhong; Sun, Caijun; Pan, Haobo; Wong, Chi-Wai; Guan, Min

    2016-11-15

    As a novel mediator of hepatic very low-density lipoproteins (VLDL) secretion, phospholipase A2 G12B (PLA2G12B) is transcriptionally regulated by hepatocyte nuclear factor-4 alpha (HNF-4α). Farnesoid X receptor (FXR) plays a critical role in maintaining bile acids and triglycerides (TG) homeostasis. Here we report that FXR regulates serum TG level in part through PLA2G12B. Activation of FXR by chenodeoxycholic acid (CDCA) or GW4064 significantly decreased PLA2G12B expression in HepG2 cells. PLA2G12B expression was transcriptionally repressed due to an FXR-mediated up-regulation of small heterodimer partner (SHP) which functionally suppresses HNF-4α activity. We found that hepatic PLA2G12B expression was suppressed and serum TG level reduced in high fat diet mice treated with CDCA. Concurrently, CDCA treatment lowered hepatic VLDL-TG secretion. Our data demonstrate that activation of FXR promotes TG lowering, not only by decreasing de novo lipogenesis but also reducing hepatic secretion of TG-rich VLDL particles in part through suppressing PLA2G12B expression. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. SH2-B promotes insulin receptor substrate 1 (IRS1)- and IRS2-mediated activation of the phosphatidylinositol 3-kinase pathway in response to leptin.

    PubMed

    Duan, Chaojun; Li, Minghua; Rui, Liangyou

    2004-10-15

    Leptin regulates energy homeostasis primarily by binding and activating its long form receptor (LRb). Deficiency of either leptin or LRb causes morbid obesity. Leptin stimulates LRb-associated JAK2, thus initiating multiple pathways including the Stat3 and phosphatidylinositol (PI) 3-kinase pathways that mediate leptin biological actions. Here we report that SH2-B, a JAK2-interacting protein, promotes activation of the PI 3-kinase pathway by recruiting insulin receptor substrate 1 (IRS1) and IRS2 in response to leptin. SH2-B directly bound, via its PH and SH2 domain, to both IRS1 and IRS2 both in vitro and in intact cells and mediated formation of a JAK2/SH2-B/IRS1 or IRS2 tertiary complex. Consequently, SH2-B dramatically enhanced leptin-stimulated tyrosine phosphorylation of IRS1 and IRS2 in HEK293 cells stably expressing LRb, thus promoting association of IRS1 and IRS2 with the p85 regulatory subunit of PI 3-kinase and phosphorylation and activation of Akt. SH2-B mutants with lower affinity for IRS1 and IRS2 exhibited reduced ability to promote association of JAK2 with IRS1, tyrosine phosphorylation of IRS1, and association of IRS1 with p85 in response to leptin. Moreover, deletion of the SH2-B gene impaired leptin-stimulated tyrosine phosphorylation of endogenous IRS1 in mouse embryonic fibroblasts (MEF), which was reversed by reintroduction of SH2-B. Similarly, SH2-B promoted growth hormone-stimulated tyrosine phosphorylation of IRS1 in both HEK293 and MEF cells. Our data suggest that SH2-B is a novel mediator of the PI 3-kinase pathway in response to leptin or other hormones and cytokines that activate JAK2.

  7. Effect of a kinin B2 receptor antagonist on LPS- and cytokine-induced neutrophil migration in rats

    PubMed Central

    Santos, Danielle R; Calixto, João B; Souza, Glória E P

    2003-01-01

    This study examines the involvement of kinins in neutrophil migration into rat subcutaneous air pouches triggered by lipopolysaccharide (LPS), as well as the putative roles played by kinin B1 and B2 receptors, tumour necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and selectins in this response. LPS (5 ng to 10 μg cavity−1) injected into the 6-day-old pouch induced a dose- and time-dependent neutrophil migration which peaked between 4 and 6 h, and was maximal following the dose of 100 ng cavity−1 (saline: 0.46±0.1; LPS: 43±3.70 × 106 cells cavity−1 at 6 h). Bradykinin (BK) (600 nmol) injected into the pouch of saline-treated rats induced only modest neutrophil migration (0.73±0.16 × 106 cells cavity−1). A more robust response to BK (3.2±0.25 × 106 cells cavity−1) was seen in animals pretreated with captopril, but this was still smaller than the responses to IL-1β or TNF-α (15 pmol: 23±2.2 × 106 and 75 pmol: 29.5±2 × 106 cells cavity−1, respectively). Nevertheless, the B1 agonist des-Arg9-BK (600 nmol) failed to induce neutrophil migration. HOE-140 (1 and 2 mg kg−1), a B2 receptor antagonist, reduced LPS-induced neutrophil migration. HOE-140 also reduced the neutrophil migration induced by BK, but had no effect on the migration promoted by IL-1β or TNF-α. des-Arg9-[Leu8]-BK, B1 receptor antagonist was ineffective in changing neutrophil migration caused by any of these stimuli. Neutrophil migration induced by LPS or BK was reduced by interleukin-1 receptor antagonist (IL-1ra) (1 mg kg−1), sheep anti-rat TNF serum (anti-TNF serum) (0.3 ml cavity−1), and the nonspecific selectin inhibitor fucoidin (10 mg kg−1). TNF-α levels in the pouch fluid were increased by LPS or BK injection, peaking at 0.5–1 h and gradually declining thereafter up to 6 h. IL-1β levels increased steadily throughout the 6 h period. HOE-140 markedly inhibited the rise in IL-1β and TNF-α levels in pouch fluid triggered by both stimuli. These

  8. VizieR Online Data Catalog: Sgr B2(N) and Sgr B2(M) IRAM 30m line survey (Belloche+, 2013)

    NASA Astrophysics Data System (ADS)

    Belloche, A.; Mueller, H. S. P.; Menten, K. M.; Schilke, P.; Comito, C.

    2013-08-01

    The list of line identifications corresponding to the blue labels in Figs. 2 to 7 where the labels are often too crowded to be easily readable are available in ASCII format. The lists are split into six files, three for Sgr B2(N) and three for Sgr B2(M). For each source, there is one file per atmospheric window (3, 2, and 1mm). Each file is ordered by increasing frequency. The observed and synthetic spectra of Sgr B2(N) and Sgr B2(M) between 80 and 116GHz are available both in ASCII and FITS formats. The synthetic spectra were resampled to the same frequency channels as the observed spectra. The blanking value is -1000K for the ASCII files. There is one ASCII file per source. There are two FITS files per source, one for the observed spectrum and one for the synthetic spectrum. The intensities are in main-beam temperature scale in K. The blanking value is 42.75234K for the observed spectrum of SgrB2(N) and 53.96533K for the observed spectrum of SgrB2(M). (9 data files).

  9. Infralimbic EphB2 Modulates Fear Extinction in Adolescent Rats

    PubMed Central

    Cruz, Emmanuel; Soler-Cedeño, Omar; Negrón, Geovanny; Criado-Marrero, Marangelie; Chompré, Gladys

    2015-01-01

    Adolescent rats are prone to impaired fear extinction, suggesting that mechanistic differences in extinction could exist in adolescent and adult rats. Since the infralimbic cortex (IL) is critical for fear extinction, we used PCR array technology to identify gene expression changes in IL induced by fear extinction in adolescent rats. Interestingly, the ephrin type B receptor 2 (EphB2), a tyrosine kinase receptor associated with synaptic development, was downregulated in IL after fear extinction. Consistent with the PCR array results, EphB2 levels of mRNA and protein were reduced in IL after fear extinction compared with fear conditioning, suggesting that EphB2 signaling in IL regulates fear extinction memory in adolescents. Finally, reducing EphB2 synthesis in IL with shRNA accelerated fear extinction learning in adolescent rats, but not in adult rats. These findings identify EphB2 in IL as a key regulator of fear extinction during adolescence, perhaps due to the increase in synaptic remodeling occurring during this developmental phase. PMID:26354908

  10. Infralimbic EphB2 Modulates Fear Extinction in Adolescent Rats.

    PubMed

    Cruz, Emmanuel; Soler-Cedeño, Omar; Negrón, Geovanny; Criado-Marrero, Marangelie; Chompré, Gladys; Porter, James T

    2015-09-09

    Adolescent rats are prone to impaired fear extinction, suggesting that mechanistic differences in extinction could exist in adolescent and adult rats. Since the infralimbic cortex (IL) is critical for fear extinction, we used PCR array technology to identify gene expression changes in IL induced by fear extinction in adolescent rats. Interestingly, the ephrin type B receptor 2 (EphB2), a tyrosine kinase receptor associated with synaptic development, was downregulated in IL after fear extinction. Consistent with the PCR array results, EphB2 levels of mRNA and protein were reduced in IL after fear extinction compared with fear conditioning, suggesting that EphB2 signaling in IL regulates fear extinction memory in adolescents. Finally, reducing EphB2 synthesis in IL with shRNA accelerated fear extinction learning in adolescent rats, but not in adult rats. These findings identify EphB2 in IL as a key regulator of fear extinction during adolescence, perhaps due to the increase in synaptic remodeling occurring during this developmental phase. Copyright © 2015 the authors 0270-6474/15/3512394-10$15.00/0.

  11. Pneumococcal polysaccharides complexed with C3d bind to human B lymphocytes via complement receptor type 2.

    PubMed Central

    Griffioen, A W; Rijkers, G T; Janssens-Korpela, P; Zegers, B J

    1991-01-01

    The immunoregulatory function of the complement system has been the focus of many investigations. In particular, fragments of complement factor C3 have been shown to play a role in B-lymphocyte activation and proliferation, lymphokine production, and the generation of in vitro antibody production. Purified pneumococcal polysaccharides (PS) can induce direct activation of C3 via the alternative pathway. Using sera of C1q-deficient patients and healthy subjects, we demonstrated that C3d, a split product of C3 that is generated after degradation of iC3b, can be bound to PS antigens. The binding of C3d to PS can occur in the absence of specific antibodies. Subsequently, we showed that PS complexed with C3d can be recognized by complement receptor type 2 that is expressed on B cells. Treatment of B cells with a monoclonal antibody recognizing the C3d-binding site of complement receptor type 2 reduces the binding of PS-C3d to the cells. In addition, we showed that PS4 complexed with C3d exerted an increased immunogenicity compared with free PS4. Our results show that the complement system plays a role in the activation of PS-specific B cells, carrying membrane receptors for C3d. Consequently, the complement system plays a regulatory role in the antibody response to T-cell-independent type 2 antigens such as PS. PMID:1826897

  12. Purinergic A2b Receptor Activation by Extracellular Cues Affects Positioning of the Centrosome and Nucleus and Causes Reduced Cell Migration*

    PubMed Central

    Ou, Young; Chan, Gordon; Zuo, Jeremy; Rattner, Jerome B.; van der Hoorn, Frans A.

    2016-01-01

    The tight, relative positioning of the nucleus and centrosome in mammalian cells is important for the regulation of cell migration. Under pathophysiological conditions, the purinergic A2b receptor can regulate cell motility, but the underlying mechanism remains unknown. Expression of A2b, normally low, is increased in tissues experiencing adverse physiological conditions, including hypoxia and inflammation. ATP is released from such cells. We investigated whether extracellular cues can regulate centrosome-nucleus positioning and cell migration. We discovered that hypoxia as well as extracellular ATP cause a reversible increase in the distance between the centrosome and nucleus and reduced cell motility. We uncovered the underlying pathway: both treatments act through the A2b receptor and specifically activate the Epac1/RapGef3 pathway. We show that cells lacking A2b do not respond in this manner to hypoxia or ATP but transfection of A2b restores this response, that Epac1 is critically involved, and that Rap1B is important for the relative positioning of the centrosome and nucleus. Our results represent, to our knowledge, the first report demonstrating that pathophysiological conditions can impact the distance between the centrosome and nucleus. Furthermore, we identify the A2b receptor as a central player in this process. PMID:27226580

  13. The B7 family of immunoregulatory receptors: A comparative and evolutionary perspective

    USGS Publications Warehouse

    Hansen, J.D.; Pasquier, L.D.; Lefranc, M.-P.; Lopez, V.; Benmansour, A.; Boudinot, P.

    2009-01-01

    In mammals, T cell activation requires specific recognition of the peptide-MHC complex by the TcR and co-stimulatory signals. Important co-stimulatory receptors expressed by T cells are the molecules of the CD28 family, that regulate T cell activation, proliferation and tolerance. These receptors recognize B7s and B7-homologous (B7H) molecules that are typically expressed by the antigen presenting cells. In teleost fish, typical T cell responses have been described and the TcR, MHC and CD28/CTLA4 genes have been characterized. In contrast, the members of the B7 gene family have only been described in mammals and birds and have yet to be addressed in lower vertebrates. To learn more about the evolution of components guiding T cell activation in vertebrates, we performed a systematic genomic survey for the B7 co-stimulatory and co-inhibitory IgSF receptors in lower vertebrates with an emphasis on teleost fish. Our search identified fish sequences that are orthologous to B7, B7-H1/B7-DC, B7-H3 and B7-H4 as defined by sequence identity, phylogeny and combinations of short or long-range syntenic relationships. However, we were unable to identify clear orthologs for B7-H2 (CD275, ICOS ligand) in bony fish, which correlates with our prior inability to find ICOS in fish. Interestingly, our results indicate that teleost fish possess a single B7.1/B7.2 (CD80/86) molecule that likely interacts with CD28/CTLA4 as the ligand-binding regions seem to be conserved in both partners. Overall, our analyses implies that gene duplication (and loss) have shaped a molecular repertoire of B7-like molecules that was recruited for the refinement of T cell activation during the evolution of the vertebrates.

  14. Deregulation of MiR-34b/Sox2 Predicts Prostate Cancer Progression.

    PubMed

    Forno, Irene; Ferrero, Stefano; Russo, Maria Veronica; Gazzano, Giacomo; Giangiobbe, Sara; Montanari, Emanuele; Del Nero, Alberto; Rocco, Bernardo; Albo, Giancarlo; Languino, Lucia R; Altieri, Dario C; Vaira, Valentina; Bosari, Silvano

    2015-01-01

    Most men diagnosed with prostate cancer will have an indolent and curable disease, whereas approximately 15% of these patients will rapidly progress to a castrate-resistant and metastatic stage with high morbidity and mortality. Therefore, the identification of molecular signature(s) that detect men at risk of progressing disease remains a pressing and still unmet need for these patients. Here, we used an integrated discovery platform combining prostate cancer cell lines, a Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model and clinically-annotated human tissue samples to identify loss of expression of microRNA-34b as consistently associated with prostate cancer relapse. Mechanistically, this was associated with epigenetics silencing of the MIR34B/C locus and increased DNA copy number loss, selectively in androgen-dependent prostate cancer. In turn, loss of miR-34b resulted in downstream deregulation and overexpression of the "stemness" marker, Sox2. These findings identify loss of miR-34b as a robust biomarker for prostate cancer progression in androgen-sensitive tumors, and anticipate a potential role of progenitor/stem cell signaling in this stage of disease.

  15. Deregulation of MiR-34b/Sox2 Predicts Prostate Cancer Progression

    PubMed Central

    Russo, Maria Veronica; Gazzano, Giacomo; Giangiobbe, Sara; Montanari, Emanuele; Del Nero, Alberto; Rocco, Bernardo; Albo, Giancarlo; Languino, Lucia R.; Altieri, Dario C.; Vaira, Valentina; Bosari, Silvano

    2015-01-01

    Most men diagnosed with prostate cancer will have an indolent and curable disease, whereas approximately 15% of these patients will rapidly progress to a castrate-resistant and metastatic stage with high morbidity and mortality. Therefore, the identification of molecular signature(s) that detect men at risk of progressing disease remains a pressing and still unmet need for these patients. Here, we used an integrated discovery platform combining prostate cancer cell lines, a Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model and clinically-annotated human tissue samples to identify loss of expression of microRNA-34b as consistently associated with prostate cancer relapse. Mechanistically, this was associated with epigenetics silencing of the MIR34B/C locus and increased DNA copy number loss, selectively in androgen-dependent prostate cancer. In turn, loss of miR-34b resulted in downstream deregulation and overexpression of the “stemness” marker, Sox2. These findings identify loss of miR-34b as a robust biomarker for prostate cancer progression in androgen-sensitive tumors, and anticipate a potential role of progenitor/stem cell signaling in this stage of disease. PMID:26107383

  16. NR2B-containing NMDA receptors promote neural progenitor cell proliferation through CaMKIV/CREB pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Mei, E-mail: limeihit@163.com; Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, Nanjing; Zhang, Dong-Qing

    2011-08-12

    Highlights: {yields} The NR2B component of the NMDARs is important for the NSPC proliferation. {yields} pCaMKIV and pCREB exist in NSPCs. {yields} The CaMKIV/CREB pathway mediates NSPC proliferation. -- Abstract: Accumulating evidence indicates the involvement of N-methyl-D-aspartate receptors (NMDARs) in regulating neural stem/progenitor cell (NSPC) proliferation. Functional properties of NMDARs can be markedly influenced by incorporating the regulatory subunit NR2B. Here, we aim to analyze the effect of NR2B-containing NMDARs on the proliferation of hippocampal NSPCs and to explore the mechanism responsible for this effect. NSPCs were shown to express NMDAR subunits NR1 and NR2B. The NR2B selective antagonist, Romore » 25-6981, prevented the NMDA-induced increase in cell proliferation. Moreover, we demonstrated that the phosphorylation levels of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein (CREB) were increased by NMDA treatment, whereas Ro 25-6981 decreased them. The role that NR2B-containing NMDARs plays in NSPC proliferation was abolished when CREB phosphorylation was attenuated by CaMKIV silencing. These results suggest that NR2B-containing NMDARs have a positive role in regulating NSPC proliferation, which may be mediated through CaMKIV phosphorylation and subsequent induction of CREB activation.« less

  17. Novel kinin B1 receptor agonists with improved pharmacological profiles.

    PubMed

    Côté, Jérôme; Savard, Martin; Bovenzi, Veronica; Bélanger, Simon; Morin, Josée; Neugebauer, Witold; Larouche, Annie; Dubuc, Céléna; Gobeil, Fernand

    2009-04-01

    There is some evidence to suggest that inducible kinin B1 receptors (B1R) may play beneficial and protecting roles in cardiovascular-related pathologies such as hypertension, diabetes, and ischemic organ diseases. Peptide B1R agonists bearing optimized pharmacological features (high potency, selectivity and stability toward proteolysis) hold promise as valuable therapeutic agents in the treatment of these diseases. In the present study, we used solid-phase methodology to synthesize a series of novel peptide analogues based on the sequence of Sar[dPhe(8)]desArg(9)-bradykinin, a relatively stable peptide agonist with moderate affinity for the human B1R. We evaluated the pharmacological properties of these peptides using (1) in vitro competitive binding experiments on recombinant human B1R and B2R (for index of selectivity determination) in transiently transfected human embryonic kidney 293 cells (HEK-293T cells), (2) ex vivo vasomotor assays on isolated human umbilical veins expressing endogenous human B1R, and (3) in vivo blood pressure tests using anesthetized lipopolysaccharide-immunostimulated rabbits. Key chemical modifications at the N-terminus, the positions 3 and 5 on Sar[dPhe(8)]desArg(9)-bradykinin led to potent analogues. For example, peptides 18 (SarLys[Hyp(3),Cha(5), dPhe(8)]desArg(9)-bradykinin) and 20 (SarLys[Hyp(3),Igl(5), dPhe(8)]desArg(9)-bradykinin) outperformed the parental molecule in terms of affinity, functional potency and duration of action in vitro and in vivo. These selective agonists should be valuable in future animal and human studies to investigate the potential benefits of B1R activation.

  18. miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism.

    PubMed

    Tang, Xian-ye; Zheng, Wei; Ding, Min; Guo, Kai-jin; Yuan, Feng; Feng, Hu; Deng, Bin; Sun, Wei; Hou, Yang; Gao, Lu

    2016-01-01

    Chondrosarcoma is the second most common type of primary bone malignancy in the United States after osteosarcoma. Surgical resections of these tumors are the only effective treatment to chondrosarcoma patients due to their resistance to conventional chemo- and radiotherapy. In this study, miR-125b was found to perform its tumor-suppressor function to inhibit glucose metabolism via the direct targeting of oncogene, ErbB2. We report miR-125b was downregulated in both chondrosarcoma patient samples and cell lines. The total 20 Asian chondrosarcoma patients showed significantly downregulated miR-125b expression compared with normal tissues. Meanwhile, miR-125 was downregulated in chondrosarcoma cells and doxorubicin resistant cells. Overexpression of miR-125 enhanced the sensitivity of both parental and doxorubicin resistant cells to doxorubicin through direct targeting on the ErbB2-mediated upregulation of glycolysis in chondrosarcoma cells. Moreover, restoration of the expression of ErbB2 and glucose metabolic enzymes in miR-125 pretransfected cells recovered the susceptibility to doxorubicin. Our study will provide a novel aspect on the overcoming chemoresistance in human chondrosarcoma cells and may help in the development of therapeutic strategies for the treatments of patients.

  19. Presynaptic Muscarinic Acetylcholine Receptors and TrkB Receptor Cooperate in the Elimination of Redundant Motor Nerve Terminals during Development.

    PubMed

    Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A; Cilleros, Victor; Tomàs, Josep

    2017-01-01

    The development of the nervous system involves the overproduction of synapses but connectivity is refined by Hebbian activity-dependent axonal competition. The newborn skeletal muscle fibers are polyinnervated but, at the end of the competition process, some days later, become innervated by a single axon. We used quantitative confocal imaging of the autofluorescent axons from transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice to investigate the possible cooperation of the muscarinic autoreceptors (mAChR, M 1 -, M 2 - and M 4 -subtypes) and the tyrosine kinase B (TrkB) receptor in the control of axonal elimination after the mice Levator auris longus (LAL) muscle had been exposed to several selective antagonist of the corresponding receptor pathways in vivo . Our previous results show that M 1 , M 2 and TrkB signaling individually increase axonal loss rate around P9. Here we show that although the M 1 and TrkB receptors cooperate and add their respective individual effects to increase axonal elimination rate even more, the effect of the M 2 receptor is largely independent of both M 1 and TrkB receptors. Thus both, cooperative and non-cooperative signaling mechanisms contribute to developmental synapse elimination.

  20. Presynaptic Muscarinic Acetylcholine Receptors and TrkB Receptor Cooperate in the Elimination of Redundant Motor Nerve Terminals during Development

    PubMed Central

    Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A.; Cilleros, Victor; Tomàs, Josep

    2017-01-01

    The development of the nervous system involves the overproduction of synapses but connectivity is refined by Hebbian activity-dependent axonal competition. The newborn skeletal muscle fibers are polyinnervated but, at the end of the competition process, some days later, become innervated by a single axon. We used quantitative confocal imaging of the autofluorescent axons from transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice to investigate the possible cooperation of the muscarinic autoreceptors (mAChR, M1-, M2- and M4-subtypes) and the tyrosine kinase B (TrkB) receptor in the control of axonal elimination after the mice Levator auris longus (LAL) muscle had been exposed to several selective antagonist of the corresponding receptor pathways in vivo. Our previous results show that M1, M2 and TrkB signaling individually increase axonal loss rate around P9. Here we show that although the M1 and TrkB receptors cooperate and add their respective individual effects to increase axonal elimination rate even more, the effect of the M2 receptor is largely independent of both M1 and TrkB receptors. Thus both, cooperative and non-cooperative signaling mechanisms contribute to developmental synapse elimination. PMID:28228723

  1. Disease-associated missense mutations in GluN2B subunit alter NMDA receptor ligand binding and ion channel properties.

    PubMed

    Fedele, Laura; Newcombe, Joseph; Topf, Maya; Gibb, Alasdair; Harvey, Robert J; Smart, Trevor G

    2018-03-06

    Genetic and bioinformatic analyses have identified missense mutations in GRIN2B encoding the NMDA receptor GluN2B subunit in autism, intellectual disability, Lennox Gastaut and West Syndromes. Here, we investigated several such mutations using a near-complete, hybrid 3D model of the human NMDAR and studied their consequences with kinetic modelling and electrophysiology. The mutants revealed reductions in glutamate potency; increased receptor desensitisation; and ablation of voltage-dependent Mg 2+ block. In addition, we provide new views on Mg 2+ and NMDA channel blocker binding sites. We demonstrate that these mutants have significant impact on excitatory transmission in developing neurons, revealing profound changes that could underlie their associated neurological disorders. Of note, the NMDAR channel mutant GluN2B V618G unusually allowed Mg 2+ permeation, whereas nearby N615I reduced Ca 2+ permeability. By identifying the binding site for an NMDAR antagonist that is used in the clinic to rescue gain-of-function phenotypes, we show that drug binding may be modified by some GluN2B disease-causing mutations.

  2. 2′,3′-cAMP, 3′-AMP, and 2′-AMP inhibit human aortic and coronary vascular smooth muscle cell proliferation via A2B receptors

    PubMed Central

    Ren, Jin; Gillespie, Delbert G.

    2011-01-01

    Rat vascular smooth muscle cells (VSMCs) from renal microvessels metabolize 2′,3′-cAMP to 2′-AMP and 3′-AMP, and these AMPs are converted to adenosine that inhibits microvascular VSMC proliferation via A2B receptors. The goal of this study was to test whether this mechanism also exists in VSMCs from conduit arteries and whether it is similarly expressed in human vs. rat VSMCs. Incubation of rat and human aortic VSMCs with 2′,3′-cAMP concentration-dependently increased levels of 2′-AMP and 3′-AMP in the medium, with a similar absolute increase in 2′-AMP vs. 3′-AMP. In contrast, in human coronary VSMCs, 2′,3′-cAMP increased 2′-AMP levels yet had little effect on 3′-AMP levels. In all cell types, 2′,3′-cAMP increased levels of adenosine, but not 5′-AMP, and 2′,3′-AMP inhibited cell proliferation. Antagonism of A2B receptors (MRS-1754), but not A1 (1,3-dipropyl-8-cyclopentylxanthine), A2A (SCH-58261), or A3 (VUF-5574) receptors, attenuated the antiproliferative effects of 2′,3′-cAMP. In all cell types, 2′-AMP, 3′-AMP, and 5′-AMP increased adenosine levels, and inhibition of ecto-5′-nucleotidase blocked this effect of 5′-AMP but not that of 2′-AMP nor 3′-AMP. Also, 2′-AMP, 3′-AMP, and 5′-AMP, like 2′,3′-cAMP, exerted antiproliferative effects that were abolished by antagonism of A2B receptors with MRS-1754. In conclusion, VSMCs from conduit arteries metabolize 2′,3′-cAMP to AMPs, which are metabolized to adenosine. In rat and human aortic VSMCs, both 2′-AMP and 3′-AMP are involved in this process, whereas, in human coronary VSMCs, 2′,3′-cAMP is mainly converted to 2′-AMP. Because adenosine inhibits VSMC proliferation via A2B receptors, local vascular production of 2′,3′-cAMP may protect conduit arteries from atherosclerosis. PMID:21622827

  3. Anticancer activity of celastrol in combination with ErbB2-targeted therapeutics for treatment of ErbB2-overexpressing breast cancers

    PubMed Central

    Clubb, Robert J; Ortega-Cava, Cesar; Williams, Stetson H; Bailey, Tameka A; Duan, Lei; Zhao, Xiangshan; Reddi, Alagarasamy L; Nyong, Abijah M; Natarajan, Amarnath; Band, Vimla

    2011-01-01

    The receptor tyrosine kinase ErbB2 is overexpressed in up to a third of breast cancers, allowing targeted therapy with ErbB2-directed humanized antibodies such as Trastuzumab. Concurrent targeting of ErbB2 stability with HSP90 inhibitors is synergistic with Trastuzumab, suggesting that pharmacological agents that can inhibit HSP90 as well as signaling pathways activated by ErbB2 could be useful against ErbB2-overexpressing breast cancers. The triterpene natural product Celastrol inhibits HSP90 and several pathways relevant to ErbB2-dependent oncogenesis including the NFκB pathway and the proteasome, and has shown promising activity in other cancer models. Here, we demonstrate that Celastrol exhibits in vitro antitumor activity against a panel of human breast cancer cell lines with selectivity towards those overexpressing ErbB2. Celastrol strongly synergized with ErbB2-targeted therapeutics Trastuzumab and Lapatinib, producing higher cytotoxicity with substantially lower doses of Celastrol. Celastrol significantly retarded the rate of growth of ErbB2-overexpressing human breast cancer cells in a mouse xenograft model with only minor systemic toxicity. Mechanistically, Celastrol not only induced the expected ubiquitinylation and degradation of ErbB2 and other HSP90 client proteins, but it also increased the levels of reactive oxygen species (ROS). Our studies show that the Michael Acceptor functionality in Celastrol is important for its ability to destabilize ErbB2 and exert its bioactivity against ErbB2-overexpressing breast cancer cells. These studies suggest the potential use of Michael acceptor-containing molecules as novel therapeutic modalities against ErbB2-driven breast cancer by targeting multiple biological attributes of the driver oncogene. PMID:21088503

  4. Synthesis and SAR studies of novel 2-(6-aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists.

    PubMed

    Napier, Susan E; Letourneau, Jeffrey J; Ansari, Nasrin; Auld, Douglas S; Baker, James; Best, Stuart; Campbell-Wan, Leigh; Chan, Ray; Craighead, Mark; Desai, Hema; Ho, Koc-Kan; MacSweeney, Cliona; Milne, Rachel; Richard Morphy, J; Neagu, Irina; Ohlmeyer, Michael H J; Pick, Jack; Presland, Jeremy; Riviello, Chris; Zanetakos, Heather A; Zhao, Jiuqiao; Webb, Maria L

    2011-06-15

    Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) antagonists are described. 2-(6-Aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and OT. Optimised compound 16 shows a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Integrating Multimodal Radiation Therapy Data into i2b2.

    PubMed

    Zapletal, Eric; Bibault, Jean-Emmanuel; Giraud, Philippe; Burgun, Anita

    2018-04-01

     Clinical data warehouses are now widely used to foster clinical and translational research and the Informatics for Integrating Biology and the Bedside (i2b2) platform has become a de facto standard for storing clinical data in many projects. However, to design predictive models and assist in personalized treatment planning in cancer or radiation oncology, all available patient data need to be integrated into i2b2, including radiation therapy data that are currently not addressed in many existing i2b2 sites.  To use radiation therapy data in projects related to rectal cancer patients, we assessed the feasibility of integrating radiation oncology data into the i2b2 platform.  The Georges Pompidou European Hospital, a hospital from the Assistance Publique - Hôpitaux de Paris group, has developed an i2b2-based clinical data warehouse of various structured and unstructured clinical data for research since 2008. To store and reuse various radiation therapy data-dose details, activities scheduling, and dose-volume histogram (DVH) curves-in this repository, we first extracted raw data by using some reverse engineering techniques and a vendor's application programming interface. Then, we implemented a hybrid storage approach by combining the standard i2b2 "Entity-Attribute-Value" storage mechanism with a "JavaScript Object Notation (JSON) document-based" storage mechanism without modifying the i2b2 core tables. Validation was performed using (1) the Business Objects framework for replicating vendor's application screens showing dose details and activities scheduling data and (2) the R software for displaying the DVH curves.  We developed a pipeline to integrate the radiation therapy data into the Georges Pompidou European Hospital i2b2 instance and evaluated it on a cohort of 262 patients. We were able to use the radiation therapy data on a preliminary use case by fetching the DVH curve data from the clinical data warehouse and displaying them in a R chart.

  6. Blockade of epidermal growth factor- or heregulin-dependent ErbB2 activation with the anti-ErbB2 monoclonal antibody 2C4 has divergent downstream signaling and growth effects.

    PubMed

    Jackson, James G; St Clair, Patricia; Sliwkowski, Mark X; Brattain, Michael G

    2004-04-01

    Due to heterodimerization and a variety of stimulating ligands, the ErbB receptor system is both diverse and flexible, which proves particularly advantageous to the aberrant signaling of cancer cells. However, specific mechanisms of how a particular receptor contributes to generating the flexibility that leads to aberrant growth regulation have not been well described. We compared the utilization of ErbB2 in response to epidermal growth factor (EGF) and heregulin stimulation in colon carcinoma cells. Anti-ErbB2 monoclonal antibody 2C4 blocked heregulin-stimulated phosphorylation of ErbB2 and ErbB3; activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3'-kinase (PI3K), and Akt; proliferation; and anchorage-independent growth. 2C4 blocked EGF-mediated phosphorylation of ErbB2 and inhibited PI3K/Akt and anchorage-independent growth but did not affect ErbB1 or MAPK. Immunoprecipitations showed that ErbB3 and Grb2-associated binder (Gab) 1 were phosphorylated and associated with PI3K activity after heregulin treatment and that Gab1 and Gab2, but not ErbB3, were phosphorylated and associated with PI3K activity after EGF treatment. These data show that monoclonal antibody 2C4 inhibited all aspects of heregulin signaling as well as anchorage-independent and monolayer growth. Furthermore, we identify ErbB2 as a critical component of EGF signaling to the Gab1/Gab2-PI3K-Akt pathway and anchorage-independent growth, but EGF stimulation of MAPK and monolayer growth can occur efficiently without the contribution of ErbB2.

  7. Clinical relevance of ErbB-2/HER2 nuclear expression in breast cancer.

    PubMed

    Schillaci, Roxana; Guzmán, Pablo; Cayrol, Florencia; Beguelin, Wendy; Díaz Flaqué, María C; Proietti, Cecilia J; Pineda, Viviana; Palazzi, Jorge; Frahm, Isabel; Charreau, Eduardo H; Maronna, Esteban; Roa, Juan C; Elizalde, Patricia V

    2012-02-22

    The biological relevance of nuclear ErbB-2/HER2 (NuclErbB-2) presence in breast tumors remains unexplored. In this study we assessed the clinical significance of ErbB-2 nuclear localization in primary invasive breast cancer. The reporting recommendations for tumor marker prognostic studies (REMARK) guidelines were used as reference. Tissue microarrays from a cohort of 273 primary invasive breast carcinomas from women living in Chile, a Latin American country, were examined for membrane (MembErbB-2) and NuclErbB-2 expression by an immunofluorescence (IF) protocol we developed. ErbB-2 expression was also evaluated by immunohistochemistry (IHC) with a series of antibodies. Correlation between NuclErbB-2 and MembErbB-2, and between NuclErbB-2 and clinicopathological characteristics of tumors was studied. The prognostic value of NuclErbB-2 in overall survival (OS) was evaluated using Kaplan-Meier method, and Cox model was used to explore NuclErbB-2 as independent prognostic factor for OS. The IF protocol we developed showed significantly higher sensitivity for detection of NuclErbB-2 than IHC procedures, while its specificity and sensitivity to detect MembErbB-2 were comparable to those of IHC procedures. We found 33.6% NuclErbB-2 positivity, 14.2% MembErbB-2 overexpression by IF, and 13.0% MembErbB-2 prevalence by IHC in our cohort. We identified NuclErbB-2 positivity as a significant independent predictor of worse OS in patients with MembErbB-2 overexpression. NuclErbB-2 was also a biomarker of lower OS in tumors that overexpress MembErbB-2 and lack steroid hormone receptors. We revealed a novel role for NuclErbB-2 as an independent prognostic factor of poor clinical outcome in MembErbB-2-positive breast tumors. Our work indicates that patients presenting NuclErbB-2 may need new therapeutic strategies involving specific blockage of ErbB-2 nuclear migration.

  8. Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

    PubMed Central

    Xia, Wenle; Gooden, David; Liu, Leihua; Zhao, Sumin; Soderblom, Erik J.; Toone, Eric J.; Beyer, Wayne F.; Walder, Harold; Spector, Neil L.

    2014-01-01

    Photo-activation of psoralen with UVA irradiation, referred to as PUVA, is used in the treatment of proliferative skin disorders. The anti-proliferative effects of PUVA have been largely attributed to psoralen intercalation of DNA, which upon UV treatment, triggers the formation of interstrand DNA crosslinks (ICL) that inhibit transcription and DNA replication. Here, we show that PUVA exerts antitumor effects in models of human breast cancer that overexpress the ErbB2 receptor tyrosine kinase oncogene, through a new mechanism. Independent of ICL formation, the antitumor effects of PUVA in ErbB2+ breast cancer models can instead be mediated through inhibition of ErbB2 activation and signaling. Using a mass spectroscopy-based approach, we show for the first time that photo-activated 8MOP (8-methoxypsoralen) interacts with the ErbB2 catalytic autokinase domain. Furthermore, PUVA can reverse therapeutic resistance to lapatinib and other ErbB2 targeted therapies, including resistance mediated via expression of a phosphorylated, truncated form of ErbB2 (p85ErbB2) that is preferentially expressed in tumor cell nuclei. Current ErbB2 targeted therapies, small molecule kinase inhibitors or antibodies, do not block the phosphorylated, activated state of p85ErbB2. Here we show that PUVA reduced p85ErbB2 phosphorylation leading to tumor cell apoptosis. Thus, in addition to its effects on DNA and the formation of ICL, PUVA represents a novel ErbB2 targeted therapy for the treatment of ErbB2+ breast cancers, including those that have developed resistance to other ErbB2 targeted therapies. PMID:24551203

  9. Synthesis and serotonergic activity of substituted 2, N-benzylcarboxamido-5-(2-ethyl-1-dioxoimidazolidinyl)-N, N-dimethyltryptamine derivatives: novel antagonists for the vascular 5-HT(1B)-like receptor.

    PubMed

    Moloney, G P; Martin, G R; Mathews, N; Milne, A; Hobbs, H; Dodsworth, S; Sang, P Y; Knight, C; Williams, M; Maxwell, M; Glen, R C

    1999-07-15

    The synthesis and vascular 5-HT(1B)-like receptor activity of a novel series of substituted 2, N-benzylcarboxamido-5-(2-ethyl-1-dioxoimidazolidinyl)-N, N-dimethyltryptamine derivatives are described. Modifications to the 5-ethylene-linked heterocycle and to substituents on the 2-benzylamide side chain have been explored. Several compounds were identified which exhibited affinity at the vascular 5-HT(1B)-like receptor of pK(B) > 7.0, up to 100-fold selectivity over alpha(1)-adrenoceptor affinity and 5-HT(2A) receptor affinity, and which exhibited a favorable pharmacokinetic profile. N-Benzyl-3-[2-(dimethylamino)ethyl]-5-[2-(4,4-dimethyl-2, 5-dioxo-1-imidazolidinyl)ethyl]-1H-indole-2-carboxamide (23) was identified as a highly potent, silent (as judged by the inability of angiotensin II to unmask 5-HT(1B)-like receptor-mediated agonist activity in the rabbit femoral artery), and competitive vascular 5-HT(1B)-like receptor antagonist with a plasma elimination half-life of approximately 4 h in dog plasma and with good oral bioavailability. The selectivity of compounds from this series for the vascular 5-HT(1B)-like receptors over other receptor subtypes is discussed as well as a proposed mode of binding to the receptor pharmacophore. It has been proposed that the aromatic ring of the 2, N-benzylcarboxamide group can occupy an aromatic binding site rather than the indole ring. The resulting conformation allows an amine-binding site to be occupied by the ethylamine nitrogen and a hydrogen-bonding site to be occupied by one of the hydantoin carbonyls. The electronic nature of the 2,N-benzylcarboxamide aromatic group as well as the size of substituents on this aromatic group is crucial for producing potent and selective antagonists. The structural requirement on the 3-ethylamine side chain incorporating the protonatable nitrogen is achieved by the bulky 2, N-benzylcarboxamide group and its close proximity to the 3-side chain.

  10. Dopamine receptor D5 deficiency results in a selective reduction of hippocampal NMDA receptor subunit NR2B expression and impaired memory.

    PubMed

    Moraga-Amaro, Rodrigo; González, Hugo; Ugalde, Valentina; Donoso-Ramos, Juan Pablo; Quintana-Donoso, Daisy; Lara, Marcelo; Morales, Bernardo; Rojas, Patricio; Pacheco, Rodrigo; Stehberg, Jimmy

    2016-04-01

    Pharmacological evidence associates type I dopamine receptors, including subtypes D1 and D5, with learning and memory. Analyses using genetic approaches have determined the relative contribution of dopamine receptor D1 (D1R) in cognitive tasks. However, the lack of drugs that can discriminate between D1R and D5R has made the pharmacological distinction between the two receptors difficult. Here, we aimed to determine the role of D5R in learning and memory. In this study we tested D5R knockout mice and wild-type littermates in a battery of behavioral tests, including memory, attention, locomotion, anxiety and motivational evaluations. Our results show that genetic deficiency of D5R significantly impairs performance in the Morris water maze paradigm, object location and object recognition memory, indicating a relevant role for D5R in spatial memory and recognition memory. Moreover, the lack of D5R resulted in decreased exploration and locomotion. In contrast, D5R deficiency had no impact on working memory, anxiety and depressive-like behavior, measured using the spontaneous alternation, open-field, tail suspension test, and forced swimming test. Electrophysiological analyses performed on hippocampal slices showed impairment in long-term-potentiation in mice lacking D5R. Further analyses at the molecular level showed that genetic deficiency of D5R results in a strong and selective reduction in the expression of the NMDA receptor subunit NR2B in the hippocampus. These findings demonstrate the relevant contribution of D5R in memory and suggest a functional interaction of D5R with hippocampal glutamatergic pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. TRAF2 multitasking in TNF receptor-induced signaling to NF-κB, MAP kinases and cell death.

    PubMed

    Borghi, Alice; Verstrepen, Lynn; Beyaert, Rudi

    2016-09-15

    Tumor Necrosis Factor (TNF) is a potent inflammatory cytokine that exerts its functions through the activation of two distinct receptors, TNFR1 and TNFR2. Both receptors can activate canonical NF-κB and JNK MAP kinase signaling, while TNFR2 can also activate non-canonical NF-κB signaling, leading to numerous changes in gene expression that drive inflammation, cell proliferation and cell survival. On the other hand, TNFR1 also activates signaling pathways leading to cell death by either apoptosis or necroptosis, depending on the cellular context. A key player in TNFR1- and TNFR2-induced signaling is the RING finger protein TRAF2, which is recruited to both receptors upon their stimulation. TRAF2 exerts multiple receptor-specific functions but also mediates cross-talk between TNFR1 and TNFR2, dictating the outcome of TNF stimulation. In this review, we provide an overview of the positive and negative regulatory role of TRAF2 in different TNFR1 and TNFR2 signaling pathways. We discuss the underlying molecular mechanism of action, distinguishing between TRAF2 scaffold and E3 ubiquitin ligase functions, and the regulation of TRAF2 by specific post-translational modifications. Finally, we elaborate on some possible strategies to modulate TRAF2 function in the context of therapeutic targeting in autoimmunity and cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Volumetric properties under pressure for 1,2-dichlorofluoroethane (R141), 1-fluoro-1,1,2-trichloroethane (R131a), and 1,2-dichloro-1,1-difluoroethane (R132b)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Malhotra, R.; Woolf, L.A.

    1997-01-01

    The effect of pressure on the volume of R141, R131, and R132b is reported as volume ratios (the volume under pressure relative to its value at atmospheric pressure) at six temperatures covering the range 278.15 to 338.13 K and pressures up to 380 MPa for R141 and R131a. For R132b the same temperature range has been used, but above its normal boiling point experimental arrangements have limited maximum pressures to below 300 MPa, with some loss of accuracy. Densities have been measured at atmospheric pressure for each liquid. The experimental data have been used to calculate isothermal compressibilities, thermal expansivities,more » and internal pressures; the change in isobaric heat capacity from its value at atmospheric pressure has also been estimated. The volume ratios for all three compounds can be represented by a version of the Tait equation based on previously reported data for 1,2-dichloroethane and 1,1,2-trichloroethane with the inclusion of allowances of the substitution in the former of chlorine or fluorine for the hydrogens on one of the carbons.« less

  13. Purinergic A2b Receptor Activation by Extracellular Cues Affects Positioning of the Centrosome and Nucleus and Causes Reduced Cell Migration.

    PubMed

    Ou, Young; Chan, Gordon; Zuo, Jeremy; Rattner, Jerome B; van der Hoorn, Frans A

    2016-07-15

    The tight, relative positioning of the nucleus and centrosome in mammalian cells is important for the regulation of cell migration. Under pathophysiological conditions, the purinergic A2b receptor can regulate cell motility, but the underlying mechanism remains unknown. Expression of A2b, normally low, is increased in tissues experiencing adverse physiological conditions, including hypoxia and inflammation. ATP is released from such cells. We investigated whether extracellular cues can regulate centrosome-nucleus positioning and cell migration. We discovered that hypoxia as well as extracellular ATP cause a reversible increase in the distance between the centrosome and nucleus and reduced cell motility. We uncovered the underlying pathway: both treatments act through the A2b receptor and specifically activate the Epac1/RapGef3 pathway. We show that cells lacking A2b do not respond in this manner to hypoxia or ATP but transfection of A2b restores this response, that Epac1 is critically involved, and that Rap1B is important for the relative positioning of the centrosome and nucleus. Our results represent, to our knowledge, the first report demonstrating that pathophysiological conditions can impact the distance between the centrosome and nucleus. Furthermore, we identify the A2b receptor as a central player in this process. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Bu-2470, a new peptide antibiotic complex. II. Structure determination of Bu-2470 A, B1, B2a and B2b.

    PubMed

    Sugawara, K; Yonemoto, T; Konishi, M; Matsumoto, K; Miyaki, T; Kawaguchi, H

    1983-06-01

    The structures of Bu-2470 A, B1, B2a, and B2b have been determined. Bu-2470 A is a simple octapeptide having no fatty acid moiety, while Bu-2470 B1, B2a and B2b are octapeptides that have been acylated with a beta-hydroxy C11 or C10 fatty acid. The octapeptide structure of Bu-2470 components was found identical with that of octapeptin C1, hence generic names of octapeptin C0, C2, C3 and C4 are proposed for Bu-2470 A, B1, B2a and B2b, respectively.

  15. Kinase activation through dimerization by human SH2-B.

    PubMed

    Nishi, Masahiro; Werner, Eric D; Oh, Byung-Chul; Frantz, J Daniel; Dhe-Paganon, Sirano; Hansen, Lone; Lee, Jongsoon; Shoelson, Steven E

    2005-04-01

    The isoforms of SH2-B, APS, and Lnk form a family of signaling proteins that have been described as activators, mediators, or inhibitors of cytokine and growth factor signaling. We now show that the three alternatively spliced isoforms of human SH2-B readily homodimerize in yeast two-hybrid and cellular transfections assays, and this is mediated specifically by a unique domain in its amino terminus. Consistent with previous reports, we further show that the SH2 domains of SH2-B and APS bind JAK2 at Tyr813. These findings suggested a model in which two molecules of SH2-B or APS homodimerize with their SH2 domains bound to two JAK2 molecules, creating heterotetrameric JAK2-(SH2-B)2-JAK2 or JAK2-(APS)2-JAK2 complexes. We further show that APS and SH2-B isoforms heterodimerize. At lower levels of SH2-B or APS expression, dimerization approximates two JAK2 molecules to induce transactivation. At higher relative concentrations of SH2-B or APS, kinase activation is blocked. SH2-B or APS homodimerization and SH2-B/APS heterodimerization thus provide direct mechanisms for activating and inhibiting JAK2 and other kinases from the inside of the cell and for potentiating or attenuating cytokine and growth factor receptor signaling when ligands are present.

  16. miR-125b-5p enhances chemotherapy sensitivity to cisplatin by down-regulating Bcl2 in gallbladder cancer

    PubMed Central

    Yang, Dong; Zhan, Ming; Chen, Tao; Chen, Wei; Zhang, Yunhe; Xu, Sunwang; Yan, Jinchun; Huang, Qihong; Wang, Jian

    2017-01-01

    Gallbladder cancer represents the most common malignancy of the biliary tract and is highly lethal with less than 5% overall 5-year survival rate. Chemotherapy remains the major treatment for late-stage patients. However, insensitivity to these chemotherapeutic agents including cisplatin is common. MicroRNAs (miRNAs) have been shown as modulators of drug resistance in many cancer types. We used genome-wide gene expression analysis in clinical samples to identify miR-125b-5p down-regulated in gallbladder cancer. miR-125b-5p up-regulation promoted cell death in gallbladder cancer cells in the presence of cisplatin. In contrast, knockdown of miR-125b-5p reduced cell death in gallbladder cancer cells treated with cisplatin. Up-regulation of miR-125b-5p significantly decreased tumor growth in combination with cisplatin in a mouse model. We identified Bcl2 as a direct target of miR-125b-5p which mediates the function of miR-125b-5p in gallbladder cancer. In clinical samples, miR-125b-5p was down-regulated in gallbladder cancer whereas Bcl2 was up-regulated and their expression was inversely correlated. Moreover, low miR-125b-5p expression or high expression of Bcl2 is correlated with poor prognosis in gallbladder cancer. Taken together, our findings indicate that miR-125b-5p is a potent chemotherapy sensitizer and may function as a new biomarker for the prognosis of gallbladder cancer patients. PMID:28256505

  17. Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

    PubMed Central

    Garay, Camilo; Judge, Gurjeet; Lucarelli, Stefanie; Bautista, Stephen; Pandey, Rohan; Singh, Tanveer; Antonescu, Costin N.

    2015-01-01

    Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation. PMID:26246598

  18. B2B Models for DoD Acquisition

    DTIC Science & Technology

    2007-07-30

    Internet, extranets, intranets, or private networks • In 2003 it represented about 11% of total B2B trade estimated at $13.5 trillion • Predicted to... B2B Models for DoD Acquisition 30 July 2007 by Magdi N. Kamel, Associate Professor Graduate School of Operational & Information Sciences...number. 1. REPORT DATE 30 JUL 2007 2. REPORT TYPE 3. DATES COVERED 00-00-2007 to 00-00-2007 4. TITLE AND SUBTITLE B2B Models for DoD

  19. MicroRNA-29b-2-5p inhibits cell proliferation by directly targeting Cbl-b in pancreatic ductal adenocarcinoma.

    PubMed

    Li, Ce; Dong, Qian; Che, Xiaofang; Xu, Ling; Li, Zhi; Fan, Yibo; Hou, Kezuo; Wang, Shuo; Qu, Jinglei; Xu, Lu; Wen, Ti; Yang, Xianghong; Qu, Xiujuan; Liu, Yunpeng

    2018-06-25

    MicroRNAs can be used in the prognosis of malignancies; however, their regulatory mechanisms are unknown, especially in pancreatic ductal adenocarcinoma (PDAC). In 120 PDAC specimens, miRNA levels were assessed by quantitative real time polymerase chain reaction (qRT-PCR). Then, the role of miR-29b-2-5p in cell proliferation was evaluated both in vitro (Trypan blue staining and cell cycle analysis in the two PDAC cell lines SW1990 and Capan-2) and in vivo using a xenograft mouse model. Next, bioinformatics methods, a luciferase reporter assay, Western blot, and immunohistochemistry (IHC) were applied to assess the biological effects of Cbl-b inhibition by miR-29b-2-5p. Moreover, the relationship between Cbl-b and p53 was evaluated by immunoprecipitation (IP), Western blot, and immunofluorescence. From the 120 PDAC patients who underwent surgical resection, ten patients with longest survival and ten with shortest survival were selected. We found that high miR-29b-2-5p expression was associated with good prognosis (p = 0.02). The validation cohort confirmed miR-29b-2-5p as an independent prognostic factor in PDAC (n = 100, 95% CI = 0.305-0.756, p = 0.002). Furthermore, miR-29b-2-5p inhibited cell proliferation, induced cell cycle arrest, and promoted apoptosis both in vivo and in vitro. Interestingly, miR-29b-2-5p directly bound the Cbl-b gene, down-regulating its expression and reducing Cbl-b-mediated degradation of p53. Meanwhile, miR-29b-2-5p expression was negatively correlated with Cbl-b in PDAC tissues (r = - 0.33, p = 0.001). Taken together, these findings indicated that miR-29b-2-5p improves prognosis in PDAC by targeting Cbl-b to promote p53 expression, and would constitute an important prognostic factor in PDAC.

  20. Activation‐Induced Killer Cell Immunoglobulin‐like Receptor 3DL2 Binding to HLA–B27 Licenses Pathogenic T Cell Differentiation in Spondyloarthritis

    PubMed Central

    Ridley, Anna; Hatano, Hiroko; Wong‐Baeza, Isabel; Shaw, Jacqueline; Matthews, Katherine K.; Al‐Mossawi, Hussein; Ladell, Kristin; Price, David A.; Bowness, Paul

    2016-01-01

    Objective In the spondyloarthritides (SpA), increased numbers of CD4+ T cells express killer cell immunoglobulin‐like receptor 3DL2 (KIR‐3DL2). The aim of this study was to determine the factors that induce KIR‐3DL2 expression, and to characterize the relationship between HLA–B27 and the phenotype and function of KIR‐3DL2–expressing CD4+ T cells in SpA. Methods In total, 34 B27+ patients with SpA, 28 age‐ and sex‐matched healthy controls (20 B27− and 8 B27+), and 9 patients with rheumatoid arthritis were studied. KIR-3DL2 expression and other phenotypic characteristics of peripheral blood and synovial fluid CD4+ T cells were studied by flow cytometry, quantitative polymerase chain reaction, and Western blotting. T cell receptor clonality was determined by template‐switch anchored reverse transcription–polymerase chain reaction and sequencing analysis. Cytokines were measured by enzyme‐linked immunosorbent assay. Results Cellular activation induced KIR‐3DL2 expression on both naive and effector CD4+ T cells. KIR‐3DL2 binding to B27+ cells promoted expression of KIR‐3DL2, the Th17‐specific transcription factor retinoic acid receptor–related orphan nuclear receptor γt, and the antiapoptotic factor B cell lymphoma 2. KIR‐3DL2+CD4+ T cells in patients with ankylosing spondylitis were oligoclonal and enriched for markers of T cell activation and for the gut homing receptor CCR9. In the presence of B27+ antigen‐presenting cells, KIR‐3DL2+CD4+ T cells produced less interleukin‐2 (IL‐2) but more IL‐17. This effect was blocked by HC10, an antibody that inhibits the binding of KIR‐3DL2 to B27 heavy chains. Conclusion KIR‐3DL2 binding to HLA–B27 licenses Th17 cell differentiation in SpA. These findings raise the therapeutic potential of targeting HLA–B27–KIR‐3DL2 interactions for the treatment of B27+ patients with SpA. PMID:26841353

  1. miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism

    PubMed Central

    Tang, Xian-ye; Zheng, Wei; Ding, Min; Guo, Kai-jin; Yuan, Feng; Feng, Hu; Deng, Bin; Sun, Wei; Hou, Yang; Gao, Lu

    2016-01-01

    Chondrosarcoma is the second most common type of primary bone malignancy in the United States after osteosarcoma. Surgical resections of these tumors are the only effective treatment to chondrosarcoma patients due to their resistance to conventional chemo- and radiotherapy. In this study, miR-125b was found to perform its tumor-suppressor function to inhibit glucose metabolism via the direct targeting of oncogene, ErbB2. We report miR-125b was downregulated in both chondrosarcoma patient samples and cell lines. The total 20 Asian chondrosarcoma patients showed significantly downregulated miR-125b expression compared with normal tissues. Meanwhile, miR-125 was downregulated in chondrosarcoma cells and doxorubicin resistant cells. Overexpression of miR-125 enhanced the sensitivity of both parental and doxorubicin resistant cells to doxorubicin through direct targeting on the ErbB2-mediated upregulation of glycolysis in chondrosarcoma cells. Moreover, restoration of the expression of ErbB2 and glucose metabolic enzymes in miR-125 pretransfected cells recovered the susceptibility to doxorubicin. Our study will provide a novel aspect on the overcoming chemoresistance in human chondrosarcoma cells and may help in the development of therapeutic strategies for the treatments of patients. PMID:26966351

  2. B2B Models for DoD Acquisition

    DTIC Science & Technology

    2008-01-15

    unlimited. Prepared for: Naval Postgraduate School, Monterey, California 93943 B2B Models for DoD Acquisition 15 January 2008 by Magdi N...TYPE AND DATES COVERED 1 Oct 2006 through 30 Sept 2007 4. TITLE AND SUBTITLE B2B Models for DoD Acquisition 5. FUNDING 6. AUTHOR (S...public release; distribution is unlimited 12b. DISTRIBUTION CODE 13. ABSTRACT (Maximum 200 words.) A central vision of B2B e-commerce is that

  3. Putative subunits of the rat mesangial KATP: a type 2B sulfonylurea receptor and an inwardly rectifying K+ channel.

    PubMed

    Szamosfalvi, Balázs; Cortes, Pedro; Alviani, Rebecca; Asano, Kenichiro; Riser, Bruce L; Zasuwa, Gary; Yee, Jerry

    2002-05-01

    Sulfonylurea agents exert their physiological effects in many cell types via binding to specific sulfonylurea receptors (SUR). SUR couple to inwardly-rectifying K+ channel (Kir6.x) to form tetradimeric ATP-sensitive K+ channels (KATP). The SUR subunits confer ATP-sensitivity on KATP and also provide the binding sites for sulfonylureas and other pharmacological agents. Our previous work demonstrated that the exposure of mesangial cells (MC) to sulfonylureas generated profound effects on MC glucose uptake and matrix metabolism and induced heightened cell contractility in association with Ca2+ transients. Because these responses likely resulted from the binding of sulfonylurea to a mesangial SUR2, we subsequently documented [3H]-glibenclamide binding to MC and the gene expression of several mesangial SUR2 transcripts. From these data, we inferred that MC expressed the components of a mesangial KATP and sought to establish their presence in primary MC. To obtain mesangial SUR2 cDNA sequences, rapid amplification of cDNA ends (RACE) was utilized. DNA sequences were established by the fluorescent dye termination method. Gene expression of mesangial SUR2 and Kir6.1/2 was examined by reverse transcription polymerase chain reaction (RT-PCR) and Northern analysis. SUR2 proteins were identified by immunoblotting of mesangial proteins from membrane-enriched fractions with polyclonal antiserum directed against SUR2. RACE cloning yielded two mesangial SUR2 cDNAs of 4.8 and 6.7 kbp whose open reading frames translated proteins of 964 and 1535 aa, respectively. Using probes specific to each cDNA, the presence of a unique, 5.5 kbp serum-regulated mesangial SUR2 splice variant was established. The sequence of this mesangial SUR2 (mcSUR2B) shares identity with the recently cloned rat SUR2B (rSUR2B), but, in comparison to rSUR2B, is truncated by 12 exons at the N-terminus where it contains a unique insert of 16 aa. Immunoblotting studies with anti-SUR2 antiserum demonstrated SUR2

  4. A human anti-neuronal autoantibody against GABA B receptor induces experimental autoimmune agrypnia.

    PubMed

    Frisullo, Giovanni; Della Marca, Giacomo; Mirabella, Massimiliano; Caggiula, Marcella; Broccolini, Aldobrando; Rubino, Marco; Mennuni, Gioacchino; Tonali, Pietro Attilio; Batocchi, Anna Paola

    2007-04-01

    In the serum and cerebrospinal fluid of a patient with recurrent acute episodes of respiratory crises, autonomic symptoms and total insomnia (agrypnia), we identified a novel anti-neural complement fixing antibody directed against GABA(B) receptor (GABA(B)R). Patient purified IgG recognized a band of approximately 110 kDa on protein extracts of mouse cerebellum, cortex and brainstem and immunolabelled cultured Chinese hamster ovary (CHO) cells, transfected with human GABA(B)R1 and rat GABA(B)R2 receptors. Western blot analysis of transfected CHO homogenates showed the same band using both patient purified IgG and anti-GABA(B)R1 antibody. In order to verify the pathogenic role of these purified antibodies, we injected patient IgG intrathecally into cisterna magna of C57BL/6 mice pre-implanted with EEG electrodes and we observed severe ataxia followed by breathing depression and total suppression of slow wave sleep, as evidenced by EEG recording, in a dose-dependent manner. Immunohistochemistry on brain sections of mice injected with patient IgG showed the simultaneous presence of bound human IgG and C5b-9 deposits on Purkinje cells and cerebellar granular layer. After incubation with anti-GABA(B)R antibody, a marked reduction of receptor immunostaining was found with relative sparing of neuronal architecture. In conclusion we recognized an anti-neuronal autoantibody directed against GABA(B)R that is associated with autoimmune agrypnia and we showed that our patient purified IgG was able to induce in mice experimental autoimmune agrypnia characterized by a complex neurological syndrome affecting several CNS functions.

  5. TNF-{alpha} upregulates the A{sub 2B} adenosine receptor gene: The role of NAD(P)H oxidase 4

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    St Hilaire, Cynthia; Koupenova, Milka; Carroll, Shannon H.

    2008-10-24

    Proliferation of vascular smooth muscle cells (VSMC), oxidative stress, and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor-alpha (TNF-{alpha}) is released during vascular injury, and contributes to lesion formation also by affecting VSMC proliferation. Recently, an A{sub 2B} adenosine receptor (A{sub 2B}AR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response following injury, including the release of TNF-{alpha}. Here, we show a regulatory loop by which TNF-{alpha} upregulates the A{sub 2B}AR in VSMC in vitromore » and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A{sub 2B}AR, and Nox inhibitors dampen the effect of TNF-{alpha}. Hence, our study is the first to show that signaling associated with Nox4 is also able to upregulate the tissue protecting A{sub 2B}AR.« less

  6. Telecom 2-B and 2-C (TC2B and TC2C)

    NASA Technical Reports Server (NTRS)

    Dulac, J.; Alvarez, H.

    1991-01-01

    The DSN (Deep Space Network) mission support requirements for Telecom 2-B and 2-C (TC2B and TC2C) are summarized. These Telecom missions will provide high-speed data link applications, telephone, and television service between France and overseas territories as a follow-on to TC2A. Mission objectives are outlined and the DSN support requirements are defined through the presentation of tables and narratives describing the spacecraft flight profile; DSN support coverage; frequency assignments; support parameters for telemetry, command and support systems; and tracking support responsibility.

  7. Association of GABA(B)R1 receptor gene polymorphism with obstructive sleep apnea syndrome.

    PubMed

    Bayazit, Yildirim A; Yilmaz, Metin; Kokturk, Oguz; Erdal, M Emin; Ciftci, Tansu; Gokdogan, Tuba; Kemaloglu, Yusuf; Ileri, Fikret

    2007-01-01

    GABA(B)R (gamma-amino butyric acid B receptor)-mediated neurotransmission has been implicated in the pathophysiology of a variety of neuropsychiatric disorders. GABA(B)R1 gene variants were identified by single-strand conformation analysis. The nucleotide exchanges cause a substitution of alanine to valine in exon 1a1 (Ala20Val), a substitution of glycine to serine in exon 7 (Gly489Ser) and a silent C to G nucleotide exchange encoding the amino acid phenylalanine in exon 11 (Phe658Phe). The significance of GABA(B)R1a gene polymorphism in obstructive sleep apnea syndrome (OSAS) as well as the association of these polymorphisms with the polysomnography findings in OSAS patients are not known. In this study, we aimed to assess the significance of 3 different GABA(B)R1 gene polymorphisms (Ala20Val, Gly489Ser and Phe658Phe) in OSAS. Seventy-five patients (23 female and 52 male) with OSAS and 99 healthy volunteers (51 female, 48 male) were included in the study to assess Ala20Val, Gly489Ser and Phe658Phe polymorphisms of the GABA(B)R1 gene. For the Ala20Val variants, there was no significant difference between the genotypes and allele frequencies of the patients and controls, nor between both genders (p > 0.05). For Phe658Phe polymorphism, there was no significant difference between genotypes and allele frequencies of the patients and controls (p > 0.05). However, the C/C genotype was overrepresented and the T/C genotype was less frequent in male than female patients (p = 0.03). The C/C genotype was overrepresented and the T/C genotype was less frequent in male patients than male controls (p = 0.01). For GABA(B)R1-Gly489Ser polymorphism, all of the patients and controls had G/G genotype. The apnea arousal index scores of the male patients with C/C genotype were significantly higher than in the patients with C/T genotype (p = 0.01). The percent total sleep time in non-REM 1 scores of the male patients with T/T genotype were significantly higher than in the patients with T

  8. The A-Current Modulates Learning via NMDA Receptors Containing the NR2B Subunit

    PubMed Central

    Fontán-Lozano, Ángela; Suárez-Pereira, Irene; González-Forero, David; Carrión, Ángel Manuel

    2011-01-01

    Synaptic plasticity involves short- and long-term events, although the molecular mechanisms that underlie these processes are not fully understood. The transient A-type K+ current (IA) controls the excitability of the dendrites from CA1 pyramidal neurons by regulating the back-propagation of action potentials and shaping synaptic input. Here, we have studied how decreases in IA affect cognitive processes and synaptic plasticity. Using wild-type mice treated with 4-AP, an IA inhibitor, and mice lacking the DREAM protein, a transcriptional repressor and modulator of the IA, we demonstrate that impairment of IA decreases the stimulation threshold for learning and the induction of early-LTP. Hippocampal electrical recordings in both models revealed alterations in basal electrical oscillatory properties toward low-theta frequencies. In addition, we demonstrated that the facilitated learning induced by decreased IA requires the activation of NMDA receptors containing the NR2B subunit. Together, these findings point to a balance between the IA and the activity of NR2B-containing NMDA receptors in the regulation of learning. PMID:21966384

  9. Detection of plant quarantine pathogen Ralstonia solanacearum r3b2 with portable POCKIT™ and BLItz® systems

    USDA-ARS?s Scientific Manuscript database

    Ralstonia solanacearum (Rs) race 3 biovar 2 (r3b2) is designated as a quarantine pathogen in many countries and additionally as a Select Agent in the United States. Rapid, sensitive and accurate detection methods are urgently needed. We report here the development of two portable platforms for r3b...

  10. Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma.

    PubMed

    Galoian, Karina; Abrahamyan, Silva; Chailyan, Gor; Qureshi, Amir; Patel, Parthik; Metser, Gil; Moran, Alexandra; Sahakyan, Inesa; Tumasyan, Narine; Lee, Albert; Davtyan, Tigran; Chailyan, Samvel; Galoyan, Armen

    2018-01-01

    Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function.

  11. Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma

    PubMed Central

    Galoian, Karina; Abrahamyan, Silva; Chailyan, Gor; Qureshi, Amir; Patel, Parthik; Metser, Gil; Moran, Alexandra; Sahakyan, Inesa; Tumasyan, Narine; Lee, Albert; Davtyan, Tigran; Chailyan, Samvel; Galoyan, Armen

    2018-01-01

    Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function. PMID:29138803

  12. Functional role of human NK cell receptor 2B4 (CD244) isoforms.

    PubMed

    Mathew, Stephen O; Rao, Krithi K; Kim, Jong R; Bambard, Nowland D; Mathew, Porunelloor A

    2009-06-01

    2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM/CD150), is expressed on all NK cells, a subpopulation of T cells, monocytes and basophils. Human NK cells express two isoforms of 2B4, h2B4-A and h2B4-B that differ in a small portion of the extracellular domain. In the present investigation, we have studied the functions of h2B4-A and h2B4-B. Our study demonstrated that these two isoforms differ in their binding affinity for CD48, which results in differential cytotoxic activity as well as intracellular calcium release by NK cells upon target cell recognition. Analysis of the predicted 3-D structure of the two isoforms showed conformational differences that could account for their differences in binding affinity to CD48. h2B4-A was able to mediate natural cytotoxicity against CD48-expressing K562 target cells and induce intracellular calcium release, whereas h2B4-B showed no effects. NK-92MI, U937, THP-1, KU812, primary monocytes, basophils and NK cells showed expression of both h2B4-A and h2B4-B whereas YT and IL-2-activated NK cells did not show any h2B4-B expression. Stimulation of NK cells through 2B4 resulted in decreased mRNA levels of both h2B4-A and h2B4-B indicating that down-regulation of 2B4 isoforms may be an important factor in controlling NK cell activation during immune responses.

  13. Characterisation of endogenous A2A and A2B receptor-mediated cyclic AMP responses in HEK 293 cells using the GloSensor™ biosensor: Evidence for an allosteric mechanism of action for the A2B-selective antagonist PSB 603.

    PubMed

    Goulding, Joelle; May, Lauren T; Hill, Stephen J

    2018-01-01

    Endogenous adenosine A 2B receptors (A 2B AR) mediate cAMP accumulation in HEK 293 cells. Here we have used a biosensor to investigate the mechanism of action of the A 2B AR antagonist PSB 603 in HEK 293 cells. The A 2A agonist CGS 21680 elicited a small response in these cells (circa 20% of that obtained with NECA), suggesting that they also contain a small population of A 2A receptors. The responses to NECA and adenosine were antagonised by PSB 603, but not by the selective A 2A AR antagonist SCH 58261. In contrast, CGS 21680 responses were not antagonised by high concentrations of PSB 603, but were sensitive to inhibition by SCH 58261. Analysis of the effect of increasing concentrations of PSB 603 on the response to NECA indicated a non-competitive mode of action yielding a marked reduction in the NECA E MAX with no significant effect on EC 50 values. Kinetics analysis of the effect of PSB 603 on the A 2B AR-mediated NECA responses confirmed a saturable effect that was consistent with an allosteric mode of antagonism. The possibility that PSB 603 acts as a negative allosteric modulator of A 2B AR suggests new approaches to the development of therapeutic agents to treat conditions where adenosine levels are high. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  14. Synaptotagmin C2B Domain Regulates Ca2+-triggered Fusion in Vitro

    PubMed Central

    Gaffaney, Jon D.; Dunning, F. Mark; Wang, Zhao; Hui, Enfu; Chapman, Edwin R.

    2008-01-01

    Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC for Ca2+ 50-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B. PMID:18784080

  15. Characterization of CCX140-B, an orally bioavailable antagonist of the CCR2 chemokine receptor, for the treatment of type 2 diabetes and associated complications.

    PubMed

    Sullivan, Timothy J; Dairaghi, Daniel J; Krasinski, Antoni; Miao, Zhenhua; Wang, Yu; Zhao, Bin N; Baumgart, Trageen; Berahovich, Rob; Ertl, Linda S; Pennell, Andrew; Seitz, Lisa; Miao, Shichang; Ungashe, Solomon; Wei, Zheng; Johnson, Dan; Boring, Landin; Tsou, Chia-Lin; Charo, Israel F; Bekker, Pirow; Schall, Thomas J; Jaen, Juan C

    2012-06-05

    The following manuscript was published as a Fast Forward article on February 29, 2012: Sullivan TJ, Dairaghi DJ, Krasinski A, Miao Z, Wang Y, Zhao BN, Baumgart T, Berahovich R, Ertl LS, Pennell A, Seitz L, Miao S, Ungashe S, Wei Z, Johnson D, Boring L, Tsou C-L, Charo IF, Bekker P, Schall TJ, and Jaen JC, Characterization of CCX140-B, an orally bioavailable antagonist of the CCR2 chemokine receptor, for the treatment of type 2 diabetes and associated complications. J Pharmacol Exp Ther jpet.111.190918; doi:10.1124/jpet.111.190918 It was later found that the chemical identity of a compound cited in the article, CCX140-B, was not sufficiently disclosed. The authors are unable, at this time, to provide the chemical identity of CCX140-B in accordance with the editorial policies of The Journal of Pharmacology and Experimental Therapeutics. As a result, the authors have voluntarily withdrawn this manuscript from publication. We apologize for any inconvenience this may cause JPET's readers.

  16. [Polymorphic Variants of Glutamate Receptor (GRIK5, GRIN2B) and Serotonin Receptor (HTR2A) Genes Are Associated with Chronic Obstructive Pulmonary Disease].

    PubMed

    Korytina, G F; Akhmadishina, L Z; Kochetova, O V; Aznabaeva, Y G; Zagidullin, Sh Z; Victorova, T V

    2017-01-01

    Chronic obstructive pulmonary disease (COPD) is a complex chronic inflammatory disease of the respiratory system that affects primarily distal respiratory pathways and lung parenchyma. Smoking tobacco is a major risk factor for COPD. The relationship of HTR4 (rs3995090), HTR2A (rs6313), GRIK5 (rs8099939), GRIN2B (rs2268132), and CHRNB4 (rs1948) gene polymorphisms and COPD, as well as the contribution of these polymorphisms to the variations in quantitative characteristics that describe respiratory function, smoking behavior, and nicotine dependence was assessed in an ethnically homogeneous Tatar population. The polymorphisms of HTR2A (rs6313) (P = 0.026, OR = 1.42 for the CC genotype) and GRIN2B (rs2268132) (P = 0.0001, OR = 2.39 for the TT genotype) were significantly associated with increased risk of COPD. The AA genotype of GRIK5 (rs8099939) had a protective effect (P = 0.02, OR = 0.61). Importantly, the HTR2A (rs6313), GRIN2B (rs2268132), and GRIK5 (rs8099939) polymorphisms were only associated with COPD in smokers. Smoking index (pack-years) was significantly higher in carriers of the GRIK5 genotype AC (rs8099939) (P = 0.0027). The TT genotype of GRIN2B (rs2268132) was associated with COPD in subjects with high nicotine dependence according to the Fagerstrõm test (P = 0.002, OR = 2.98). The TT genotype of HTR2A (rs6313) was associated with a reduced risk of the disease in the group with moderate nicotine dependence (P = 0.02, OR = 0.22). The CC genotype of HTR2A (rs6313) and the TT genotype of GRIN2B (rs2268132) were associated with higher levels of nicotine dependence according to the Fagerstrõm test (P = 0.0011 and P = 0.037). Our results may provide insight into potential molecular mechanisms that involve the glutamate (GRIK5, GRIN2B) and serotonin (HTR2A) receptor genes in the pathogenesis of COPD.

  17. Spinophilin Is Indispensable for the α2B Adrenergic Receptor-Elicited Hypertensive Response.

    PubMed

    Che, Pulin; Chen, Yunjia; Lu, Roujian; Peng, Ning; Gannon, Mary; Wyss, J Michael; Jiao, Kai; Wang, Qin

    2015-01-01

    The α2 adrenergic receptor (AR) subtypes are important for blood pressure control. When activated, the α2A subtype elicits a hypotensive response whereas the α2B subtype mediates a hypertensive effect that counteracts the hypotensive response by the α2A subtype. We have previously shown that spinophilin attenuates the α2AAR-dependent hypotensive response; in spinophilin null mice, this response is highly potentiated. In this study, we demonstrate that spinophilin impedes arrestin-dependent phosphorylation and desensitization of the α2BAR subtype by competing against arrestin binding to this receptor subtype. The Del301-303 α2BAR, a human variation that shows impaired phosphorylation and desensitization and is linked to hypertension in certain populations, exhibits preferential interaction with spinophilin over arrestin. Furthermore, Del301-303 α2BAR-induced ERK signaling is quickly desensitized in cells without spinophilin expression, showing a profile similar to that induced by the wild type receptor in these cells. Together, these data suggest a critical role of spinophilin in sustaining α2BAR signaling. Consistent with this notion, our in vivo study reveals that the α2BAR-elicited hypertensive response is diminished in spinophilin deficient mice. In arrestin 3 deficient mice, where the receptor has a stronger binding to spinophilin, the same hypertensive response is enhanced. These data suggest that interaction with spinophilin is indispensable for the α2BAR to elicit the hypertensive response. This is opposite of the negative role of spinophilin in regulating α2AAR-mediated hypotensive response, suggesting that spinophilin regulation of these closely related receptor subtypes can result in distinct functional outcomes in vivo. Thus, spinophilin may represent a useful therapeutic target for treatment of hypertension.

  18. Vps26B-retromer negatively regulates plasma membrane resensitization of PAR-2.

    PubMed

    Bugarcic, Andrea; Vetter, Irina; Chalmers, Silke; Kinna, Genevieve; Collins, Brett M; Teasdale, Rohan D

    2015-11-01

    Retromer is a trimeric complex composed of Vps26, Vps29, and Vps35 and has been shown to be involved in trafficking and sorting of transmembrane proteins within the endosome. The Vps26 paralog, Vps26B, defines a distinct retromer complex (Vps26B-retromer) in vivo and in vitro. Although endosomally associated, Vps26B-retromer does not bind the established retromer transmembrane cargo protein, cation-independent mannose 6-phosphate receptor (CI-M6PR), indicating it has a distinct role to retromer containing the Vps26A paralog. In the present study we use the previously established Vps26B-expressing HEK293 cell model to address the role of Vps26B-retromer in trafficking of the protease activated G-protein coupled receptor PAR-2 to the plasma membrane. In these cells there is no apparent defect in the initial activation of the receptor, as evidenced by release of intracellular calcium, ERK1/2 signaling and endocytosis of activated receptor PAR-2 into degradative organelles. However, we observe a significant delay in plasma membrane repopulation of the protease activated G protein-coupled receptor PAR-2 following stimulation, resulting in a defect in PAR-2 activation after resensitization. Here we propose that PAR-2 plasma membrane repopulation is regulated by Vps26B-retromer, describing a potential novel role for this complex. © 2015 International Federation for Cell Biology.

  19. Induction of nuclear factor kappaB by the CD30 receptor is mediated by TRAF1 and TRAF2.

    PubMed Central

    Duckett, C S; Gedrich, R W; Gilfillan, M C; Thompson, C B

    1997-01-01

    CD30 is a lymphoid cell-specific surface receptor which was originally identified as an antigen expressed on Hodgkin's lymphoma cells. Activation of CD30 induces the nuclear factor kappaB (NF-kappaB) transcription factor. In this study, we define the domains in CD30 which are required for NF-kappaB activation. Two separate elements of the cytoplasmic domain which were capable of inducing NF-kappaB independently of one another were identified. The first domain (domain 1) mapped to a approximately 120-amino-acid sequence in the membrane-proximal region of the CD30 cytoplasmic tail, between residues 410 and 531. A second, more carboxy-terminal region (domain 2) was identified between residues 553 and 595. Domain 2 contains two 5- to 10-amino-acid elements which can mediate the binding of CD30 to members of the tumor necrosis factor receptor-associated factor (TRAF) family of signal transducing proteins. Coexpression of CD30 with TRAF1 or TRAF2 but not TRAF3 augmented NF-kappaB activation through domain 2 but not domain 1. NF-kappaB induction through domain 2 was inhibited by coexpression of either full-length TRAF3 or dominant negative forms of TRAF1 or TRAF2. In contrast, NF-kappaB induction by domain 1 was not affected by alterations in TRAF protein levels. Together, these data support a model in which CD30 can induce NF-kappaB by both TRAF-dependent and -independent mechanisms. TRAF-dependent induction of NF-kappaB appears to be regulated by the relative levels of individual TRAF proteins in the cell. PMID:9032281

  20. Molecular Docking and Prediction of Pharmacokinetic Properties of Dual Mechanism Drugs that Block MAO-B and Adenosine A2A Receptors for the Treatment of Parkinson's Disease

    PubMed Central

    Azam, Faizul; Madi, Arwa M.; Ali, Hamed I.

    2012-01-01

    Monoamine oxidase B (MAO-B) inhibitory potential of adenosine A2A receptor (AA2AR) antagonists has raised the possibility of designing dual-target–directed drugs that may provide enhanced symptomatic relief and that may also slow the progression of Parkinson's disease (PD) by protecting against further neurodegeneration. To explain the dual inhibition of MAO-B and AA2AR at the molecular level, molecular docking technique was employed. Lamarckian genetic algorithm methodology was used for flexible ligand docking studies. A good correlation (R2= 0.524 and 0.627 for MAO-B and AA2AR, respectively) was established between docking predicted and experimental Ki values, which confirms that the molecular docking approach is reliable to study the mechanism of dual interaction of caffeinyl analogs with MAO-B and AA2AR. Parameters for Lipinski's “Rule-of-Five” were also calculated to estimate the pharmacokinetic properties of dual-target–directed drugs where both MAO-B inhibition and AA2AR antagonism exhibited a positive correlation with calculated LogP having a correlation coefficient R2 of 0.535 and 0.607, respectively. These results provide some beneficial clues in structural modification for designing new inhibitors as dual-target–directed drugs with desired pharmacokinetic properties for the treatment of PD. PMID:23112538

  1. Genotype and environment effects on the contents of vitamins B1, B2, B3, and B6 in wheat grain.

    PubMed

    Shewry, Peter R; Van Schaik, Frank; Ravel, Catherine; Charmet, Gilles; Rakszegi, Mariann; Bedo, Zoltan; Ward, Jane L

    2011-10-12

    The total contents of thiamine (vitamin B1), riboflavin (B2), and pyridoxine (B6) and the bioavailable forms of niacin (B3) were determined on wholemeal flours of 24 winter wheat varieties grown on four sites (United Kingdom, Poland, France, and Hungary) in 2007 and of two spring varieties grown on the same sites with the exception of Poland. The contents of vitamins B1 (5.53-13.55 μg/g dw), B2 (0.77-1.40 μg/g dw), and B6 (1.27-2.97 μg/g dw) were within the ranges reported previously, while the content of bioavailable vitamin B3 (0.16-1.74 μg/g dw) was about 10-15% of the total contents of vitamin B3 reported in previous studies. Strong correlations were observed between the contents of vitamins B1, B3, and B6, and partitioning of the variance in the contents of these three B vitamins showed that between 48 and 70% was accounted for by the environment. By contrast, the content of vitamin B2 was not correlated with the contents of other B vitamins, and 73% of the variance was ascribed to the error term, which suggests that this trait may be influenced by genotype × environment interactions. Whereas the contents of vitamins B1, B3, and B6 were correlated positively with the mean temperature from heading to harvest (r > 0.8), the content of vitamin B2 was positively correlated with precipitation during the 3 months prior to heading. These results are discussed in relation to the development of new wheat varieties with enhanced health benefits.

  2. 2B4-SAP signaling is required for the priming of naive CD8+ T cells by antigen-expressing B cells and B lymphoma cells

    PubMed Central

    2017-01-01

    ABSTRACT Mutations in SH2D1A gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency disease defined by exquisite sensitivity to the B-lymphotropic Epstein–Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we investigate the hypothesis that SAP is critical for CD8+ T cell immune surveillance of antigen (Ag)-expressing B cells or B lymphoma cells under conditions of defined T cell receptor (TCR) signaling. Sh2d1a−/− CD8+ T cells exhibited greatly diminished proliferation relative to wild type when Ag-presenting-B cells or -B lymphoma cells served as the primary Ag-presenting cell (APC). By contrast, Sh2d1a−/− CD8+ T cells responded equivalently to wild-type CD8+ T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed Ag presentation. Through application of signaling lymphocyte activation molecule (SLAM) family receptor blocking antibodies or SLAM family receptor-deficient CD8+ T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the Ag-driven CD8+ T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naive CD8+ T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas. PMID:28344876

  3. 2B4-SAP signaling is required for the priming of naive CD8+ T cells by antigen-expressing B cells and B lymphoma cells.

    PubMed

    Huang, Yu-Hsuan; Tsai, Kevin; Tan, Sara Y; Kang, Sohyeong; Ford, Mandy L; Harder, Kenneth W; Priatel, John J

    2017-01-01

    Mutations in SH2D1A gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency disease defined by exquisite sensitivity to the B-lymphotropic Epstein-Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we investigate the hypothesis that SAP is critical for CD8 + T cell immune surveillance of antigen (Ag)-expressing B cells or B lymphoma cells under conditions of defined T cell receptor (TCR) signaling. Sh2d1a - / - CD8 + T cells exhibited greatly diminished proliferation relative to wild type when Ag-presenting-B cells or -B lymphoma cells served as the primary Ag-presenting cell (APC). By contrast, Sh2d1a - / - CD8 + T cells responded equivalently to wild-type CD8 + T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed Ag presentation. Through application of signaling lymphocyte activation molecule (SLAM) family receptor blocking antibodies or SLAM family receptor-deficient CD8 + T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the Ag-driven CD8 + T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naive CD8 + T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas.

  4. Discovery of sodium R-(+)-4-{2-[5-(2-fluoro-3-methoxyphenyl)-3-(2-fluoro-6-[trifluoromethyl]benzyl)-4-methyl-2,6-dioxo-3,6-dihydro-2H-pyrimidin-1-yl]-1-phenylethylamino}butyrate (elagolix), a potent and orally available nonpeptide antagonist of the human gonadotropin-releasing hormone receptor.

    PubMed

    Chen, Chen; Wu, Dongpei; Guo, Zhiqiang; Xie, Qiu; Reinhart, Greg J; Madan, Ajay; Wen, Jenny; Chen, Takung; Huang, Charles Q; Chen, Mi; Chen, Yongsheng; Tucci, Fabio C; Rowbottom, Martin; Pontillo, Joseph; Zhu, Yun-Fei; Wade, Warren; Saunders, John; Bozigian, Haig; Struthers, R Scott

    2008-12-11

    The discovery of novel uracil phenylethylamines bearing a butyric acid as potent human gonadotropin-releasing hormone receptor (hGnRH-R) antagonists is described. A major focus of this optimization was to improve the CYP3A4 inhibition liability of these uracils while maintaining their GnRH-R potency. R-4-{2-[5-(2-fluoro-3-methoxyphenyl)-3-(2-fluoro-6-[trifluoromethyl]benzyl)-4-methyl-2,6-dioxo-3,6-dihydro-2H-pyrimidin-1-yl]-1-phenylethylamino}butyric acid sodium salt, 10b (elagolix), was identified as a potent and selective hGnRH-R antagonist. Oral administration of 10b suppressed luteinizing hormone in castrated macaques. These efforts led to the identification of 10b as a clinical compound for the treatment of endometriosis.

  5. The hybrid molecule, VCP746, is a potent adenosine A2B receptor agonist that stimulates anti-fibrotic signalling.

    PubMed

    Vecchio, Elizabeth A; Chuo, Chung Hui; Baltos, Jo-Anne; Ford, Leigh; Scammells, Peter J; Wang, Bing H; Christopoulos, Arthur; White, Paul J; May, Lauren T

    2016-10-01

    We have recently described the rationally-designed adenosine receptor agonist, 4-(5-amino-4-benzoyl-3-(3-(trifluoromethyl)phenyl)thiophen-2-yl)-N-(6-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxylmethyl)tetrahydro-furan-2-yl)-9H-purin-6-ylamino)hexyl)benzamide (VCP746), a hybrid molecule consisting of an adenosine moiety linked to an adenosine A1 receptor (A1AR) allosteric modulator moiety. At the A1AR, VCP746 mediated cardioprotection in the absence of haemodynamic side effects such as bradycardia. The current study has now identified VCP746 as an important pharmacological tool for the adenosine A2B receptor (A2BAR). The binding and function of VCP746 at the A2BAR was rigorously characterised in a heterologous expression system, in addition to examination of its anti-fibrotic signalling in cardiac- and renal-derived cells. In FlpInCHO cells stably expressing the human A2BAR, VCP746 was a high affinity, high potency A2BAR agonist that stimulated Gs- and Gq-mediated signal transduction, with an apparent lack of system bias relative to prototypical A2BAR agonists. The distinct agonist profile may result from an atypical binding mode of VCP746 at the A2BAR, which was consistent with a bivalent mechanism of receptor interaction. In isolated neonatal rat cardiac fibroblasts (NCF), VCP746 stimulated potent inhibition of both TGF-β1- and angiotensin II-mediated collagen synthesis. Similar attenuation of TGF-β1-mediated collagen synthesis was observed in renal mesangial cells (RMC). The anti-fibrotic signalling mediated by VCP746 in NCF and RMC was selectively reversed in the presence of an A2BAR antagonist. Thus, we believe, VCP746 represents an important tool to further investigate the role of the A2BAR in cardiac (patho)physiology. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Aberrant gonadotropin-releasing hormone receptor (GnRHR) expression and its regulation of CYP11B2 expression and aldosterone production in adrenal aldosterone-producing adenoma (APA).

    PubMed

    Nakamura, Yasuhiro; Hattangady, Namita G; Ye, Ping; Satoh, Fumitoshi; Morimoto, Ryo; Ito-Saito, Takako; Sugawara, Akira; Ohba, Koji; Takahashi, Kazuhiro; Rainey, William E; Sasano, Hironobu

    2014-03-25

    Aberrant expression of gonadotropin-releasing hormone receptor (GnRHR) has been reported in human adrenal tissues including aldosterone-producing adenoma (APA). However, the details of its expression and functional role in adrenals are still not clear. In this study, quantitative RT-PCR analysis revealed the mean level of GnRHR mRNA was significantly higher in APAs than in human normal adrenal (NA) (P=0.004). GnRHR protein expression was detected in human NA and neoplastic adrenal tissues. In H295R cells transfected with GnRHR, treatment with GnRH resulted in a concentration-dependent increase in CYP11B2 reporter activity. Chronic activation of GnRHR with GnRH (100nM), in a cell line with doxycycline-inducible GnRHR (H295R-TR/GnRHR), increased CYP11B2 expression and aldosterone production. These agonistic effects were inhibited by blockers for the calcium signaling pathway, KN93 and calmidazolium. These results suggest GnRH, through heterotopic expression of its receptor, may be a potential regulator of CYP11B2 expression levels in some cases of APA. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  7. IRE1α links Nck1 deficiency to attenuated PTP1B expression in HepG2 cells.

    PubMed

    Li, Hui; Li, Bing; Larose, Louise

    2017-08-01

    PTP1B, a prototype of the non-receptor subfamily of the protein tyrosine phosphatase superfamily, plays a key role in regulating intracellular signaling from various receptor and non-receptor protein tyrosine kinases. Previously, we reported that silencing Nck1 in human hepatocellular carcinoma HepG2 cells enhances basal and growth factor-induced activation of the PI3K-Akt pathway through attenuating PTP1B expression. However, the underlying mechanism by which Nck1 depletion represses PTP1B expression remains unclear. In this study, we found that silencing Nck1 attenuates PTP1B expression in HepG2 cells through down-regulation of IRE1α. Indeed, we show that silencing Nck1 in HepG2 cells leads to decreased IRE1α expression and signaling. Accordingly, IRE1α depletion using siRNA in HepG2 cells enhances PI3K-dependent basal and growth factor-induced Akt activation, reproducing the effects of silencing Nck1 on activation of this pathway. In addition, depletion of IRE1α also leads to reduced PTP1B expression, which was rescued by ectopic expression of IRE1α in Nck1-depleted cells. Mechanistically, we found that silencing either Nck1 or IRE1α in HepG2 cells decreases PTP1B mRNA levels and stability. However, despite miR-122 levels, a miRNA targeting PTP1B 3' UTR and inducing PTP1B mRNA degradation in HepG2 cells, are increased in both Nck1- and IRE1α-depleted HepG2 cells, a miR-122 antagomir did not rescue PTP1B expression in these cells. Overall, this study highlights an important role for Nck1 in fine-tuning IRE1α expression and signaling that regulate PTP1B expression and subsequent activation of the PI3K-Akt pathway in HepG2 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling.

    PubMed

    Mundell, S J; Matharu, A-L; Nisar, S; Palmer, T M; Benovic, J L; Kelly, E

    2010-02-01

    We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A(2B) adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5'-(N-ethylcarboxamido)-adenosine. The trafficking of the wild type A(2B) adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy. The wild type A(2B) adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln(325)-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu(330)-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln(325)-stop, Ser(326)-stop and Phe(328)-stop receptors. Following internalization, the wild type A(2B) adenosine receptor recycled rapidly to the cell surface, whereas the Gln(325)-stop receptor did not recycle. Deletion of the COOH-terminus of the A(2B) adenosine receptor beyond Leu(330) switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A(2B) adenosine receptor following prolonged agonist addition.

  9. Cloning and functional analysis of P2X1b, a new variant in rat optic nerve that regulates the P2X1 receptor in a use-dependent manner.

    PubMed

    Rangel-Yescas, Gisela E; Vazquez-Cuevas, Francisco G; Garay, Edith; Arellano, Rogelio O

    2012-01-01

    P2X receptors are trimeric, ATP-gated cation channels. In mammals seven P2X subtypes have been reported (P2X1-P2X7), as well as several variants generated by alternative splicing. Variants confer to the homomeric or heteromeric channels distinct functional and/or pharmacological properties. Molecular biology, biochemical, and functional analysis by electrophysiological methods were used to identify and study a new variant of the P2X1 receptor named P2X1b. This new variant, identified in rat optic nerve, was also expressed in other tissues. P2X1b receptors lack amino acids 182 to 208 of native P2X1, a region that includes residues that are highly conserved among distinct P2X receptors. When expressed in Xenopus oocytes, P2X1b was not functional as a homomer; however, when co-expressed with P2X1, it downregulated the electrical response generated by ATP compared with that of oocytes expressing P2X1 alone, and it seemed to form heteromeric channels with a modestly enhanced ATP potency. A decrease in responses to ATP in oocytes co-expressing different ratios of P2X1b to P2X1 was completely eliminated by overnight pretreatment with apyrase. Thus, it is suggested that P2X1b regulates, through a use-dependent mechanism, the availability, in the plasma membrane, of receptor channels that can be operated by ATP.

  10. In-situ X-ray diffraction studies of the phase transformations and structural states of B2, R and B19′ phases in Ti{sub 49.5}Ni{sub 50.5} alloy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ostapenko, Marina G., E-mail: artifakt@ispms.tsc.ru; National Research Tomsk Polytechnic University, Tomsk, 634050; Meisner, Ludmila L., E-mail: llm@ispms.tsc.ru

    2015-10-27

    The martensitic transformation, Debye–Waller factor, mean-square atomic displacements and the coefficient of thermal expansion on cooling of the Ti{sub 49.5}Ni{sub 50.5} shape memory alloy were examined using in-situ X-ray diffraction. It was revealed B2R (T{sub R} ≡ T = 273 ± 10 K) along with B2B19’ (M{sub s} ≡ T = 273 ± 10 K) transitions occur. It was found that Debye–Waller factor and mean-square displacement of B2 phase undergo significant increase as functions of temperature when phase transition B2R and B2B19’ take place. The analysis of the thermal expansion coefficient of the B2 phase indicates that the value of a increasesmore » almost linearly while cooling.« less

  11. Impaired discrimination learning in interneuronal NMDAR-GluN2B mutant mice.

    PubMed

    Brigman, Jonathan L; Daut, Rachel A; Saksida, Lisa; Bussey, Timothy J; Nakazawa, Kazu; Holmes, Andrew

    2015-06-17

    Previous studies have established a role for N-methyl-D-aspartate receptor (NMDAR) containing the GluN2B subunit in efficient learning behavior on a variety of tasks. Recent findings have suggested that NMDAR on GABAergic interneurons may underlie the modulation of striatal function necessary to balance efficient action with cortical excitatory input. Here we investigated how loss of GluN2B-containing NMDAR on GABAergic interneurons altered corticostriatal-mediated associative learning. Mutant mice (floxed-GluN2B×Ppp1r2-Cre) were generated to produce loss of GluN2B on forebrain interneurons and phenotyped on a touchscreen-based pairwise visual learning paradigm. We found that the mutants showed normal performance during Pavlovian and instrumental pretraining, but were significantly impaired on a discrimination learning task. Detailed analysis of the microstructure of discrimination performance revealed reduced win→stay behavior in the mutants. These results further support the role of NMDAR, and GluN2B in particular, on modulation of striatal function necessary for efficient choice behavior and suggest that NMDAR on interneurons may play a critical role in associative learning.

  12. Magnetic behavior of R2Fe14B hydrides (R = Gd, Tb, Dy, Ho and Er)

    NASA Astrophysics Data System (ADS)

    Zhang, L. Y.; Pourarian, F.; Wallace, W. E.

    1988-01-01

    R 2Fe 14B systems, with R = Gd, Tb, By, HoandEr were hydrogenated to the composition R 2Fe 14BH x where x ranges from 3.7 to 5.4. The pressure-composition isotherms (PCIs) of the hydrides showed only a solid solution behavior. No plateau pressure region was observed between room temperature and 300° C and at pressures down to 10 -2 atm. The absorbed hydrogen leads to an increase of 2.6 to 3.4% in unit cell volume, without a change in crystal structure. Magnetic characteristics of the present compounds were investigated over the temperature range 4.2 to 1100 K and at applied field up to 20 kOe. Saturation magnetization, Ms, and magnetic ordering temperature, Tc, were enhanced upon hydrogenation. Tc, is found to be dependent on the hydriding composition. Hydrogen induces a spin-reorientation effect (SR) in Gd- and Dy-containing compounds, while it has a marked influence in raising the spin-reorientation temperature, TSR, in the Er 2Fe 14B compound. The hydride involving Tb appears to remain uniaxial to the lowest temperature studied. In all cases the anisotropy fields, HA/' were significally reduced by hydrogen absorption. These varied magnetic behaviors can be ascribed to the effects: (1) variations in the interatomic distances, (2) strengthening the 3d-3d and weakening the 4f-3d exchange interactions and (3) the interstitial site occupations of hydrogen in the lattice. The spin-reorientation phenomena observed for Gd 2Fe 14BH x suggest that there is competition among the 6 Fe sublattices in regard to the sign and temperature coefficient of anisotropy.

  13. Impacts of the Glucuronidase Genotypes UGT1A4, UGT2B7, UGT2B15 and UGT2B17 on Tamoxifen Metabolism in Breast Cancer Patients

    PubMed Central

    Romero-Lorca, Alicia; Novillo, Apolonia; Gaibar, María; Bandrés, Fernando; Fernández-Santander, Ana

    2015-01-01

    Tamoxifen is used to prevent and treat estrogen-dependent breast cancer. It is described as a prodrug since most of its antiestrogen effects are exerted through its hydroxylated metabolites 4-OH-tamoxifen and endoxifen. In prior work, we correlated optimal plasma levels of these metabolites with certain genotypes of CYP2D6 and SULT1A2. This descriptive study examines correlations between concentrations of tamoxifen's glucuronide metabolites and genotypes UGT1A4 Pro24Thr, UGT1A4 Leu48Val, UGT2B7 His268Tyr, UGT2B15 Asp85YTyr UGT2B15 Lys523Thr and UGT2B17del in 132 patients with estrogen receptor-positive breast cancer under treatment with tamoxifen. Patients were genotyped by real-time and conventional PCR-RFLP. The glucuronides 4-OH-tamoxifen-N-glucuronide, 4-OH-tamoxifen-O-glucuronide and endoxifen-O-glucuronide were isolated from blood plasma and quantified using a high-pressure liquid chromatography-tandem mass spectrometry system. Individuals who were homozygous for UGT1A448VAL showed significantly lower mean concentrations of both glucuronide metabolites compared to subjects genotyped as wt/wt plus wt/48Val (p=0.037 and p=0.031, respectively). Women homozygous for UGT2B7268Tyr also showed mean substrate/product ratios of 4-OH-tamoxifen/4-OH-tamoxifen-O-glucuronide and 4-OH-tamoxifen/4-OH-tamoxifen-N-glucuronide indicative of reduced glucuronidase activity compared to wt homozygotes or to heterozygotes for the polymorphism (p=0.005 and p=0.003, respectively). In contrast, UGT2B15 Lys523Thr and UGT2B17del were associated with possibly increased enzyme activity. Patients with at least one variant allele UGT2B15523Thr showed significantly higher 4-OH-tamoxifen-O-glucuronide and endoxifen-glucuronide levels (p=0.023 and p=0.025, respectively) indicating a variant gene-dose effect. Higher 4-OH-tamoxifen-N-glucuronide levels observed in UGT2B17del genotypes (p=0.042) could be attributed to a mechanism that compensates for the greater expression of other genes in UGT2B

  14. Induced Pluripotent Stem Cell‐Derived Endothelial Cells Overexpressing Interleukin‐8 Receptors A/B and/or C‐C Chemokine Receptors 2/5 Inhibit Vascular Injury Response

    PubMed Central

    Giordano, Samantha; Zhao, Xiangmin; Chen, Yiu‐Fai; Litovsky, Silvio H.; Hage, Fadi G.; Townes, Tim M.; Sun, Chiao‐Wang; Wu, Li‐Chen; Oparil, Suzanne

    2017-01-01

    Abstract Recruitment of neutrophils and monocytes/macrophages to the site of vascular injury is mediated by binding of chemoattractants to interleukin (IL) 8 receptors RA and RB (IL8RA/B) C‐C chemokine receptors (CCR) 2 and 5 expressed on neutrophil and monocyte/macrophage membranes. Endothelial cells (ECs) derived from rat‐induced pluripotent stem cells (RiPS) were transduced with adenovirus containing cDNA of IL8RA/B and/or CCR2/5. We hypothesized that RiPS‐ECs overexpressing IL8RA/B (RiPS‐IL8RA/B‐ECs), CCR2/5 (RiPS‐CCR2/5‐ECs), or both receptors (RiPS‐IL8RA/B+CCR2/5‐ECs) will inhibit inflammatory responses and neointima formation in balloon‐injured rat carotid artery. Twelve‐week‐old male Sprague‐Dawley rats underwent balloon injury of the right carotid artery and intravenous infusion of (a) saline vehicle, (b) control RiPS‐Null‐ECs (ECs transduced with empty virus), (c) RiPS‐IL8RA/B‐ECs, (d) RiPS‐CCR2/5‐ECs, or (e) RiPS‐IL8RA/B+CCR2/5‐ECs. Inflammatory mediator expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 hours postinjury by enzyme‐linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. Neointima formation was assessed at 14 days postinjury. RiPS‐ECs expressing the IL8RA/B or CCR2/5 homing device targeted the injured arteries and decreased injury‐induced inflammatory cytokine expression, neutrophil/macrophage infiltration, and neointima formation. Transfused RiPS‐ECs overexpressing IL8RA/B and/or CCR2/5 prevented inflammatory responses and neointima formation after vascular injury. Targeted delivery of iPS‐ECs with a homing device to inflammatory mediators in injured arteries provides a novel strategy for the treatment of cardiovascular diseases. Stem Cells Translational Medicine 2017;6:1168–1177 PMID:28233474

  15. Biological Roles of Hydroxysteroid (11-Beta) Dehydrogenase 1 (HSD11B1), HSD11B2, and Glucocorticoid Receptor (NR3C1) in Sheep Conceptus Elongation.

    PubMed

    Brooks, Kelsey; Burns, Gregory; Spencer, Thomas E

    2015-08-01

    In sheep, the elongating conceptus synthesizes and secretes interferon tau (IFNT) as well as prostaglandins (PGs) and cortisol. The enzymes, hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1) and HSD11B2 interconvert cortisone and cortisol. In sheep, HSD11B1 is expressed and active in the conceptus trophectoderm as well as in the endometrial luminal epithelia; in contrast, HSD11B2 expression is most abundant in conceptus trophectoderm. Cortisol is a biologically active glucocorticoid and ligand for the glucocorticoid receptor (NR3C1 or GR) and mineralocorticoid receptor (NR3C2 or MR). Expression of MR is not detectable in either the ovine endometrium or conceptus during early pregnancy. In tissues that do not express MR, HSD11B2 protects cells from the growth-inhibiting and/or proapoptotic effects of cortisol, particularly during embryonic development. In study one, an in utero loss-of-function analysis of HSD11B1 and HSD11B2 was conducted in the conceptus trophectoderm using morpholino antisense oligonucleotides (MAOs) that inhibit mRNA translation. Elongating, filamentous conceptuses were recovered on Day 14 from ewes infused with control morpholino or HSD11B2 MAO. In contrast, HSD11B1 MAO resulted in severely growth-retarded conceptuses or conceptus fragments with apoptotic trophectoderm. In study two, clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing was used to determine the role of GR in conceptus elongation and development. Elongating, filamentous-type conceptuses (12-14 cm in length) were recovered from ewes gestating control embryos (n = 7/7) and gestating GR-edited embryos (n = 6/7). These results support the idea that the effects of HSD11B1-derived cortisol on conceptus elongation are indirectly mediated by the endometrium and are not directly mediated through GR in the trophectoderm. © 2015 by the Society for the Study of Reproduction, Inc.

  16. Exposure to PM2.5 induces aberrant activation of NF-κB in human airway epithelial cells by downregulating miR-331 expression.

    PubMed

    Song, Lei; Li, Dan; Li, Xiaoping; Ma, Lianjun; Bai, Xiaoxue; Wen, Zhongmei; Zhang, Xiufang; Chen, Dong; Peng, Liping

    2017-03-01

    Exposure to particulate matter (PM) with an aerodynamic diameter≤2.5μm (PM2.5) induces reactive oxygen species (ROS) and pro-inflammatory cytokine production, leading to airway epithelial injury. However, the mechanisms underlying the toxicity of PM2.5 have not been clarified. Here, we show that exposure to PM2.5 induces sustained activation of the nuclear factor kappa B (NF-κB) signaling in human airway epithelial Beas-2B (B2B) cells. In addition, PM2.5 exposure significantly decreased miR-331 expression in B2B cells, which was abrogated by inhibition of ROS or phosphoinositide 3-kinase (PI3K)/Akt pathway. Induction of miR-331 overexpression attenuated the PM2.5 exposure-induced NF-kBp65 nuclear translocation, IL-6 and IL-8 expression in B2B cells. Furthermore, miR-331 targeted the inhibitor of NF-κB kinase beta (IKK-β) by down-regulating the IKK-β-regulated luciferase activity in HEK293 cells. Moreover, induction of miR-331 over-expression inhibited IKK-β expression while induction of IKK-β over-expression prevented the inhibition of miR-331 on the PM2.5 exposure-induced NF-kBp65 nuclear translocation, IL-6 and IL-8 expression in B2B cells. Therefore, PM2.5 exposure decreased miR-331 expression via the ROS/PI3K/Akt pathway, resulting in an increase in the IKK-β expression and sustained NF-κB activation in human airway epithelial cells. Our findings may provide new insights into the molecular mechanisms underlying the toxicity of PM2.5 exposure and aid in design of new therapeutic strategies to prevent PM2.5-induced toxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Endogenous bradykinin activates ischaemically sensitive cardiac visceral afferents through kinin B2 receptors in cats

    PubMed Central

    Tjen-A-Looi, Stephanie C; Pan, Hui-Lin; Longhurst, John C

    1998-01-01

    Activity of ischaemically sensitive cardiac visceral afferents during myocardial ischaemia induces both angina and cardiovascular reflexes. Increased production of bradykinin (BK) and cyclo-oxygenase products (i.e. prostaglandins (PGs)) occurs during myocardial ischaemia. However, the role of these agents in activation of ischaemically sensitive cardiac afferents has not been established. The present study tested the hypothesis that BK produced during ischaemia activates cardiac afferents through kinin B2 receptors. Single-unit activity of cardiac afferents innervating the left ventricle was recorded from the left thoracic sympathetic chain (T1–T4) of anaesthetized cats. Ischaemically sensitive cardiac afferents were identified according to their response to 5 min of myocardial ischaemia. The mechanism of BK in activation of ischaemically sensitive cardiac afferents was determined by injection of BK (1 μg kg−1 i.a.), des-Arg9-BK (1 μg kg−1 i.a., a specific kinin B1 receptor agonist), kinin B2 receptor antagonists: HOE140 (30 μg kg−1 i.v.) and NPC-17731 (40 μg kg−1 i.v.), cyclo-oxygenase inhibition with indomethacin (5 mg kg−1 i.v.) and NPC-17731 (40 μg kg−1 i.v.) after pretreatment with indomethacin (5 mg kg−1 i.v.). We observed that BK increased the discharge rate of all eleven ischaemically sensitive cardiac afferents from 0.39 ± 0.12 to 1.47 ± 0.37 impulses s−1 (P < 0.05). Conversely, des-Arg9-BK did not significantly increase the activity of eleven ischaemically sensitive fibres (0.58 ± 0.02 vs. 0.50 ± 0.18 impulses s−1). HOE140 significantly attenuated the response of twelve afferents to ischaemia (0.61 ± 0.22 to 1.85 ± 0.5 vs. 0.53 ± 0.16 to 1.09 ± 0.4 impulses s−1). NPC-17731, another kinin B2 receptor antagonist, had similar inhibitory effects on six other ischaemically sensitive cardiac afferents (0.35 ± 0.14 to 1.19 ± 0.29 vs. 0.22 ± 0.08 to 0.23 ± 0.07 impulses s−1). Indomethacin significantly reduced the

  18. Differential functions of NR2A and NR2B in short-term and long-term memory in rats.

    PubMed

    Jung, Ye-Ha; Suh, Yoo-Hun

    2010-08-23

    N-methyl-D-aspartate receptors (NMDARs) are glutamate receptors implicated in synaptic plasticity and memory function. The specific functions of NMDA receptor subunits NR2A and NR2B have not yet been fully determined in the different types of memory. Nine Wistar rats (8-weeks-old) were subjected to the Morris water maze task to evaluate the memory behaviorally. Quantitative analysis of NR1, NR2A, and NR2B levels in the right and left forebrain of rats was performed and subunit associations with different types of memory were investigated using the Morris water maze task. Right forebrain NR2A expression was significantly increased and correlated with faster escape time onto a hidden platform, indicating involvement of short-term memory, because of the training time interval. Right forebrain NR2B expression was positively associated with long-term memory lasting 24-h (h). In the left forebrain, NR2B expression was positively related to 72-h long-term memory. In conclusion, the functions of NR2A and NR2B receptors were differentially specialized in short-term and long-term memory, depending on the right or left forebrain.

  19. PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine

    PubMed Central

    Okazaki, Taku; Maeda, Akito; Nishimura, Hiroyuki; Kurosaki, Tomohiro; Honjo, Tasuku

    2001-01-01

    PD-1 is an immunoreceptor that belongs to the immunoglobulin (Ig) superfamily and contains two tyrosine residues in the cytoplasmic region. Studies on PD-1-deficient mice have shown that PD-1 plays critical roles in establishment and/or maintenance of peripheral tolerance, but the mode of action is totally unknown. To study the molecular mechanism for negative regulation of lymphocytes through the PD-1 receptor, we generated chimeric molecules composed of the IgG Fc receptor type IIB (FcγRIIB) extracellular region and the PD-1 cytoplasmic region and expressed them in a B lymphoma cell line, IIA1.6. Coligation of the cytoplasmic region of PD-1 with the B cell receptor (BCR) in IIA1.6 transformants inhibited BCR-mediated growth retardation, Ca2+ mobilization, and tyrosine phosphorylation of effector molecules, including Igβ, Syk, phospholipase C-γ2 (PLCγ2), and ERK1/2, whereas phosphorylation of Lyn and Dok was not affected. Mutagenesis studies indicated that these inhibitory effects do not require the N-terminal tyrosine in the immunoreceptor tyrosine-based inhibitory motif-like sequence, but do require the other tyrosine residue in the C-terminal tail. This tyrosine was phosphorylated and recruited src homology 2-domain-containing tyrosine phosphatase 2 (SHP-2) on coligation of PD-1 with BCR. These results show that PD-1 can inhibit BCR signaling by recruiting SHP-2 to its phosphotyrosine and dephosphorylating key signal transducers of BCR signaling. PMID:11698646

  20. Staufen 2 regulates mGluR long-term depression and Map1b mRNA distribution in hippocampal neurons.

    PubMed

    Lebeau, Geneviève; Miller, Linda C; Tartas, Maylis; McAdam, Robyn; Laplante, Isabel; Badeaux, Frédérique; DesGroseillers, Luc; Sossin, Wayne S; Lacaille, Jean-Claude

    2011-01-01

    The two members of the Staufen family of RNA-binding proteins, Stau1 and Stau2, are present in distinct ribonucleoprotein complexes and associate with different mRNAs. Stau1 is required for protein synthesis-dependent long-term potentiation (L-LTP) in hippocampal pyramidal cells. However, the role of Stau2 in synaptic plasticity remains unexplored. We found that unlike Stau1, Stau2 is not required for L-LTP. In contrast, Stau2, but not Stau1, is necessary for DHPG-induced protein synthesis-dependent long-term depression (mGluR-LTD). While Stau2 is involved in early development of spines, its down-regulation does not alter spine morphology or spontaneous miniature synaptic activity in older cultures where LTD occurs. In addition, Stau2, but not Stau1, knockdown reduces the dendritic localization of Map1b mRNA, a specific transcript involved in mGluR-LTD. Moreover, mGluR stimulation with DHPG induces Map1b, but not Map2, mRNA dissociation from mRNA granules containing Stau2 and the ribosomal protein P0. This dissociation was not observed in cells in which Stau2 was depleted. Finally, Stau2 knockdown reduces basal Map1b protein expression in dendrites and prevents DHPG-induced increases in dendritic Map1b protein level. We suggest a role for Stau2 in the generation and regulation of Map1b mRNA containing granules that are required for mGluR-LTD.

  1. TRAF2-binding BIR1 domain of c-IAP2/MALT1 fusion protein is essential for activation of NF-kappaB.

    PubMed

    Garrison, J B; Samuel, T; Reed, J C

    2009-04-02

    Marginal zone mucosa-associated lymphoid tissue (MALT) B-cell lymphoma is the most common extranodal non-Hodgkin lymphoma. The t(11;18)(q21;q21) translocation occurs frequently in MALT lymphomas and creates a chimeric NF-kappaB-activating protein containing the baculoviral IAP repeat (BIR) domains of c-IAP2 (inhibitor of apoptosis protein 2) fused with portions of the MALT1 protein. The BIR1 domain of c-IAP2 interacts directly with TRAF2 (TNFalpha-receptor-associated factor-2), but its role in NF-kappaB activation is still unclear. Here, we investigated the role of TRAF2 in c-IAP2/MALT1-induced NF-kappaB activation. We show the BIR1 domain of c-IAP2 is essential for NF-kappaB activation, whereas BIR2 and BIR3 domains are not. Studies of c-IAP2/MALT1 BIR1 mutant (E47A/R48A) that fails to activate NF-kappaB showed loss of TRAF2 binding, but retention of TRAF6 binding, suggesting that interaction of c-IAP2/MALT1 with TRAF6 is insufficient for NF-kappaB induction. In addition, a dominant-negative TRAF2 mutant or downregulation of TRAF2 achieved by small interfering RNA inhibited NF-kappaB activation by c-IAP2/MALT1 showing that TRAF2 is indispensable. Comparisons of the bioactivity of intact c-IAP2/MALT1 oncoprotein and BIR1 E47A/R48A c-IAP2/MALT1 mutant that cannot bind TRAF2 in a lymphoid cell line provided evidence that TRAF2 interaction is critical for c-IAP2/MALT1-mediated increases in the NF-kappaB activity, increased expression of endogenous NF-kappaB target genes (c-FLIP, TRAF1), and resistance to apoptosis.

  2. Propofol effectively inhibits lithium-pilocarpine- induced status epilepticus in rats via downregulation of N-methyl-D-aspartate receptor 2B subunit expression

    PubMed Central

    Wang, Henglin; Wang, Zhuoqiang; Mi, Weidong; Zhao, Cong; Liu, Yanqin; Wang, Yongan; Sun, Haipeng

    2012-01-01

    Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine. The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior, electroencephalography and 24-hour survival rate. Propofol (12.5–100 mg/kg) improved status epilepticus in a dose-dependent manner, and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection. Western blot results showed that, 24 hours after induction of status epilepticus, the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly increased in rat cerebral cortex and hippocampus. Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels, but not the increase in N-methyl-D-aspartate receptor 2A subunit levels. The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine. This effect may be associated with downregulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures. PMID:25737709

  3. Upregulation of MicroRNA-15a Contributes to Pathogenesis of Abdominal Aortic Aneurysm (AAA) by Modulating the Expression of Cyclin-Dependent Kinase Inhibitor 2B (CDKN2B).

    PubMed

    Gao, Peng; Si, Jiyuan; Yang, Bin; Yu, Jixiang

    2017-02-18

    BACKGROUND The objective of the present study was to identify the association between miR-15a-5p and CDKN2B, and their roles in regulating the development of abdominal aortic aneurysm (AAA). MATERIAL AND METHODS We searched the miRNA database online (www.mirdb.org) and used a luciferase reporter assay system to study the regulatory relationship between miR-15a-5p and CDKN2B. We also conducted real-time PCR and Western blot analysis to study the mRNA and protein expression level of CDKN2B among different patient groups (participants with abdominal aortic aneurysm (AAA) and normal controls) or cells treated with scramble control, miR-15a-5p mimics, CDKN2B siRNA, and miR-15a-5p inhibitors. RESULTS We found that CDKN2B was a virtual target of miR-15a-5p with potential binding sites in the 3'UTR of CDKN2B (77-83 bp). We also showed that miR-15a-5p could bind to the CDKN2B 3'UTR, resulting in a significant decrease in luciferase activity compared with the scramble control. Furthermore, we found that the cells isolated from AAA participants showed an over-expression of miR-15a-5p compared to the normal controls, while the CDKN2B mRNA and protein expression level of the AAA group were much lower than the normal control group. Additionally, the expression of CDKN2B mRNA and the protein of the cells transfected with miR-15a-5p mimics and CDKN2B siRNA was downregulated, while the cells showed upregulated expression subsequent to transfection with miR-15a-5p inhibitors compared to the scramble control. CONCLUSIONS The data revealed a negative regulatory role of miR-15a-5p in the apoptosis of smooth muscle cells via targeting CDKN2B, and showed that miR-15a-5p could be a novel therapeutic target of AAA.

  4. The effect of vasopressin 1b receptor (V1bR) blockade on HPA axis activity in rats exposed to acute heat stress.

    PubMed

    Jasnic, Nebojsa; Djordjevic, Jelena; Vujovic, Predrag; Lakic, Iva; Djurasevic, Sinisa; Cvijic, Gordana

    2013-06-15

    Thermal stressors such as low and high ambient temperature elicit an abundance of neuroendocrine responses including activation of the hypothalamo-pituitary-adrenal (HPA) axis and arginine vasopressin (AVP) release. The exposure to heat is a particularly interesting model for studying AVP action because this kind of stressor represents not only an unpleasant experience but also a threat to osmotic homeostasis. As AVP has long been recognized as a hormone involved in the modulation of HPA axis activity, the aim of this study was to elucidate the role of AVP in acutely heat-exposed rats using Nelivaptan, a selective vasopressin 1b receptor (V1bR) antagonist. Rats were exposed to high ambient temperature (38°C) for 60 min. The circulating hormones were determined by ELISA or chemiluminescence, and intrapituitary adrenocorticotropic hormone (ACTH) and V1bR level were determined by western blot. The results obtained show that V1bR blockade negatively affected the increase in blood ACTH caused by heat exposure. This treatment alone, or in combination with Nelivaptan, decreased intrapituitary V1bR levels while circulating AVP concentration was increased under the same conditions. Furthermore, a strong correlation was observed between blood ACTH and corticosterone concentration. In conclusion, our results directly confirm the positive role of AVP in the regulation of ACTH secretion from the pituitary in animals exposed to heat. Moreover, the results suggest that AVP from the general circulation influences pituitary V1bR.

  5. Stimulation of EphB2/ephrin-B1 signalling by tumour necrosis factor alpha in human dental pulp stem cells.

    PubMed

    Zhu, Lifang; Dissanayaka, Waruna Lakmal; Green, David William; Zhang, Chengfei

    2015-04-01

    The aim of this study was to investigate whether in vitro stimulation of dental pulp stem cells (DPSCs) by tumour necrosis factor alpha (TNF-α) would induce secretion of EphB2/ephrin-B1 signalling. Dental pulp stem cells isolated from human dental pulp were treated with TNF-α (5-100 ng/ml) over 2-48 h. EphB2/ephrin-B1 mRNA and protein levels were measured by real-time polymerase chain reaction (RT-PCR) and western blot analysis respectively. Additionally, DPSCs were pre-incubated with TNF-α receptor neutralizing antibodies or infected with nuclear factor-kappa B (NF-ĸB) inhibitor, p38 MAPK inhibitor, Jun N-terminal kinase (JNK) inhibitor and MEK inhibitor before TNF-α treatment. Results were analysed by one-way ANOVA. Tumour necrosis factor alpha increased EphB2 mRNA expression in DPSCs at concentrations up to 20 ng/ml and ephrin-B1 at concentrations up to 40 ng/ml (P < 0.05). Its mRNA expression reached maximum at 24 h when treated with TNF-α at 20 ng/ml (P < 0.05). EphB2/ephrin-B1 protein expression levels were high at 16 and 24 h as shown by western blotting. Neutralizing antibodies for TNFR1/2 receptors down-regulated EphB2/ephrin-B1 mRNA expression (P < 0.05) and ephrin-B1 protein expression, but not EphB2 protein expression. JNK-inhibitor inhibited EphB2 mRNA expression only (P < 0.05). EphB2/ephrin-B1 were invoked in DPSCs with TNF-α treatment via the JNK-dependent pathway, but not NF-ĸB, p38 MAPK or MEK signalling. © 2015 John Wiley & Sons Ltd.

  6. Attenuation of reserpine-induced pain/depression dyad by gentiopicroside through downregulation of GluN2B receptors in the amygdala of mice.

    PubMed

    Liu, Shui-bing; Zhao, Rong; Li, Xu-sheng; Guo, Hong-ju; Tian, Zhen; Zhang, Nan; Gao, Guo-dong; Zhao, Ming-gao

    2014-06-01

    Epidemiological studies demonstrate that pain frequently occurs comorbid with depression. Gentiopicroside (Gent) is a secoiridoid compound isolated from Gentiana lutea that exhibits analgesic properties and inhibits the expression of GluN2B-containing N-methyl-D-aspartate (NMDA) receptors in the anterior cingulate cortex of mice. However, the effects of Gent on the reserpine-induced pain/depression dyad and its underlying mechanisms are unclear. Reserpine administration (1 mg/kg subcutaneous daily for 3 days) caused a significant decrease in the nociceptive threshold as evidenced by the reduced paw withdrawal latency in response to a radiant heat source and mechanical allodynia. Behavioral detection indicated a significant increase in immobility time during a forced swim test, as well as decreased time in the central area and total travel distance in an open field test. Furthermore, reserpinized animals exhibited increased oxidative stress. Systemic Gent administration dose-dependently ameliorated the behavioral deficits associated with reserpine-induced pain/depression dyad. At the same time, the decrease in biogenic amine levels (norepinephrine, dopamine, and serotonin) was integrated with the increase in caspase-3 levels and GluN2B-containing NMDA receptors in the amygdala of the reserpine-injected mice. Gent significantly reversed the changes in the levels of biogenic amines, caspase-3, and GluN2B-containing NMDA receptors in amygdala. However, Gent did not affect the expression of GluN2A-containing NMDA receptors. The inhibitory effects of Gent on oxidative stress were occluded by simultaneous treatment of GluN2B receptors antagonist Ro25-6981. Our study provides strong evidence that Gent inhibits reserpine-induced pain/depression dyad by downregulating GluN2B receptors in the amygdala.

  7. BAG3 increases the invasiveness of uterine corpus carcinoma cells by suppressing miR-29b and enhancing MMP2 expression.

    PubMed

    Habata, Shutaro; Iwasaki, Masahiro; Sugio, Asuka; Suzuki, Miwa; Tamate, Masato; Satohisa, Seiro; Tanaka, Ryoichi; Saito, Tsuyoshi

    2015-05-01

    Approximately 30% of uterine corpus carcinomas are diagnosed at an advanced stage and have a poor prognosis. Our previous study indicated that BCL2-associated athanogene 3 (BAG3) enhances matrix metalloproteinase-2 (MMP2) expression and binds to MMP2 to positively regulate the process of cell invasion in ovarian cancer cells. Recently, altered miRNA expression patterns were observed in several groups of patients with endometrial cancers. One of the altered miRNAs, miR-29b, reportedly reduces tumor invasiveness by suppressing MMP2 expression. Our aim in the present study was to examine the relationships among BAG3, miR-29b and MMP2 in endometrioid adenocarcinoma cells. We found that BAG3 suppresses miR-29b expression and enhances MMP2 expression, which in turn increases cell motility and invasiveness. Moreover, restoration of miR-29b through BAG3 knockdown reduced MMP2 expression, as well as cell motility and invasiveness. Collectively, our findings indicate that BAG3 enhances MMP2 expression by suppressing miR-29b, thereby increasing the metastatic potential of endometrioid adenocarcinomas.

  8. Copine-III interacts with ErbB2 and promotes tumor cell migration.

    PubMed

    Heinrich, C; Keller, C; Boulay, A; Vecchi, M; Bianchi, M; Sack, R; Lienhard, S; Duss, S; Hofsteenge, J; Hynes, N E

    2010-03-18

    ErbB2 amplification and overexpression in breast cancer correlates with aggressive disease and poor prognosis. To find novel ErbB2-interacting proteins, we used stable isotope labeling of amino acids in cell culture followed by peptide affinity pull-downs and identified specific binders using relative quantification by mass spectrometry. Copine-III, a member of a Ca(2+)-dependent phospholipid-binding protein family, was identified as binding to phosphorylated Tyr1248 of ErbB2. In breast cancer cells, Copine-III requires Ca(2+) for binding to the plasma membrane, where it interacts with ErbB2 upon receptor stimulation, an interaction that is dependent on receptor activity. Copine-III also binds receptor of activated C kinase 1 and colocalizes with phosphorylated focal adhesion kinase at the leading edge of migrating cells. Importantly, knockdown of Copine-III in T47D breast cancer cells causes a decrease in Src kinase activation and ErbB2-dependent wound healing. Our data suggest that Copine-III is a novel player in the regulation of ErbB2-dependent cancer cell motility. In primary breast tumors, high CPNE3 RNA levels significantly correlate with ERBB2 amplification. Moreover, in an in situ tissue microarray analysis, we detected differential protein expression of Copine-III in normal versus breast, prostate and ovarian tumors, suggesting a more general role for Copine-III in carcinogenesis.

  9. Enhanced attention and impulsive action following NMDA receptor GluN2B-selective antagonist pretreatment.

    PubMed

    Higgins, Guy A; Silenieks, Leo B; MacMillan, Cam; Sevo, Julia; Zeeb, Fiona D; Thevarkunnel, Sandy

    2016-09-15

    NMDA GluN2B (NR2B) subtype selective antagonists are currently in clinical development for a variety of indications, including major depression. We previously reported the selective NMDA GluN2B antagonists Ro 63-1908 and traxoprodil, increase premature responding in a 5-choice serial reaction time task (5-CSRTT) suggesting an effect on impulsive action. The present studies extend these investigations to a Go-NoGo and delay discounting task, and the 5-CSRTT under test conditions of both regular (5s) and short (2-5s) multiple ITI (Intertrial interval). Dizocilpine was included for comparison. Both Ro 63-1908 (0.1-1mg/kg SC) and traxoprodil (0.3-3mg/kg SC) increased premature and perseverative responses in both 5-CSRT tasks and improved attention when tested under a short ITI test condition. Ro 63-1908 but not traxoprodil increased motor impulsivity (false alarms) in a Go-NoGo task. Dizocilpine (0.01-0.06mg/kg SC) affected both measures of motor impulsivity and marginally improved attention. In a delay discounting test of impulsive choice, both dizocilpine and Ro 63-1908 decreased impulsive choice (increased choice for the larger, delayed reward), while traxoprodil showed a similar trend. Motor stimulant effects were evident following Ro 63-1908, but not traxoprodil treatment - although no signs of motor stereotypy characteristic of dizocilpine (>0.1mg/kg) were noted. The findings of both NMDA GluN2B antagonists affecting measures of impulsive action and compulsive behavior may underpin emerging evidence to suggest glutamate signaling through the NMDA GluN2B receptor plays an important role in behavioural flexibility. The profiles between Ro 63-1908 and traxoprodil were not identical, perhaps suggesting differences between members of this drug class. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. NR2A- and NR2B-NMDA receptors and drebrin within postsynaptic spines of the hippocampus correlate with hunger-evoked exercise.

    PubMed

    Chen, Yi-Wen; Actor-Engel, Hannah; Sherpa, Ang Doma; Klingensmith, Lauren; Chowdhury, Tara G; Aoki, Chiye

    2017-07-01

    Hunger evokes foraging. This innate response can be quantified as voluntary wheel running following food restriction (FR). Paradoxically, imposing severe FR evokes voluntary FR, as some animals choose to run rather than eat, even during limited periods of food availability. This phenomenon, called activity-based anorexia (ABA), has been used to identify brain changes associated with FR and excessive exercise (EX), two core symptoms of anorexia nervosa (AN), and to explore neurobiological bases of AN vulnerability. Previously, we showed a strong positive correlation between suppression of FR-evoked hyperactivity, i.e., ABA resilience, and levels of extra-synaptic GABA receptors in stratum radiatum (SR) of hippocampal CA1. Here, we tested for the converse: whether animals with enhanced expression of NMDA receptors (NMDARs) exhibit greater levels of FR-evoked hyperactivity, i.e., ABA vulnerability. Four groups of animals were assessed for NMDAR levels at CA1 spines: (1) ABA, in which 4 days of FR was combined with wheel access to allow voluntary EX; (2) FR only; (3) EX only; and (4) control (CON) that experienced neither EX nor FR. Electron microscopy revealed that synaptic NR2A-NMDARs and NR2B-NMDARs levels are significantly elevated, relative to CONs'. Individuals' ABA severity, based on weight loss, correlated with synaptic NR2B-NMDAR levels. ABA resilience, quantified as suppression of hyperactivity, correlated strongly with reserve pools of NR2A-NMDARs in spine cytoplasm. NR2A- and NR2B-NMDAR measurements correlated with spinous prevalence of an F-actin binding protein, drebrin, suggesting that drebrin enables insertion of NR2B-NMDAR to and retention of NR2A-NMDARs away from synaptic membranes, together influencing ABA vulnerability.

  11. CDKL5 controls postsynaptic localization of GluN2B-containing NMDA receptors in the hippocampus and regulates seizure susceptibility.

    PubMed

    Okuda, Kosuke; Kobayashi, Shizuka; Fukaya, Masahiro; Watanabe, Aya; Murakami, Takuto; Hagiwara, Mai; Sato, Tempei; Ueno, Hiroe; Ogonuki, Narumi; Komano-Inoue, Sayaka; Manabe, Hiroyuki; Yamaguchi, Masahiro; Ogura, Atsuo; Asahara, Hiroshi; Sakagami, Hiroyuki; Mizuguchi, Masashi; Manabe, Toshiya; Tanaka, Teruyuki

    2017-10-01

    Mutations in the Cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders accompanied by intractable epilepsies, i.e. West syndrome or atypical Rett syndrome. Here we report generation of the Cdkl5 knockout mouse and show that CDKL5 controls postsynaptic localization of GluN2B-containing N-methyl-d-aspartate (NMDA) receptors in the hippocampus and regulates seizure susceptibility. Cdkl5 -/Y mice showed normal sensitivity to kainic acid; however, they displayed significant hyperexcitability to NMDA. In concordance with this result, electrophysiological analysis in the hippocampal CA1 region disclosed an increased ratio of NMDA/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (EPSCs) and a significantly larger decay time constant of NMDA receptor-mediated EPSCs (NMDA-EPSCs) as well as a stronger inhibition of the NMDA-EPSCs by the GluN2B-selective antagonist ifenprodil in Cdkl5 -/Y mice. Subcellular fractionation of the hippocampus from Cdkl5 -/Y mice revealed a significant increase of GluN2B and SAP102 in the PSD (postsynaptic density)-1T fraction, without changes in the S1 (post-nuclear) fraction or mRNA transcripts, indicating an intracellular distribution shift of these proteins to the PSD. Immunoelectron microscopic analysis of the hippocampal CA1 region further confirmed postsynaptic overaccumulation of GluN2B and SAP102 in Cdkl5 -/Y mice. Furthermore, ifenprodil abrogated the NMDA-induced hyperexcitability in Cdkl5 -/Y mice, suggesting that upregulation of GluN2B accounts for the enhanced seizure susceptibility. These data indicate that CDKL5 plays an important role in controlling postsynaptic localization of the GluN2B-SAP102 complex in the hippocampus and thereby regulates seizure susceptibility, and that aberrant NMDA receptor-mediated synaptic transmission underlies the pathological mechanisms of the CDKL5 loss-of-function. Copyright © 2017 Elsevier Inc. All rights

  12. Human Freud-2/CC2D1B: a novel repressor of postsynaptic serotonin-1A receptor expression.

    PubMed

    Hadjighassem, Mahmoud R; Austin, Mark C; Szewczyk, Bernadeta; Daigle, Mireille; Stockmeier, Craig A; Albert, Paul R

    2009-08-01

    Altered expression of serotonin-1A (5-HT1A) receptors, both presynaptic in the raphe nuclei and post-synaptic in limbic and cortical target areas, has been implicated in mood disorders such as major depression and anxiety. Within the 5-HT1A receptor gene, a powerful dual repressor element (DRE) is regulated by two protein complexes: Freud-1/CC2D1A and a second, unknown repressor. Here we identify human Freud-2/CC2D1B, a Freud-1 homologue, as the second repressor. Freud-2 distribution was examined with Northern and Western blot, reverse transcriptase polymerase chain reaction, and immunohistochemistry/immunofluorescence; Freud-2 function was examined by electrophoretic mobility shift, reporter assay, and Western blot. Freud-2 RNA was widely distributed in brain and peripheral tissues. Freud-2 protein was enriched in the nuclear fraction of human prefrontal cortex and hippocampus but was weakly expressed in the dorsal raphe nucleus. Freud-2 immunostaining was co-localized with 5-HT1A receptors, neuronal and glial markers. In prefrontal cortex, Freud-2 was expressed at similar levels in control and depressed male subjects. Recombinant hFreud-2 protein bound specifically to 5' or 3' human DRE adjacent to the Freud-1 site. Human Freud-2 showed strong repressor activity at the human 5-HT1A or heterologous promoter in human HEK-293 5-HT1A-negative cells and neuronal SK-N-SH cells, a model of postsynaptic 5-HT1A receptor-positive cells. Furthermore, small interfering RNA knockdown of endogenous hFreud-2 expression de-repressed 5-HT1A promoter activity and increased levels of 5-HT1A receptor protein in SK-N-SH cells. Human Freud-2 binds to the 5-HT1A DRE and represses the human 5-HT1A receptor gene to regulate its expression in non-serotonergic cells and neurons.

  13. Involvement of microRNA miR-2b-3p in regulation of metabolic resistance to insecticides in Plutella xylostella.

    PubMed

    Etebari, K; Afrad, M H; Tang, B; Silva, R; Furlong, M J; Asgari, S

    2018-03-24

    The diamondback moth, Plutella xylostella, has developed extremely high levels of resistance to chlorantraniliprole and other classes of insecticides in the field. As microRNAs (miRNAs) play important roles in various biological processes through gene regulation, we examined the miRNA profile of P. xylostella in response to chlorantraniliprole exposure. RNA sequencing analysis showed that insecticide treatment caused significant changes in the abundance of some miRNAs. Increasing exposure time and insecticide concentration induced more dysregulated miRNAs in P. xylostella larvae. We also screened potential target genes for some of the differentially expressed miRNAs (such as miR-2b-3p, miR-14b-5p and let-7-5p), which may play important roles in insecticide resistance development. Exposure of P. xylostella larvae to chlorantraniliprole caused considerable overexpression in the transcript levels of potential target genes cytochrome P450 9f2 (CYP9F2) and 307a1 (CYP307a1). Application of miR-2b-3p and miR-14b-5p mimics significantly suppressed the relative transcript levels of CYP9F2 and CYP307a1, respectively, in a P. xylostella cell line. Furthermore, enrichment of P. xylostella diet with miR-2b-3p mimics significantly increased mortality in deltamethrin-resistant larvae when exposed to deltamethrin. The results suggest that miR-2b-3p may suppress CYP9F2 transcript levels in P. xylostella and consequently inhibit larval detoxification pathways. The findings provide an insight into possible role of miRNAs in regulation of metabolic resistance of insects to insecticides. © 2018 The Royal Entomological Society.

  14. FOXP2-positive diffuse large B-cell lymphomas exhibit a poor response to R-CHOP therapy and distinct biological signatures

    PubMed Central

    Wong, Kah Keng; Gascoyne, Duncan M.; Soilleux, Elizabeth J.; Lyne, Linden; Spearman, Hayley; Roncador, Giovanna; Pedersen, Lars M.; Møller, Michael B.; Green, Tina M.; Banham, Alison H.

    2016-01-01

    FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL. PMID:27224915

  15. FOXP2-positive diffuse large B-cell lymphomas exhibit a poor response to R-CHOP therapy and distinct biological signatures.

    PubMed

    Wong, Kah Keng; Gascoyne, Duncan M; Soilleux, Elizabeth J; Lyne, Linden; Spearman, Hayley; Roncador, Giovanna; Pedersen, Lars M; Møller, Michael B; Green, Tina M; Banham, Alison H

    2016-08-16

    FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL.

  16. Differential agonist sensitivity of glycine receptor α2 subunit splice variants

    PubMed Central

    Miller, Paul S; Harvey, Robert J; Smart, Trevor G

    2004-01-01

    The glycine receptor (GlyR) α2A and α2B splice variants differ by a dual, adjacent amino acid substitution from α2AV58,T59 to α2BI58,A59 in the N-terminal extracellular domain. Comparing the effects of the GlyR agonists, glycine, β-alanine and taurine, on the GlyR α2 isoforms, revealed a significant increase in potency for all three agonists at the α2B variant. The sensitivities of the splice variants to the competitive antagonist, strychnine, and to the biphasic modulator Zn2+, were comparable. In contrast, the allosteric inhibitor picrotoxin was more potent on GlyR α2A compared to GlyR α2B receptors. Coexpression of α2A or α2B subunits with the GlyR β subunit revealed that the higher agonist potencies observed with the α2B homomer were retained for the α2Bβ heteromer. The identical sensitivity to strychnine combined with a reduction in the maximum current induced by the partial agonist taurine at the GlyR α2A homomer, suggested that the changed sensitivity to agonists is in accordance with a modulation of agonist efficacy rather than agonist affinity. An effect on agonist efficacy was also supported by using a structural model of the GlyR, localising the region of splice variation to the proposed docking region between GlyR loop 2 and the TM2-3 loop, an area associated with channel activation. The existence of a spasmodic mouse phenotype linked to a GlyR α1A52S mutation, the equivalent position to the source of the α2 splice variation, raises the possibility that the GlyR α2 splice variants may be responsible for distinct roles in neuronal function. PMID:15302677

  17. NMDA receptor subunits and associated signaling molecules mediating antidepressant-related effects of NMDA-GluN2B antagonism

    PubMed Central

    Kiselycznyk, Carly; Jury, Nicholas; Halladay, Lindsay; Nakazawa, Kazu; Mishina, Masayoshi; Sprengel, Rolf; Grant, Seth G.N.; Svenningsson, Per; Holmes, Andrew

    2015-01-01

    Drugs targeting the glutamate N-methyl-D-aspartate receptor (NMDAR) may be efficacious for treating mood disorders, as exemplified by the rapid antidepressant effects produced by single administration of the NMDAR antagonist ketamine. Though the precise mechanisms underlying the antidepressant-related effects of NMDAR antagonism remain unclear, recent studies implicate specific NMDAR subunits, including GluN2A and GluN2B, as well as the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunit glutamate receptor interacting molecule, PSD-95. Here, integrating mutant and pharmacological in mice, we investigated the contribution of these subunits and molecules to antidepressant-related behaviors and the antidepressant-related effects of the GluN2B blocker, Ro 25-6981. We found that global deletion of GluA1 or PSD-95 reduced forced swim test (FST) immobility, mimicking the antidepressant-related effect produced by systemically administered Ro 25-6981 in C57BL/6J mice. Moreover, the FST antidepressant-like effects of systemic Ro 25-6981 were intact in mutants with global GluA1 deletion or GluN1 deletion in forebrain interneurons, but were absent in mutants constitutively lacking GluN2A or PSD-95. Next, we found that microinfusing Ro 25-6981 into the medial prefrontal cortex (mPFC), but not basolateral amygdala, of C57BL/6J mice was sufficient to produce an antidepressant-like effect. Together, these findings extend and refine current understanding of the mechanisms mediating antidepressant-like effects produced by NMDAR-GluN2B antagonists, and may inform the development of a novel class of medications for treating depression that target the GluN2B subtype of NMDAR. PMID:25800971

  18. Identification of ectodomain regions contributing to gating, deactivation, and resensitization of purinergic P2X receptors.

    PubMed

    Zemkova, Hana; He, Mu-Lan; Koshimizu, Taka-aki; Stojilkovic, Stanko S

    2004-08-04

    The P2X receptors (P2XRs) are a family of ligand-gated channels activated by extracellular ATP through a sequence of conformational transitions between closed, open, and desensitized states. In this study, we examined the dependence of the activity of P2XRs on ectodomain structure and agonist potency. Experiments were done in human embryonic kidney 293 cells expressing rat P2X2aR, P2X2bR, and P2X3R, and chimeras having the V60-R180 or V60-F301 ectodomain sequences of P2X3R instead of the I66-H192 or I66-Y310 sequences of P2X2aR and P2X2bR. Chimeric P2X2a/V60-F301X3R and P2X2b/V60-F301X3R inherited the P2X3R ligand-selective profile, whereas the potency of agonists for P2X2a/V60-R180X3R was in between those observed at parental receptors. Furthermore, P2X2a/V60-F301X3R and P2X2a/V60-R180X3R desensitized in a P2X2aR-specific manner, and P2X2b/V60-F301X3R desensitized with rates comparable with those of P2X2bR. In striking contrast to parental receptors, the rates of decay in P2X2a/V60-F301X3R and P2X2b/V60-F301X3R currents after agonist withdrawal were 15- to 200-fold slower. For these chimeras, the decays in currents were not dependent on duration of stimuli and reflected both continuous desensitization and deactivation of receptors. Also, participation of deactivation in closure of channels inversely correlated with potency of agonists to activate receptors. The delay in deactivation was practically abolished in P2X2a/V60-R180X3R-expressing cells. However, the recovery from desensitization of P2X2a/V60-F301X3R and P2X2a/V60-R180X3R was similar and substantially delayed compared with that of parental receptors. These results indicate that both ectodomain halves participate in gating, but that the C and N halves influence the stability of open and desensitized conformation states, respectively, which in turn reflects on rates of receptor deactivation and resensitization.

  19. gC1q-R/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes.

    PubMed

    Braun, L; Ghebrehiwet, B; Cossart, P

    2000-04-03

    InlB is a Listeria monocytogenes protein that promotes entry of the bacterium into mammalian cells by stimulating tyrosine phosphorylation of the adaptor proteins Gab1, Cbl and Shc, and activation of phosphatidyl- inositol (PI) 3-kinase. Using affinity chromatography and enzyme-linked immunosorbent assay, we demonstrate a direct interaction between InlB and the mammalian protein gC1q-R, the receptor of the globular part of the complement component C1q. Soluble C1q or anti-gC1q-R antibodies impair InlB-mediated entry. Transient transfection of GPC16 cells, which are non-permissive to InlB-mediated entry, with a plasmid-expressing human gC1q-R promotes entry of InlB-coated beads. Furthermore, several experiments indicate that membrane recruitment and activation of PI 3-kinase involve an InlB-gC1q-R interaction and that gC1q-R associates with Gab1 upon stimulation of Vero cells with InlB. Thus, gC1q-R constitutes a cellular receptor involved in InlB-mediated activation of PI 3-kinase and tyrosine phosphorylation of the adaptor protein Gab1. After E-cadherin, the receptor for internalin, gC1q-R is the second identified mammalian receptor promoting entry of L. monocytogenes into mammalian cells.

  20. Immunometric assays of ras and c-erbB-2/neu overexpression in breast cancer: a pilot study.

    PubMed

    Pelizzola, D; Malagutti, R; Giovannini, G; Indelli, M; Carcoforo, P; Piffanelli, A

    1999-01-01

    The expression of the ras and c-erbB2 oncoproteins (p21 and p185, respectively), together with estrogen receptor (ER) and progesterone receptor (PgR) determination, has been retrospectively analyzed in 68 primary breast carcinomas and in 19 normal breast tissue samples. The aims of this study were: a) to explore the association between ras and c-erbB2 expression; b) to evaluate the relationship between ras and c-erbB2 expression and both steroid receptor status and the classical clinical and pathological parameters; and c) to compare two different methods for p185 determination. p185 and p21 were measured by enzyme immunoassay (EIA); p185 was also determined by Western blotting (WB); ER and PgR were assayed by radioligand binding assay. The highest value of p185 in benign breast lesions was used as the threshold to distinguish between positive and negative samples. With this threshold the c-erbB2 oncoprotein was overexpressed in 41.2% (with EIA) and in 50% (with WB) of cancer samples. The concordance rate between the two methods was 79.4. No significant association was found between p21 and p185 levels either in cancer or in normal breast tissue samples. Increasing levels of tumor p21 were associated with a shorter time to recurrence and overall survival. Increasing levels of p185 were associated with a significantly shorter time to recurrence (p185 EIA: p = 0.04, p185 WB: p = 0.029) and overall survival (p185 EIA: p = 0.04, p185 WB: p = 0.029).

  1. CC2D1A and CC2D1B regulate degradation and signaling of EGFR and TLR4.

    PubMed

    Deshar, Rakesh; Cho, Eun-Bee; Yoon, Sungjoo Kim; Yoon, Jong-Bok

    2016-11-11

    Signaling through many transmembrane receptors is terminated by their sorting to the intraluminal vesicles (ILVs) of multivescular bodies (MVBs) and subsequent lysosomal degradation. ILV formation requires the endosomal sorting complex required for transport (ESCRT) machinery. CC2D1A and CC2D1B interact with the CHMP4 family of proteins, the major subunit of the ESCRT-III complex, however, their roles in receptor degradation and signaling are poorly defined. Here, we report that CC2D1A binds to CHMP4B polymers formed on endosomes to regulate the endosomal sorting pathway. We show that depletion of CC2D1A and B accelerates degradation of EGFR and elicits rapid termination of its downstream signaling through ERK1 and 2. Depletion of CC2D1A and B promotes sorting of EGFR to ILV leading to its rapid lysosomal degradation. In addition, we show that knockdown of CC2D1A and B has similar effects on degradation and downstream signaling of another membrane receptor, TLR4. Thus, these findings suggest that CC2D1A and B may have broad effects on transmembrane receptors by preventing premature ILV sorting and termination of signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Effects of the recombinant crustacean hyperglycemic hormones rCHH-B1 and rCHH-B2 on the osmo-ionic regulation of the shrimp Litopenaeus vannamei exposed to acute salinity stress.

    PubMed

    Camacho-Jiménez, Laura; Díaz, Fernando; Sánchez-Castrejón, Edna; Ponce-Rivas, Elizabeth

    2018-03-26

    The Pacific white shrimp Litopenaeus vannamei is a euryhaline organism that copes with salinity fluctuations in the environment; therefore, its osmotic and ionic regulation abilities are vital. Osmoregulation may be controlled by the crustacean hyperglycemic hormone (CHH), a neuropeptide mainly expressed in the eyestalks. In L. vannamei, CHH-B1 and CHH-B2 are CHH isoforms isolated from the eyestalks whose expression is influenced by environmental salinity. It has been suggested that they are involved in the response to salinity stress. To clarify this, we investigated the effect of the recombinant peptides, rCHH-B1 and rCHH-B2, on the osmo-ionic regulation of shrimp acutely exposed to different salinity conditions (8, 26 and 45‰). Both rCHHs promoted differential effects on the osmoregulatory capacity (OC) and the ionoregulatory capacity (IC) for hemolymph Na + and Cl - during iso-osmotic (26‰) and hyper-osmotic (45‰) transfers. These changes were linked to the changes observed in Na + /K + ATPase and carbonic anhydrase gene expression in gills, especially under high salinity conditions, suggesting that the hormones may regulate the expression of these genes. Glucose and protein levels measured during acute salinity transfer suggest their roles as sources of metabolic energy for osmotic regulation or as organic osmolytes. These results taken together suggest that both the CHH-B1 and CHH-B2 peptides are important regulators of the physiological response of L. vannamei to acute salinity fluctuations.

  3. The role of MiR-324-3p in polycystic ovary syndrome (PCOS) via targeting WNT2B.

    PubMed

    Jiang, Y-C; Ma, J-X

    2018-06-01

    To investigate the expression of microRNA-324-3p (miR-324-3p) in polycystic ovary syndrome (PCOS) and its effects on the proliferation and apoptosis of ovarian granulosa cells. A total of 60 Sprague-Dawley (SD) rats were randomly divided into normal group (n=30) and experimental group (n=30). Rats in the experimental group were intramuscularly injected with dehydroepiandrosterone (DHEA) (6 mg/100 g of body weight) and 0.2 mL oil for injection, while those in normal group were intramuscularly injected with 0.2 mL oil for injection. The ovarian tissues of PCOS model rats were removed to extract the total ribose nucleic acid (RNA). The expression of miR-324-3p was detected via reverse transcription-polymerase chain reaction (RT-PCR). Primary ovarian granulosa cells were isolated and cultured, and NC-miRNA and miR-324-3p mimic were transfected into cells. After 48 h, cell proliferation and apoptosis were detected via cell counting kit 8 (CCK-8) and flow cytometry assay, respectively. The targeted molecule of miR-324-3p was explored using bioinformatics, and dual-luciferase assay was performed to verify the effect of miR-324-3p on WNT2B expression. Granulosa cells were co-transfected with WNT2B-small-interfering RNA (siRNA) and miR-324-3p mimic, and then cell proliferation and apoptosis were detected via CCK-8 and flow cytometry assay, respectively. The expression of miR-324-3p in ovarian tissues of PCOS group was significantly lower than that of normal group (p < 0.01). After transfection with miR-324-3p mimic into granulosa cells, cell proliferation was significantly inhibited and cell apoptosis was promoted (p < 0.01). MiR-324-3p exerted its effect on granulosa cells by directly targeting WNT2B. Silencing WNT2B expression could reverse the effects of miR-324-3p on proliferation and apoptosis of granulosa cells (p < 0.05). The expression of miR-324-3p in the ovary of PCOS rats is decreased significantly. Overexpression of miR-324-3p can reduce the proliferation and

  4. Overexpression of miR-26b-5p regulates the cell cycle by targeting CCND2 in GC-2 cells under exposure to extremely low frequency electromagnetic fields.

    PubMed

    Liu, Yong; Liu, Wen-Bin; Liu, Kai-Jun; Ao, Lin; Cao, Jia; Zhong, Julia Li; Liu, Jin-Yi

    2016-01-01

    The increasing prevalence of extremely low frequency electromagnetic fields (ELF-EMFs) exposure has raised considerable public concern regarding the potential hazardous effects of ELF-EMFs on male reproductive function. Increasing evidence indicates that miRNAs are necessary for spermatogenesis and male fertility. However, the regulation of miRNA expression and the roles of miRNAs in response to ELF-EMFs remain unclear. In our study, mouse spermatocyte-derived GC-2 cells were intermittently exposed to a 50 Hz ELF-EMF for 72 h (5 min on/10 min off) at magnetic field intensities of 1 mT, 2 mT and 3 mT. MiR-26b-5p was differentially expressed in response to different magnetic field intensities of ELF-EMFs. The host gene CTDSP1 showed an unmethylation status in GC-2 cells at different magnetic field intensities of ELF-EMF exposure. MiR-26b-5p had no significant, obvious influence on the cell viability, apoptosis or cell cycle of GC-2 cells. However, the overexpression of miR-26b-5p significantly decreased the percentage of G0/G1 phase cells and slightly increased the percentage of S phase cells compared to the sham group that was exposed to a 50 Hz ELF-EMF. Computational algorithms identified Cyclin D2 (CCND2) as a direct target of miR-26b-5p. MiR-26b-5p and a 50 Hz ELF-EMF altered the expression of CCND2 at both the mRNA and protein levels. Overexpressed miR-26b-5p in GC-2 cells can change the mRNA expression of CCND2 following 50 Hz ELF-EMF at 3 mT. These findings demonstrate that miR-26b-5p could serve as a potential biomarker following 50 Hz ELF-EMF exposure, and miR-26b-5p-CCND2-mediated cell cycle regulation might play a pivotal role in the biological effects of ELF-EMFs.

  5. Labeling of SR 46349B, a potent and selective 5-HT{sub 2} receptor antagonist

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tan, P.; Fowler, J.S.; Ding, Y.S.

    1995-05-01

    SR 46349B is a potent and selective 5-HT{sub 2} receptor antagonist (Kd =1.2 nM) which is currently being evaluated as an antidepressant. We labeled SR46349B with F-18 for PET studies via the nitro-for-fluorine exchange reaction. Among the five nitro-precursors (o-nitroacetophenone) examined for nucleophilic aromatic substitution ({sup 18}F{sup {minus}}, K{sub 2}CO{sub 3}, kryptofix-222, 120{degrees}C, 6 min), only phenol protected ether proceeded well and gave 36.4 {plus_minus} 14.3%(n=19) yield of which was directly hydrolyzed (Hcl, 90{degrees}C, 10 min) to afford. Removal of the nitro-precursor, which was generated in situ during hydrolysis was critical in the purification of the final product and wasmore » accomplished using a combination of C-18 Sep-Pak and silica gel column chromatography. The condensation of {sup 18}F- ketone with Me{sub 2}NCH{sub 2}CH{sub 2}ONH{sub 2}HCl in 2-(2{prime}-methoxyethoxy)ethanol (p-TsOH, 165{degrees}C, 10 min) gave a mixture of [{sup 18}F]SR 46349B and its geometric isomer with ca 1:1 ratio in quantitative yield. [{sup 18}F]SR 46349B was separated from its geometric isomer and other by-products by HPLC [Econosil C-18 semi-prep column, MeOH:MeCN:0.1 MK{sub 2}HPO{sub 4}(27.5:27.5:45), 5 ml/min]. The three step hot synthesis required 170 min and gave a specific activity of 1.14 Ci/{mu}mol, 5% radiochemical yield (EOB) and 96% radiochemical purity.« less

  6. Functional ET(A)-ET(B) Receptor Cross-talk in Basilar Artery In Situ From ET(B) Receptor Deficient Rats.

    PubMed

    Yoon, SeongHun; Gariepy, Cheryl E; Yanagisawa, Masashi; Zuccarello, Mario; Rapoport, Robert M

    2016-03-01

    The role of endothelin (ET)(A)-ET(B) receptor cross-talk in limiting the ET(A) receptor antagonist inhibition of ET-1 constriction is revealed by the partial or complete dependency of the ET(A) receptor antagonist inhibition on functional removal of the ET(B) receptor. Although functional removal of the ET(B) receptor is generally accomplished with ET(B) receptor antagonist, a novel approach using rats containing a naturally occurring deletion mutation in the ET(B) receptor [rescued "spotting lethal" (sl) rats; ET(B)(sl/sl)] demonstrated increased ET(A) receptor antagonist inhibition of ET-1 constriction in vena cava. We investigated whether this deletion mutation was also sufficient to remove the ET(B) receptor dependency of the ET(A) receptor antagonist inhibition of ET-1 constriction in the basilar artery. Consistent with previous reports, ET-1 plasma levels were elevated in ET(B)(sl/sl) as compared with ET(B)(+/+) rats. ET(B) receptor antagonist failed to relax the ET-1 constricted basilar artery from ET(B)(+/+) and ET(B)(sl/sl) rats. Relaxation to combined ET(A) and ET(B) receptor antagonist was greater than relaxation to ET(A) receptor antagonist in the basilar artery from ET(B)(+/+) and, unexpectedly, ET(B)(sl/sl) rats. These findings confirm the presence of ET(A)-ET(B) receptor cross-talk in the basilar artery. We speculate that mutant ET(B) receptor expression produced by alternative splicing may be sufficient to allow cross-talk.

  7. ORM-Based Semantics of B2B Transactions

    NASA Astrophysics Data System (ADS)

    Balsters, H.; van Blommestein, F.

    After widespread implementation of Enterprise Resource Planning and Personal Information Management, the next wave in the application of ICT is headed towards business to business (B2B) communication. B2B has a number of specific aspects, one of them being negotiation. This aspect has been largely neglected by present implementations of standard EDI- or XML-messaging and by B2B webservice implementations. In this paper a precise model is given of the negotiation process. The requirements of a potential Buyer and the offer of a potential Seller are matched and, if the negotiation is successful, a contract is concluded. The negotiation process model is represented in ORM, extended with dynamic constraints. Our model may be implemented in the databases of the trading partners and in message- or service definitions.

  8. Structure and signalling functions of C3 receptors on human B cells.

    PubMed

    Frade, R

    1990-03-01

    CR1 (C3b receptor) and CR2 (C3d/EBV receptor) are two C3 receptors expressed on B lymphocytes. CR1 and CR2 have structural similarities and their cross-linking at the B cell surface by antibodies or specific ligands in multimeric forms induce B cell activation. However, activation of human B cells through cell surface interactions or by intracellular protein kinase C activators leads to phosphorylation of CR2 but not CR1. CR2 is phosphorylated on serine and tyrosine residues. Analysis of post-membrane events associated with CR2 revealed intracellular interactions of CR2 with p53, a plasma membrane anti-oncogene-encoded phosphoprotein, and with p120, a nuclear phosphoribonucleoprotein. These intracellular interactions probably represent important steps in the signalling functions of CR2.

  9. miR-302b inhibits tumorigenesis by targeting EphA2 via Wnt/ β-catenin/EMT signaling cascade in gastric cancer.

    PubMed

    Huang, Jin; He, Yijing; Mcleod, Howard L; Xie, Yanchun; Xiao, Desheng; Hu, Huabin; Chen, Pan; Shen, Liangfang; Zeng, Shan; Yin, Xianli; Ge, Jie; Li, Li; Tang, Lanhua; Ma, Jian; Chen, Zihua

    2017-12-22

    EphA2 is a crucial oncogene in gastric cancer (GC) development and metastasis, this study aims to identify microRNAs that target it and serve as key regulators of gastric carcinogenesis. We identified several potential microRNAs targeting EphA2 by bioinformatics websites and then analyzed the role of miR-302b in modulating EphA2 in vitro and in vivo of GC, and it's mechanism. Our analysis identified miR-302b, a novel regulator of EphA2, as one of the most significantly downregulated microRNA (miRNA) in GC tissues. Overexpression of miR-302b impaired GC cell migratory and invasive properties robustly and suppressed cell proliferation by arresting cells at G0-G1 phase in vitro. miR-302b exhibited anti-tumor activity by reversing EphA2 regulation, which relayed a signaling transduction cascade that attenuated the functions of N-cadherin, β-catenin, and Snail (markers of Wnt/β-catenin and epithelial-mesenchymal transition, EMT). This modulation of EphA2 also had distinct effects on cell proliferation and migration in GC in vivo. miR-302b serves as a critical suppressor of GC cell tumorigenesis and metastasis by targeting the EphA2/Wnt/β-catenin/EMT pathway.

  10. MicroRNA miR-27b rescues bone marrow-derived angiogenic cell function and accelerates wound healing in type 2 diabetes mellitus.

    PubMed

    Wang, Jie-Mei; Tao, Jun; Chen, Dan-Dan; Cai, Jing-Jing; Irani, Kaikobad; Wang, Qinde; Yuan, Hong; Chen, Alex F

    2014-01-01

    Vascular precursor cells with angiogenic potentials are important for tissue repair, which is impaired in diabetes mellitus. MicroRNAs are recently discovered key regulators of gene expression, but their role in vascular precursor cell-mediated angiogenesis in diabetes mellitus is unknown. We tested the hypothesis that the microRNA miR-27b rescues impaired bone marrow-derived angiogenic cell (BMAC) function in vitro and in vivo in type 2 diabetic mice. BMACs from adult male type 2 diabetic db/db and from normal littermate db/+ mice were used. miR-27b expression was decreased in db/db BMACs. miR-27b mimic improved db/db BMAC function, including proliferation, adhesion, tube formation, and delayed apoptosis, but it did not affect migration. Elevated thrombospondin-1 (TSP-1) protein in db/db BMACs was suppressed on miR-27b mimic transfection. Inhibition of miR-27b in db/+ BMACs reduced angiogenesis, which was reversed by TSP-1 small interfering RNA (siRNA). miR-27b suppressed the pro-oxidant protein p66(shc) and mitochondrial oxidative stress, contributing to its protection of BMAC function. miR-27b also suppressed semaphorin 6A to improve BMAC function in diabetes mellitus. Luciferase binding assay suggested that miR-27b directly targeted TSP-1, TSP-2, p66(shc), and semaphorin 6A. miR-27b improved topical cell therapy of diabetic BMACs on diabetic skin wound closure, with a concomitant augmentation of wound perfusion and capillary formation. Normal BMAC therapy with miR-27b inhibition demonstrated reduced efficacy in wound closure, perfusion, and capillary formation. Local miR-27b delivery partly improved wound healing in diabetic mice. miR-27b rescues impaired BMAC angiogenesis via TSP-1 suppression, semaphorin 6A expression, and p66shc-dependent mitochondrial oxidative stress and improves BMAC therapy in wound healing in type 2 diabetic mice.

  11. The loss of Gnai2 and Gnai3 in B cells eliminates B lymphocyte compartments and leads to a hyper-IgM like syndrome.

    PubMed

    Hwang, Il-Young; Park, Chung; Luong, Thuyvi; Harrison, Kathleen A; Birnbaumer, Lutz; Kehrl, John H

    2013-01-01

    B lymphocytes are compartmentalized within lymphoid organs. The organization of these compartments depends upon signaling initiated by G-protein linked chemoattractant receptors. To address the importance of the G-proteins Gαi2 and Gαi3 in chemoattractant signaling we created mice lacking both proteins in their B lymphocytes. While bone marrow B cell development and egress is grossly intact; mucosal sites, splenic marginal zones, and lymph nodes essentially lack B cells. There is a partial block in splenic follicular B cell development and a 50-60% reduction in splenic B cells, yet normal numbers of splenic T cells. The absence of Gαi2 and Gαi3 in B cells profoundly disturbs the architecture of lymphoid organs with loss of B cell compartments in the spleen, thymus, lymph nodes, and gastrointestinal tract. This results in a severe disruption of B cell function and a hyper-IgM like syndrome. Beyond the pro-B cell stage, B cells are refractory to chemokine stimulation, and splenic B cells are poorly responsive to antigen receptor engagement. Gαi2 and Gαi3 are therefore critical for B cell chemoattractant receptor signaling and for normal B cell function. These mice provide a worst case scenario of the consequences of losing chemoattractant receptor signaling in B cells.

  12. Electronic Commerce in Tourism in China: B2B or B2C?

    NASA Astrophysics Data System (ADS)

    Li, Hongxiu; Suomi, Reima

    E-commerce has significantly changed the distribution channels of travel products in the world including China. Online channels are growing important in travel service distribution. In China tourism industry has been developed rapidly with the economic development, more and more international travel service providers are trying to expand their Chinese market through the Internet. This paper sheds lights on the e-commerce development models in China for international travel service providers. It explores the current e-tourism in China from the three different participants in the value chain in tourism industry - consumer, travel agent and travel service provider. The paper also identifies the barriers in B2C arena in international outbound travel market, and discusses the possible approaches for international travel service providers to develop their e-commerce in the huge Chinese market. The results in this study reveal that international travel service providers should focus on B2B model to expand their electronic market in China. B2C development in tourism largely depends on the change of Chinese customers' behavior and the change of international tourism regulations. The findings of the study are expected to assist international travel service providers to understand current e-tourism in China and to support their planning for future e-commerce development in China.

  13. Lattice Thermal Conductivity from Atomistic Simulations: ZrB2 and HfB2

    NASA Technical Reports Server (NTRS)

    Lawson, John W.; Daw, Murray S.; Bauschlicher, Charles W.

    2012-01-01

    Ultra high temperature ceramics (UHTC) including ZrB2 and HfB2 have a number of properties that make them attractive for applications in extreme environments. One such property is their high thermal conductivity. Computational modeling of these materials will facilitate understanding of fundamental mechanisms, elucidate structure-property relationships, and ultimately accelerate the materials design cycle. Progress in computational modeling of UHTCs however has been limited in part due to the absence of suitable interatomic potentials. Recently, we developed Tersoff style parameterizations of such potentials for both ZrB2 and HfB2 appropriate for atomistic simulations. As an application, Green-Kubo molecular dynamics simulations were performed to evaluate the lattice thermal conductivity for single crystals of ZrB2 and HfB2. The atomic mass difference in these binary compounds leads to oscillations in the time correlation function of the heat current, in contrast to the more typical monotonic decay seen in monoatomic materials such as Silicon, for example. Results at room temperature and at elevated temperatures will be reported.

  14. Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kyotani, Yoji, E-mail: cd147@naramed-u.ac.jp; Department of Pharmacy, Nara Medical University Hospital, Kashihara 634-8522; Ota, Hiroyo

    Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned mediummore » significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation. - Highlights: ●In vitro system for intermittent hypoxia (IH) and sustained hypoxia (SH). ●IH, but not SH, induces the proliferation of rat vascular smooth muscle cell.

  15. Salt-loading increases vasopressin and vasopressin 1b receptor mRNA in the hypothalamus and choroid plexus.

    PubMed

    Zemo, D A; McCabe, J T

    2001-01-01

    The choroid plexus plays a pivotal role in the production of cerebrospinal fluid (CSF). Messenger RNA (mRNA) transcripts encoding arginine vasopressin (AVP) and the vasopressin 1b receptor (V(1b)R) are found in various structures of the central nervous system, including the choroid plexus. The present study measured AVP and V(1b)R mRNA production in response to plasma hyperosmolality. Compared to rats maintained on water, 2% salt-drinking rats had increased levels of AVP and V(1b)R mRNAs in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus and in the choroid plexus. The increase in V(1b)R mRNA in the SON and PVN as a result of plasma hyperosmolality may reflect changes in receptor production that, in turn, have a role in AVP autoregulation of hypothalamic magnocellular neurons. The increase of AVP and V(1b)R mRNAs in the choroid plexus further shows the involvement of AVP in the regulation of brain water content and cerebral edema. Copyright 2001 Harcourt Publishers Ltd.

  16. Electrocardiographic Characterization of Cardiac Hypertrophy in Mice that Overexpress the ErbB2 Receptor Tyrosine Kinase

    PubMed Central

    Sysa-Shah, Polina; Sørensen, Lars L; Abraham, M Roselle; Gabrielson, Kathleen L

    2015-01-01

    Electrocardiography is an important method for evaluation and risk stratification of patients with cardiac hypertrophy. We hypothesized that the recently developed transgenic mouse model of cardiac hypertrophy (ErbB2tg) will display distinct ECG features, enabling WT (wild type) mice to be distinguished from transgenic mice without using conventional PCR genotyping. We evaluated more than 2000 mice and developed specific criteria for genotype determination by using cageside ECG, during which unanesthetized mice were manually restrained for less than 1 min. Compared with those from WT counterparts, the ECG recordings of ErbB2tg mice were characterized by higher P- and R-wave amplitudes, broader QRS complexes, inverted T waves, and ST interval depression. Pearson's correlation matrix analysis of combined WT and ErbB2tg data revealed significant correlation between heart weight and the ECG parameters of QT interval (corrected for heart rate), QRS interval, ST height, R amplitude, P amplitude, and PR interval. In addition, the left ventricular posterior wall thickness as determined by echocardiography correlated with ECG-determined ST height, R amplitude, QRS interval; echocardiographic left ventricular mass correlated with ECG-determined ST height and PR interval. In summary, we have determined phenotypic ECG criteria to differentiate ErbB2tg from WT genotypes in 98.8% of mice. This inexpensive and time-efficient ECG-based phenotypic method might be applied to differentiate between genotypes in other rodent models of cardiac hypertrophy. Furthermore, with appropriate modifications, this method might be translated for use in other species. PMID:26310459

  17. Identification of UGT2B9*2 and UGT2B33 isolated from female rhesus monkey liver.

    PubMed

    Dean, Brian; Arison, Byron; Chang, Steve; Thomas, Paul E; King, Christopher

    2004-06-01

    Two UDP-glucuronosyltransferases (UGT2B9(*)2 and UGT2B33) have been isolated from female rhesus monkey liver. Microsomal preparations of the cell lines expressing the UGTs catalyzed the glucuronidation of the general substrate 7-hydroxy-4-(trifluoromethyl)coumarin in addition to selected estrogens (beta-estradiol and estriol) and opioids (morphine, naloxone, and naltrexone). UGT2B9(*)2 displayed highest efficiency for beta-estradiol-17-glucuronide production and did not catalyze the glucuronidation of naltrexone. UGT2B33 displayed highest efficiency for estriol and did not catalyze the glucuronidation of beta-estradiol. UGT2B9(*)2 was found also to catalyze the glucuronidation of 4-hydroxyestrone, 16-epiestriol, and hyodeoxycholic acid, while UGT2B33 was capable of conjugating 4-hydroxyestrone, androsterone, diclofenac, and hyodeoxycholic acid. Three glucocorticoids (cortisone, cortisol, and corticosterone) were not substrates for glucuronidation by liver or kidney microsomes or any expressed UGTs. Our current data suggest the use of beta-estradiol-3-glucuronidation, beta-estradiol-17-glucuronidation, and estriol-17-glucuronidation to assay UGT1A01, UGT2B9(*)2, and UGT2B33 activity in rhesus liver microsomes, respectively.

  18. Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai

    2012-02-21

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology ofmore » the ECD and a distinct mode of ligand recognition. Here we report a 1.9 {angstrom} crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.« less

  19. Kinin B1 Receptor Promotes Neurogenic Hypertension Through Activation of Centrally Mediated Mechanisms.

    PubMed

    Sriramula, Srinivas; Lazartigues, Eric

    2017-12-01

    Hypertension is associated with increased activity of the kallikrein-kinin system. Kinin B1 receptor (B1R) activation leads to vasoconstriction and inflammation. Despite evidence supporting a role for the B1R in blood pressure regulation, the mechanisms by which B1R could alter autonomic function and participate in the pathogenesis of hypertension remain unidentified. We sought to explore whether B1R-mediated inflammation contributes to hypertension and investigate the molecular mechanisms involved. In this study, we tested the hypothesis that activation of B1R in the brain is involved in the pathogenesis of hypertension, using the deoxycorticosterone acetate-salt model of neurogenic hypertension in wild-type and B1R knockout mice. Deoxycorticosterone acetate-salt treatment in wild-type mice led to significant increases in B1R mRNA and protein levels and bradykinin levels, enhanced gene expression of carboxypeptidase N supporting an increase in the B1R ligand, associated with enhanced blood pressure, inflammation, sympathoexcitation, autonomic dysfunction, and impaired baroreflex sensitivity, whereas these changes were blunted or prevented in B1R knockout mice. B1R stimulation was further shown to involve activation of the ASK1-JNK-ERK1/2 and NF-κB pathways in the brain. To dismiss potential developmental alterations in knockout mice, we further used B1R blockade selectively in the brain of wild-type mice. Supporting the central origin of this mechanism, intracerebroventricular infusion of a specific B1R antagonist, attenuated the deoxycorticosterone acetate-salt-induced increase in blood pressure in wild-type mice. Our data provide the first evidence of a central role for B1R-mediated inflammatory pathways in the pathogenesis of deoxycorticosterone acetate-salt hypertension and offer novel insights into possible B1R-targeted therapies for the treatment of neurogenic hypertension. © 2017 American Heart Association, Inc.

  20. Combustion synthesis of AlB2-Al2O3 composite powders with AlB2 nanowire structures

    NASA Astrophysics Data System (ADS)

    Yang, Pan; Xiao, Guoqing; Ding, Donghai; Ren, Yun; Yang, Shoulei; Lv, Lihua; Hou, Xing

    2018-05-01

    Using of Al and B2O3 powders as starting materials, and Mg-Al alloy as additives, AlB2-Al2O3 composite powders with AlB2 nanowire structures were successfully fabricated via combustion synthesis method in Ar atmosphere at a pressure of 1.5 MPa. The effect of different amount of Mg-Al alloy on the phase compositions and morphology of the combustion products was investigated. The results revealed that AlB2 and Al2O3 increased, whereas Al decreased with the content of Mg-Al alloy increasing. The impurities MgAl2O4 and AlB12 would exist in the sample with adding of 18 wt% Mg-Al alloy. Interestingly, FESEM/TEM/EDS results showed that AlB2 nanowires were observed in the products when the content of Mg-Al alloy is 6 wt% and 12 wt%. The more AlB2 nanowires can be found as the content of Mg-Al alloy increased. And the yield of AlB2 nanowires with the diameter of about 200 nanometers (nm) and the length up to several tens of micrometers (μm) in the combustion product is highest when the content of Mg-Al alloy is 12 wt%. The vapor, such as Mg-Al (g), B2O2 (g), AlO (g) and Al2O (g), produced during the process of combustion synthesis, reacted with each other to yield AlB2 nanowires by vapor-solid (VS) mechanism and the corresponding model was also proposed.

  1. Freud-2/CC2D1B mediates dual repression of the serotonin-1A receptor gene.

    PubMed

    Hadjighassem, Mahmoud R; Galaraga, Kimberly; Albert, Paul R

    2011-01-01

    The serotonin-1A (5-HT1A) receptor functions as a pre-synaptic autoreceptor in serotonin neurons that regulates their activity, and is also widely expressed on non-serotonergic neurons as a post-synaptic heteroreceptor to mediate serotonin action. The 5-HT1A receptor gene is strongly repressed by a dual repressor element (DRE), which is recognized by two proteins: Freud-1/CC2D1A and another unknown protein. Here we identify mouse Freud-2/CC2D1B as the second repressor of the 5-HT1A-DRE. Freud-2 shares 50% amino acid identity with Freud-1, and contains conserved structural domains. Mouse Freud-2 bound specifically to the rat 5-HT1A-DRE adjacent to, and partially overlapping, the Freud-1 binding site. By supershift assay using nuclear extracts from L6 myoblasts, Freud-2-DRE complexes were distinguished from Freud-1-DRE complexes. Freud-2 mRNA and protein were detected throughout mouse brain and peripheral tissues. Freud-2 repressed 5-HT1A promoter-reporter constructs in a DRE-dependent manner in non-neuronal (L6) or 5-HT1A-expressing neuronal (NG108-15, RN46A) cell models. In NG108-15 cells, knockdown of Freud-2 using a specific short-interfering RNA reduced endogenous Freud-2 protein levels and decreased Freud-2 bound to the 5-HT1A-DRE as detected by chromatin immunoprecipitation assay, but increased 5-HT1A promoter activity and 5-HT1A protein levels. Taken together, these data show that Freud-2 is the second component that, with Freud-1, mediates dual repression of the 5-HT1A receptor gene at the DRE. © 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  2. Immunohistochemical Localization of AT1a, AT1b, and AT2 Angiotensin II Receptor Subtypes in the Rat Adrenal, Pituitary, and Brain with a Perspective Commentary

    PubMed Central

    Premer, Courtney; Lamondin, Courtney; Mitzey, Ann; Speth, Robert C.; Brownfield, Mark S.

    2013-01-01

    Angiotensin II increases blood pressure and stimulates thirst and sodium appetite in the brain. It also stimulates secretion of aldosterone from the adrenal zona glomerulosa and epinephrine from the adrenal medulla. The rat has 3 subtypes of angiotensin II receptors: AT1a, AT1b, and AT2. mRNAs for all three subtypes occur in the adrenal and brain. To immunohistochemically differentiate these receptor subtypes, rabbits were immunized with C-terminal fragments of these subtypes to generate receptor subtype-specific antibodies. Immunofluorescence revealed AT1a and AT2 receptors in adrenal zona glomerulosa and medulla. AT1b immunofluorescence was present in the zona glomerulosa, but not the medulla. Ultrastructural immunogold labeling for the AT1a receptor in glomerulosa and medullary cells localized it to plasma membrane, endocytic vesicles, multivesicular bodies, and the nucleus. AT1b and AT2, but not AT1a, immunofluorescence was observed in the anterior pituitary. Stellate cells were AT1b positive while ovoid cells were AT2 positive. In the brain, neurons were AT1a, AT1b, and AT2 positive, but glia was only AT1b positive. Highest levels of AT1a, AT1b, and AT2 receptor immunofluorescence were in the subfornical organ, median eminence, area postrema, paraventricular nucleus, and solitary tract nucleus. These studies complement those employing different techniques to characterize Ang II receptors. PMID:23573410

  3. Evidence for the η(b)(2S) and observation of h(b)(1P)→η(b)(1S)γ and h(b)(2P)→η(b)(1S)γ.

    PubMed

    Mizuk, R; Asner, D M; Bondar, A; Pedlar, T K; Adachi, I; Aihara, H; Arinstein, K; Aulchenko, V; Aushev, T; Aziz, T; Bakich, A M; Bay, A; Belous, K; Bhardwaj, V; Bhuyan, B; Bischofberger, M; Bonvicini, G; Bozek, A; Bračko, M; Brodzicka, J; Browder, T E; Chekelian, V; Chen, A; Chen, P; Cheon, B G; Chilikin, K; Chistov, R; Cho, I-S; Cho, K; Choi, S-K; Choi, Y; Dalseno, J; Danilov, M; Doležal, Z; Drásal, Z; Drutskoy, A; Eidelman, S; Epifanov, D; Fast, J E; Gaur, V; Gabyshev, N; Garmash, A; Golob, B; Haba, J; Hara, T; Hayasaka, K; Hayashii, H; Horii, Y; Hoshi, Y; Hou, W-S; Hsiung, Y B; Hyun, H J; Iijima, T; Ishikawa, A; Itoh, R; Iwabuchi, M; Iwasaki, Y; Iwashita, T; Jaegle, I; Julius, T; Kang, J H; Kapusta, P; Kawasaki, T; Kim, H J; Kim, H O; Kim, J H; Kim, K T; Kim, M J; Kim, Y J; Kinoshita, K; Ko, B R; Koblitz, S; Kodyš, P; Korpar, S; Kouzes, R T; Križan, P; Krokovny, P; Kuhr, T; Kumita, T; Kuzmin, A; Kwon, Y-J; Lange, J S; Lee, S-H; Li, J; Libby, J; Liu, C; Liu, Y; Liu, Z Q; Liventsev, D; Louvot, R; Matvienko, D; McOnie, S; Miyabayashi, K; Miyata, H; Mohanty, G B; Mohapatra, D; Moll, A; Muramatsu, N; Mussa, R; Nakao, M; Natkaniec, Z; Ng, C; Nishida, S; Nishimura, K; Nitoh, O; Nozaki, T; Ohshima, T; Okuno, S; Olsen, S L; Onuki, Y; Pakhlov, P; Pakhlova, G; Park, C W; Park, H; Pestotnik, R; Petrič, M; Piilonen, L E; Poluektov, A; Röhrken, M; Sakai, Y; Sandilya, S; Santel, D; Sanuki, T; Sato, Y; Schneider, O; Schwanda, C; Senyo, K; Seon, O; Sevior, M E; Shapkin, M; Shen, C P; Shibata, T-A; Shiu, J-G; Shwartz, B; Sibidanov, A; Simon, F; Smerkol, P; Sohn, Y-S; Sokolov, A; Solovieva, E; Stanič, S; Starič, M; Sumihama, M; Sumiyoshi, T; Tanida, K; Tatishvili, G; Teramoto, Y; Tikhomirov, I; Trabelsi, K; Tsuboyama, T; Uchida, M; Uehara, S; Uglov, T; Unno, Y; Uno, S; Vanhoefer, P; Varner, G; Varvell, K E; Vinokurova, A; Vorobyev, V; Wang, C H; Wang, M-Z; Wang, P; Wang, X L; Watanabe, M; Watanabe, Y; Williams, K M; Won, E; Yabsley, B D; Yamaoka, J; Yamashita, Y; Yuan, C Z; Zhang, Z P; Zhilich, V

    2012-12-07

    We report the first evidence for the η(b)(2S) using the h(b)(2P)→η(b)(2S)γ transition and the first observation of the h(b)(1P)→η(b)(1S)γ and h(b)(2P)→η(b)(1S)γ transitions. The mass and width of the η(b)(1S) and η(b)(2S) are measured to be m(η(b)(1S))=(9402.4±1.5±1.8) MeV/c(2), m(η(b)(2S))=(9999.0±3.5(-1.9)(+2.8)) MeV/c(2), and Γ(η(b)(1S))=(10.8(-3.7-2.0)(+4.0+4.5)) MeV. We also update the h(b)(1P) and h(b)(2P) mass measurements. We use a 133.4 fb(-1) data sample collected at energies near the Υ(5S) resonance with the Belle detector at the KEKB asymmetric-energy e(+)e(-) collider.

  4. A systematic understanding of signaling by ErbB2 in cancer using phosphoproteomics.

    PubMed

    Sidhanth, C; Manasa, P; Krishnapriya, S; Sneha, S; Bindhya, S; Nagare, R P; Garg, M; Ganesan, T S

    2018-06-01

    ErbB2 is an important receptor tyrosine kinase and a member of the ErbB family. Although it does not have a specific ligand, it transmits signals downstream by heterodimerization with other receptors in the family. It plays a major role in a variety of cellular responses like proliferation, differentiation, and adhesion. ErbB2 is amplified at the DNA level in breast cancer (20%-30%) and gastric cancer (10%-20%), and trastuzumab is effective as a therapeutic antibody. This review is a critical analysis of the currently published data on the signaling pathways of ErbB2 and the interacting proteins. It also focuses on the techniques that are currently available to evaluate the entire phosphoproteome following activation of ErbB2. Identification of new and relevant phosphoproteins can not only serve as new therapeutic targets but also as a surrogate marker in patients to assess the activity of compounds that inhibit ErbB2. Overall, such analysis will improve understanding of signaling by ErbB2.

  5. Recognition of the Class Ib Molecule Qa-1b by Putative Activating Receptors Cd94/Nkg2c and Cd94/Nkg2e on Mouse Natural Killer Cells

    PubMed Central

    Vance, Russell E.; Jamieson, Amanda M.; Raulet, David H.

    1999-01-01

    The heterodimeric CD94/NKG2A receptor, expressed by mouse natural killer (NK) cells, transduces inhibitory signals upon recognition of its ligand, Qa-1b, a nonclassical major histocompatibility complex class Ib molecule. Here we clone and express two additional receptors, CD94/NKG2C and CD94/NKG2E, which we show also bind to Qa-1b. Within their extracellular carbohydrate recognition domains, NKG2C and NKG2E share extensive homology with NKG2A (93–95% amino acid similarity); however, NKG2C/E receptors differ from NKG2A in their cytoplasmic domains (only 33% similarity) and contain features that suggest that CD94/NKG2C and CD94/NKG2E may be activating receptors. We employ a novel blocking anti-NKG2 monoclonal antibody to provide the first direct evidence that CD94/NKG2 molecules are the only Qa-1b receptors on NK cells. Molecular analysis reveals that NKG2C and NKG2E messages are extensively alternatively spliced and ∼20-fold less abundant than NKG2A message in NK cells. The organization of the mouse Cd94/Nkg2 gene cluster, presented here, shows striking similarity with that of the human, arguing that the entire CD94/NKG2 receptor system is relatively primitive in origin. Analysis of synonymous substitution frequencies suggests that within a species, NKG2 genes may maintain similarities with each other by concerted evolution, possibly involving gene conversion–like events. These findings have implications for understanding NK cells and also raise new possibilities for the role of Qa-1 in immune responses. PMID:10601355

  6. Exploring neuroprotective potential of Withania somnifera phytochemicals by inhibition of GluN2B-containing NMDA receptors: An in silico study.

    PubMed

    Kumar, Gaurav; Patnaik, Ranjana

    2016-07-01

    N-methyl-d-aspartate receptors (NMDARs) mediated excitotoxicity has been implicated in multi-neurodegenerative diseases. Due to lack of efficacy and adverse effects of NMDA receptor antagonists, search for herbal remedies that may act as therapeutic agents is an active area of research to combat these diseases. Withania somnifera (WS) is being used for centuries as a nerve tonic and Nootropic agents. The present study targets the in silico evaluation of the neuroprotective efficacy of W. somnifera phytochemicals by inhibition of NMDA receptor-mediated excitotoxicity through allosteric inhibition of the GluN2B containing NMDARs. We predict Blood Brain Barrier (BBB) penetration, mutagenicity, drug-likeness and Human Intestinal Absorption properties of 25 WS phytochemicals. Further, molecular docking was performed to know whether these phytochemicals inhibit the GluN2B containing NMDARs or not. The results suggest that Anaferine, Beta-Sitosterol, Withaferin A, Withanolide A, Withanolide B and Withanolide D inhibit GluN2B containing NMDARs through allosteric mode similar to the well-known selective antagonist Ifenprodil. These phytochemicals have potential as an essentially useful oral drug to counter NMDARs mediated excitotoxicity and to treat multi-neurodegenerative diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Methadone pharmacogenetics: CYP2B6 polymorphisms determine plasma concentrations, clearance and metabolism

    PubMed Central

    Kharasch, Evan D.; Regina, Karen J.; Blood, Jane; Friedel, Christina

    2015-01-01

    Background Interindividual variability in methadone disposition remains unexplained, and methadone accidental overdose in pain therapy is a significant public health problem. Cytochrome P4502B6 (CYP2B6) is the principle determinant of clinical methadone elimination. The CYP2B6 gene is highly polymorphic, with several variant alleles. CYP2B6.6, the protein encoded by the CYP2B6*6 polymorphism, deficiently catalyzes methadone metabolism in vitro. This investigation determined the influence of CYP2B6*6, and other allelic variants encountered, on methadone concentrations, clearance, and metabolism. Methods Healthy volunteers in genotype cohorts CYP2B6*1/*1 (n=21), CYP2B6*1/*6 (n=20), and CYP2B6*6/*6 (n=17), and also CYP2B6*1/*4 (n=1), CYP2B6*4/*6 (n=3), CYP2B6*5/*5 (n=2) subjects received single doses of intravenous and oral methadone. Plasma and urine methadone and metabolite concentrations were determined by tandem mass spectrometry. Results Average S-methadone apparent oral clearance was 35 and 45% lower in CYP2B6*1/*6 and CYP2B6*6/*6 genotypes, respectively, compared with CYP2B6*1/*1, and R-methadone apparent oral clearance was 25 and 30% lower. R- and S-methadone apparent oral clearance was 3- and 4-fold greater in CYP2B6*4 carriers. Intravenous and oral R- and S-methadone metabolism was significantly lower in CYP2B6*6 carriers compared with CYP2B6*1 homozygotes, and greater in CYP2B6*4 carriers. Methadone metabolism and clearance were lower in African-Americans due to the CYP2B6*6 genetic polymorphism. Conclusions CYP2B6 polymorphisms influence methadone plasma concentrations, due to altered methadone metabolism and thus clearance. Genetic influence is greater for oral than intravenous, and S- than R-methadone. CYP2B6 pharmacogenetics explains, in part, interindividual variability in methadone elimination. CYP2B6 genetic effects on methadone metabolism and clearance may identify subjects at risk for methadone toxicity and drug interactions. PMID:26389554

  8. Silencing miR-106b accelerates osteogenesis of mesenchymal stem cells and rescues against glucocorticoid-induced osteoporosis by targeting BMP2.

    PubMed

    Liu, Ke; Jing, Ying; Zhang, Wen; Fu, Xuejie; Zhao, Huan; Zhou, Xichao; Tao, Yunxia; Yang, Huilin; Zhang, Yan; Zen, Ke; Zhang, Chenyu; Li, Donghai; Shi, Qin

    2017-04-01

    Osteoporosis is a serious health problem worldwide. MicroRNA is a post-transcriptional regulator of gene expression by either promoting mRNA degradation or interfering with mRNA translation of specific target genes. It plays a significant role in the pathogenesis of osteoporosis. Here, we first demonstrated that miR-106b (miR-106b-5p) negatively regulated osteogenic differentiation of mesenchymal stem cells in vitro. Then, we found that miR-106b expression increased in C57BL/6 mice with glucocorticoid-induced osteoporosis (GIOP), and that silencing of miR-106b signaling protected mice against GIOP through promoting bone formation and inhibiting bone resorption. At last, we showed that miR-106b inhibited osteoblastic differentiation and bone formation partly through directly targeting bone morphogenetic protein 2 (BMP2) both in vitro and in the GIOP model. Together, our findings have identified the role and mechanism of miR-106b in negatively regulating osteogenesis. Inhibition of miR-106b might be a potential new strategy for treating osteoporosis and bone defects. Copyright © 2017. Published by Elsevier Inc.

  9. Neurochemical Correlates of Accumbal Dopamine D2 and Amygdaloid 5-HT1B Receptor Densities on Observational Learning of Aggression

    PubMed Central

    Suzuki, Hideo; Lucas, Louis R.

    2015-01-01

    Social learning theory postulates that individuals learn to engage in aggressive behavior through observing an aggressive social model. Prior studies have shown that repeatedly observing aggression, also called “chronic passive exposure to aggression,” changes accumbal dopamine D2 receptor (D2R) and amygdaloid 5-HT1B receptor (5-HT1BR) densities in observers. But, the association between these outcomes remains unknown. Thus, our study used a rat paradigm to comprehensively examine the linkage between aggression, D2R density in the nucleus accumbens core (AcbC) and shell (AcbSh), and 5-HT1BR density in the medial (MeA), basomedial (BMA), and basolateral (BLA) amygdala following chronic passive exposure to aggression. Male Sprague-Dawley rats (N = 72) were passively exposed to either aggression or non-aggression acutely (1 day) or chronically (23 days). When observer rats were exposed to aggression chronically, they showed increased aggressive behavior and reduced D2R density in the bilateral AcbSh. On the other hand, exposure to aggression, regardless of exposure length, increased 5-HT1BR density in the bilateral BLA. Finally, low D2R in the AcbSh significantly interacted with high 5-HT1BR density in the BLA in predicting high levels of aggression in observer rats. Our results advance our understanding of the neurobiological mechanisms for observational learning of aggression, highlighting that dopamine-serotonin interaction, or AcbSh-BLA interaction, may contribute to a risk factor for aggression in observers who chronically witness aggressive interactions. PMID:25650085

  10. Deoxycholic Acid-Mediated Sphingosine-1-Phosphate Receptor 2 Signaling Exacerbates DSS-Induced Colitis through Promoting Cathepsin B Release.

    PubMed

    Zhao, Shengnan; Gong, Zizhen; Du, Xixi; Tian, Chunyan; Wang, Lingyu; Zhou, Jiefei; Xu, Congfeng; Chen, Yingwei; Cai, Wei; Wu, Jin

    2018-01-01

    We recently have proved that excessive fecal DCA caused by high-fat diet may serve as an endogenous danger-associated molecular pattern to activate NLRP3 inflammasome and thus contributes to the development of inflammatory bowel disease (IBD). Moreover, the effect of DCA on inflammasome activation is mainly mediated through bile acid receptor sphingosine-1-phosphate receptor 2 (S1PR2); however, the intermediate process remains unclear. Here, we sought to explore the detailed molecular mechanism involved and examine the effect of S1PR2 blockage in a colitis mouse model. In this study, we found that DCA could dose dependently upregulate S1PR2 expression. Meanwhile, DCA-induced NLRP3 inflammasome activation is at least partially achieved through stimulating extracellular regulated protein kinases (ERK) signaling pathway downstream of S1PR2 followed by promoting of lysosomal cathepsin B release. DCA enema significantly aggravated DSS-induced colitis in mice and S1PR2 inhibitor as well as inflammasome inhibition by cathepsin B antagonist substantially reducing the mature IL-1 β production and alleviated colonic inflammation superimposed by DCA. Therefore, our findings suggest that S1PR2/ERK1/2/cathepsin B signaling plays a critical role in triggering inflammasome activation by DCA and S1PR2 may represent a new potential therapeutic target for the management of intestinal inflammation in individuals on a high-fat diet.

  11. Design and Synthesis of a Series of l-trans-4-Substituted Prolines as Selective Antagonists for the Ionotropic Glutamate Receptors Including Functional and X-ray Crystallographic Studies of New Subtype Selective Kainic Acid Receptor Subtype 1 (GluK1) Antagonist (2S,4R)-4-(2-Carboxyphenoxy)pyrrolidine-2-carboxylic Acid.

    PubMed

    Krogsgaard-Larsen, Niels; Delgar, Claudia G; Koch, Karina; Brown, Patricia M G E; Møller, Charlotte; Han, Liwei; Huynh, Tri H V; Hansen, Stinne W; Nielsen, Birgitte; Bowie, Derek; Pickering, Darryl S; Kastrup, Jette Sandholm; Frydenvang, Karla; Bunch, Lennart

    2017-01-12

    Ionotropic glutamate receptor antagonists are valuable tool compounds for studies of neurological pathways in the central nervous system. On the basis of rational ligand design, a new class of selective antagonists, represented by (2S,4R)-4-(2-carboxyphenoxy)pyrrolidine-2-carboxylic acid (1b), for cloned homomeric kainic acid receptors subtype 1 (GluK1) was attained (K i = 4 μM). In a functional assay, 1b displayed full antagonist activity with IC 50 = 6 ± 2 μM. A crystal structure was obtained of 1b when bound in the ligand binding domain of GluK1. A domain opening of 13-14° was seen compared to the structure with glutamate, consistent with 1b being an antagonist. A structure-activity relationship study showed that the chemical nature of the tethering atom (C, O, or S) linking the pyrrolidine ring and the phenyl ring plays a key role in the receptor selectivity profile and that substituents on the phenyl ring are well accommodated by the GluK1 receptor.

  12. An Empirical Examination of Reverse Auction Appropriateness in B2B Source Selection

    DTIC Science & Technology

    2006-01-01

    Measures of Marketing Constructs," Journal of Marketing Research , Vol. 16, No.2, pp. 64 - 73. Cohn, L. (2000), "B2B: The Hottest Net Bet Yet...34 Psycholgical Bulletin, Vol. 103, No.3, pp. 411-423. Armstrong, J.S. and T.S. Overton (1977), "Estimating Nonresponse Bias in Mail Surveys," Journal of Marketing ... Research , Vol.14, No.3, pp. 396-402. Bagozzi, R. (1983), "Issues in the Application of Covariance Structure Analysis: A Further Comment," Journal of

  13. Overexpression of human NR2B receptor subunit in LMAN causes stuttering and song sequence changes in adult zebra finches.

    PubMed

    Chakraborty, Mukta; Chen, Liang-Fu; Fridel, Emma E; Klein, Marguerita E; Senft, Rebecca A; Sarkar, Abhra; Jarvis, Erich D

    2017-04-21

    Zebra finches (Taeniopygia guttata) learn to produce songs in a manner reminiscent of spoken language development in humans. One candidate gene implicated in influencing learning is the N-methyl-D-aspartate (NMDA) subtype 2B glutamate receptor (NR2B). Consistent with this idea, NR2B levels are high in the song learning nucleus LMAN (lateral magnocellular nucleus of the anterior nidopallium) during juvenile vocal learning, and decreases to low levels in adults after learning is complete and the song becomes more stereotyped. To test for the role of NR2B in generating song plasticity, we manipulated NR2B expression in LMAN of adult male zebra finches by increasing its protein levels to those found in juvenile birds, using a lentivirus containing the full-length coding sequence of the human NR2B subunit. We found that increased NR2B expression in adult LMAN induced increases in song sequence diversity and slower song tempo more similar to juvenile songs, but also increased syllable repetitions similar to stuttering. We did not observe these effects in control birds with overexpression of NR2B outside of LMAN or with the green fluorescent protein (GFP) in LMAN. Our results suggest that low NR2B subunit expression in adult LMAN is important in conserving features of stereotyped adult courtship song.

  14. B cell receptor editing in tolerance and autoimmunity

    PubMed Central

    Luning Prak, Eline T.; Monestier, Marc; Eisenberg, Robert A.

    2010-01-01

    Receptor editing is the process of ongoing antibody gene rearrangement in a lymphocyte that already has a functional antigen receptor. The expression of a functional antigen receptor will normally terminate further rearrangement (allelic exclusion). However, lymphocytes with autoreactive receptors have a chance at escaping negative regulation by “editing” the specificities of their receptors with additional antibody gene rearrangements. Nemazee points out, “receptor editing separates receptor selection from cellular selection.”1 As such, editing complicates the Clonal Selection Hypothesis, because edited cells are not simply endowed for life with a single, invariant antigen receptor.2 For example, an edited B cell changes the specificity of its B cell receptor (BCR), and if the initial immunoglobulin gene is not inactivated during the editing process, allelic exclusion is violated, and the B cell can exhibit two specificities. Here we will describe the discovery of editing, the pathways of receptor editing at the heavy (H) and light (L) chain loci, and current evidence regarding how and where editing happens and what effects it has on the antibody repertoire. PMID:21251012

  15. Differential expression of miR-34b and androgen receptor pathway regulate prostate cancer aggressiveness between African-Americans and Caucasians

    PubMed Central

    Kato, Taku; Yamamura, Soichiro; Tanaka, Yuichiro; Majid, Shahana; Saini, Sharanjot; Varahram, Shahryari; Kulkarni, Priyanka; Dasgupta, Pritha; Mitsui, Yozo; Sumida, Mitsuho; Tabatabai, Laura; Deng, Guoren; Kumar, Deepak; Dahiya, Rajvir

    2017-01-01

    African-Americans are diagnosed with more aggressive prostate cancers and have worse survival than Caucasians, however a comprehensive understanding of this health disparity remains unclear. To clarify the mechanisms leading to this disparity, we analyzed the potential involvement of miR-34b expression in African-Americans and Caucasians. miR-34b functions as a tumor suppressor and has a multi-functional role, through regulation of cell proliferation, cell cycle and apoptosis. We found that miR-34b expression is lower in human prostate cancer tissues from African-Americans compared to Caucasians. DNA hypermethylation of the miR-34b-3p promoter region showed significantly higher methylation in prostate cancer compared to normal samples. We then sequenced the promoter region of miR-34b-3p and found a chromosomal deletion in miR-34b in African-American prostate cancer cell line (MDA-PCA-2b) and not in Caucasian cell line (DU-145). We found that AR and ETV1 genes are differentially expressed in MDA-PCa-2b and DU-145 cells after overexpression of miR-34b. Direct interaction of miR-34b with the 3’ untranslated region of AR and ETV1 was validated by luciferase reporter assay. We found that miR-34b downregulation in African-Americans is inversely correlated with high AR levels that lead to increased cell proliferation. Overexpression of miR-34b in cell lines showed higher inhibition of cell proliferation, apoptosis and G1 arrest in the African-American cells (MDA-PCa-2b) compared to Caucasian cell line (DU-145). Taken together, our results show that differential expression of miR-34b and AR are associated with prostate cancer aggressiveness in African-Americans. PMID:28039468

  16. Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling

    PubMed Central

    Kimura, Yuriko; Fujita, Yuki; Shibata, Kumi; Mori, Megumi; Yamashita, Toshihide

    2013-01-01

    Sigma-1 receptor (Sig-1R) is an integral membrane protein predominantly expressed in the endoplasmic reticulum. Sig-1R demonstrates a high affinity to various synthetic compounds including well-known psychotherapeutic drugs in the central nervous system (CNS). For that, it is considered as an alternative target for psychotherapeutic drugs. On the cellular level, when Sig-1R is activated, it is known to play a role in neuroprotection and neurite elongation. These effects are suggested to be mediated by its ligand-operated molecular chaperone activity, and/or upregulation of various Ca2+ signaling. In addition, recent studies show that Sig-1R activation induces neurite outgrowth via neurotrophin signaling. Here, we tested the hypothesis that Sig-1R activation promotes neurite elongation through activation of tropomyosin receptor kinase (Trk), a family of neurotrophin receptors. We found that 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (PRE-084), a selective Sig-1R agonist, significantly promoted neurite outgrowth, and K252a, a Trk inhibitor, attenuated Sig-1R-mediated neurite elongation in cerebellar granule neurons (CGNs). Moreover, we revealed that Sig-1R interacts with TrkB, and PRE-084 treatment enhances phosphorylation of Y515, but not Y706. Thus, our results indicate that Sig-1R activation promotes neurite outgrowth in CGNs through Y515 phosphorylation of TrkB. PMID:24116072

  17. Sigma-1 receptor enhances neurite elongation of cerebellar granule neurons via TrkB signaling.

    PubMed

    Kimura, Yuriko; Fujita, Yuki; Shibata, Kumi; Mori, Megumi; Yamashita, Toshihide

    2013-01-01

    Sigma-1 receptor (Sig-1R) is an integral membrane protein predominantly expressed in the endoplasmic reticulum. Sig-1R demonstrates a high affinity to various synthetic compounds including well-known psychotherapeutic drugs in the central nervous system (CNS). For that, it is considered as an alternative target for psychotherapeutic drugs. On the cellular level, when Sig-1R is activated, it is known to play a role in neuroprotection and neurite elongation. These effects are suggested to be mediated by its ligand-operated molecular chaperone activity, and/or upregulation of various Ca(2+) signaling. In addition, recent studies show that Sig-1R activation induces neurite outgrowth via neurotrophin signaling. Here, we tested the hypothesis that Sig-1R activation promotes neurite elongation through activation of tropomyosin receptor kinase (Trk), a family of neurotrophin receptors. We found that 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (PRE-084), a selective Sig-1R agonist, significantly promoted neurite outgrowth, and K252a, a Trk inhibitor, attenuated Sig-1R-mediated neurite elongation in cerebellar granule neurons (CGNs). Moreover, we revealed that Sig-1R interacts with TrkB, and PRE-084 treatment enhances phosphorylation of Y515, but not Y706. Thus, our results indicate that Sig-1R activation promotes neurite outgrowth in CGNs through Y515 phosphorylation of TrkB.

  18. Group 4 metal mono-dicarbollide piano stool complexes. Synthesis, structure, and reactivity of ({eta}{sup 5}-C{sub 2}B{sub 9}H{sub 11})M(NR{sub 2}){sub 2}(NHR{sub 2}) (M = Zr, R = Et; M = Ti, R = Me, Et)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bowen, D.E.; Jordan, R.F.; Rogers, R.D.

    1995-08-01

    The amine elimination reaction of C{sub 2}B{sub 9}H{sub 13} and Zr(NEt{sub 2}){sub 4} yields the mono-dicarbollide complex ({eta}{sup 5}-C{sub 2}B{sub 9}H{sub 11})Zr(NEt{sub 2}){sub 2}(NHEt{sub 2}), (1), which has been shown to adopt a three-legged piano stool structure by X-ray crystallography. Crystal data for 1: space group P2{sub 1}/c, a = 10.704(4) A, b = 11.066(3) A, c = 20.382(8) A, {beta} = 99.20(3){degree}, V = 2383(1) A{sup 3}, Z = 4. Complex 1 undergoes facile ligand substitution by THF and 4-picoline, yielding ({eta}{sup 5}-C{sub 2}B{sub 9}H{sub 11})Zr(NEt{sub 2}){sub 2}-(THF) (2) and ({eta}{sup 5}-C{sub 2}B{sub 9}H{sub 11})Zr(NEt{sub 2}){sub 2}(4-picoline){sub 2} (3).more » Compound 3 exists as the four-coordinate species ({eta}{sup 5}-C{sub 2}B{sub 9}H{sub 11})Zr(NEt{sub 2}){sub 2}(4-picoline) in CH{sub 2}Cl{sub 2} solution. Complex 1 reacts selectively with 2 equiv of [NH{sub 2}ET{sub 2}]Cl, yielding ({eta}{sup 5}-C{sub 2}B{sub 9}H{sub 11})ZrCl{sub 2}(NHEt{sub 2}){sub 2} (4). Similarly, the reaction of C{sub 2}B{sub 9}H{sub 13} and Ti(NR{sub 2}){sub 4} yields ({eta}{sup 5}-C{sub 2}B{sub 9}H{sub 11})Ti(NR{sub 2}){sub 2}(NHR{sub 2}) (5, R = Me; 6, R = Et). Compounds 1-6 are potential precursors to group 4 metal ({eta}{sup 5}-C{sub 2}B{sub 9}H{sub 11})MR{sub 2}L{sub n} alkyl species. 25 refs., 3 figs., 3 tabs.« less

  19. Phase and Physicochemical Properties Diagrams of Quaternary System Li2B4O7 + Na2B4O7 + Mg2B6O11 + H2O

    NASA Astrophysics Data System (ADS)

    Wang, Shi-qiang; Du, Xue-min; Jing, Yan; Guo, Ya-fei; Deng, Tian-long

    2017-12-01

    The phase and physicochemical properties diagrams of the quaternary system (Li2B4O7 + Na2B4O7 + Mg2B6O11) at 288.15 K and 0.1 MPa were constructed using the solubilities, densities, and refractive indices measured. In the phase diagrams of the system there are one invariant point, three univariant isothermic dissolution curves, and three crystallization regions corresponding to Li2B4O7 · 3H2O, Na2B4O7 · 10H2O, and Mg2B6O11 · 15H2O, respectively. The solution density, refractive index of the quaternary system changes regularly with the increasing of Li2B4O7 concentration. The calculated values of density and refractive index using empirical equations of the quaternary system are in good agreement with the experimental values.

  20. Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia

    PubMed Central

    Silva, Ricardo Jorge; Cruz, Ana Rita; Mano, Miguel

    2017-01-01

    MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells. PMID:28394930

  1. Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia.

    PubMed

    Sunkavalli, Ushasree; Aguilar, Carmen; Silva, Ricardo Jorge; Sharan, Malvika; Cruz, Ana Rita; Tawk, Caroline; Maudet, Claire; Mano, Miguel; Eulalio, Ana

    2017-04-01

    MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells.

  2. Ecdysone receptor isoform-B mediates soluble trehalase expression to regulate growth and development in the mirid bug, Apolygus lucorum (Meyer-Dür).

    PubMed

    Tan, Y-A; Xiao, L-B; Zhao, J; Xiao, Y-F; Sun, Y; Bai, L-X

    2015-12-01

    Ecdysone receptor (EcR) is the hormonal receptor of ecdysteroids and strictly regulates growth and development in insects. However, the action mechanism of EcR is not very clear. In this study, the cDNA of EcR isoform-B was cloned from Apolygus lucorum (AlEcR-B) and its expression profile was investigated. We reduced AlEcR-B mRNA expression using systemic RNA interference in vivo, and obtained knockdown specimens. Examination of these specimens indicated that AlEcR-B is required for nymphal survival, and that reduced expression is associated with longer development time and lower nymphal weight. To investigate the underlying molecular mechanism of the observed suppression effects, we selected trehalase for a detailed study. Transcript encoding soluble trehalase (AlTre-1) was up-regulated by 20-hydroxyecdysone and in agreement with the mRNA expression of AlEcR-B. The expression profile of AlTre-1, soluble trehalase activity and translated protein level in the midgut of surviving nymphs were down-regulated, compared with controls, after the knockdown expression of AlEcR-B. By contrast, membrane-bound trehalase activity, the related gene expression and translated protein level remained at their initial levels. However, trehalose content significantly increased and the glucose content significantly decreased under the same conditions. We propose that AlEcR-B controls normal carbohydrate metabolism by mediating the expression of AlTre-1 to regulate the growth and development in A. lucorum, which provide an extended information into the functions of AlEcR-B. © 2015 The Royal Entomological Society.

  3. Prostaglandin E2 regulates B cell proliferation through a candidate tumor suppressor, Ptger4.

    PubMed

    Murn, Jernej; Alibert, Olivier; Wu, Ning; Tendil, Simon; Gidrol, Xavier

    2008-12-22

    B cell receptor (BCR) signaling contributes to the pathogenesis of B cell malignancies, and most B cell lymphomas depend on BCR signals for survival. Identification of genes that restrain BCR-mediated proliferation is therefore an important goal toward improving the therapy of B cell lymphoma. Here, we identify Ptger4 as a negative feedback regulator of proliferation in response to BCR signals and show that its encoded EP4 receptor is a principal molecule conveying the growth-suppressive effect of prostaglandin E2 (PGE2). Stable knockdown of Ptger4 in B cell lymphoma markedly accelerated tumor spread in mice, whereas Ptger4 overexpression yielded significant protection. Mechanistically, we show that the intrinsic activity of Ptger4 and PGE2-EP4 signaling target a similar set of activating genes, and find Ptger4 to be significantly down-regulated in human B cell lymphoma. We postulate that Ptger4 functions in B cells as a candidate tumor suppressor whose activity is regulated by PGE2 in the microenvironment. These findings suggest that targeting EP4 receptor for prostaglandin may present a novel strategy for treatment of B cell malignancies.

  4. Prostaglandin E2 regulates B cell proliferation through a candidate tumor suppressor, Ptger4

    PubMed Central

    Murn, Jernej; Alibert, Olivier; Wu, Ning; Tendil, Simon; Gidrol, Xavier

    2008-01-01

    B cell receptor (BCR) signaling contributes to the pathogenesis of B cell malignancies, and most B cell lymphomas depend on BCR signals for survival. Identification of genes that restrain BCR-mediated proliferation is therefore an important goal toward improving the therapy of B cell lymphoma. Here, we identify Ptger4 as a negative feedback regulator of proliferation in response to BCR signals and show that its encoded EP4 receptor is a principal molecule conveying the growth-suppressive effect of prostaglandin E2 (PGE2). Stable knockdown of Ptger4 in B cell lymphoma markedly accelerated tumor spread in mice, whereas Ptger4 overexpression yielded significant protection. Mechanistically, we show that the intrinsic activity of Ptger4 and PGE2–EP4 signaling target a similar set of activating genes, and find Ptger4 to be significantly down-regulated in human B cell lymphoma. We postulate that Ptger4 functions in B cells as a candidate tumor suppressor whose activity is regulated by PGE2 in the microenvironment. These findings suggest that targeting EP4 receptor for prostaglandin may present a novel strategy for treatment of B cell malignancies. PMID:19075289

  5. The role of complement receptor positive and complement receptor negative B cells in the primary and secondary immune response to thymus independent type 2 and thymus dependent antigens.

    PubMed

    Lindsten, T; Yaffe, L J; Thompson, C B; Guelde, G; Berning, A; Scher, I; Kenny, J J

    1985-05-01

    Both complement receptor positive (CR+) and complement receptor negative (CR-) B cells have been shown to be involved in the primary immune response to PC-Hy (phosphocholine conjugated hemocyanin), a thymus dependent (TD) antigen which preferentially induces antibody secretion in Lyb-5+ B cells during a primary adoptive transfer assay. CR+ and CR- B cells also responded in a primary adoptive transfer assay to TNP-Ficoll, a thymus independent type 2 (TI-2) antigen which activates only Lyb-5+ B cells. When the secondary immune response to PC-Hy and TNP-Ficoll were analyzed, it was found that most of the immune memory to both antigens was present in the CR- B cell subset. The CR- B cell subset also dominated the secondary immune response to PC-Hy in immune defective (CBA/N X DBA/2N)F1 male mice. These data indicate that CR- B cells dominate the memory response in both the Lyb-5+ and Lyb-5- B cell subsets of normal and xid immune defective mice and suggest that Lyb-5+ and Lyb-5- B cells can be subdivided into CR+ and CR- subsets.

  6. Periaqueductal gray knockdown of V2, not V1a and V1b receptor influences nociception in the rat. yj6676@yahoo.com.

    PubMed

    Yang, Jun; Yang, Yu; Chen, Jian-Min; Wang, Gen; Xu, Hong-Tao; Liu, Wen-Yan; Lin, Bao-Cheng

    2007-01-01

    Our pervious study has proved that arginine vasopressin (AVP) in periaqueductal gray (PAG) plays a role in antinociception. After establishing a model of local special gene knockdown, the nociceptive effect of vasopressin receptor subunit in PAG was investigated in the rat. Microinjection of short-interfering RNA (siRNA) into PAG, which targeted vasopressin receptor subtypes (V(1a), V(1b) and V(2)), locally weakened the associated mRNA expression and depressed the related receptor synthesis in a dose-dependent manner, in which the significant inhibitive effect occurred on from 7th day to 14th day following 1microg or 2microg siRNA administration. PAG knockdown of V(2) receptor gene markedly decreased pain threshold in from 6th day to 13th day after siRNA administration, whereas local knockdown of either V(1a) or V(1b) receptor gene could not influence pain threshold. The data suggest that V(2) rather than V(1a) and V(1b) receptor in PAG involves in nociception.

  7. Contribution of CYP2B6 alleles in explaining extreme (S)-methadone plasma levels: a CYP2B6 gene resequencing study.

    PubMed

    Dobrinas, Maria; Crettol, Séverine; Oneda, Beatrice; Lahyani, Rachel; Rotger, Margalida; Choong, Eva; Lubomirov, Rubin; Csajka, Chantal; Eap, Chin B

    2013-02-01

    (S)-Methadone, metabolized mainly by CYP2B6, shows a wide interindividual variability in its pharmacokinetics and pharmacodynamics. Resequencing of the CYP2B6 gene was performed in 12 and 35 selected individuals with high (S)-methadone plasma exposure and low (S)-methadone plasma exposure, respectively, from a previously described cohort of 276 patients undergoing methadone maintenance treatment. Selected genetic polymorphisms were then analyzed in the complete cohort. The rs35303484 (*11; c136A>G; M46V) polymorphism was overrepresented in the high (S)-methadone level group, whereas the rs3745274 (*9; c516G>T; Q172H), rs2279344 (c822+183G>A), and rs8192719 (c1294+53C>T) polymorphisms were underrepresented in the low (S)-methadone level group, suggesting an association with decreased CYP2B6 activity. Conversely, the rs3211371 (*5; c1459C>T; R487C) polymorphism was overrepresented in the low-level group, indicating an increased CYP2B6 activity. A higher allele frequency was found in the high-level group compared with the low-level group for rs3745274 (*9; c516G>T; Q172H), rs2279343 (*4; c785A>G; K262R) (together representing CYP2B6*6), rs8192719 (c1294+53C>T), and rs2279344 (c822+183G>A), suggesting their involvement in decreased CYP2B6 activity. These results should be replicated in larger independent cohorts. Known genetic polymorphisms in CYP2B6 contribute toward explaining extreme (S)-methadone plasma levels observed in a cohort of patients following methadone maintenance treatment.

  8. An Investigation of Hierachical Protein Recruitment to the Inhibitory Platelet Receptor, G6B-b

    PubMed Central

    Coxon, Carmen H.; Sadler, Amanda J.; Huo, Jiandong; Campbell, R. Duncan

    2012-01-01

    Platelet activation is regulated by both positive and negative signals. G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC50 for both CRP and collagen. PMID:23185356

  9. Antagonism at the NR2B subunit of NMDA receptors induces increased connectivity of the prefrontal and subcortical regions regulating reward behavior.

    PubMed

    Gass, Natalia; Becker, Robert; Sack, Markus; Schwarz, Adam J; Reinwald, Jonathan; Cosa-Linan, Alejandro; Zheng, Lei; von Hohenberg, Christian Clemm; Inta, Dragos; Meyer-Lindenberg, Andreas; Weber-Fahr, Wolfgang; Gass, Peter; Sartorius, Alexander

    2018-04-01

    Evidence indicates that ketamine's rapid antidepressant efficacy likely results from its antagonism of NR2B-subunit-containing NMDA receptors (NMDAR). Since ketamine equally blocks NR2A- and NR2B-containing NMDAR, and has affinity to other receptors, NR2B-selective drugs might have improved therapeutic efficiency and side effect profile. We aimed to compare the effects of (S)-ketamine and two different types of NR2B-selective antagonists on functional brain networks in rats, in order to find common circuits, where their effects intersect, and that might explain their antidepressant action. The experimental design comprised four parallel groups of rats (N = 37), each receiving (S)-Ketamine, CP-101,606, Ro 25-6981 or saline. After compound injection, we acquired resting-state functional magnetic resonance imaging time series. We used graph theoretical approach to calculate brain network properties. Ketamine and CP-101,606 diminished the global clustering coefficient and small-worldness index. At the nodal level, all compounds induced increased connectivity of the regions mediating reward and cognitive aspects of emotional processing, such as ventromedial prefrontal cortex, septal nuclei, and nucleus accumbens. The dorsal hippocampus and regions involved in sensory processing and aversion, such as superior and inferior colliculi, exhibited an opposite effect. The effects common to ketamine and NR2B-selective compounds were localized to the same brain regions as those reported in depression, but in the opposite direction. The upregulation of the reward circuitry might partially underlie the antidepressant and anti-anhedonic effects of the antagonists and could potentially serve as a translational imaging phenotype for testing putative antidepressants, especially those targeting the NR2B receptor subtype.

  10. Deliberate and Crisis Action Planning and Execution Segments Increment 2B (DCAPES Inc 2B)

    DTIC Science & Technology

    2016-03-01

    2016 Major Automated Information System Annual Report Deliberate and Crisis Action Planning and Execution Segments Increment 2B (DCAPES Inc 2B...Defense Acquisition Management Information Retrieval (DAMIR) UNCLASSIFIED DCAPES Inc 2B 2016 MAR UNCLASSIFIED 2 Table of Contents Common...Logistics DCAPES Inc 2B 2016 MAR UNCLASSIFIED 3 Lt Col Christopher Thrower 201 East Moore Drive Building 856, Room 154 Maxwell Air Force Base-Gunter

  11. Role of PTP1B in HER2 Signaling in Breast Cancer

    DTIC Science & Technology

    2012-10-01

    AD_________________ Award Number: W81XWH-10-1-1005 TITLE: Role of PTP1B in HER2 Signaling in...AND SUBTITLE 5a. CONTRACT NUMBER Role of PTP1B in HER2 Signaling in Breast Cancer 5b. GRANT NUMBER W81XWH-10-1-1005 5c. PROGRAM ELEMENT NUMBER...2009, Postdoctoral Fellowship Award W81XWH-10-1-1005, “Role of PTP1B in HER2 Signaling in Breast Cancer" INTRODUCTION  The receptor tyrosine

  12. Cholinergic chemosensory cells of the thymic medulla express the bitter receptor Tas2r131.

    PubMed

    Soultanova, Aichurek; Voigt, Anja; Chubanov, Vladimir; Gudermann, Thomas; Meyerhof, Wolfgang; Boehm, Ulrich; Kummer, Wolfgang

    2015-11-01

    The thymus is the site of T cell maturation which includes positive selection in the cortex and negative selection in the medulla. Acetylcholine is locally produced in the thymus and cholinergic signaling influences the T cell development. We recently described a distinct subset of medullary epithelial cells in the murine thymus which express the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT) and components of the canonical taste transduction cascade, i.e. transient receptor potential melastatin-like subtype 5 channel (TRPM5), phospholipase Cβ(2), and Gα-gustducin. Such a chemical phenotype is characteristic for chemosensory cells of mucosal surfaces which utilize bitter receptors for detection of potentially hazardous compounds and cholinergic signaling to initiate avoidance reflexes. We here demonstrate mRNA expression of bitter receptors Tas2r105, Tas2r108, and Tas2r131 in the murine thymus. Using a Tas2r131-tauGFP reporter mouse we localized the expression of this receptor to cholinergic cells expressing the downstream elements of the taste transduction pathway. These cells are distinct from the medullary thymic epithelial cells which promiscuously express tissue-restricted self-antigens during the process of negative selection, since double-labeling immunofluorescence showed no colocalization of autoimmune regulator (AIRE), the key mediator of negative selection, and TRPM5. These data demonstrate the presence of bitter taste-sensing signaling in cholinergic epithelial cells in the thymic medulla and opens a discussion as to what is the physiological role of this pathway. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Experiences and Future Expectations towards Online Courses--An Empirical Study of the B2C-and B2B-Segments

    ERIC Educational Resources Information Center

    Krämer, Andreas; Böhrs, Sandra

    2016-01-01

    This article explores the future potential for the development of online courses. The findings are based on an empirical study with 3 sample groups: (1) B2C segment in Germany, (2) B2C segment in the United States, and (3) B2B segment (international). In the first step the status quo of the use of e-learning in general and online courses in…

  14. B cell IFN-γ receptor signaling promotes autoimmune germinal centers via cell-intrinsic induction of BCL-6

    PubMed Central

    Jackson, Shaun W.; Jacobs, Holly M.; Arkatkar, Tanvi; Dam, Elizabeth M.; Scharping, Nicole E.; Kolhatkar, Nikita S.; Hou, Baidong; Buckner, Jane H.

    2016-01-01

    Dysregulated germinal center (GC) responses are implicated in the pathogenesis of human autoimmune diseases, including systemic lupus erythematosus (SLE). Although both type 1 and type 2 interferons (IFNs) are involved in lupus pathogenesis, their respective impacts on the establishment of autoimmune GCs has not been addressed. In this study, using a chimeric model of B cell-driven autoimmunity, we demonstrate that B cell type 1 IFN receptor signals accelerate, but are not required for, lupus development. In contrast, B cells functioning as antigen-presenting cells initiate CD4+ T cell activation and IFN-γ production, and strikingly, B cell–intrinsic deletion of the IFN-γ receptor (IFN-γR) abrogates autoimmune GCs, class-switched autoantibodies (auto-Abs), and systemic autoimmunity. Mechanistically, although IFN-γR signals increase B cell T-bet expression, B cell–intrinsic deletion of T-bet exerts an isolated impact on class-switch recombination to pathogenic auto-Ab subclasses without impacting GC development. Rather, in both mouse and human B cells, IFN-γ synergized with B cell receptor, toll-like receptor, and/or CD40 activation signals to promote cell-intrinsic expression of the GC master transcription factor, B cell lymphoma 6 protein. Our combined findings identify a novel B cell–intrinsic mechanism whereby IFN signals promote lupus pathogenesis, implicating this pathway as a potential therapeutic target in SLE. PMID:27069113

  15. Characterization of B61, the ligand for the Eck receptor protein-tyrosine kinase.

    PubMed

    Shao, H; Pandey, A; O'Shea, K S; Seldin, M; Dixit, V M

    1995-03-10

    B61 was originally described as a novel secreted tumor necrosis factor-alpha-inducible gene product in endothelial cells (Holzman, L. B., Marks, R. M., and Dixit, V. M. (1990) Mol. Cell. Biol. 10, 5830-5838). It was recently discovered that soluble recombinant B61 could serve as a ligand for the Eck receptor protein-tyrosine kinase, a member of the Eph/Eck subfamily of receptor protein-tyrosine kinases (Bartley, T.D., Hunt, R. W., Welcher, A. A., Boyle, W. J., Parker, V. P., Lindberg, R. A., Lu, H. S., Colombero, A. M., Elliott, R. L., Guthrie, R. A., Holst, P. L., Skrine, J. D., Toso, R. J., Zhang, M., Fernandez, E., Trail, G., Yarnum, B., Yarden, Y., Hunter, T., and Fox, G. M. (1994) Nature 368, 558-560). We now show that B61 can also exist as a cell surface glycosylphosphatidyl-inositol-linked protein that is capable of activating the Eck receptor protein-tyrosine kinase, the first such report of a receptor protein-tyrosine kinase ligand that is glycosylphosphatidylinositol-linked. In addition, the expression patterns of B61 and Eck during mouse ontogeny were determined by in situ hybridization. Both were found to be highly expressed in the developing lung and gut, while Eck was preferentially expressed in the thymus. Finally, the gene for B61 was localized to a specific position on mouse chromosome 3 by interspecific back-cross analysis.

  16. Intrinsic fluorescence spectra characteristics of vitamin B1, B2, and B6

    NASA Astrophysics Data System (ADS)

    Yang, Hui; Xiao, Xue; Zhao, Xuesong; Hu, Lan; Lv, Caofang; Yin, Zhangkun

    2015-11-01

    This paper presents the intrinsic fluorescence characteristics of vitamin B1, B2 and B6 measured with 3D fluorescence Spectrophotometer. Three strong fluorescence areas of vitamin B2 locate at λex/λem=270/525nm, 370/525nm and 450/525nm, one fluorescence areas of vitamin B1 locates at λex/λem=370/460nm, two fluorescence areas of vitamin B6 locates at λex/λem=250/370nm and 325/370nm were found. The influence of pH of solution to the fluorescence profile was also discussed. Using the PARAFAC algorithm, 10 vitamin B1, B2 and B6 mixed solutions were successfully decomposed, and the emission profiles, excitation profiles, central wavelengths and the concentration of the three components were retrieved precisely through about 5 iteration times.

  17. THE UNCOVERING OF A NOVEL REGULATORY MECHANISM FOR PLD2: FORMATION OF A TERNARY COMPLEX WITH PROTEIN TYROSINE PHOSPHATASE PTP1B AND GROWTH FACTOR RECEPTOR-BOUND PROTEIN GRB2

    PubMed Central

    Horn, Jeff; Lopez, Isabel; Miller, Mill; Gomez-Cambronero, Julian

    2011-01-01

    The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2 and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase. PMID:15896299

  18. Retinoic Acid-inducible Gene I-inducible miR-23b Inhibits Infections by Minor Group Rhinoviruses through Down-regulation of the Very Low Density Lipoprotein Receptor*

    PubMed Central

    Ouda, Ryota; Onomoto, Koji; Takahasi, Kiyohiro; Edwards, Michael R.; Kato, Hiroki; Yoneyama, Mitsutoshi; Fujita, Takashi

    2011-01-01

    In mammals, viral infections are detected by innate immune receptors, including Toll-like receptor and retinoic acid inducible gene I (RIG-I)-like receptor (RLR), which activate the type I interferon (IFN) system. IFN essentially activates genes encoding antiviral proteins that inhibit various steps of viral replication as well as facilitate the subsequent activation of acquired immune responses. In this study, we investigated the expression of non-coding RNA upon viral infection or RLR activation. Using a microarray, we identified several microRNAs (miRNA) specifically induced to express by RLR signaling. As suggested by Bioinformatics (miRBase Target Data base), one of the RLR-inducible miRNAs, miR-23b, actually knocked down the expression of very low density lipoprotein receptor (VLDLR) and LDLR-related protein 5 (LRP5). Transfection of miR-23b specifically inhibited infection of rhinovirus 1B (RV1B), which utilizes the low density lipoprotein receptor (LDLR) family for viral entry. Conversely, introduction of anti-miRNA-23b enhanced the viral yield. Knockdown experiments using small interfering RNA (siRNA) revealed that VLDLR, but not LRP5, is critical for an efficient infection by RV1B. Furthermore, experiments with the transfection of infectious viral RNA revealed that miR-23b did not affect post-entry viral replication. Our results strongly suggest that RIG-I signaling results in the inhibitions of infections of RV1B through the miR-23b-mediated down-regulation of its receptor VLDLR. PMID:21642441

  19. Retinoic acid-inducible gene I-inducible miR-23b inhibits infections by minor group rhinoviruses through down-regulation of the very low density lipoprotein receptor.

    PubMed

    Ouda, Ryota; Onomoto, Koji; Takahasi, Kiyohiro; Edwards, Michael R; Kato, Hiroki; Yoneyama, Mitsutoshi; Fujita, Takashi

    2011-07-22

    In mammals, viral infections are detected by innate immune receptors, including Toll-like receptor and retinoic acid inducible gene I (RIG-I)-like receptor (RLR), which activate the type I interferon (IFN) system. IFN essentially activates genes encoding antiviral proteins that inhibit various steps of viral replication as well as facilitate the subsequent activation of acquired immune responses. In this study, we investigated the expression of non-coding RNA upon viral infection or RLR activation. Using a microarray, we identified several microRNAs (miRNA) specifically induced to express by RLR signaling. As suggested by Bioinformatics (miRBase Target Data base), one of the RLR-inducible miRNAs, miR-23b, actually knocked down the expression of very low density lipoprotein receptor (VLDLR) and LDLR-related protein 5 (LRP5). Transfection of miR-23b specifically inhibited infection of rhinovirus 1B (RV1B), which utilizes the low density lipoprotein receptor (LDLR) family for viral entry. Conversely, introduction of anti-miRNA-23b enhanced the viral yield. Knockdown experiments using small interfering RNA (siRNA) revealed that VLDLR, but not LRP5, is critical for an efficient infection by RV1B. Furthermore, experiments with the transfection of infectious viral RNA revealed that miR-23b did not affect post-entry viral replication. Our results strongly suggest that RIG-I signaling results in the inhibitions of infections of RV1B through the miR-23b-mediated down-regulation of its receptor VLDLR.

  20. Crosstalk between ERK2 and RXR regulates nuclear import of transcription factor NGFI-B

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jacobs, Chris M.; Paulsen, Ragnhild E.

    2005-10-21

    Transcription factor NGFI-B initiates apoptosis when allowed to translocate to mitochondria. Retinoid-X receptor (RXR), another member of the nuclear receptor family, regulates NGFI-B signaling through heterodimerization and nuclear export. Growth factor EGF activates ERK2, which phosphorylates NGFI-B and determines if NGFI-B is allowed to translocate to mitochondria. In the present study, EGF treatment resulted in an increased nuclear import of NGFI-B. Likewise, active ERK2 resulted in a preferential nuclear localization of NGFI-B. When coexpressed with RXR the nuclear import and nuclear localization induced by active ERK2 were strongly reduced. In the presence of its ligand 9-cis-retinoic acid, RXR no longermore » inhibited ERK2-induced nuclear import. Thus, RXR serves a permissive role for ERK2-mediated nuclear accumulation of NGFI-B. This finding represents a novel crosstalk between ERK2 and RXR signaling pathways, and explains how two independent inhibitors of apoptosis (EGF and 9-cis-retinoic acid) may cooperate to regulate nuclear targeting of apoptosis inducer NGFI-B.« less

  1. A physiological role of AMP-activated protein kinase in phenobarbital-mediated constitutive androstane receptor activation and CYP2B induction

    PubMed Central

    Shindo, Sawako; Numazawa, Satoshi; Yoshida, Takemi

    2006-01-01

    CAR (constitutive androstane receptor) is a nuclear receptor that regulates the transcription of target genes, including CYP (cytochrome P450) 2B and 3A. The transactivation by CAR is regulated by its subcellular localization; however, the mechanism that governs nuclear translocation has yet to be clarified. It has been reported recently that AMPK (AMP-activated protein kinase) is involved in phenobarbital-mediated CYP2B induction in a particular culture system. We therefore investigated in vivo whether AMPK is involved in the activation of CAR-dependent gene expression. Immunoblot analysis using an antibody which recognizes Thr-172-phosphorylated AMPKα1/2 revealed phenobarbital-induced AMPK activation in rat and mouse livers as well. Phenobarbital, however, failed to increase the liver phospho-AMPK level of tumour-bearing rats in which CAR nuclear translocation had been impaired. In in vivo reporter gene assays employing PBREM (phenobarbital-responsive enhancer module) from CYP2B1, an AMPK inhibitor 8-bromo-AMP abolished phenobarbital-induced transactivation. In addition, Cyp2b10 gene expression was attenuated by 8-bromo-AMP. Forced expression of a dominant-negative mutant and the wild-type of AMPKα2 in the mouse liver suppressed and further enhanced phenobarbital-induced PBREM-reporter activity respectively. Moreover, the AMPK activator AICAR (5-amino-4-imidazolecarboxamide riboside) induced PBREM transactivation and an accumulation of CAR in the nuclear fraction of the mouse liver. However, AICAR and metformin, another AMPK activator, failed to induce hepatic CYP2B in mice and rats. These observations suggest that AMPK is at least partly involved in phenobarbital-originated signalling, but the kinase activation by itself is not sufficient for CYP2B induction in vivo. PMID:17032173

  2. Genetic variations in UGT2B28, UGT2B17, UGT2B15 genes and the risk of prostate cancer: A case-control study.

    PubMed

    Habibi, Mohsen; Mirfakhraie, Reza; Khani, Maryam; Rakhshan, Azadeh; Azargashb, Eznollah; Pouresmaeili, Farkhondeh

    2017-11-15

    Glucuronidation is a major pathway for elimination of exogenous and endogenous compounds such as environmental carcinogens and androgens from the body. This biochemical pathway is mediated by enzymes called uridine diphosphoglucuronosyltransferases (UGTs). Null (del/del) genes polymorphisms in UGT2B17, and UGT2B28 and D85Y single-nucleotide polymorphism (SNP) of UGT2B15 have been reported to increase the risk of prostate cancer. The goal of this study was to determine the association of mentioned genetic variants with the risk of prostate cancer. We investigated the copy number variations (CNVs) of UGT2B17 and UGT2B28 loci and the association between rs1902023 polymorphism of UGT2B15 gene in 360 subjects consisted of 120 healthy controls, 120 prostate cancer (PC) patients and 120 benign prostatic hyperplasia (BPH) patients. No association was detected for the mentioned polymorphisms and the risk of PC. However, a significant association was detected between UGT2B17 copy number variation and BPH risk (OR=2.189; 95% CI, 1.303-3.675; p=0.003). Furthermore, we observed that the D85Y polymorphism increases the risk of BPH when analyzed in combination with the copy number variation of UGT2B17 gene (OR=0.135; 95% CI, 0.036-0.512; p=0.003). Our findings suggest that the D85Y polymorphism of UGT2B15 and CNVs in UGT2B28 and UGT2B17 genes is not associated with prostate cancer risk in Iranian patients. To our knowledge, this is the first report that implicates the role of CNV of UGT2B17 gene in BPH. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Could the 5-HT1B receptor inverse agonism affect learning consolidation?

    PubMed

    Meneses, A

    2001-03-01

    Diverse evidence indicates that, the 5-HT system might play a role in learning and memory, since it occurs in brain areas mediating such processes and 5-HT drugs modulate them. Hence in this work, in order to explore further 5-HT involvement on learning and memory 5-HT1B receptors' role is investigated. Evidence indicates that SB-224289 (a 5-HT1B receptor inverse agonist) post-training injection facilitated learning consolidation in an associative autoshaping learning task, this effect was partially reversed by GR 127935 (a 5-HT1B/1D receptor antagonist), but unaffected by MDL 100907 (a 5-HT2A receptor antagonist) or ketanserin (a 5-HT1D/2A/7 receptor antagonist) at low doses. Moreover, SB-224289 antagonized the learning deficit produced by TFMPP (a 5-HT1A/1B/1D/2A/2C receptor agonist), GR 46611 (a 5-HT1A/1B/1D receptor agonist), mCPP (a 5-HT2A/2C/3/7 receptor agonist/antagonist) or GR 127935 (at low dose). SB-224289 did not alter the 8-OH-DPAT (a 5-HT1A/7 receptor agonist) learning facilitatory effect. SB-224289 eliminated the deficit learning produced by the anticholinergic muscarinic scopolamine or the glutamatergic antagonist dizocilpine. Administration of both, GR 127935 (5mg/kg) plus ketanserin (0.01 mg/kg) did not modify learning consolidation; nevertheless, when ketanserin dose was increased (0.1-1.0mg/kg) and SB-224289 dose was maintained constant, a learning facilitation effect was observed. Notably, SB-224289 at 1.0mg/kg potentiated a subeffective dose of the 5-HT1B/1D receptor agonist/antagonist mixed GR 127935, which facilitated learning consolidation and this effect was abolished by ketanserin at a higher dose. Collectively, the data confirm and extend the earlier findings with GR 127935 and the effects of non-selective 5-HT(1B) receptor agonists. Clearly 5-HT1B agonists induced a learning deficit which can be reversed with SB-224289. Perhaps more importantly, SB-224289 enhances learning consolidation when given alone and can reverse the deficits

  4. Memory Enhancement by Targeting Cdk5 Regulation of NR2B

    PubMed Central

    Plattner, Florian; Hernandéz, Adan; Kistler, Tara M.; Pozo, Karine; Zhong, Ping; Yuen, Eunice Y.; Tan, Chunfeng; Hawasli, Ammar H.; Cooke, Sam F.; Nishi, Akinori; Guo, Ailan; Wiederhold, Thorsten; Yan, Zhen; Bibb, James A.

    2014-01-01

    SUMMARY Many psychiatric and neurological disorders are characterized by learning and memory deficits, for which cognitive enhancement is considered a valid treatment strategy. The N-methyl-D-aspartate receptor (NMDAR) is a prime target for the development of cognitive enhancers due to its fundamental role in learning and memory. In particular, the NMDAR subunit NR2B improves synaptic plasticity and memory when over-expressed in neurons. However, NR2B regulation is not well understood and no therapies potentiating NMDAR function have been developed. Here, we show that serine 1116 of NR2B is phosphorylated by cyclin-dependent kinase 5 (Cdk5). Cdk5-dependent NR2B phosphorylation is regulated by neuronal activity and controls the receptor’s cell surface expression. Disrupting NR2B-Cdk5 interaction using a small interfering peptide (siP) increases NR2B surface levels, facilitates synaptic transmission, and improves memory formation in vivo. Our results reveal a novel regulatory mechanism critical to NR2B function that can be targeted for the development of cognitive enhancers. PMID:24607229

  5. Gab-family adapter proteins act downstream of cytokine and growth factor receptors and T- and B-cell antigen receptors.

    PubMed

    Nishida, K; Yoshida, Y; Itoh, M; Fukada, T; Ohtani, T; Shirogane, T; Atsumi, T; Takahashi-Tezuka, M; Ishihara, K; Hibi, M; Hirano, T

    1999-03-15

    We previously found that the adapter protein Gab1 (110 kD) is tyrosine-phosphorylated and forms a complex with SHP-2 and PI-3 kinase upon stimulation through either the interleukin-3 receptor (IL-3R) or gp130, the common receptor subunit of IL-6-family cytokines. In this report, we identified another adapter molecule (100 kD) interacting with SHP-2 and PI-3 kinase in response to various stimuli. The molecule displays striking homology to Gab1 at the amino acid level; thus, we named it Gab2. It contains a PH domain, proline-rich sequences, and tyrosine residues that bind to SH2 domains when they are phosphorylated. Gab1 is phosphorylated on tyrosine upon stimulation through the thrombopoietin receptor (TPOR), stem cell factor receptor (SCFR), and T-cell and B-cell antigen receptors (TCR and BCR, respectively), in addition to IL-3R and gp130. Tyrosine phosphorylation of Gab2 was induced by stimulation through gp130, IL-2R, IL-3R, TPOR, SCFR, and TCR. Gab1 and Gab2 were shown to be substrates for SHP-2 in vitro. Overexpression of Gab2 enhanced the gp130 or Src-related kinases-mediated ERK2 activation as that of Gab1 did. These data indicate that Gab-family molecules act as adapters for transmitting various signals.

  6. From small sweeteners to sweet proteins: anatomy of the binding sites of the human T1R2_T1R3 receptor.

    PubMed

    Morini, Gabriella; Bassoli, Angela; Temussi, Piero A

    2005-08-25

    The sweet taste receptor, a heterodimeric G protein coupled receptor (GPCR) protein, formed by the T1R2 and T1R3 subunits, recognizes several sweet compounds including carbohydrates, amino acids, peptides, proteins, and synthetic sweeteners. Its similarity with the metabotropic glutamate mGluR1 receptor allowed us to build homology models. All possible dimers formed by combinations of the human T1R2 and T1R3 subunits, modeled on the A (closed) or B (open) chains of the extracellular ligand binding domain of the mGluR1 template, yield four ligand binding sites for low-molecular-weight sweeteners. These sites were probed by docking a set of molecules representative of all classes of sweet compounds and calculating the free energy of ligand binding. These sites are not easily accessible to sweet proteins, but docking experiments in silico showed that sweet proteins can bind to a secondary site without entering the deep cleft. Our models account for many experimental observations on the tastes of sweeteners, including sweetness synergy, and can help to design new sweeteners.

  7. ADAM10-mediated ephrin-B2 shedding promotes myofibroblast activation and organ fibrosis

    PubMed Central

    Lagares, David; Ghassemi-Kakroodi, Parisa; Tremblay, Caroline; Santos, Alba; Probst, Clemens K.; Franklin, Alicia; Santos, Daniela M.; Grasberger, Paula; Ahluwalia, Neil; Montesi, Sydney B.; Shea, Barry S.; Black, Katharine E.; Knipe, Rachel; Blati, Meryem; Baron, Murray; Wu, Brian; Fahmi, Hassan; Gandhi, Rajiv; Pardo, Annie; Selman, Moisés; Wu, Jiangping; Pelletier, Jean-Pierre; Martel-Pelletier, Johanne; Tager, Andrew M.; Kapoor, Mohit

    2017-01-01

    Maladaptive wound healing responses to chronic tissue injury result in organ fibrosis. Fibrosis, which entails excessive extracellular matrix (ECM) deposition and tissue remodelling by activated myofibroblasts, leads to loss of proper tissue architecture and organ function; however the molecular mediators of myofibroblast activation remain to be fully identified. Here we identify soluble ephrin-B2 as a novel pro-fibrotic mediator in lung and skin fibrosis. We provide molecular, functional and translational evidence that the ectodomain of membrane-bound ephrin-B2 is shed from fibroblasts into the alveolar airspace after lung injury. Shedding of soluble ephrin-B2 (sEphrin-B2) promotes fibroblast chemotaxis and activation via EphB3/EphB4 receptor signaling. We found that mice lacking ephrin-B2 in fibroblasts are protected from skin and lung fibrosis and that a distintegrin and metalloproteinase 10 (ADAM10) is the major ephrin-B2 sheddase in fibroblasts. ADAM10 is induced by transforming growth factor-β1 (TGF-β1), and ADAM10-mediated sEphrin-B2 generation is required for TGF-β1–induced myofibroblast activation. Pharmacological inhibition of ADAM10 reduces sEphrin-B2 levels in bronchoalveolar lavage and prevents lung fibrosis in mice. Consistent with the mouse data, ADAM10/sEphrin-B2 signaling is upregulated in fibroblasts from human subjects with idiopathic pulmonary fibrosis. These results uncover a new molecular mechanism of tissue fibrogenesis and identify sEphrin-B2, its receptors Eph3/Eph4, and ADAM10 as potential therapeutic targets in the treatment of fibrotic diseases. PMID:29058717

  8. Interferon Alfa-2b Injection

    MedlinePlus

    Interferon alfa-2b injection is used to treat a number of conditions.Interferon alfa-2b injection is used alone or in combination ... lymphoma (NHL; a slow-growing blood cancer). Interferon alfa-2b is in a class of medications called ...

  9. Mechanistic and signaling analysis of Muc4-ErbB2 signaling module: new insights into the mechanism of ligand-independent ErbB2 activity.

    PubMed

    Kozloski, Goldi A; Carraway, Coralie A Carothers; Carraway, Kermit L

    2010-09-01

    The membrane mucin Muc4 is aberrantly expressed in numerous epithelial carcinomas and is currently used as a cancer diagnostic and prognostic tool. Muc4 can also potentiate signal transduction by modulating differential ErbB2 phosphorylation in the absence and in the presence of the ErbB3 soluble ligand heregulin (HRG-beta1). These features of Muc4 suggest that Muc4 is not merely a cancer marker, but an oncogenic factor with a unique-binding/activation relationship with the receptor ErbB2. In the present study, we examined the signaling mechanisms that are associated with the Muc4-ErbB2 module by analyzing ErbB2 differential signaling in response to Muc4 expression. Our study was carried out in the A375 human melanoma and BT-474 breast cancer cell lines as our model systems. Quantitative and comparative signaling modulations were evaluated by immunoblot using phospho-specific antibodies, and densitometry analysis. Signaling complex components were identified by chemical cross-linking, fractionation by gel filtration, immunoprecipitation, and immunoblotting. Activated downstream signaling pathways were analyzed by an antibody microarray screen and immunoblot analyses. Our results indicate that Muc4 modulates ErbB2 signaling potential significantly by stabilizing and directly interacting with the ErbB2-ErbB3 heterodimer. Further analyses indicate that Muc4 promotes ErbB2 autocatalysis, but it has no effect on ErbB3 phosphorylation, although the chemical cross-linking data indicated that the signaling module is composed of Muc4, ErbB2, and ErbB3. Our microarray analysis indicates that Muc4 expression promotes cell migration by increasing the phosphorylation of the focal adhesion kinase and also through an increase in the levels of beta-catenin. (c) 2010 Wiley-Liss, Inc.

  10. Alterations of the PPP2R1B gene located at 11q23 in human colorectal cancers

    PubMed Central

    Takagi, Y; Futamura, M; Yamaguchi, K; Aoki, S; Takahashi, T; Saji, S

    2000-01-01

    BACKGROUND/AIMS—In 1998 the PPP2R1B gene encoding the A subunit of the serine/threonine protein phosphatase was identified as a putative tumour suppressor gene in lung and colon cancer in the chromosome region 11q22-24. The aim of the present study was to determine the type of alterations in primary rectal cancers as well as colon cancers and the correlation between these alterations and clinicopathological data.
METHODS—Mutation analyses of the PPP2R1B gene sequence encoding the binding sites of the catalytic C subunit (Huntington elongation A subunit TOR (HEAT) repeats 11-15) and partial binding sites of the regulatory B subunit were carried out on cDNA samples from 30 primary colorectal cancer specimens and corresponding normal tissues using a combination of the polymerase chain reaction and subsequent direct DNA sequencing.
RESULTS—Five missense mutations producing amino acid substitutions were detected in the four colon cancer cases (13.3%; four of 30 colorectal cancers): 15glycine (GGT) to alanine (GCT) and 499leucine (TTA) to isoleucine (ATA) in the same case, and 498valine (GTG) to glutamic acid (GAG), 500valine (GTA) to glycine (GGA), and 365serine (TCT) to proline (CCT). Of these five mutations, three (60%) were located in HEAT repeat 13 and four (80%) showed T to other nucleotide substitutions. In addition, a normal polymorphism, 478leucine, was found. No correlation was found between these mutations and clinicopathological data.
CONCLUSION—Our results suggest that the PPP2R1B gene is one of the true targets at 11q23, and its inactivation is involved in the development of all types of colorectal cancers.


Keywords: PPP2R1B gene; colorectal cancer; tumour suppressor gene; protein phosphatase PMID:10896920

  11. Investigation of the Application of miR10b and miR135b in the Identification of Semen Stains

    PubMed Central

    Xue, Tianyu; Ma, Xiaoyan; Zhang, Jinxiang; Ou, Xueling; Cheng, Jianding; Sun, Hongyu

    2015-01-01

    To evaluate the identification method using the microRNA markers miR10b and miR135b to distinguish semen stains from menstrual blood, peripheral blood, vaginal fluid and so on body fluid stains. The expression levels of miR10b and miR35b in semen stains and menstrual blood and so on were detected utilizing a real-time quantitative PCR technique with a specific fluorescence-labeled TaqMan probe. RNU6b was used as the internal reference gene; the difference in their expression was analyzed, and the specificity, sensitivity, and detection capability of the techniques were evaluated. The expression of miR10b and miR135b in semen stains was significantly higher than that of other body fluid stains, with a mean value of ΔCт from-6 to-7. However, it ranged from-2 to-4 for other body fluid stains. The initial criteria for judging which semen stains can be identified were determined by analyzing the research results. When the threshold value was set to 0.04, the CT value could be detected in the target genes miR10b, miR135b and in the internal reference gene RNU6b, and CT values are<40, ΔCT[10b-U6]<-5.5, and ΔCT[135b-U6]<-6, respectively, and the semen stain could be identified. The expression levels of miR10b and miR135b are higher in semen with strong tissue specificity; thus, they can be used to differentiate semen stains from other body fluid stains in forensic science. PMID:26355456

  12. Inhibition of monoamine oxidase A and B activities by imidazol(ine)/guanidine drugs, nature of the interaction and distinction from I2-imidazoline receptors in rat liver

    PubMed Central

    Ozaita, Andrés; Olmos, Gabriel; Assumpció Boronat, M; Miguel Lizcano, José; Unzeta, Mercedes; García-Sevilla, Jesús A

    1997-01-01

    mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the drug inhibition constants (Ki values) were similar to the IC50 values displayed for the inhibition of MAO-A or MAO-B activities. In fact, very good correlations were obtained when the affinities of drugs at MAO-A or MAO-B catalytic sites were correlated with their potencies in inhibiting MAO-A (r=0.92) or MAO-B (r=0.99) activity. This further suggested a direct drug interaction with the catalytic sites of MAO-A and MAO-B isoforms. No significant correlations were found when the potencies of imidazol(ine)/guanidine drugs at the high affinity site (pKiH, nanomolar range) or the low-affinity site (pKiL, micromolar range) of I2-imidazoline receptors labelled with [3H]-clonidine were correlated with the pIC50 values of the same drugs for inhibition of MAO-A or MAO-B activity. These discrepancies indicated that I2-imidazoline receptors are not directly related to the site of action of these drugs on MAO activity in rat liver mitochondrial fractions. Although these studies cannot exclude the presence of additional binding sites on MAO that do not affect the activity of the enzyme, they would suggest that I2-imidazoline receptors represent molecular species that are distinct from MAO. PMID:9222546

  13. Observation of CH A (sup 2)Delta approaches X (sup 2)Pi(sub r) and B (sup 2)Sigma(sup -) approaches X (sup 2)Pi(sub r) emissions in gas-phase collisions of fast O((sup 3)P) atoms with acetylene

    NASA Technical Reports Server (NTRS)

    Orient, O. J.; Chutjian, A.; Murad, E.

    1995-01-01

    Optical emissions in single-collision, beam-beam reactions of fast (3-22 eV translational energy) O(P-3) atoms with C2H2 have been measured in the wavelength range 300-850 nm. Two features were observed, one with a peak wavelength at 431 nm, corresponding to the CH A (sup 2)Delta yields X (sup 2)Pi(sub r) transition, and a second weaker emission in the range 380-400 nm corresponding to the B (sup 2)Sigma(sup -) yields X (sup 2)Pi(sub r) transition. Both the A yields X and B yields X emissions were fit to a synthetic spectrum of CH(A) at a vibrational temperature T(sub v) of 10,000 K (0.86 eV) and a rotational temperature T(r) of approximately 5000 K (0.43 eV); and CH(B) to T(sub v) = 2500 K (0.22 eV) and T(sub r) = 1000 K (0.09 eV). The energy threshold for the A yields X emission was measured to be 7.3 +/- 0.4 eV (lab) or 4.5 +/- 0.2 eV (c.m.). This agrees with the energy threshold of 7.36 eV (lab) for the reaction O(P-3) + C2H2 yields CH(A) + HCO.

  14. The role of GluN2A and GluN2B NMDA receptor subunits in AgRP and POMC neurons on body weight and glucose homeostasis.

    PubMed

    Üner, Aykut; Gonçalves, Gabriel H M; Li, Wenjing; Porceban, Matheus; Caron, Nicole; Schönke, Milena; Delpire, Eric; Sakimura, Kenji; Bjørbæk, Christian

    2015-10-01

    Hypothalamic agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) expressing neurons play critical roles in control of energy balance. Glutamatergic input via n-methyl-d-aspartate receptors (NMDARs) is pivotal for regulation of neuronal activity and is required in AgRP neurons for normal body weight homeostasis. NMDARs typically consist of the obligatory GluN1 subunit and different GluN2 subunits, the latter exerting crucial differential effects on channel activity and neuronal function. Currently, the role of specific GluN2 subunits in AgRP and POMC neurons on whole body energy and glucose balance is unknown. We used the cre-lox system to genetically delete GluN2A or GluN2B only from AgRP or POMC neurons in mice. Mice were then subjected to metabolic analyses and assessment of AgRP and POMC neuronal function through morphological studies. We show that loss of GluN2B from AgRP neurons reduces body weight, fat mass, and food intake, whereas GluN2B in POMC neurons is not required for normal energy balance control. GluN2A subunits in either AgRP or POMC neurons are not required for regulation of body weight. Deletion of GluN2B reduces the number of AgRP neurons and decreases their dendritic length. In addition, loss of GluN2B in AgRP neurons of the morbidly obese and severely diabetic leptin-deficient Lep (ob/ob) mice does not affect body weight and food intake but, remarkably, leads to full correction of hyperglycemia. Lep (ob/ob) mice lacking GluN2B in AgRP neurons are also more sensitive to leptin's anti-obesity actions. GluN2B-containing NMDA receptors in AgRP neurons play a critical role in central control of body weight homeostasis and blood glucose balance via mechanisms that likely involve regulation of AgRP neuronal survival and structure, and modulation of hypothalamic leptin action.

  15. Insulin receptor isoforms A and B as well as insulin receptor substrates-1 and -2 are differentially expressed in prostate cancer.

    PubMed

    Heni, Martin; Hennenlotter, Jörg; Scharpf, Marcus; Lutz, Stefan Z; Schwentner, Christian; Todenhöfer, Tilman; Schilling, David; Kühs, Ursula; Gerber, Valentina; Machicao, Fausto; Staiger, Harald; Häring, Hans-Ulrich; Stenzl, Arnulf

    2012-01-01

    In different cancers types, insulin receptor isoform composition or insulin receptor substrate (IRS) isoforms are different to healthy tissue. This may be a molecular link to increased cancer risk in diabetes and obesity. Since this is yet unclear for prostate cancer, we investigated IR isoform composition and IRS balance in prostate cancer compared to benign and tumor adjacent benign prostate tissue and brought this into relation to cell proliferation. We studied 23 benign prostate samples from radical cystectomy or benign prostatic hyperplasia surgery, 30 samples from benign tissue directly adjacent to prostate cancer foci and 35 cancer samples from different patients. RNA expression levels for insulin receptor isoforms A and B, IRS-1, IRS-2, and IGF-1 receptor were assessed by quantitative real-time RT-PCR. In addition, RNA- and protein expression of the cell cycle regulator p27(Kip1) was quantified by real-time RT-PCR and immunohistochemistry. Insulin receptor isoform A to B ratio was significantly higher in cancer as well as in tumor adjacent benign prostate tissue compared to purely benign prostates (p<0.05). IRS-1 to IRS-2 ratios were lower in malignant than in benign prostatic tissue (p<0.05). These altered ratios both in cancer and adjacent tissue were significantly associated with reduced p27(Kip1) content (p<0.02). Interestingly, IGF-1 receptor levels were significantly lower in patients with type 2 diabetes (p = 0.0019). We found significant differences in the insulin signaling cascade between benign prostate tissue and prostate cancer. Histological benign tissue adjacent to cancer showed expression patterns similar to the malignancies. Our findings suggest a role of the insulin signaling pathway in prostate cancer and surrounding tissue and can hence be relevant for both novel diagnostic and therapeutic approaches in this malignancy.

  16. Investigation of the cardiomyocyte dysfunction in bradykinin type 2 receptor knockout mice.

    PubMed

    Roman-Campos, Danilo; Duarte, Hugo Leonardo; Gomes, Enéas Ricardo; Castro, Carlos Henrique; Guatimosim, Silvia; Natali, Antonio José; Almeida, Alvair Pinto; Pesquero, João Bosco; Pesquero, Jorge Luiz; Cruz, Jader Santos

    2010-12-18

    Bradykinin type 2 receptor (B(2)R) is the key component to trigger the intracellular signaling pathway in response to bradykinin under physiological conditions. The present study sought to investigate whether the B(2)R gene deletion will have an impact on myocardial function. Isolated cell shortening, patch-clamp technique, Western blot and confocal microscopy. Isolated cell shortening measurements showed significant reduction in B(2)R knockout (B(2)R(-/-)) left ventricular cardiac myocytes' shortening. Whole-cell recordings were used to study the electrophysiological aspects of the left ventricular B(2)R(-/-) cardiomyocytes. Results showed: 1) action potential lengthening; 2) unchanged inwardly rectifying K(+) current; 3) reduced transient outward K(+) (I(to)) and L-type Ca(2+) current densities; 5) changes in kinetic properties related to I(to) and I(Ca,L). In addition, transient sarcoplasmic reticulum (SR) Ca(2+) release was found to be smaller in B(2)R(-/-) cardiomyocytes. Importantly, evidence is provided that NO constitutive production is, at least in part, responsible for the reported electrophysiological modifications observed in cardiomyocytes from B(2)R(-/-) mice. Surprisingly, NO is not involved in the SR Ca(2+) release reduction as demonstrated in the present study. Taken together, our findings indicate that B(2)R plays a fundamental role in the regulation of cardiac function and Ca(2+) homeostasis, probably through a NO dependent pathway. These results may contribute to our understanding of the kinins participation in the control of cardiac function. Copyright © 2010 Elsevier Inc. All rights reserved.

  17. CXCL13 regulates the trafficking of GluN2B-containing NMDA receptor via IL-17 in the development of remifentanil-induced hyperalgesia in rats.

    PubMed

    Zhu, M; Yuan, S T; Yu, W L; Jia, L L; Sun, Y

    2017-05-01

    This study aimed to investigate whether CXCL13 modulated the trafficking of NMDA receptor via interleukin (IL)-17 in a rat model of remifentanil-induced hyperalgesia (RIH).Although chemokines are crucial regulators of neuroinflammation, spinal N-methyl-d-aspartate (NMDA) receptor activation, and development of hypernociceptive process, little is known about specific pathogenesis and effective treatment. Inflammatory mediators are required for excitatory synaptic transmission in pathologic pain. A neutralizing antibody against CXCL13 (anti-CXCL13), antiserum against IL-17 (anti-IL-17), and recombinant CXCL13 and IL-17 were administered intrathecally to explore the roles of CXCL13, IL-17, and NMDA receptor, as well as the prevention of hyperalgesia. Paw withdrawal threshold and paw withdrawal latency were employed to record mechanical allodynia and thermal hyperalgesia. Reverse transcriptase quantitative polymerase chain reaction was used to evaluate the levels of CXCL13/CXCR5 and IL-17/IL-17RA in the spinal dorsal horn. The trafficking of spinal GluN2B-containing NMDA receptor was assessed by Western blot after nociceptive testing. This study found mechanical allodynia and thermal hyperalgesia with a remarkable increase in the expression of spinal CXCL13/CXCR5 and IL-17/IL-17RA and trafficking of GluN2B-containing NMDA receptor after remifentanil exposure. Behavioral RIH and elevated GluN2B trafficking were dampened by intrathecal anti-CXCL13 and anti-IL-17, respectively. The delivery of exogenous CXCL13 dose-dependently generated a rapid nociceptive hypersensitivity in naïve rats, which was prevented by coadministering anti-IL-17. CXCL13-induced IL-17RA overproduction and GluN2B trafficking were reversed by anti-IL-17 treatment. GluN2B antagonist also blocked CXCL13, and IL-17 directly induced hyperalgesia. This study highlighted the contribution of IL-17 pathway in the trafficking of CXCL13-induced GluN2B-containing NMDA receptor in the pathogenesis of RIH

  18. Identification and in vitro pharmacological characterization of a novel and selective α7 nicotinic acetylcholine receptor agonist, Br-IQ17B.

    PubMed

    Tang, Jing-shu; Xie, Bing-xue; Bian, Xi-ling; Xue, Yu; Wei, Ning-ning; Zhou, Jing-heng; Hao, Yu-chen; Li, Gang; Zhang, Liang-ren; Wang, Ke-wei

    2015-07-01

    Alpha7-nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated Ca(2+)-permeable ion channel implicated in cognition and neuropsychiatric disorders. Activation of α7 nAChR improves learning, memory, and sensory gating in animal models. To identify novel α7 nAChR agonists, we synthesized a series of small molecules and characterized a representative compound, Br-IQ17B, N-[(3R)-1-azabicyclo[2,2,2]oct-3-yl]-5-bromoindolizine-2-carboxamide, which specifically activates α7 nAChR. Two-electrode voltage clamp (TEVC) recordings were primarily used for screening in Xenopus oocytes expressing human α7 nAChR. Assays, including radioisotope ligand binding, Western blots, whole-cell recordings of hippocampal culture neurons, and spontaneous IPSC recordings of brain slices, were also utilized to evaluate and confirm the specific activation of α7 nAChR by Br-IQ17B. Br-IQ17B potently activates α7 nAChR with an EC50 of 1.8±0.2 μmol/L. Br-IQ17B is selective over other subtypes such as α4β2 and α3β4, but it blocks 5-HT3A receptors. Br-IQ17B displaced binding of the α7 blocker [(3)H]-MLA to hippocampal crude membranes with a Ki of 14.9±3.2 nmol/L. In hippocampal neurons, Br-IQ17B evoked α7-like currents that were inhibited by MLA and enhanced in the presence of the α7 PAM PNU-120596. In brain slice recordings, Br-IQ17B enhanced GABAergic synaptic transmission in CA1 neurons. Mechanistically, Br-IQ17B increased ERK1/2 phosphorylation that was MLA-sensitive. We identified the novel, potent, and selective α7 agonist Br-IQ17B, which enhances synaptic transmission. Br-IQ17B may be a helpful tool to understand new aspects of α7 nAChR function, and it also has potential for being developed as therapy for schizophrenia and cognitive deficits.

  19. Identification and in vitro pharmacological characterization of a novel and selective α7 nicotinic acetylcholine receptor agonist, Br-IQ17B

    PubMed Central

    Tang, Jing-shu; Xie, Bing-xue; Bian, Xi-ling; Xue, Yu; Wei, Ning-ning; Zhou, Jing-heng; Hao, Yu-chen; Li, Gang; Zhang, Liang-ren; Wang, Ke-wei

    2015-01-01

    Aim: Alpha7-nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated Ca2+-permeable ion channel implicated in cognition and neuropsychiatric disorders. Activation of α7 nAChR improves learning, memory, and sensory gating in animal models. To identify novel α7 nAChR agonists, we synthesized a series of small molecules and characterized a representative compound, Br-IQ17B, N-[(3R)-1-azabicyclo[2,2,2]oct-3-yl]-5-bromoindolizine-2-carboxamide, which specifically activates α7 nAChR. Methods: Two-electrode voltage clamp (TEVC) recordings were primarily used for screening in Xenopus oocytes expressing human α7 nAChR. Assays, including radioisotope ligand binding, Western blots, whole-cell recordings of hippocampal culture neurons, and spontaneous IPSC recordings of brain slices, were also utilized to evaluate and confirm the specific activation of α7 nAChR by Br-IQ17B. Results: Br-IQ17B potently activates α7 nAChR with an EC50 of 1.8±0.2 μmol/L. Br-IQ17B is selective over other subtypes such as α4β2 and α3β4, but it blocks 5-HT3A receptors. Br-IQ17B displaced binding of the α7 blocker [3H]-MLA to hippocampal crude membranes with a Ki of 14.9±3.2 nmol/L. In hippocampal neurons, Br-IQ17B evoked α7-like currents that were inhibited by MLA and enhanced in the presence of the α7 PAM PNU-120596. In brain slice recordings, Br-IQ17B enhanced GABAergic synaptic transmission in CA1 neurons. Mechanistically, Br-IQ17B increased ERK1/2 phosphorylation that was MLA-sensitive. Conclusion: We identified the novel, potent, and selective α7 agonist Br-IQ17B, which enhances synaptic transmission. Br-IQ17B may be a helpful tool to understand new aspects of α7 nAChR function, and it also has potential for being developed as therapy for schizophrenia and cognitive deficits. PMID:25948478

  20. Electronic and elastic properties of new semiconducting oP(12)-type RuB(2) and OsB(2).

    PubMed

    Hao, Xianfeng; Xu, Yuanhui; Gao, Faming

    2011-03-30

    Using first-principles total energy calculations we investigate the structural, elastic and electronic properties of new hypothetical oP(12)-type phase RuB(2) and OsB(2). The calculations indicate that the oP(12)-type phase RuB(2) and OsB(2) are thermodynamically and mechanically stable. Remarkably, the new phases RuB(2) and OsB(2) are predicted to be semiconductors, and the appearance of band gaps is ascribed to the enhanced B-B covalent hybridization. Compared to metallic oP(6)-type RuB(2) and OsB(2) phases, the new phases possess similar mechanical properties and hardness. The combination of the probability of tunable electronic properties, strong stiffness and high hardness make RuB(2) and OsB(2) attractive and interesting for advanced applications. © 2011 IOP Publishing Ltd

  1. Scratch Testing of Hot-Pressed Monolithic Chromium Diboride (CrB2) and CrB2 + MoSi2 Composite

    NASA Astrophysics Data System (ADS)

    Bhatt, B.; Murthy, T. S. R. Ch.; Singh, K.; Sashanka, A.; Vishwanadh, B.; Sonber, J. K.; Sairam, K.; Nageswara Rao, G. V. S.; Srinivasa Rao, T.; Kain, Vivekanand

    2017-10-01

    The tribological performance of hot-pressed monolithic CrB2 and a newly developed CrB2 + 20 vol.% MoSi2 composite was investigated by using scratch test. The test was carried out under progressive loading ranging from 0.9 to 30 N over a scratch distance of 3 mm. In situ values of coefficient of friction (COF), depth of penetration and acoustic emission were recorded. The wear volume and fracture toughness were also calculated. COF of both materials is increased with increasing the scratch length and progressive load. COF of the composite was observed to be slightly higher compared to the monolithic CrB2. The wear volume of the composite is 60% higher compared to monolithic CrB2. Fracture toughness values of 2.48 and 2.81 MPa m1/2 were calculated for monolithic CrB2 and CrB2 + 20 vol.% MoSi2 composite, respectively. Microstructural characterization indicates that the abrasive wear is the dominant wear mechanism in both the materials.

  2. Opiate-induced motor stimulation is regulated by gamma-aminobutyric acid type B receptors found in the ventral tegmental area in mice.

    PubMed

    Leite-Morris, Kimberly A; Fukudome, Eugene Y; Kaplan, Gary B

    2002-01-14

    Recent studies suggest that gamma-aminobutyric acid type B (GABA(B)) receptors located on dopaminergic cells in the ventral tegmental area (VTA) regulate mesolimbic dopaminergic (A10) activity. In the current study, we identified GABA(B) receptor subtypes in the area of the VTA and examined their role in modulating acute opiate actions. We studied the effects of intra-VTA infusions of the selective GABA(B) agonist baclofen on morphine-induced locomotor stimulation and A10 neuronal activation. Drug treatments were followed by ambulatory activity monitoring for 180 min. Intra-VTA baclofen treatment produced a 70% inhibition of morphine-stimulated locomotor activity. Furthermore, functional activation of A10 neurons was assessed by immunohistochemical staining of c-Fos in the nucleus accumbens (NAc), where A10 neurons terminate. We found that morphine treatment increased the levels of Fos-positive nuclei in the NAc, while intra-VTA baclofen treatment reversed morphine's effects. Finally, GABA(B) receptor subtypes and isoforms were identified in the ventromedial mesencephalon using immunoblotting. We demonstrated the presence of GABA(B)R1a (130 kDa), GABA(B)R1b (100 kDa), and GABA(B)R2 (120 kDa) receptor subtypes in this region. These results suggest that GABA(B) receptor isoforms are found in the VTA and their activation results in the blockade of behavioral effects of opiates via inhibition of dopaminergic neurotransmission.

  3. Structure investigation of maltacine B1a, B1b, B2a and B2b: cyclic peptide lactones of the maltacine complex from Bacillus subtilis.

    PubMed

    Hagelin, Gunnar

    2005-04-01

    A new complex of cyclic peptide lactone antibiotics from Bacillus subtilis, which we named maltacines, has recently been described. The structure elucidation of four of them is reported in this paper. The amino acid sequences and structures of the peptides were found by MSn of the ring-opened linear peptides that gave uninterrupted sequences of Bn and Y''n ions. The identities of three unknown residues in the sequences were solved by a combination of derivatization with phenyl isothiocyanate (PITC), high-resolution mass spectrometry and H/D exchange. The nature and position of the cyclic structure were revealed by a chemoselective ring opening with Na18OH and was found to be a lactone formed between a hydroxyl of residue number 4 and the C-terminal amino acid number 12. For verification of the structure of the B2+ ion, peptides with different combinations of P/Q and P/K at the N-terminus were synthesized. The structures of the four peptides were found to be as follows: B1a/B2a, cyclo-4,12(P-Q-Y-HNLeu-A-E-T-Y-Orn-103-Y-I-OH); and B1b/B2b, cyclo-4,12(P-Q-Y-HNLeu-A-E-T-Y-K-103-Y-I-OH). Copyright 2005 John Wiley & Sons, Ltd.

  4. A Rare Variant Identified Within the GluN2B C-Terminus in a Patient with Autism Affects NMDA Receptor Surface Expression and Spine Density.

    PubMed

    Liu, Shuxi; Zhou, Liang; Yuan, Hongjie; Vieira, Marta; Sanz-Clemente, Antonio; Badger, John D; Lu, Wei; Traynelis, Stephen F; Roche, Katherine W

    2017-04-12

    NMDA receptors (NMDARs) are ionotropic glutamate receptors that are crucial for neuronal development and higher cognitive processes. NMDAR dysfunction is involved in a variety of neurological and psychiatric diseases; however, the mechanistic link between the human pathology and NMDAR dysfunction is poorly understood. Rare missense variants within NMDAR subunits have been identified in numerous patients with mental or neurological disorders. We specifically focused on the GluN2B NMDAR subunit, which is highly expressed in the hippocampus and cortex throughout development. We analyzed several variants located in the GluN2B C terminus and found that three variants in patients with autism (S1415L) or schizophrenia (L1424F and S1452F) (S1413L, L1422F, and S1450F in rodents, respectively) displayed impaired binding to membrane-associated guanylate kinase (MAGUK) proteins. In addition, we observed a deficit in surface expression for GluN2B S1413L. Furthermore, there were fewer dendritic spines in GluN2B S1413L-expressing neurons. Importantly, synaptic NMDAR currents in neurons transfected with GluN2B S1413L in GluN2A/B-deficient mouse brain slices revealed only partial rescue of synaptic current amplitude. Functional properties of GluN2B S1413L in recombinant systems revealed no change in receptor properties, consistent with synaptic defects being the result of reduced trafficking and targeting of GluN2B S1413L to the synapse. Therefore, we find that GluN2B S1413L displays deficits in NMDAR trafficking, synaptic currents, and spine density, raising the possibility that this mutation may contribute to the phenotype in this autism patient. More broadly, our research demonstrates that the targeted study of certain residues in NMDARs based on rare variants identified in patients is a powerful approach to studying receptor function. SIGNIFICANCE STATEMENT We have used a "bedside-to-bench" approach to investigate the functional regulation of NMDA receptors (NMDARs). Using

  5. Modulation of E2F activity in primary mouse B cells following stimulation via surface IgM and CD40 receptors.

    PubMed

    Lam, E W; Glassford, J; van der Sman, J; Banerji, L; Pizzey, A R; Shaun, N; Thomas, B; Klaus, G G

    1999-10-01

    Since signals via CD40 and the B cell receptor are known to synergize to induce B cell activation, we have analyzed the pocket protein/E2F complexes in mouse B lymphocytes following stimulation by anti-IgM, anti-CD40, alone or together. We find that E2F4 and DP1 form the predominant E2F heterodimers in the G0 and G1 phases of the cell cycle, complexed with hypophosphorylated p130. During late G1 and S phase this complex is replaced by at least three different E2F complexes, one of which is an E2F complex containing p107 or pRB as well as two "free" E2F complexes consisting of E2F4/DP1 and E2F1-3/DP1. These effects were mirrored by the levels and phosphorylation status of the three pocket proteins. We also observed an increase in electrophoretic mobility of DP1 and E2F4 as B cells progressed from G0 into early G1, resulting from their dephosphorylation. This is known to correlate with a decrease in DNA binding capacity of these proteins and could also be important for derepression of genes negatively regulated through E2F sites in their promoters. These results therefore indicate that the pRB/E2F pathway integrates proliferative signals emanating from the sIgM and CD40 receptors.

  6. GABA(B) receptor modulation of feedforward inhibition through hippocampal neurogliaform cells.

    PubMed

    Price, Christopher J; Scott, Ricardo; Rusakov, Dmitri A; Capogna, Marco

    2008-07-02

    Feedforward inhibition of neurons is a fundamental component of information flow control in the brain. We studied the roles played by neurogliaform cells (NGFCs) of stratum lacunosum moleculare of the hippocampus in providing feedforward inhibition to CA1 pyramidal cells. We recorded from synaptically coupled pairs of anatomically identified NGFCs and CA1 pyramidal cells and found that, strikingly, a single presynaptic action potential evoked a biphasic unitary IPSC (uIPSC), consisting of two distinct components mediated by GABA(A) and GABA(B) receptors. A GABA(B) receptor-mediated unitary response has not previously been observed in hippocampal excitatory neurons. The decay of the GABA(A) receptor-mediated response was slow (time constant = 50 ms), and was tightly regulated by presynaptic GABA(B) receptors. Surprisingly, the GABA(B) receptor ligands baclofen and (2S)-3-{[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl}(phenylmethyl)phosphinic acid (CGP55845), while affecting the NGFC-mediated uIPSCs, had no effect on action potential-evoked presynaptic Ca2+ signals monitored in individual axonal boutons of NGFCs with two-photon microscopy. In contrast, baclofen clearly depressed presynaptic Ca2+ transients in non-NGF interneurons. Changes in extracellular Ca2+ concentration that mimicked the effects of baclofen or CGP55845 on uIPSCs significantly altered presynaptic Ca2+ transients. Electrophysiological data suggest that GABA(B) receptors expressed by NGFCs contribute to the dynamic control of the excitatory input to CA1 pyramidal neurons from the temporoammonic path. The NGFC-CA1 pyramidal cell connection therefore provides a unique and subtle mechanism to shape the integration time domain for signals arriving via a major excitatory input to CA1 pyramidal cells.

  7. [Receptors of selected cytokines and angiokine bFGF in patients with colorectal cancer (a preliminary study)].

    PubMed

    Grotowski, M; Piechota, W

    2001-11-01

    The aim of the study was to examine the frequency of increased serum levels of the soluble receptors TNF and IL-2 and angiokine bFGF in colorectal cancer patients. Also correlation between their concentrations and stage of the tumor was made. The study was done on group consisted of 30 diagnosed colorectal cancer patients, with different location and stage of the tumor. The used classification of stage of the tumor was described by Dukes. The results were compared with control group consisted of 10 healthy persons. The examined factors were assayed by ELISA method (R&D Systems Minneapolis). In colorectal cancer group the serum levels of sTNFRI were increased 1.8 times, sTNFRII 1.4 times, sIL-2R 2.2 times and bFGF 5.3 times in comparison with control group. The serum levels of sTNFRI and sTNFRII showed increased tendency in stage D of colorectal cancer. The serum levels of sIL-2R were the highest in stage D. The serum levels of bFGF showed increased tendency in stage A and B and correlated with stage D of the tumor. This results permit for further study on usefulness of sTNFRI, sTNFRII, sIL-2R and bFGF as a markers for colorectal cancer in clinical use.

  8. Crystal growth, crystal structure of new polymorphic modification, β-Bi 2B 8O 15 and thermal expansion of α-Bi 2B 8O 15

    NASA Astrophysics Data System (ADS)

    Bubnova, R. S.; Alexandrova, J. V.; Krivovichev, S. V.; Filatov, S. K.; Egorysheva, A. V.

    2010-02-01

    Single crystals of α- and β-polymorphs of Bi 2B 8O 15 were grown by Czochralski method from a charge of the stoichiometric composition. The crystal structure of β-Bi 2B 8O 15 was solved by direct methods from a twinned crystal and refined to R1=0.081 (w R=0.198) on the basis of 1584 unique observed reflections ( I>2 σ( I)). The compound is triclinic, space group P1¯, a=4.3159(8), b=6. 4604(12), c=22.485(4) Å, α=87.094(15)°, β=86.538(15)°, γ=74.420(14)°, V=602.40(19) Å 3, Z=2. The B-O layered anion of β-Bi 2B 8O 15 is topologically identical to the anion of α-Bi 2B 8O 15 but the orientation of neighboring layers is different. Thermal expansion of α-Bi 2B 8O 15 has been investigated by X-ray powder diffraction in air in temperature range from 20 to 700 °C. It is strongly anisotropic, which can be explained by the hinge mechanism applied to chains of Bi-O polyhedra. While the anisotropy of thermal expansion is rather high, the volume thermal expansion coefficient α V=40×10 6 °C -1 for α-Bi 2B 8O 15 is close to those of other bismuth borates.

  9. NR2A- and NR2B-Containing NMDA Receptors in the Prelimbic Medial Prefrontal Cortex Differentially Mediate Trace, Delay, and Contextual Fear Conditioning

    ERIC Educational Resources Information Center

    Gilmartin, Marieke R.; Kwapis, Janine L.; Helmstetter, Fred J.

    2013-01-01

    Activation of "N"-methyl-D-aspartate receptors (NMDAR) in the prelimbic medial prefrontal cortex (PL mPFC) is necessary for the acquisition of both trace and contextual fear memories, but it is not known how specific NR2 subunits support each association. The NR2B subunit confers unique properties to the NMDAR and may differentially…

  10. SMART-on-FHIR implemented over i2b2

    PubMed Central

    Mandel, Joshua C; Klann, Jeffery G; Wattanasin, Nich; Mendis, Michael; Chute, Christopher G; Mandl, Kenneth D; Murphy, Shawn N

    2017-01-01

    We have developed an interface to serve patient data from Informatics for Integrating Biology and the Bedside (i2b2) repositories in the Fast Healthcare Interoperability Resources (FHIR) format, referred to as a SMART-on-FHIR cell. The cell serves FHIR resources on a per-patient basis, and supports the “substitutable” modular third-party applications (SMART) OAuth2 specification for authorization of client applications. It is implemented as an i2b2 server plug-in, consisting of 6 modules: authentication, REST, i2b2-to-FHIR converter, resource enrichment, query engine, and cache. The source code is freely available as open source. We tested the cell by accessing resources from a test i2b2 installation, demonstrating that a SMART app can be launched from the cell that accesses patient data stored in i2b2. We successfully retrieved demographics, medications, labs, and diagnoses for test patients. The SMART-on-FHIR cell will enable i2b2 sites to provide simplified but secure data access in FHIR format, and will spur innovation and interoperability. Further, it transforms i2b2 into an apps platform. PMID:27274012

  11. SCFAtPP2-B11 modulates ABA signaling by facilitating SnRK2.3 degradation in Arabidopsis thaliana

    PubMed Central

    Ren, Ziyin; Zhi, Liya; Yao, Bin; Su, Chao; Liu, Liu; Li, Xia

    2017-01-01

    The phytohormone abscisic acid (ABA) is an essential part of the plant response to abiotic stressors such as drought. Upon the perception of ABA, pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) proteins interact with co-receptor protein phosphatase type 2Cs to permit activation Snf1-related protein kinase2 (SnRK2) kinases, which switch on ABA signaling by phosphorylating various target proteins. Thus, SnRK2 kinases are central regulators of ABA signaling. However, the mechanisms that regulate SnRK2 degradation remain elusive. Here, we show that SnRK2.3 is degradated by 26S proteasome system and ABA promotes its degradation. We found that SnRK2.3 interacts with AtPP2-B11 directly. AtPP2-B11 is an F-box protein that is part of a SKP1/Cullin/F-box E3 ubiquitin ligase complex that negatively regulates plant responses to ABA by specifically promoting the degradation of SnRK2.3. AtPP2-B11 was induced by ABA, and the knockdown of AtPP2-B11 expression markedly increased the ABA sensitivity of plants during seed germination and postgerminative development. Overexpression of AtPP2-B11 does not affect ABA sensitivity, but inhibits the ABA hypersensitive phenotypes of SnRK2.3 overexpression lines. These results reveal a novel mechanism through which AtPP2-B11 specifically degrades SnRK2.3 to attenuate ABA signaling and the abiotic stress response in Arabidopsis. PMID:28787436

  12. Beneficial Role of Erythrocyte Adenosine A2B Receptor-Mediated AMP-Activated Protein Kinase Activation in High-Altitude Hypoxia.

    PubMed

    Liu, Hong; Zhang, Yujin; Wu, Hongyu; D'Alessandro, Angelo; Yegutkin, Gennady G; Song, Anren; Sun, Kaiqi; Li, Jessica; Cheng, Ning-Yuan; Huang, Aji; Edward Wen, Yuan; Weng, Ting Ting; Luo, Fayong; Nemkov, Travis; Sun, Hong; Kellems, Rodney E; Karmouty-Quintana, Harry; Hansen, Kirk C; Zhao, Bihong; Subudhi, Andrew W; Jameson-Van Houten, Sonja; Julian, Colleen G; Lovering, Andrew T; Eltzschig, Holger K; Blackburn, Michael R; Roach, Robert C; Xia, Yang

    2016-08-02

    High altitude is a challenging condition caused by insufficient oxygen supply. Inability to adjust to hypoxia may lead to pulmonary edema, stroke, cardiovascular dysfunction, and even death. Thus, understanding the molecular basis of adaptation to high altitude may reveal novel therapeutics to counteract the detrimental consequences of hypoxia. Using high-throughput, unbiased metabolomic profiling, we report that the metabolic pathway responsible for production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity, was significantly induced in 21 healthy humans within 2 hours of arrival at 5260 m and further increased after 16 days at 5260 m. This finding led us to discover that plasma adenosine concentrations and soluble CD73 activity rapidly increased at high altitude and were associated with elevated erythrocyte 2,3-BPG levels and O2 releasing capacity. Mouse genetic studies demonstrated that elevated CD73 contributed to hypoxia-induced adenosine accumulation and that elevated adenosine-mediated erythrocyte A2B adenosine receptor activation was beneficial by inducing 2,3-BPG production and triggering O2 release to prevent multiple tissue hypoxia, inflammation, and pulmonary vascular leakage. Mechanistically, we demonstrated that erythrocyte AMP-activated protein kinase was activated in humans at high altitude and that AMP-activated protein kinase is a key protein functioning downstream of the A2B adenosine receptor, phosphorylating and activating BPG mutase and thus inducing 2,3-BPG production and O2 release from erythrocytes. Significantly, preclinical studies demonstrated that activation of AMP-activated protein kinase enhanced BPG mutase activation, 2,3-BPG production, and O2 release capacity in CD73-deficient mice, in erythrocyte-specific A2B adenosine receptor knockouts, and in wild-type mice and in turn reduced tissue hypoxia and inflammation. Together, human and mouse studies reveal novel

  13. The zinc finger gene Krox20 regulates HoxB2 (Hox2.8) during hindbrain segmentation.

    PubMed

    Sham, M H; Vesque, C; Nonchev, S; Marshall, H; Frain, M; Gupta, R D; Whiting, J; Wilkinson, D; Charnay, P; Krumlauf, R

    1993-01-29

    The zinc finger gene Krox20 and many Hox homeobox genes are expressed in segment-restricted domains in the hindbrain. The restricted expression patterns appear before morphological segmentation, suggesting that these transcription factors may play an early role in the establishment and identity of rhombomeric segments. In this paper, we show that the HoxB2 (Hox2.8) gene is normally upregulated in rhombomeres (r) 3, 4, and 5, and we identify an enhancer region upstream of the gene that imposes r3/r5 expression in transgenic mice. This enhancer contains three Krox20-binding sites required in vitro for complex formation with Krox20 protein and in vivo for rhombomere-restricted expression. In transgenic mice, Krox20 expressed in ectopic domains can transactivate a reporter construct containing the HoxB2 r3/r5 enhancer. These data demonstrate that Krox20 is a part of the upstream transcriptional cascade that directly regulates HoxB2 expression during hindbrain segmentation.

  14. Recombinant human B7.2 IgV-like domain expressed in bacteria maintains its co-stimulatory activity in vitro.

    PubMed

    Yan, Xiaocai; Ma, Jun; Zheng, Jin; Lai, Baochang; Geng, Yiping; Wang, Yili; Si, Lüsheng

    2002-07-01

    To investigate which of the two immunoglobulin (Ig)-like domains, the immunoglobulin variable region homologous domain IgV (hB7.2 IgV) and the immunoglobulin constant region homologous domain IgC (hB7.2 IgC) on the human B7.2 molecule contains receptor binding sites, and to evaluate whether the B7.2 protein expressed in bacteria has biological activity in vitro. Three fragments of hB7.2 IgV,hB7.2 IgC and the complete extracellular region of human B7.2 containing both the IgV and IgC domains,hB7.2 Ig (V+C), were amplified by PCR and subcloned into pGEM-Teasy. Three recombinants,pGEX-4T-3-hB7.2 IgV,pGEX-4T-3-hB7.2 IgC and pGEX-4T-3-hB7.2 Ig (V+C), were generated by cloning the fragments into a prokaryote expression plasmid (pGEX-4T-3) and transformed into the host strain E. coli DH5alpha. The relevant target fusion proteins consisting of GST and hB7.2 IgV,hB7.2 IgC and hB7.2 Ig (V+C), were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7.2 fusion proteins by [(3)H]-TdR incorporation. Three recombinant fusion proteins of human B7.2, GST-hB7.2 IgV, GST-hB7.2 IgC and GST-hB7.2 Ig (V+C) were produced and detected in inclusion body form from engineered bacteria. With the first signal present,T lymphocytes proliferated when co-stimulated by bacterially-produced either GST-hB7.2 Ig (V+C) or GST-hB7.2 IgV fusion proteins, but not by GST-hB7.2 IgC. Functional human B7.2 fusion protein can be produced in bacteria. The IgV-like domain of human B7.2 is sufficient for B7.2 to interact with its counter-receptors and co-stimulate T lymphocytes.

  15. In vitro and in vivo biotransformation of WMS-1410, a potent GluN2B selective NMDA receptor antagonist.

    PubMed

    Falck, Evamaria; Begrow, Frank; Verspohl, Eugen J; Wünsch, Bernhard

    2014-06-01

    Structural modification of the GluN2B selective NMDA receptor antagonist ifenprodil led to the 3-benzazepine WMS-1410 with similar GluN2B affinity but higher receptor selectivity. Herein the in vitro and in vivo biotransformation of WMS-1410 is reported. Incubation of WMS-1410 with rat liver microsomes and different cofactors resulted in four hydroxylated phase I metabolites, two phase II metabolites and five combined phase I/II metabolites. With exception of catechol 4, these metabolites were also identified in the urine of a rat treated with WMS-1410. However the metabolites 7, 8 and 12 clearly show that the catechol metabolite 4 was also formed in vivo. As shown for ifenprodil the phenol of WMS-1410 represents the metabolically most reactive structural element. The biotransformation of WMS-1410 is considerably slower than the biotransformation of ifenprodil indicating a higher metabolic stability. From the viewpoint of metabolic stability the bioisosteric replacement of the phenol of WMS-1410 by a metabolically more stable moiety should be favourable. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. GluN2B-containing NMDA receptors and AMPA receptors in medial prefrontal cortex are necessary for odor span in rats.

    PubMed

    Davies, Don A; Greba, Quentin; Howland, John G

    2013-01-01

    Working memory is a type of short-term memory involved in the maintenance and manipulation of information essential for complex cognition. While memory span capacity has been extensively studied in humans as a measure of working memory, it has received considerably less attention in rodents. Our aim was to examine the role of the N-methyl-D-aspartate (NMDA) and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors in odor span capacity using systemic injections or infusions of receptor antagonists into the medial prefrontal cortex (mPFC). Long Evans rats were trained on a well-characterized odor span task (OST). Initially, rats were trained to dig for a food reward in sand followed by training on a non-match to sample discrimination using sand scented with household spices. The rats were then required to perform a serial delayed non-match to sample procedure which was their odor span. Systemic injection of the broad spectrum NMDA receptor antagonist 3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) (10 mg/kg) or the GluN2B-selective antagonist Ro 25-6981 (10 mg/kg but not 6 mg/kg) significantly reduced odor span capacity. Infusions of the GluN2B- selective antagonist Ro 25-6981 (2.5 μg/hemisphere) into mPFC reduced span capacity, an effect that was nearly significant (p = 0.069). Infusions of the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (1.25 μg/hemisphere) into mPFC reduced span capacity and latency for the rats to make a choice in the task. These results demonstrate span capacity in rats depends on ionotropic glutamate receptor activation in the mPFC. Further understanding of the circuitry underlying span capacity may aid in the novel therapeutic drug development for persons with working memory impairments as a result of disorders such as schizophrenia and Alzheimer's disease.

  17. Growth inhibitory effects of the dual ErbB1/ErbB2 tyrosine kinase inhibitor PKI-166 on human prostate cancer xenografts.

    PubMed

    Mellinghoff, Ingo K; Tran, Chris; Sawyers, Charles L

    2002-09-15

    Experiments with human prostate cancer cell lines have shown that forced overexpression of the ErbB2-receptor tyrosine kinase (RTK) promotes androgen-independent growth and increases androgen receptor-transcriptional activity in a ligand-independent fashion. To investigate the relationship between ErbB-RTK signaling and androgen in genetically unmanipulated human prostate cancer, we performed biochemical and biological studies with the dual ErbB1/ErbB2 RTK inhibitor PKI-166 using human prostate cancer xenograft models with isogenic sublines reflecting the transition from androgen-dependent to androgen-independent growth. In the presence of low androgen concentrations, PKI-166 showed profound growth-inhibitory effects on tumor growth, which could be partially reversed by androgen add-back. At physiological androgen concentrations, androgen withdrawal greatly enhanced the ability of PKI-166 to retard tumor growth. The level of extracellular signal-regulated kinase activation correlated with the response to PKI-166 treatment, whereas the expression levels of ErbB1 and ErbB2 did not. These results suggest that ErbB1/ErbB2 RTKs play an important role in the biology of androgen-independent prostate cancer and provide a rationale for clinical evaluation of inhibitors targeted to this pathway.

  18. Neratinib, an irreversible ErbB receptor tyrosine kinase inhibitor, in patients with advanced ErbB2-positive breast cancer.

    PubMed

    Burstein, Harold J; Sun, Yan; Dirix, Luc Y; Jiang, Zefei; Paridaens, Robert; Tan, Antoinette R; Awada, Ahmad; Ranade, Anantbhushan; Jiao, Shunchang; Schwartz, Gary; Abbas, Richat; Powell, Christine; Turnbull, Kathleen; Vermette, Jennifer; Zacharchuk, Charles; Badwe, Rajendra

    2010-03-10

    Neratinib is an oral, irreversible pan-ErbB receptor tyrosine kinase inhibitor. The efficacy and safety of neratinib were evaluated in two cohorts of patients with advanced ErbB2-positive breast cancer-those with and those without prior trastuzumab treatment-in an open-label, multicenter, phase II trial. Patients in the two cohorts (prior trastuzumab, n = 66; no prior trastuzumab, n = 70) received oral neratinib 240 mg once daily. The primary end point was the 16-week progression-free survival (PFS) rate for the evaluable population (prior trastuzumab, n = 63; no prior trastuzumab, n = 64), as assessed by independent review. The 16-week PFS rates were 59% for patients with prior trastuzumab treatment and 78% for patients with no prior trastuzumab treatment. Median PFS was 22.3 and 39.6 weeks, respectively. Objective response rates were 24% among patients with prior trastuzumab treatment and 56% in the trastuzumab-naïve cohort. The most common adverse events were diarrhea, nausea, vomiting, and fatigue. Diarrhea was the most frequent grades 3 to 4 adverse event, occurring in 30% of patients with prior trastuzumab treatment and in 13% of patients with no prior trastuzumab treatment, which prompted dose reductions in 29% and 4% of patients, respectively, but treatment discontinuation in only one patient. No neratinib-related, grades 3 or 4 cardiotoxicity was reported. Oral neratinib showed substantial clinical activity and was reasonably well tolerated among both heavily pretreated and trastuzumab-naïve patients who had advanced, ErbB2-positive breast cancer. Diarrhea was the most common adverse effect but was manageable with antidiarrheal agents and dose modification.

  19. Soliton cellular automaton associated with Dn(1)-crystal B2,s

    NASA Astrophysics Data System (ADS)

    Misra, Kailash C.; Wilson, Evan A.

    2013-04-01

    A solvable vertex model in ferromagnetic regime gives rise to a soliton cellular automaton which is a discrete dynamical system in which site variables take on values in a finite set. We study the scattering of a class of soliton cellular automata associated with the U_q(D_n^{(1)})-perfect crystal B2, s. We calculate the combinatorial R matrix for all elements of B2, s ⊗ B2, 1. In particular, we show that the scattering rule for our soliton cellular automaton can be identified with the combinatorial R matrix for U_q(A_1^{(1)}) oplus U_q(D_{n-2}^{(1)})-crystals.

  20. ErbB2 and EGFR are downmodulated during the differentiation of 3T3-L1 preadipocytes.

    PubMed

    Pagano, Eleonora; Calvo, Juan Carlos

    2003-10-15

    The expression of receptors belonging to the epidermal growth factor receptor subfamily has been largely studied these last years in epithelial cells mainly as involved in cell proliferation and malignant progression. Although much work has focused on the role of these growth factor receptors in the differentiation of a variety of tissues, there is little information in regards to normal stromal cells. We investigated erbB2 expression in the murine fibroblast cell line Swiss 3T3L1, which naturally or hormonally induced undergoes adipocyte differentiation. We found that the Swiss 3T3-L1 fibroblasts express erbB2, in addition to EGFR, and in a quantity comparable to or even greater than the breast cancer cell line T47D. Proliferating cells increased erbB2 and EGFR levels when reaching confluence up to 4- and 10-fold, respectively. This expression showed a significant decrease when growth-arrested cells were stimulated to differentiate with dexamethasone and isobutyl-methylxanthine. Differentiated cells presented a decreased expression of both erbB2 and EGFR regardless of whether the cells were hormonally or spontaneously differentiated. EGF stimulation of serum-starved cells increased erbB2 tyrosine phosphorylation and retarded erbB2 migration in SDS-PAGE, suggesting receptor association and activation. Heregulin-alpha1 and -beta1, two EGF related factors, had no effect on erbB2 or EGFR phosphorylation. Although 3T3-L1 cells expressed heregulin, its specific receptors, erbB3 and erbB4, were not found. This is the first time in which erbB2 is reported to be expressed in an adipocytic cell line which does not depend on non EGF family growth factors (thyroid hormone, growth hormone, etc.) to accomplish adipose differentiation. Since erbB2 and EGFR expression were downmodulated as differentiation progressed it is conceivable that a mechanism of switching from a mitogenic to a differentiating signaling pathway may be involved, through regulation of the expression of these

  1. Amygdala EphB2 Signaling Regulates Glutamatergic Neuron Maturation and Innate Fear.

    PubMed

    Zhu, Xiao-Na; Liu, Xian-Dong; Zhuang, Hanyi; Henkemeyer, Mark; Yang, Jing-Yu; Xu, Nan-Jie

    2016-09-28

    The amygdala serves as emotional center to mediate innate fear behaviors that are reflected through neuronal responses to environmental aversive cues. However, the molecular mechanism underlying the initial neuron responses is poorly understood. In this study, we monitored the innate defensive responses to aversive stimuli of either elevated plus maze or predator odor in juvenile mice and found that glutamatergic neurons were activated in amygdala. Loss of EphB2, a receptor tyrosine kinase expressed in amygdala neurons, suppressed the reactions and led to defects in spine morphogenesis and fear behaviors. We further found a coupling of spinogenesis with these threat cues induced neuron activation in developing amygdala that was controlled by EphB2. A constitutively active form of EphB2 was sufficient to rescue the behavioral and morphological defects caused by ablation of ephrin-B3, a brain-enriched ligand to EphB2. These data suggest that kinase-dependent EphB2 intracellular signaling plays a major role for innate fear responses during the critical developing period, in which spinogenesis in amygdala glutamatergic neurons was involved. Generation of innate fear responses to threat as an evolutionally conserved brain feature relies on development of functional neural circuit in amygdala, but the molecular mechanism remains largely unknown. We here identify that EphB2 receptor tyrosine kinase, which is specifically expressed in glutamatergic neurons, is required for the innate fear responses in the neonatal brain. We further reveal that EphB2 mediates coordination of spinogenesis and neuron activation in amygdala during the critical period for the innate fear. EphB2 catalytic activity plays a major role for the behavior upon EphB-ephrin-B3 binding and transnucleus neuronal connections. Our work thus indicates an essential synaptic molecular signaling within amygdala that controls synapse development and helps bring about innate fear emotions in the postnatal

  2. Dephosphorylation of GluN2B C-Terminal Tyrosine Residues Does Not Contribute to Acute Ethanol Inhibition of Recombinant NMDA Receptors

    PubMed Central

    Hughes, Benjamin A.; Smothers, Corigan T.; Woodward, John J.

    2013-01-01

    N-methyl-D-aspartate (NMDA) receptors are ion channels activated by the neurotransmitter glutamate and are highly expressed by neurons. These receptors are critical for excitatory synaptic signaling and inhibition of NMDA receptors leads to impaired cognition and learning. Ethanol inhibits NMDA currents at concentrations associated with intoxication and this action may underlie some of the behavioral effects of ethanol. Although numerous sites and mechanisms of action have been tested, the manner in which ethanol inhibits NMDA receptors remains unclear. Recent findings in the literature suggest that ethanol, via facilitation of tyrosine phosphatase activity, may dephosphorylate key tyrosine residues in the C-terminus of GluN2B subunits resulting in diminished channel function. To directly test this hypothesis, we engineered GluN2B mutants that contained phenylalanine in place of tyrosine at three different sites and transiently expressed them with the GluN1 subunit in human embryonic kidney (HEK) cells. Whole-cell patch clamp electrophysiology was used to record glutamate-activated currents in the absence and presence of ethanol (10–600 mM). All mutants were functional and did not differ from one another with respect to current amplitude, steady-state to peak ratio, or magnesium block. Analysis of ethanol dose-response curves showed no significant difference in IC50 values between wild-type receptors and Y1252F, Y1336F, Y1472F or triple Y-F mutants. These findings suggest that dephosphorylation of C-terminal tyrosine residues does not account for ethanol inhibition of GluN2B receptors. PMID:23357553

  3. The 2√{3}×2√{3}R30 surface reconstruction of alkali/Si(1 1 1):B semiconducting surfaces

    NASA Astrophysics Data System (ADS)

    Tournier-Colletta, C.; Chaput, L.; Tejeda, A.; Cardenas, L. A.; Kierren, B.; Malterre, D.; Fagot-Revurat, Y.; Fèvre, P. Le; Bertran, F.; Taleb-Ibrahimi, A.

    2013-02-01

    The surface structure of alkali doped Si(1 1 1):B ultra-thin films has been studied by low-energy electron diffraction (LEED), X-ray photoemission spectroscopy (XPS) and scanning tunneling microscopy (STM). A comparative study of K/Si(1 1 1)-3 × 1 and K/Si(1 1 1):B-2√{3}×2√{3}R30 interfaces allowed us to determine the saturation coverage to be 0.5 monolayer in the later case. The 2√{3}-surface reconstruction is shown to be a common property of pure K, Rb, Cs materials and K0.4Rb0.6 alloys but progressively disappears if Rb is replaced by Ca. Taking into account the existence of two distinct boron sites in the ratio 1/3 as seen from B-1s core levels spectra, LAPW-DFT calculations have been carried out in order to optimize the atomic structure. As a result, alkali adatoms are shown to form trimers leading to a large modulation of the Sisbnd B bonds accompanied by an inhomogeneous doping of the dangling bonds in agreement with voltage dependent STM images.

  4. Selective Inhibition of Tumor Growth by Clonal NK Cells Expressing an ErbB2/HER2-Specific Chimeric Antigen Receptor

    PubMed Central

    Schönfeld, Kurt; Sahm, Christiane; Zhang, Congcong; Naundorf, Sonja; Brendel, Christian; Odendahl, Marcus; Nowakowska, Paulina; Bönig, Halvard; Köhl, Ulrike; Kloess, Stephan; Köhler, Sylvia; Holtgreve-Grez, Heidi; Jauch, Anna; Schmidt, Manfred; Schubert, Ralf; Kühlcke, Klaus; Seifried, Erhard; Klingemann, Hans G; Rieger, Michael A; Tonn, Torsten; Grez, Manuel; Wels, Winfried S

    2015-01-01

    Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy. PMID:25373520

  5. Studies on the biosynthesis of vitamin B sub 2 and vitamin B sub 12

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, H.C.

    1988-01-01

    Feeding experiments with Ashbya gossypii followed by NMR analysis of the resulting riboflavin showed incorporation of deuterium from D-(2-{sup 2}H)ribose at C-2{prime} and from D-(1-{sup 2}H)ribose in the pro-R position at C-1{prime} of the ribityl side chain. The results rule out an Amadori rearrangement mechanism for the reduction of the ribosylamino to the ribitylamino linkage and point to formation of a Schiff base that is reduced stereospecifically opposite to the face from which the oxygen has departed. As prerequisite for the analysis, the {sup 1}H NMR signals for the pro-R and pro-S hydrogens at C-1{prime} of riboflavin and its tetraacetatemore » were assigned with the aid of synthetic stereospecifically deuteriated samples. Feeding experiments with Propionibacterium shermianii followed by NMR analysis of the resulting vitamin B{sub 12} showed: (1) 5-methylbenzimidazole (5MBI) incorporated and only one regioisomer (B6-demethylcyanocobalamin)formed. (2) 8-demethylriboflavin incorporated and the same regioisomer was obtained as 5MBI experiment. (3) (1{prime}-{sup 13}C, 5-{sup 15}N)riboflavin incorporated and {sup 13}C-NMR showed that {sup 13}C at the B2 position of cyanocobalamin coupled to both adjacent nitrogen-15 atoms at about the same ratio.« less

  6. Enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7.

    PubMed

    Fang, Zhong-Ze; Wang, Haina; Cao, Yun-Feng; Sun, Dong-Xue; Wang, Li-Xuan; Hong, Mo; Huang, Ting; Chen, Jian-Xing; Zeng, Jia

    2015-03-01

    UDP-glucuronosyltransferases (UGTs)-catalyzed glucuronidation conjugation reaction plays an important role in the elimination of many important clinical drugs and endogenous substances. The present study aims to investigate the enantioselective inhibition of carprofen towards UGT isoforms. In vitro a recombinant UGT isoforms-catalyzed 4-methylumbelliferone (4-MU) glucuronidation incubation mixture was used to screen the inhibition potential of (R)-carprofen and (S)-carprofen towards multiple UGT isoforms. The results showed that (S)-carprofen exhibited stronger inhibition potential than (R)-carprofen towards UGT2B7. However, no significant difference was observed for the inhibition of (R)-carprofen and (S)-carprofen towards other UGT isoforms. Furthermore, the inhibition kinetic behavior was compared for the inhibition of (S)-carprofen and (R)-carprofen towards UGT2B7. A Lineweaver-Burk plot showed that both (S)-carprofen and (R)-carprofen exhibited competitive inhibition towards UGT2B7-catalyzed 4-MU glucuronidation. The inhibition kinetic parameter (Ki ) was calculated to be 7.0 μM and 31.1 μM for (S)-carprofen and (R)-carprofen, respectively. Based on the standard for drug-drug interaction, the threshold for (S)-carprofen and (R)-carprofen to induce a drug-drug interaction is 0.7 μM and 3.1 μM, respectively. In conclusion, enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7 was demonstrated in the present study. Using the in vitro inhibition kinetic parameter, the concentration threshold of (S)-carprofen and (R)-carprofen to possibly induce the drug-drug interaction was obtained. Therefore, clinical monitoring of the plasma concentration of (S)-carprofen is more important than (R)-carprofen to avoid a possible drug-drug interaction between carprofen and the drugs mainly undergoing UGT2B7-catalyzed metabolism. © 2014 Wiley Periodicals, Inc.

  7. miR-150 influences B-cell receptor signaling in chronic lymphocytic leukemia by regulating expression of GAB1 and FOXP1

    PubMed Central

    Mraz, Marek; Chen, Liguang; Rassenti, Laura Z.; Ghia, Emanuela M.; Li, Hongying; Jepsen, Kristen; Smith, Erin N.; Messer, Karen; Frazer, Kelly A.; Kipps, Thomas J.

    2014-01-01

    We examined the microRNAs (miRNAs) expressed in chronic lymphocytic leukemia (CLL) and identified miR-150 as the most abundant, but with leukemia cell expression levels that varied among patients. CLL cells that expressed ζ-chain–associated protein of 70 kDa (ZAP-70) or that used unmutated immunoglobulin heavy chain variable (IGHV) genes, each had a median expression level of miR-150 that was significantly lower than that of ZAP-70–negative CLL cells or those that used mutated IGHV genes. In samples stratified for expression of miR-150, CLL cells with low-level miR-150 expressed relatively higher levels of forkhead box P1 (FOXP1) and GRB2-associated binding protein 1 (GAB1), genes with 3′ untranslated regions having evolutionary-conserved binding sites for miR-150. High-level expression of miR-150 could repress expression of these genes, which encode proteins that enhance B-cell receptor signaling, a putative CLL-growth/survival signal. Also, high-level expression of miR-150 was a significant independent predictor of longer treatment-free survival or overall survival, whereas an inverse association was observed for high-level expression of GAB1 or FOXP1 for overall survival. This study demonstrates that expression of miR-150 can influence the relative expression of GAB1 and FOXP1 and the signaling potential of the B-cell receptor, thereby possibly accounting for the noted association of expression of miR-150 and disease outcome. PMID:24787006

  8. miR-150 influences B-cell receptor signaling in chronic lymphocytic leukemia by regulating expression of GAB1 and FOXP1.

    PubMed

    Mraz, Marek; Chen, Liguang; Rassenti, Laura Z; Ghia, Emanuela M; Li, Hongying; Jepsen, Kristen; Smith, Erin N; Messer, Karen; Frazer, Kelly A; Kipps, Thomas J

    2014-07-03

    We examined the microRNAs (miRNAs) expressed in chronic lymphocytic leukemia (CLL) and identified miR-150 as the most abundant, but with leukemia cell expression levels that varied among patients. CLL cells that expressed ζ-chain-associated protein of 70 kDa (ZAP-70) or that used unmutated immunoglobulin heavy chain variable (IGHV) genes, each had a median expression level of miR-150 that was significantly lower than that of ZAP-70-negative CLL cells or those that used mutated IGHV genes. In samples stratified for expression of miR-150, CLL cells with low-level miR-150 expressed relatively higher levels of forkhead box P1 (FOXP1) and GRB2-associated binding protein 1 (GAB1), genes with 3' untranslated regions having evolutionary-conserved binding sites for miR-150. High-level expression of miR-150 could repress expression of these genes, which encode proteins that enhance B-cell receptor signaling, a putative CLL-growth/survival signal. Also, high-level expression of miR-150 was a significant independent predictor of longer treatment-free survival or overall survival, whereas an inverse association was observed for high-level expression of GAB1 or FOXP1 for overall survival. This study demonstrates that expression of miR-150 can influence the relative expression of GAB1 and FOXP1 and the signaling potential of the B-cell receptor, thereby possibly accounting for the noted association of expression of miR-150 and disease outcome. © 2014 by The American Society of Hematology.

  9. Olfactomedin-like 2 A and B (OLFML2A and OLFML2B) expression profile in primates (human and baboon).

    PubMed

    Pérez-Ibave, Diana Cristina; González-Alvarez, Rafael; de La Luz Martinez-Fierro, Margarita; Ruiz-Ayma, Gabriel; Luna-Muñoz, Maricela; Martínez-De-Villarreal, Laura Elia; De Lourdes Garza-Rodríguez, María; Reséndez-Pérez, Diana; Mohamed-Noriega, Jibran; Garza-Guajardo, Raquel; Bautista-De-Lucío, Víctor Manuel; Mohamed-Noriega, Karim; Barboza-Quintana, Oralia; Arámburo-De-La-Hoz, Carlos; Barrera-Saldaña, Hugo Alberto; Rodríguez-Sánchez, Irám Pablo

    2016-11-08

    The olfactomedin-like domain (OLFML) is present in at least four families of proteins, including OLFML2A and OLFML2B, which are expressed in adult rat retina cells. However, no expression of their orthologous has ever been reported in human and baboon. The aim of this study was to investigate the expression of OLFML2A and OLFML2B in ocular tissues of baboons (Papio hamadryas) and humans, as a key to elucidate OLFML function in eye physiology. OLFML2A and OLFML2B cDNA detection in ocular tissues of these species was performed by RT-PCR. The amplicons were cloned and sequenced, phylogenetically analyzed and their proteins products were confirmed by immunofluorescence assays. OLFML2A and OLFML2B transcripts were found in human cornea, lens and retina and in baboon cornea, lens, iris and retina. The baboon OLFML2A and OLFML2B ORF sequences have 96% similarity with their human's orthologous. OLFML2A and OLFML2B evolution fits the hypothesis of purifying selection. Phylogenetic analysis shows clear orthology in OLFML2A genes, while OLFML2B orthology is not clear. Expression of OLFML2A and OLFML2B in human and baboon ocular tissues, including their high similarity, make the baboon a powerful model to deduce the physiological and/or metabolic function of these proteins in the eye.

  10. 8-Substituted 1,3-dimethyltetrahydropyrazino[2,1-f]purinediones: Water-soluble adenosine receptor antagonists and monoamine oxidase B inhibitors.

    PubMed

    Brunschweiger, Andreas; Koch, Pierre; Schlenk, Miriam; Rafehi, Muhammad; Radjainia, Hamid; Küppers, Petra; Hinz, Sonja; Pineda, Felipe; Wiese, Michael; Hockemeyer, Jörg; Heer, Jag; Denonne, Frédéric; Müller, Christa E

    2016-11-01

    Multitarget approaches, i.e., addressing two or more targets simultaneously with a therapeutic agent, are hypothesized to offer additive therapeutic benefit for the treatment of neurodegenerative diseases. Validated targets for the treatment of Parkinson's disease are, among others, the A 2A adenosine receptor (AR) and the enzyme monoamine oxidase B (MAO-B). Additional blockade of brain A 1 ARs may also be beneficial. We recently described 8-benzyl-substituted tetrahydropyrazino[2,1-f]purinediones as a new lead structure for the development of such multi-target drugs. We have now designed a new series of tetrahydropyrazino[2,1-f]purinediones to extensively explore their structure-activity-relationships. Several compounds blocked human and rat A 1 and A 2A ARs at similar concentrations representing dual A 1 /A 2A antagonists with high selectivity versus the other AR subtypes. Among the best dual A 1 /A 2A AR antagonists were 8-(3-(4-chlorophenyl)propyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (41, K i human A 1 : 65.5nM, A 2A : 230nM; K i rat A 1 : 352nM, A 2A : 316nM) and 1,3-dimethyl-8-((2-(thiophen-2-yl)thiazol-4-yl)methyl)-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (57, K i human A 1 : 642nM, A 2A : 203nM; K i rat A 1 : 166nM, A 2A : 121nM). Compound 57 was found to be well water-soluble (0.7mg/mL) at a physiological pH value of 7.4. One of the new compounds showed triple-target inhibition: (R)-1,3-dimethyl-8-(2,1,3,4-tetrahydronaphthalen-1-yl)-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (49) was about equipotent at A 1 and A 2A ARs and at MAO-B (K i human A 1 : 393nM, human A 2A : 595nM, IC 50 human MAO-B: 210nM) thus allowing future in vivo explorations of the intended multi-target approach. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Synthesis of Substituted 2,3,5,6-tetraarylbenzo(1,2-b:5,4-b')difurans

    NASA Technical Reports Server (NTRS)

    Abdul-Aziz, Mahmoud; Auping, Judith V.; Meador, Michael A.

    1995-01-01

    A series of substituted 2,3,5,6-tetraarylbenzo(l,2-b:5,4-b')difurans 1 was synthesized. This synthesis is based upon the photocyclization of 2,5-dibenzoylresorcinol dibenzyl ethers to the corresponding tetrahydrobenzo(1,2-b:5,4-b')difurans. Treatment of the photoproducts with methanesulfonyl chloride in pyridine afforded 1 in overall yields ranging from 30-72%. A number of these compounds have high fluorescence quantum yields (of phi(sub f) = 0.76-0.90), and their fluorescence spectra exhibit large solvatochromic shifts. These compounds may be suitable for use as fluorescent probes.

  12. Adenosine A(2B) receptor antagonist PSB603 suppresses tumor growth and metastasis by inhibiting induction of regulatory T cells.

    PubMed

    Kaji, Wakako; Tanaka, Satomi; Tsukimoto, Mitsutoshi; Kojima, Shuji

    2014-04-01

    Regulatory T cells (Treg) play a role in suppression of immune response, including anti-tumor immunity. We have recently reported that treatment of naïve CD4 T cells with adenosine A(2B) receptor antagonist PSB603 under Treg-skewing conditions inhibits expression of Foxp3, a marker of differentiation to Treg, without blocking IL-2 production or CD25 expression, which are activation markers, in CD4 T cells. We hypothesized that PSB603 suppresses cancer growth and metastasis by inhibiting induction of Treg, thereby facilitating anti-tumor immunity. In this study, we first examined the effect of PSB603 on tumor growth in B16 melanoma-bearing C57BL/6 mice. Administration of PSB603 significantly suppressed the increase of tumor volume as well as the increase of Treg population in these mice. The populations of CD4 and CD8 T cells were higher and splenic lymphocyte-mediated cytotoxicity towards B16 melanoma was significantly increased in PSB603-treated mice. We confirmed that PSB603 did not reduce the viability of B16 melanoma cells in vitro. Moreover, we also examined the effect of PSB603 on tumor metastasis in pulmonary metastasis model mice intravenously injected with B16 melanoma cells. The metastasis was also suppressed in PSB603-treated mice, in which the population of Treg was significantly lower. Overall, our results suggest that A(2B) receptor antagonist PSB603 enhances anti-tumor immunity by inhibiting differentiation to Treg, resulting in a delay of tumor growth and a suppression of metastasis.

  13. ɛ '/ ɛ anomaly and neutron EDM in SU(2) L × SU(2) R × U(1) B- L model with charge symmetry

    NASA Astrophysics Data System (ADS)

    Haba, Naoyuki; Umeeda, Hiroyuki; Yamada, Toshifumi

    2018-05-01

    The Standard Model prediction for ɛ '/ ɛ based on recent lattice QCD results exhibits a tension with the experimental data. We solve this tension through W R + gauge boson exchange in the SU(2) L × SU(2) R × U(1) B- L model with `charge symmetry', whose theoretical motivation is to attribute the chiral structure of the Standard Model to the spontaneous breaking of SU(2) R × U(1) B- L gauge group and charge symmetry. We show that {M_W}{_R}<58 TeV is required to account for the ɛ' /ɛ anomaly in this model. Next, we make a prediction for the neutron EDM in the same model and study a correlation between ɛ ' /ɛ and the neutron EDM. We confirm that the model can solve the ɛ ' /ɛ anomaly without conflicting the current bound on the neutron EDM, and further reveal that almost all parameter regions in which the ɛ ' /ɛ anomaly is explained will be covered by future neutron EDM searches, which leads us to anticipate the discovery of the neutron EDM.

  14. Direct targeting of SUZ12/ROCK2 by miR-200b/c inhibits cholangiocarcinoma tumourigenesis and metastasis

    PubMed Central

    Peng, F; Jiang, J; Yu, Y; Tian, R; Guo, X; Li, X; Shen, M; Xu, M; Zhu, F; Shi, C; Hu, J; Wang, M; Qin, R

    2013-01-01

    Background: The multidrug resistance and distant metastasis of cholangiocarcinoma result in high postoperative recurrence and low long-term survival rates. It has been demonstrated that the ectopic expression of miR-200 suppresses the multidrug resistance and metastasis of cancer. However, the expression and function of miR-200 in cholangiocarcinoma has not yet been described. Methods: In this study, we identified dysregulated microRNAs (miRNAs, miR) in cholangiocarcinoma tissue by microarray analysis, and subsequent real-time PCR and northern blot analyses validated the expression of candidate miR. We performed functional analyses and investigated the relationship between miR-200b/c expression and the properties of cholangiocarcinoma cells. A dual luciferase assay was applied to examine the effect of miRNAs on the 3′-UTR of target genes, and we demonstrated the function of the target gene by siRNA transfection identifying the downstream pathway via western blotting. Results: We found significantly downregulated expression of four miR-200 family members (miR-200a/b/c/429) and then confirmed that ectopic miR-200b/200c inhibits the migration and invasion of cholangiocarcinoma cells both in vitro and in vivo. We found that miR-200b/c influenced the tumourigenesis of cholangiocarcinoma cells including their tumour-initiating capacity, sphere formation, and drug resistance. We further found that miR-200b/c regulated migration and invasion capacities by directly targeting rho-kinase 2 and regulated tumorigenic properties by directly targeting SUZ12 (a subunit of a polycomb repressor complex). Conclusion: Our study shows that miR-200b/c has a critical role in the regulation of the tumorigenic and metastatic capacity of cholangiocarcinoma and reveals the probable underlying mechanisms. PMID:24169343

  15. CD22 ligand-binding and signaling domains reciprocally regulate B-cell Ca2+ signaling

    PubMed Central

    Müller, Jennifer; Obermeier, Ingrid; Wöhner, Miriam; Brandl, Carolin; Mrotzek, Sarah; Angermüller, Sieglinde; Maity, Palash C.; Reth, Michael; Nitschke, Lars

    2013-01-01

    A high proportion of human B cells carry B-cell receptors (BCRs) that are autoreactive. Inhibitory receptors such as CD22 can downmodulate autoreactive BCR responses. With its extracellular domain, CD22 binds to sialic acids in α2,6 linkages in cis, on the surface of the same B cell or in trans, on other cells. Sialic acids are self ligands, as they are abundant in vertebrates, but are usually not expressed by pathogens. We show that cis-ligand binding of CD22 is crucial for the regulation of B-cell Ca2+ signaling by controlling the CD22 association to the BCR. Mice with a mutated CD22 ligand-binding domain of CD22 showed strongly reduced Ca2+ signaling. In contrast, mice with mutated CD22 immunoreceptor tyrosine-based inhibition motifs have increased B-cell Ca2+ responses, increased B-cell turnover, and impaired survival of the B cells. Thus, the CD22 ligand-binding domain has a crucial function in regulating BCR signaling, which is relevant for controlling autoimmunity. PMID:23836650

  16. CD22 ligand-binding and signaling domains reciprocally regulate B-cell Ca2+ signaling.

    PubMed

    Müller, Jennifer; Obermeier, Ingrid; Wöhner, Miriam; Brandl, Carolin; Mrotzek, Sarah; Angermüller, Sieglinde; Maity, Palash C; Reth, Michael; Nitschke, Lars

    2013-07-23

    A high proportion of human B cells carry B-cell receptors (BCRs) that are autoreactive. Inhibitory receptors such as CD22 can downmodulate autoreactive BCR responses. With its extracellular domain, CD22 binds to sialic acids in α2,6 linkages in cis, on the surface of the same B cell or in trans, on other cells. Sialic acids are self ligands, as they are abundant in vertebrates, but are usually not expressed by pathogens. We show that cis-ligand binding of CD22 is crucial for the regulation of B-cell Ca(2+) signaling by controlling the CD22 association to the BCR. Mice with a mutated CD22 ligand-binding domain of CD22 showed strongly reduced Ca(2+) signaling. In contrast, mice with mutated CD22 immunoreceptor tyrosine-based inhibition motifs have increased B-cell Ca(2+) responses, increased B-cell turnover, and impaired survival of the B cells. Thus, the CD22 ligand-binding domain has a crucial function in regulating BCR signaling, which is relevant for controlling autoimmunity.

  17. Antihyperalgesic activity of a novel nonpeptide bradykinin B1 receptor antagonist in transgenic mice expressing the human B1 receptor

    PubMed Central

    Fox, Alyson; Kaur, Satbir; Li, Bifang; Panesar, Moh; Saha, Uma; Davis, Clare; Dragoni, Ilaria; Colley, Sian; Ritchie, Tim; Bevan, Stuart; Burgess, Gillian; McIntyre, Peter

    2005-01-01

    We describe the properties of a novel nonpeptide kinin B1 receptor antagonist, NVP-SAA164, and demonstrate its in vivo activity in models of inflammatory pain in transgenic mice expressing the human B1 receptor. NVP-SAA164 showed high affinity for the human B1 receptor expressed in HEK293 cells (Ki 8 nM), and inhibited increases in intracellular calcium induced by desArg10kallidin (desArg10KD) (IC50 33 nM). While a similar high affinity was observed in monkey fibroblasts (Ki 7.7 nM), NVP-SAA164 showed no affinity for the rat B1 receptor expressed in Cos-7 cells. In transgenic mice in which the native B1 receptor was deleted and the gene encoding the human B1 receptor was inserted (hB1 knockin, hB1-KI), hB1 receptor mRNA was induced in tissues following LPS treatment. No mRNA encoding the mouse or human B1 receptor was detected in mouse B1 receptor knockout (mB1-KO) mice following LPS treatment. Freund's complete adjuvant-induced mechanical hyperalgesia was similar in wild-type and hB1-KI mice, but was significantly reduced in mB1-KO animals. Mechanical hyperalgesia induced by injection of the B1 agonist desArg10KD into the contralateral paw 24 h following FCA injection was similar in wild-type and hB1-KI mice, but was absent in mB1-KO animals. Oral administration of NVP-SAA164 produced a dose-related reversal of FCA-induced mechanical hyperalgesia and desArg10KD-induced hyperalgesia in hB1-KI mice, but was inactive against inflammatory pain in wild-type mice. These data demonstrate the use of transgenic technology to investigate the in vivo efficacy of species selective agents and show that NVP-SAA164 is a novel orally active B1 receptor antagonist, providing further support for the utility of B1 receptor antagonists in inflammatory pain conditions in man. PMID:15685199

  18. The antigenic complex in HIT binds to B cells via complement and complement receptor 2 (CD21)

    PubMed Central

    Khandelwal, Sanjay; Lee, Grace M.; Hester, C. Garren; Poncz, Mortimer; McKenzie, Steven E.; Sachais, Bruce S.; Rauova, Lubica; Kelsoe, Garnett; Cines, Douglas B.; Frank, Michael

    2016-01-01

    Heparin-induced thrombocytopenia is a prothrombotic disorder caused by antibodies to platelet factor 4 (PF4)/heparin complexes. The mechanism that incites such prevalent anti-PF4/heparin antibody production in more than 50% of patients exposed to heparin in some clinical settings is poorly understood. To investigate early events associated with antigen exposure, we first examined the interaction of PF4/heparin complexes with cells circulating in whole blood. In healthy donors, PF4/heparin complexes bind preferentially to B cells (>90% of B cells bind to PF4/heparin in vitro) relative to neutrophils, monocytes, or T cells. Binding of PF4 to B cells is heparin dependent, and PF4/heparin complexes are found on circulating B cells from some, but not all, patients receiving heparin. Given the high proportion of B cells that bind PF4/heparin, we investigated complement as a mechanism for noncognate antigen recognition. Complement is activated by PF4/heparin complexes, co-localizes with antigen on B cells from healthy donors, and is present on antigen-positive B cells in patients receiving heparin. Binding of PF4/heparin complexes to B cells is mediated through the interaction between complement and complement receptor 2 (CR2 [CD21]). To the best of our knowledge, these are the first studies to demonstrate complement activation by PF4/heparin complexes, opsonization of PF4/heparin to B cells via CD21, and the presence of complement activation fragments on circulating B cells in some patients receiving heparin. Given the critical contribution of complement to humoral immunity, our observations provide new mechanistic insights into the immunogenicity of PF4/heparin complexes. PMID:27412887

  19. Atom-based 3D-QSAR, induced fit docking, and molecular dynamics simulations study of thieno[2,3-b]pyridines negative allosteric modulators of mGluR5.

    PubMed

    Vijaya Prabhu, Sitrarasu; Singh, Sanjeev Kumar

    2018-05-28

    Atom-based three dimensional-quantitative structure-activity relationship (3D-QSAR) model was developed on the basis of 5-point pharmacophore hypothesis (AARRR) with two hydrogen bond acceptors (A) and three aromatic rings for the derivatives of thieno[2,3-b]pyridine, which modulates the activity to inhibit the mGluR5 receptor. Generation of a highly predictive 3D-QSAR model was performed using the alignment of predicted pharmacophore hypothesis for the training set (R 2  = 0.84, SD = 0.26, F = 45.8, N = 29) and test set (Q 2  = 0.74, RMSE = 0.235, Pearson-R = 0.94, N = 9). The best pharmacophore hypothesis AARRR was selected, and developed three dimensional-quantitative structure activity relationship (3D-QSAR) model also supported the outcome of this study by means of favorable and unfavorable electron withdrawing group and hydrophobic regions of most active compound 42d and least active compound 18b. Following, induced fit docking and binding free energy calculations reveals the reliable binding orientation of the compounds. Finally, molecular dynamics simulations for 100 ns were performed to depict the protein-ligand stability. We anticipate that the resulted outcome could be supportive to discover potent negative allosteric modulators for metabotropic glutamate receptor 5 (mGluR5).

  20. The physical and chemical structure of Sagittarius B2. II. Continuum millimeter emission of Sgr B2(M) and Sgr B2(N) with ALMA

    NASA Astrophysics Data System (ADS)

    Sánchez-Monge, Á.; Schilke, P.; Schmiedeke, A.; Ginsburg, A.; Cesaroni, R.; Lis, D. C.; Qin, S.-L.; Müller, H. S. P.; Bergin, E.; Comito, C.; Möller, Th.

    2017-07-01

    Context. The two hot molecular cores Sgr B2(M) and Sgr B2(N), which are located at the center of the giant molecular cloud complex Sagittarius B2, have been the targets of numerous spectral line surveys, revealing a rich and complex chemistry. Aims: We seek to characterize the physical and chemical structure of the two high-mass star-forming sites Sgr B2(M) and Sgr B2(N) using high-angular resolution observations at millimeter wavelengths, reaching spatial scales of about 4000 au. Methods: We used the Atacama Large Millimeter/submillimeter Array (ALMA) to perform an unbiased spectral line survey of both regions in the ALMA band 6 with a frequency coverage from 211 GHz to 275 GHz. The achieved angular resolution is 0.̋4, which probes spatial scales of about 4000 au, I.e., able to resolve different cores and fragments. In order to determine the continuum emission in these line-rich sources, we used a new statistical method, STATCONT, which has been applied successfully to this and other ALMA datasets and to synthetic observations. Results: We detect 27 continuum sources in Sgr B2(M) and 20 sources in Sgr B2(N). We study the continuum emission variation across the ALMA band 6 (I.e., spectral index) and compare the ALMA 1.3 mm continuum emission with previous SMA 345 GHz and VLA 40 GHz observations to study the nature of the sources detected. The brightest sources are dominated by (partially optically thick) dust emission, while there is an important degree of contamination from ionized gas free-free emission in weaker sources. While the total mass in Sgr B2(M) is distributed in many fragments, most of the mass in Sgr B2(N) arises from a single object, with filamentary-like structures converging toward the center. There seems to be a lack of low-mass dense cores in both regions. We determine H2 volume densities for the cores of about 107-109 cm-3 (or 105-107 M⊙ pc-3), I.e., one to two orders of magnitude higher than the stellar densities of super star clusters. We

  1. Substrate stiffness affects epithelial-mesenchymal transition of cervical cancer cells through miR-106b and its target protein DAB2.

    PubMed

    Piao, Jinlan; You, Ke; Guo, Yanli; Zhang, Youyi; Li, Zijian; Geng, Li

    2017-06-01

    The effects of different substrate stiffness were investigated on epithelial-mesenchymal transition (EMT) of cervical cancer cell lines and the role of miR-106b and its target protein DAB2 therein. Cervical cancer cell lines HeLa and SiHa were cultured on artificial substrates with different stiffness prepared using different ratios of acrylamide and bis-acrylamide. Changes of microRNA profiles were detected using microRNA chip analysis, and the expression levels of EMT-related markers E-cadherin and vimentin were detected using western blotting and real-time PCR. In addition, the effects of miR-106b overexpression as well as miR-106b and DAB2 knockdown on expression of E-cadherin and vimentin were also examined using western blotting and real-time PCR. The results showed that i) cervical cancer cell lines SiHa and HeLa cultured on substrate with stiffness of 20 kPa had the strongest EMT ability, showed the highest levels of vimentin and lowest levels of E-cadherin, compared with cells cultured on substrate with stiffness of 1 kPa; ii) miR-106b knockdown reversed the effects of substrate stiffness on EMT of cervical cancer cells, while miR-106 overexpression and DAB2 knockdown induced EMT of cervical cancer cells cultured on substrate with stiffness of 20 kPa. Overall, the results indicated that substrate stiffness could regulate EMT of cervical cancer cell lines HeLa and SiHa at least partially through miR-106b and its downstream target DAB2.

  2. Minoxidil-induced hair growth is mediated by adenosine in cultured dermal papilla cells: possible involvement of sulfonylurea receptor 2B as a target of minoxidil.

    PubMed

    Li, M; Marubayashi, A; Nakaya, Y; Fukui, K; Arase, S

    2001-12-01

    The mechanism by which minoxidil, an adenosine-triphosphate-sensitive potassium channel opener, induces hypertrichosis remains to be elucidated. Minoxidil has been reported to stimulate the production of vascular endothelial growth factor, a possible promoter of hair growth, in cultured dermal papilla cells. The mechanism of production of vascular endothelial growth factor remains unclear, however. We hypothesize that adenosine serves as a mediator of vascular endothelial growth factor production. Minoxidil-induced increases in levels of intracellular Ca(2+) and vascular endothelial growth factor production in cultured dermal papilla cells were found to be inhibited by 8-sulfophenyl theophylline, a specific antagonist for adenosine receptors, suggesting that dermal papilla cells possess adenosine receptors and sulfonylurea receptors, the latter of which is a well-known target receptor for adenosine-triphosphate-sensitive potassium channel openers. The expression of sulfonylurea receptor 2B and of the adenosine A1, A2A, and A2B receptors was detected in dermal papilla cells by means of reverse transcription polymerase chain reaction analysis. In order to determine which of the adenosine receptor subtypes contribute to minoxidil-induced hair growth, the effects of subtype-specific antagonists for adenosine receptors were investigated. Significant inhibition in increase in intracellular calcium level by minoxidil or adenosine was observed as the result of pretreatment with 8-cyclopentyl-1,3-dipropylxanthine, an antagonist for adenosine A1 receptor, but not by 3,7-dimethyl-1-propargyl-xanthine, an antagonist for adenosine A2 receptor, whereas vascular endothelial growth factor production was blocked by both adenosine A1 and A2 receptor antagonists. These results indicate that the effect of minoxidil is mediated by adenosine, which triggers intracellular signal transduction via both adenosine A1 and A2 receptors, and that the expression of sulfonylurea receptor 2B in

  3. Structure of the Zinc-Bound Amino-Terminal Domain of the NMDA Receptor NR2B Subunit

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Karakas, E.; Simorowski, N; Furukawa, H

    2009-01-01

    N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino-terminal domain (ATD) distinct from the L-glutamate-binding domain. The molecular basis for the ATD-mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc-freemore » and zinc-bound states. The structures reveal the overall clamshell-like architecture distinct from the non-NMDA receptor ATDs and molecular determinants for the zinc-binding site, ion-binding sites, and the architecture of the putative phenylethanolamine-binding site.« less

  4. Alternative translation initiation of Caveolin-2 desensitizes insulin signaling through dephosphorylation of insulin receptor by PTP1B and causes insulin resistance.

    PubMed

    Kwon, Hayeong; Jang, Donghwan; Choi, Moonjeong; Lee, Jaewoong; Jeong, Kyuho; Pak, Yunbae

    2018-06-01

    Insulin resistance, defined as attenuated sensitivity responding to insulin, impairs insulin action. Direct causes and molecular mechanisms of insulin resistance have thus far remained elusive. Here we show that alternative translation initiation (ATI) of Caveolin-2 (Cav-2) regulates insulin sensitivity. Cav-2β isoform yielded by ATI desensitizes insulin receptor (IR) via dephosphorylation by protein-tyrosine phosphatase 1B (PTP1B), and subsequent endocytosis and lysosomal degradation of IR, causing insulin resistance. Blockage of Cav-2 ATI protects against insulin resistance by preventing Cav-2β-PTP1B-directed IR desensitization, thereby normalizing insulin sensitivity and glucose uptake. Our findings show that Cav-2β is a negative regulator of IR signaling, and identify a mechanism causing insulin resistance through control of insulin sensitivity via Cav-2 ATI. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Agent-based services for B2B electronic commerce

    NASA Astrophysics Data System (ADS)

    Fong, Elizabeth; Ivezic, Nenad; Rhodes, Tom; Peng, Yun

    2000-12-01

    The potential of agent-based systems has not been realized yet, in part, because of the lack of understanding of how the agent technology supports industrial needs and emerging standards. The area of business-to-business electronic commerce (b2b e-commerce) is one of the most rapidly developing sectors of industry with huge impact on manufacturing practices. In this paper, we investigate the current state of agent technology and the feasibility of applying agent-based computing to b2b e-commerce in the circuit board manufacturing sector. We identify critical tasks and opportunities in the b2b e-commerce area where agent-based services can best be deployed. We describe an implemented agent-based prototype system to facilitate the bidding process for printed circuit board manufacturing and assembly. These activities are taking place within the Internet Commerce for Manufacturing (ICM) project, the NIST- sponsored project working with industry to create an environment where small manufacturers of mechanical and electronic components may participate competitively in virtual enterprises that manufacture printed circuit assemblies.

  6. Curcumin by down-regulating NF-kB and elevating Nrf2, reduces brain edema and neurological dysfunction after cerebral I/R.

    PubMed

    Li, Wei; Suwanwela, Nijasri C; Patumraj, Suthiluk

    2016-07-01

    Oxidation, inflammation, and apoptosis are three critical factors for the pathogenic mechanism of cerebral ischemia/reperfusion (I/R) injury. Curcumin exhibits substantial biological properties via anti-oxidation, anti-inflammation and anti-apoptotic effects; however, the molecular mechanism underlying the effects of curcumin against cerebral I/R injury remains unclear. To investigate the effects of curcumin on cerebral I/R injury associated with water content, infarction volume, and the expression of nuclear factor-kappa-B (NF-κB) and nuclear factor-erythroid-related factor-2 (Nrf2). Middle cerebral artery occlusion (MCAO, 1-hour occlusion and 24-hour reperfusion) was performed in male Wistar rats (n=64) as a cerebral I/R injury model. In the MCAO+CUR group, the rats were administered curcumin (300mg/kg BW, i.p.) at 30min after occlusion. The same surgical procedures were performed in SHAM rats without MCAO occlusion. At 24h post-operation, the parameters, including neurological deficit scores, blood brain barrier (BBB) disruption, water content, and infarction volume, were determined. Brain tissue NF-κB and Nrf2 expression levels were assayed through immunohistochemistry. Compared with the SHAM group, BBB disruption, neurological deficit, and increased brain water content and infarction volume were markedly demonstrated in the MCAO group. NF-κB expression was enhanced in the MCAO group. However, in the MCAO+CUR group, the upregulation of Nrf2, an anti-oxidation related protein, was consistent with a significant decline in the water content, infarction volume, and NF-κB expression. The protective effects of curcumin against cerebral I/R injury reflect anti-oxidation, anti-inflammation and anti-apoptotic activities, resulting in the elevation of Nrf2 and down-regulation of NF-κB. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Mg2+ activates the ryanodine receptor type 2 (RyR2) at intermediate Ca2+ concentrations.

    PubMed

    Chugun, Akihito; Sato, Osamu; Takeshima, Hiroshi; Ogawa, Yasuo

    2007-01-01

    To clarify whether activity of the ryanodine receptor type 2 (RyR2) is reduced in the sarcoplasmic reticulum (SR) of cardiac muscle, as is the case with the ryanodine receptor type 1 (RyR1), Ca(2+)-dependent [(3)H]ryanodine binding, a biochemical measure of Ca(2+)-induced Ca(2+) release (CICR), was determined using SR vesicle fractions isolated from rabbit and rat cardiac muscles. In the absence of an adenine nucleotide or caffeine, the rat SR showed a complicated Ca(2+) dependence, instead of the well-documented biphasic dependence of the rabbit SR. In the rat SR, [(3)H]ryanodine binding initially increased as [Ca(2+)] increased, with a plateau in the range of 10-100 microM Ca(2+), and thereafter further increased to an apparent peak around 1 mM Ca(2+), followed by a decrease. In the presence of these modulators, this complicated dependence prevailed, irrespective of the source. Addition of 0.3-1 mM Mg(2+) unexpectedly increased the binding two- to threefold and enhanced the affinity for [(3)H]ryanodine at 10-100 microM Ca(2+), resulting in the well-known biphasic dependence. In other words, the partial suppression of RyR2 is relieved by Mg(2+). Ca(2+) could be a substitute for Mg(2+). Mg(2+) also amplifies the responses of RyR2 to inhibitory and stimulatory modulators. This stimulating effect of Mg(2+) on RyR2 is entirely new, and is referred to as the third effect, in addition to the well-known dual inhibitory effects. This effect is critical to describe the role of RyR2 in excitation-contraction coupling of cardiac muscle, in view of the intracellular Mg(2+) concentration.

  8. Prokaryotic 2-component systems and the OmpR/PhoB superfamily.

    PubMed

    Nguyen, Minh-Phuong; Yoon, Joo-Mi; Cho, Man-Ho; Lee, Sang-Won

    2015-11-01

    In bacteria, 2-component regulatory systems (TCSs) are the critical information-processing pathways that link stimuli to specific adaptive responses. Signals perceived by membrane sensors, which are generally histidine kinases, are transmitted by response regulators (RRs) to allow cells to cope rapidly and effectively with environmental challenges. Over the past few decades, genes encoding components of TCSs and their responsive proteins have been identified, crystal structures have been described, and signaling mechanisms have been elucidated. Here, we review recent findings and interesting breakthroughs in bacterial TCS research. Furthermore, we discuss structural features, mechanisms of activation and regulation, and cross-regulation of RRs, with a focus on the largest RR family, OmpR/PhoB, to provide a comprehensive overview of these critically important signaling molecules.

  9. Amygdala EphB2 Signaling Regulates Glutamatergic Neuron Maturation and Innate Fear

    PubMed Central

    Zhu, Xiao-Na; Liu, Xian-Dong; Zhuang, Hanyi; Henkemeyer, Mark

    2016-01-01

    The amygdala serves as emotional center to mediate innate fear behaviors that are reflected through neuronal responses to environmental aversive cues. However, the molecular mechanism underlying the initial neuron responses is poorly understood. In this study, we monitored the innate defensive responses to aversive stimuli of either elevated plus maze or predator odor in juvenile mice and found that glutamatergic neurons were activated in amygdala. Loss of EphB2, a receptor tyrosine kinase expressed in amygdala neurons, suppressed the reactions and led to defects in spine morphogenesis and fear behaviors. We further found a coupling of spinogenesis with these threat cues induced neuron activation in developing amygdala that was controlled by EphB2. A constitutively active form of EphB2 was sufficient to rescue the behavioral and morphological defects caused by ablation of ephrin-B3, a brain-enriched ligand to EphB2. These data suggest that kinase-dependent EphB2 intracellular signaling plays a major role for innate fear responses during the critical developing period, in which spinogenesis in amygdala glutamatergic neurons was involved. SIGNIFICANCE STATEMENT Generation of innate fear responses to threat as an evolutionally conserved brain feature relies on development of functional neural circuit in amygdala, but the molecular mechanism remains largely unknown. We here identify that EphB2 receptor tyrosine kinase, which is specifically expressed in glutamatergic neurons, is required for the innate fear responses in the neonatal brain. We further reveal that EphB2 mediates coordination of spinogenesis and neuron activation in amygdala during the critical period for the innate fear. EphB2 catalytic activity plays a major role for the behavior upon EphB–ephrin-B3 binding and transnucleus neuronal connections. Our work thus indicates an essential synaptic molecular signaling within amygdala that controls synapse development and helps bring about innate fear emotions

  10. Enhanced phagocytic activity of HIV-specific antibodies correlates with natural production of immunoglobulins with skewed affinity for FcγR2a and FcγR2b.

    PubMed

    Ackerman, Margaret E; Dugast, Anne-Sophie; McAndrew, Elizabeth G; Tsoukas, Stephen; Licht, Anna F; Irvine, Darrell J; Alter, Galit

    2013-05-01

    While development of an HIV vaccine that can induce neutralizing antibodies remains a priority, decades of research have proven that this is a daunting task. However, accumulating evidence suggests that antibodies with the capacity to harness innate immunity may provide some protection. While significant research has focused on the cytolytic properties of antibodies in acquisition and control, less is known about the role of additional effector functions. In this study, we investigated antibody-dependent phagocytosis of HIV immune complexes, and we observed significant differences in the ability of antibodies from infected subjects to mediate this critical effector function. We observed both quantitative differences in the capacity of antibodies to drive phagocytosis and qualitative differences in their FcγR usage profile. We demonstrate that antibodies from controllers and untreated progressors exhibit increased phagocytic activity, altered Fc domain glycosylation, and skewed interactions with FcγR2a and FcγR2b in both bulk plasma and HIV-specific IgG. While increased phagocytic activity may directly influence immune activation via clearance of inflammatory immune complexes, it is also plausible that Fc receptor usage patterns may regulate the immune response by modulating downstream signals following phagocytosis--driving passive degradation of internalized virus, release of immune modulating cytokines and chemokines, or priming of a more effective adaptive immune response.

  11. A PCV2 vaccine based on genotype 2b is more effective than a 2a-based vaccine to protect against PCV2b or combined PCV2a/2b viremia in pigs with concurrent PCV2, PRRSV and PPV infection.

    PubMed

    Opriessnig, Tanja; O'Neill, Kevin; Gerber, Priscilla F; de Castro, Alessandra M M G; Gimenéz-Lirola, Luis G; Beach, Nathan M; Zhou, Lei; Meng, Xiang-Jin; Wang, Chong; Halbur, Patrick G

    2013-01-07

    The predominant genotype of porcine circovirus (PCV) in the pig population today is PCV2b yet PCV2a-based commercial vaccines are considered effective in protecting against porcine circovirus associated disease. The objective of this study was to compare the ability of PCV2a- and PCV2b-based vaccines to control PCV2b viremia in a challenge model that mimics the U.S. field situation. Sixty-three pigs were randomly assigned to one of eight groups. Sixteen pigs were vaccinated with an experimental live-attenuated chimeric PCV1-2a vaccine based on genotype 2a and another 16 pigs with a chimeric PCV1-2b vaccine based on genotype 2b. Challenge was done 28 days post vaccination (dpv) using PCV2b (or a combination of PCV2a and PCV2b), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus (PPV) to mimic what commonly occurs in the field. The experiment was terminated 21 days post challenge (dpc) or 49dpv. Pigs vaccinated with the chimeric PCV1-2b vaccine had significantly higher levels of PCV1-2b viremia and shedding of the PCV1-2b vaccine virus in feces and nasal secretions but also a more robust humoral immune response as evidenced by significantly higher ELISA S/P ratios compared to the PCV1-2a vaccination. Regardless of challenge, the PCV1-2b vaccination significantly reduced the prevalence and amount of PCV2 viremia compared to the PCV1-2a vaccination. Interestingly, in the non-vaccinated pigs concurrent PCV2a infection resulted in clinical disease and increased macroscopic lung lesions compared to pigs challenged with PCV2b alone, further supporting the idea that concurrent PCV2a/PCV2b infection is necessary for optimal PCV2 replication. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Elevated Adenosine Induces Placental DNA Hypomethylation Independent of A2B Receptor Signaling in Preeclampsia.

    PubMed

    Huang, Aji; Wu, Hongyu; Iriyama, Takayuki; Zhang, Yujin; Sun, Kaiqi; Song, Anren; Liu, Hong; Peng, Zhangzhe; Tang, Lili; Lee, Minjung; Huang, Yun; Ni, Xin; Kellems, Rodney E; Xia, Yang

    2017-07-01

    Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear. Using 2 independent experimental models of preeclampsia, adenosine deaminase-deficient mice and a pathogenic autoantibody-induced mouse model of preeclampsia, we demonstrate that elevated placental adenosine not only induces hallmark features of preeclampsia but also causes placental DNA hypomethylation. The use of genetic approaches to express an adenosine deaminase minigene specifically in placentas, or adenosine deaminase enzyme replacement therapy, restored placental adenosine to normal levels, attenuated preeclampsia features, and abolished placental DNA hypomethylation in adenosine deaminase-deficient mice. Genetic deletion of CD73 (an ectonucleotidase that converts AMP to adenosine) prevented the elevation of placental adenosine in the autoantibody-induced preeclampsia mouse model and ameliorated preeclampsia features and placental DNA hypomethylation. Immunohistochemical studies revealed that elevated placental adenosine-mediated DNA hypomethylation predominantly occurs in spongiotrophoblasts and labyrinthine trophoblasts and that this effect is independent of A2B adenosine receptor activation in both preeclampsia models. Extending our mouse findings to humans, we used cultured human trophoblasts to demonstrate that adenosine functions intracellularly and induces DNA hypomethylation without A2B adenosine receptor activation. Altogether, both mouse and human studies reveal novel mechanisms underlying placental DNA hypomethylation and potential therapeutic approaches for preeclampsia. © 2017 American Heart Association, Inc.

  13. The A2b adenosine receptor antagonist PSB-603 promotes oxidative phosphorylation and ROS production in colorectal cancer cells via adenosine receptor-independent mechanism.

    PubMed

    Mølck, Christina; Ryall, James; Failla, Laura M; Coates, Janine L; Pascussi, Jean-Marc; Heath, Joan K; Stewart, Gregory; Hollande, Frédéric

    2016-12-01

    Adenosine is a multifaceted regulator of tumor progression. It modulates immune cell activity as well as acting directly on tumor cells. The A 2b adenosine receptor (A 2b -AR) is thought to be an important mediator of these effects. In this study we sought to analyze the contribution of the A 2b -AR to the behavior of colorectal cancer cells. The A 2b -AR antagonist PSB-603 changed cellular redox state without affecting cellular viability. Quantification of cellular bioenergetics demonstrated that PSB-603 increased basal oxygen consumption rates, indicative of enhanced mitochondrial oxidative phosphorylation. Unexpectedly, pharmacological and genetic approaches to antagonize AR-related signalling of PSB-603 did not abolish the response, suggesting that it was AR-independent. PSB-603 also induced acute increases in reactive oxygen species, and PSB-603 synergized with chemotherapy treatment to increase colorectal cancer cell death, consistent with the known link between cellular metabolism and chemotherapy response. PSB-603 alters cellular metabolism in colorectal cancer cells and increases their sensitivity to chemotherapy. Although requiring more mechanistic insight into its A 2b -AR-independent activity, our results show that PSB-603 may have clinical value as an anti-colorectal cancer therapeutic. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Thermal stability of hexagonal OsB2

    NASA Astrophysics Data System (ADS)

    Xie, Zhilin; Blair, Richard G.; Orlovskaya, Nina; Cullen, David A.; Andrew Payzant, E.

    2014-11-01

    The synthesis of novel hexagonal ReB2-type OsB2 ceramic powder was performed by high energy ball milling of elemental Os and B powders. Two different sources of B powder have been used for this mechanochemical synthesis. One B powder consisted of a mixture of amorphous and crystalline phases and a mixture of 10B and 11B isotopes with a fine particle size, while another B powder was a purely crystalline (rhombohedral) material consisting of enriched 11B isotope with coarse particle size. The same Os powder was used for the synthesis in both cases. It was established that, in the first case, the hexagonal OsB2 phase was the main product of synthesis with a small quantity of Os2B3 phase present after synthesis as an intermediate product. In the second case, where coarse crystalline 11B powder was used as a raw material, only Os2B3 boride was synthesized mechanochemically. The thermal stability of hexagonal OsB2 powder was studied by heating under argon up to 876 °C and cooling in vacuo down to -225 °C. During the heating, the sacrificial reaction 2OsB2+3O22Os+2B2O3 took place due to presence of O2/water vapor molecules in the heating chamber, resulting in the oxidation of B atoms and formation of B2O3 and precipitation of Os metal out of the OsB2 lattice. As a result of such phase changes during heating, the lattice parameters of hexagonal OsB2 changed significantly. The shrinkage of the a lattice parameter was recorded in 276-426 °C temperature range upon heating, which was attributed to the removal of B atoms from the OsB2 lattice due to oxidation followed by the precipitation of Os atoms and formation of Os metal. While significant structural changes occurred upon heating due to presence of O2, the hexagonal OsB2 ceramic demonstrated good phase stability upon cooling in vacuo with linear shrinkage of the lattice parameters and no phase changes detected during cooling.

  15. Selenidation of epitaxial silicene on ZrB2

    NASA Astrophysics Data System (ADS)

    Wiggers, F. B.; Yamada-Takamura, Y.; Kovalgin, A. Y.; de Jong, M. P.

    2018-01-01

    The deposition of elemental Se on epitaxial silicene on ZrB2 thin films was investigated with synchrotron-based core-level photoelectron spectroscopy and low-energy electron diffraction. The deposition of Se at room temperature caused the appearance of Si 2p peaks with chemical shifts of n × 0.51 ± 0.04 eV (n = 1-4), suggesting the formation of SiSe2. This shows that capping the silicene monolayer, without affecting its structural and electronic properties, is not possible with Se. The annealing treatments that followed caused the desorption of Se and Si, resulting in the etching of the Si atoms formerly part of the silicene layer, and the formation of bare ZrB2(0001) surface area. In addition, a ZrB2(0001)-(√7 × 3)R40.9° surface reconstruction was observed, attributed to a Se-termination of the surface of the transition metal diboride thin film.

  16. Activation of the P2X₇ receptor induces migration of glial cells by inducing cathepsin B degradation of tissue inhibitor of metalloproteinase 1.

    PubMed

    Murphy, Niamh; Lynch, Marina A

    2012-12-01

    The P2X(7) receptor is an ion-gated channel, which is activated by high extracellular concentrations of adenosine triphosphate (ATP). Activation of P2X(7) receptors has been shown to induce neuroinflammatory changes associated with several neurological conditions. The matrix metalloproteinases (MMPs) are a family of endopeptidases that have several functions including degradation of the extracellular matrix, cell migration and modulation of bioactive molecules. The actions of MMPs are prevented by a family of protease inhibitors called tissue inhibitors of metalloproteinases (TIMPs). In this study, we show that ATP-treated glial cultures from neonatal C57BL/6 mice release and increase MMP-9 activity, which is coupled with a decrease in release of TIMP-1 and an increase in activated cathepsin B within the extracellular space. This process occurs independently of NLRP3-inflammasome formation. Treatment with a P2X(7) receptor antagonist prevents ATP-induced MMP-9 activity, inhibition of active cathepsin B release and allows for TIMP-1 to be released from the cell. We have shown that cathepsin B degrades TIMP-1, and inhibition of cathepsin B allows for release of TIMP-1 and inhibits MMP-9 activity. We also present data that indicate that ATP or cell damage induces glial cell migration, which is inhibited by P2X(7) antagonism, depletion of MMP-9 or inhibition of cathepsin B. © 2012 International Society for Neurochemistry.

  17. Antinociceptive effects of oxymatrine from Sophora flavescens, through regulation of NR2B-containing NMDA receptor-ERK/CREB signaling in a mice model of neuropathic pain.

    PubMed

    Wang, Haiyan; Li, Yuxiang; Dun, Linglu; Xu, Yaqiong; Jin, Shaojv; Du, Juan; Ma, Lin; Li, Juan; Zhou, Ru; He, Xiaoliang; Sun, Tao; Yu, Jianqiang

    2013-08-15

    In this study we investigated antinociceptive effects of oxymatrine through regulation of NR2B-containing NMDA receptor-ERK/CREB signaling in a chronic neuropathic pain model induced by chronic constrictive injury (CCI) of the sciatic nerve. The von Frey and plantar tests were performed to assess the degree of mechanical and thermal changes respectively. Immunohistochemistry assay was used to evaluate the expressions of NR2B. Western blotting assay were used to evaluate the expressions of NR2B, tERK, p-ERK, tCREB and p-CREB. The intraperitoneal administration of OMT (160, 80 mg/kg) could prevent the development of mechanical allodynia, thermal hyperalgesia induced by CCI. Intraperitoneal administration of OMT decreased the mean IOD of NR2B in the dorsal horn and expression of NR2B, p-ERK and p-CREB protein. Regulation of NMDA NR2B receptor-ERK/CREB signaling maybe the targets for the antinociceptive effects of OMT on a chronic neuropathic pain model induced by chronic constrictive injury of the sciatic nerve. Copyright © 2013 Elsevier GmbH. All rights reserved.

  18. Regulation of Bach2 by the aryl hydrocarbon receptor as a mechanism for suppression of B-cell differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    De Abrew, K. Nadira; Phadnis, Ashwini S.; Genetics Program, Michigan State University, East Lansing, MI 48824

    2011-04-15

    Exposure to the aryl hydrocarbon receptor (AHR) agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters B-cell differentiation and suppresses antibody production. Previous genomic studies in mouse B cells identified Bach2 as a direct target of the AHR. Bach2 is known to repress expression of Prdm1, a key transcription factor involved in B-cell differentiation, by binding to Maf elements (MAREs) in the regulatory regions of the gene. Chromatin immunoprecipitation followed by quantitative PCR in TCDD-treated lipopolysaccharide (LPS)-activated B cells showed increased binding of the AHR within the first intron in the Bach2 gene. The binding was further confirmed by electrophoretic mobility shift assay (EMSA). TCDDmore » also induced expression of Bach2 in activated as well as resting B cells from 2 to 24 h post-treatment in a time- and concentration-dependent manner. Expression of Prdm1 was decreased by TCDD at 24 h and was consistent with repression by Bach2. Increased DNA binding activity to the intron 5 MARE with increasing TCDD concentrations was observed by EMSA. Supershifts identified the presence of Bach2 in the DNA binding complex associated with the intron 5 MARE of Prdm1. Functional validation of the role of Bach2 in the suppression of B-cell differentiation by TCDD was performed using RNA interference (RNAi). Knockdown of Bach2 showed approximately 40% reversal in the TCDD-induced suppression of IgM secretion when compared to controls. The results suggest that the transcriptional regulation of Bach2 by the AHR is one of the mechanisms involved in the suppression of B-cell differentiation by TCDD.« less

  19. Regulation of Bach2 by the Aryl-hydrocarbon Receptor as a Mechanism for Suppression of B-cell Differentiation by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

    PubMed Central

    De Abrew, K. Nadira; Phadnis, Ashwini S.; Crawford, Robert B.; Kaminski, Norbert E.; Thomas, Russell S.

    2011-01-01

    Exposure to the aryl hydrocarbon receptor (AHR) agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), alters B cell differentiation and suppresses antibody production. Previous genomic studies in mouse B cells identified Bach2 as a direct target of the AHR. Bach2 is known to repress expression of Prdm1, a key transcription factor involved in B cell differentiation, by binding to Maf elements (MAREs) in the regulatory regions of the gene. Chromatin immunopreciptiation followed by quantitative PCR in TCDD-treated lipopolysaccharide (LPS)-activated B cells showed increased binding of the AHR within the first intron in the Bach2 gene. The binding was further confirmed by electrophoretic mobility shift assay (EMSA). TCDD also induced expression of Bach2 in activated as well as resting B cells from 2 to 24 h post-treatment in a time and concentration-dependent manner. Expression of Prdm1 was decreased by TCDD at 24 h and was consistent with repression by Bach2. Increased DNA binding activity to the intron 5 MARE with increasing TCDD concentration was observed by EMSA. Supershifts identified the presence of Bach2 in the DNA binding complex associated with the intron5 MARE of Prdm1. Functional validation of the role of Bach2 in the suppression of B-cell differentiation by TCDD was performed using RNAi. Knockdown of Bach2 showed approximately 40% reversal in the TCDD-induced suppression of IgM secretion when compared to controls. The results suggest that the transcriptional regulation of Bach2 by the AHR is one of the mechanisms involved in the suppression of B-cell differentiation by TCDD. PMID:21296099

  20. 75 FR 34062 - Airworthiness Directives; Eurocopter France Model AS 350 B, BA, B1, B2, B3, and D, and Model...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-16

    ... Airworthiness Directives; Eurocopter France Model AS 350 B, BA, B1, B2, B3, and D, and Model AS355 E, F, F1, F2... AS 350 B, BA, B1, B2, B3, and D, and Model AS355 E, F, F1, F2, and N helicopters, with certain main... an unsafe condition for certain Eurocopter France Model AS 350 B, BA, BB, B1, B2, B3, and D, and...

  1. Molecular characterization of the receptor binding structure-activity relationships of influenza B virus hemagglutinin.

    PubMed

    Carbone, V; Kim, H; Huang, J X; Baker, M A; Ong, C; Cooper, M A; Li, J; Rockman, S; Velkov, T

    2013-01-01

    Selectivity of α2,6-linked human-like receptors by B hemagglutinin (HA) is yet to be fully understood. This study integrates binding data with structure-recognition models to examine the impact of regional-specific sequence variations within the receptor-binding pocket on selectivity and structure activity relationships (SAR). The receptor-binding selectivity of influenza B HAs corresponding to either B/Victoria/2/1987 or the B/Yamagata/16/88 lineages was examined using surface plasmon resonance, solid-phase ELISA and gel-capture assays. Our SAR data showed that the presence of asialyl sugar units is the main determinant of receptor preference of α2,6 versus α2,3 receptor binding. Changes to the type of sialyl-glycan linkage present on receptors exhibit only a minor effect upon binding affinity. Homology-based structural models revealed that structural properties within the HA pocket, such as a glyco-conjugate at Asn194 on the 190-helix, sterically interfere with binding to avian receptor analogs by blocking the exit path of the asialyl sugars. Similarly, naturally occurring substitutions in the C-terminal region of the 190-helix and near the N-terminal end of the 140-loop narrows the horizontal borders of the binding pocket, which restricts access of the avian receptor analog LSTa. This study helps bridge the gap between ligand structure and receptor recognition for influenza B HA; and provides a consensus SAR model for the binding of human and avian receptor analogs to influenza B HA.

  2. Oxidation of ZrB2-and HfB2-Based Ultra-High Temperature Ceramics: Effects of Ta Additions

    NASA Technical Reports Server (NTRS)

    Opila, Elizabeth; Levine, Stanley; Lorinez, Jonathan

    2003-01-01

    Several compositions of ZrB2- and HfB2-based Ultra-High Temperature Ceramics (UHTC) were oxidized in stagnant air at 1627 C in ten minute cycles for times up to 100 minutes. These compositions include: ZrB2 - 20v% SiC, HfB2 - 20v% SiC, ZrB2 - 20v% SiC - 20v% TaSi2, ZrB2 - 33v% SiC, HfB2 - 20v% SiC - 20v% TaSi2, and ZrB2 - 20v% SiC - 20v% TaC. The weight change due to oxidation was recorded. The ZrB2 - 20v% SiC - 20v% TaSi2 composition was also oxidized in stagnant air at 1927 C and in an arc jet atmosphere. Samples were analyzed after oxidation by x-ray diffraction, field emission scanning electron microscopy, and energy dispersive spectroscopy to determine the reaction products and to observe the microstructure. The ZrB2 - 20v% SiC - 20v% TaSi2 showed the lowest oxidation rate at 1627 C, but performed poorly under the more extreme tests due to liquid phase formation. Effects of Ta-additions on the oxidation of the diboride-based UHTC are discussed.

  3. Effects of the Sazetidine-a Family of Compounds on the Body Temperature in Wildtype, Nicotinic Receptor B2(-/-) and a7(-/-) Mice

    EPA Science Inventory

    Nicotine elicits hypothermic responses in rodents. This effect appears to be related to nicotinic receptor desensitization because sazetidine-A, an a4B2 nicotinic receptor desensitizing agent, produces marked hypothermia and potentiates nicotine-induced hypothermia in mice. To de...

  4. Photoelectron Spectroscopy Study of [Ta2B6]-: a Hexagonal Bipyramdial Cluster

    NASA Astrophysics Data System (ADS)

    Jian, Tian; Li, Weili; Romanescu, Constantin; Wang, Lai-Sheng

    2014-06-01

    It has been a long-sought goal in cluster science to discover stable atomic clusters as building blocks for cluster-assembled nanomaterials, as exemplified by the fullerenes and their subsequent bulk syntheses.[1,2] Clusters have also been considered as models to understand bulk properties, providing a bridge between molecular and solid-state chemistry.[3] Herein we report a joint photoelectron spectroscopy and theoretical study on the [Ta2B6]- and [Ta2B6] clusters.[4] The photoelectron spectrum of [Ta2B6]- displays a simple spectral pattern and a large HOMO-LUMO gap, suggesting its high symmetry. Theoretical calculations show that both the neutral and anion are D6h pyramidal. The chemical bonding analyses for [Ta2B6] revealed the nature of the B6 and Ta interactions and uncovered strong covalent bonding between B6 and Ta. The D6h-[TaB6Ta] gaseous cluster is reminiscent of the structural pattern in the ReB6X6Re core in the [(Cp*Re)2B6H4Cl2] and the TiB6Ti motif in the newly synthesized Ti7Rh4Ir2B8 solid-state compound.[5,6] The current work provides an intrinsic link between a gaseous cluster and motifs for solid materials. Continued investigations of the transition-metal boron clusters may lead to the discovery of new structural motifs involving pure boron clusters for the design of novel boride materials. Reference [1] H.W. Kroto, J. R. Heath, S. C. OBrien, R. F. Curl, R. E. Smalley, Nature 1985, 318, 162 - 163. [2] W. Krtschmer, L. D. Lamb, K. Fostiropoulos, D. R. Huffman, Nature 1990, 347, 354 - 358. [3] T. P. Fehlner, J.-F. Halet, J.-Y. Saillard, Molecular Clusters: A Bridge to Solid-State Chemitry, Cambridge University Press, UK, 2007. [4] W. L. Li, L. Xie, T. Jian, C. Romanescu, X. Huang, L.-S. Wang, Angew. Chem. Int. Ed. 2014, 126, 1312 - 1316. [5] B. Le Guennic, H. Jiao, S. Kahlal, J.-Y. Saillard, J.-F. Halet, S. Ghosh, M. Shang, A. M. Beatty, A. L. Rheingold, T. P. Fehlner, J. Am. Chem. Soc. 2004, 126, 3203 - 3217. [6] B. P. T. Fokwa, M. Hermus, Angew

  5. B-Cell Activation and Tolerance Mediated by B-Cell Receptor, Toll-Like Receptor, and Survival Signal Crosstalk in SLE Pathogenesis

    DTIC Science & Technology

    2016-09-01

    from adaptive and innate receptors, including the BCR and TLRs, as well as signals from T follicular helper (TFH) cells . In this regard, several TH1...IFN-g that, in concert with innate sensors, controls T-bet and CD11c expression in B cells . Materials and Methods Mice Tbx212/2, Stat62/2, Tbx21f...O’Neill, M. S. Naradikian, J. L. Scholz, and M. P. Cancro. 2011. A B- cell subset uniquely responsive to innate stimuli accumulates in aged mice. Blood 118

  6. Enhanced expression of rat hepatic CYP2B1/2B2 and 2E1 by pyridine: differential induction kinetics and molecular basis of expression.

    PubMed

    Kim, H; Putt, D; Reddy, S; Hollenberg, P F; Novak, R F

    1993-11-01

    Expression of the cytochrome P450 (CYP) 2B subfamily in rat and rabbit hepatic tissues after pyridine (PY) treatment has been examined, and the molecular basis for enhanced 2B1/2B2 expression has been determined. P450 expression was monitored using metabolic activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses, and the identity of the proteins was confirmed through N-terminus microsequence analysis. PY caused a dose-dependent elevation of hepatic CYP2B1/B2B levels in rats, which ranged from 4- to 22-fold over the dosing regimen of 100 to 400 mg PY/kg/day, for 3 days, respectively. PY at low dose failed to induce CYP2B in rabbit hepatic tissue, suggesting a species-dependent response in 2B expression. Anti-2B1 IgG addition to PY-induced microsomes inhibited benzphetamine N-demethylase activity by only approximately 15%, in sharp contrast to the approximately 73% inhibition observed for phenobarbital-induced microsomes, suggesting the induction of other form(s) of P450 having benzphetamine N-demethylase activity. Northern blot analysis revealed that PY treatment increased 2B1 and 2B2 poly(A)+ RNA levels approximately 69- and approximately 34-fold, respectively, whereas the 2E1 poly(A)+ RNA levels failed to increase. The results of this study show that PY induces CYP2B1/2B2 and that induction is species-dependent and kinetically distinguishable from 2E1 induction. Moreover, 2B1/2B2 induction occurs as a result of elevated mRNA levels associated with either transcriptional activation or mRNA stabilization, and it differs from the mechanism of hepatic 2E1 induction by PY.

  7. Elevated expression of activated forms of Neu/ErbB-2 and ErbB-3 are involved in the induction of mammary tumors in transgenic mice: implications for human breast cancer.

    PubMed Central

    Siegel, P M; Ryan, E D; Cardiff, R D; Muller, W J

    1999-01-01

    To assess the importance of Neu activation during mammary tumorigenesis, altered receptors harboring in-frame deletions within the extracellular domain were expressed in transgenic mice. Females from several independent lines develop multiple mammary tumors that frequently metastasize to the lung. Tumor progression in these strains was associated with elevated levels of tyrosine-phosphorylated Neu and ErbB-3. Consistent with these observations, a survey of primary human breast tumors revealed frequent co-expression of both erbB-2 and erbB-3 transcripts. The ability of altered Neu receptors to induce mammary tumorigenesis in transgenic mice prompted us to examine whether similar mutations occurred in ErbB-2 during human breast cancer progression. Interestingly, an alternatively spliced form of erbB-2, closely resembling spontaneous activated forms of neu, was detected in human breast tumors. The ErbB-2 receptor encoded by this novel transcript harbors an in-frame deletion of 16 amino acids in the extracellular domain and can transform Rat-1 fibroblasts. Together, these observations argue that co-expression of ErbB-2 and ErbB-3 may play a critical role in the induction of human breast tumors, and raise the possibility that activating mutations in the ErbB-2 receptor may also contribute to this process. PMID:10205169

  8. Adhesive and Cohesive Strength in FeB/Fe2B Systems

    NASA Astrophysics Data System (ADS)

    Meneses-Amador, A.; Blancas-Pérez, D.; Corpus-Mejía, R.; Rodríguez-Castro, G. A.; Martínez-Trinidad, J.; Jiménez-Tinoco, L. F.

    2018-05-01

    In this work, FeB/Fe2B systems were evaluated by the scratch test. The powder-pack boriding process was performed on the surface of AISI M2 steel. The mechanical parameters, such as yield stress and Young's modulus of the boride layer, were obtained by the instrumented indentation technique. Residual stresses produced on the boride layer were estimated by using the x-ray diffraction (XRD) technique. The scratch test was performed in order to evaluate the cohesive/adhesive strength of the FeB/Fe2B coating. In addition, a numerical evaluation of the scratch test on boride layers was performed by the finite element method. Maximum principal stresses were related to the failure mechanisms observed by the experimental scratch test. Shear stresses at the interfaces of the FeB/Fe2B/substrate system were also evaluated. Finally, the results obtained provide essential information about the effect of the layer thickness, the residual stresses, and the resilience modulus on the cohesive/adhesive strength in FeB/Fe2B systems.

  9. Neuronal SH2B1 is essential for controlling energy and glucose homeostasis.

    PubMed

    Ren, Decheng; Zhou, Yingjiang; Morris, David; Li, Minghua; Li, Zhiqin; Rui, Liangyou

    2007-02-01

    SH2B1 (previously named SH2-B), a cytoplasmic adaptor protein, binds via its Src homology 2 (SH2) domain to a variety of protein tyrosine kinases, including JAK2 and the insulin receptor. SH2B1-deficient mice are obese and diabetic. Here we demonstrated that multiple isoforms of SH2B1 (alpha, beta, gamma, and/or delta) were expressed in numerous tissues, including the brain, hypothalamus, liver, muscle, adipose tissue, heart, and pancreas. Rat SH2B1beta was specifically expressed in neural tissue in SH2B1-transgenic (SH2B1(Tg)) mice. SH2B1(Tg) mice were crossed with SH2B1-knockout (SH2B1(KO)) mice to generate SH2B1(TgKO) mice expressing SH2B1 only in neural tissue but not in other tissues. Systemic deletion of the SH2B1 gene resulted in metabolic disorders in SH2B1(KO) mice, including hyperlipidemia, leptin resistance, hyperphagia, obesity, hyperglycemia, insulin resistance, and glucose intolerance. Neuron-specific restoration of SH2B1beta not only corrected the metabolic disorders in SH2B1(TgKO) mice, but also improved JAK2-mediated leptin signaling and leptin regulation of orexigenic neuropeptide expression in the hypothalamus. Moreover, neuron-specific overexpression of SH2B1 dose-dependently protected against high-fat diet-induced leptin resistance and obesity. These observations suggest that neuronal SH2B1 regulates energy balance, body weight, peripheral insulin sensitivity, and glucose homeostasis at least in part by enhancing hypothalamic leptin sensitivity.

  10. Negative Allosteric Modulators Selective for The NR2B Subtype of The NMDA Receptor Impair Cognition in Multiple Domains

    PubMed Central

    Weed, Michael R; Bookbinder, Mark; Polino, Joseph; Keavy, Deborah; Cardinal, Rudolf N; Simmermacher-Mayer, Jean; Cometa, Fu-ni L; King, Dalton; Thangathirupathy, Srinivasan; Macor, John E; Bristow, Linda J

    2016-01-01

    Antidepressant activity of N-methyl-D-aspartate (NMDA) receptor antagonists and negative allosteric modulators (NAMs) has led to increased investigation of their behavioral pharmacology. NMDA antagonists, such as ketamine, impair cognition in multiple species and in multiple cognitive domains. However, studies with NR2B subtype-selective NAMs have reported mixed results in rodents including increased impulsivity, no effect on cognition, impairment or even improvement of some cognitive tasks. To date, the effects of NR2B-selective NAMs on cognitive tests have not been reported in nonhuman primates. The current study evaluated two selective NR2B NAMs, CP101,606 and BMT-108908, along with the nonselective NMDA antagonists, ketamine and AZD6765, in the nonhuman primate Cambridge Neuropsychological Test Automated Battery (CANTAB) list-based delayed match to sample (list-DMS) task. Ketamine and the two NMDA NR2B NAMs produced selective impairments in memory in the list-DMS task. AZD6765 impaired performance in a non-specific manner. In a separate cohort, CP101,606 impaired performance of the nonhuman primate CANTAB visuo-spatial Paired Associates Learning (vsPAL) task with a selective impairment at more difficult conditions. The results of these studies clearly show that systemic administration of a selective NR2B NAM can cause transient cognitive impairment in multiple cognitive domains. PMID:26105137

  11. V2 Vasopressin Receptor (V2R) Mutations in Partial Nephrogenic Diabetes Insipidus Highlight Protean Agonism of V2R Antagonists*

    PubMed Central

    Takahashi, Kazuhiro; Makita, Noriko; Manaka, Katsunori; Hisano, Masataka; Akioka, Yuko; Miura, Kenichiro; Takubo, Noriyuki; Iida, Atsuko; Ueda, Norishi; Hashimoto, Makiko; Fujita, Toshiro; Igarashi, Takashi; Sekine, Takashi; Iiri, Taroh

    2012-01-01

    Inactivating mutations of the V2 vasopressin receptor (V2R) cause cross-linked congenital nephrogenic diabetes insipidus (NDI), resulting in renal resistance to the antidiuretic hormone AVP. In two families showing partial NDI, characterized by an apparently normal response to diagnostic tests and an increase in the basal ADH levels suggesting AVP resistance, we have identified two V2R mutations, Ser-333del and Y128S. Both mutant V2Rs, when expressed in COS-7 cells, show partial defects in vasopressin-stimulated cAMP accumulation and intracellular localization. The inhibition of internalization does not rescue their localization. In contrast, the non-peptide V2R antagonists OPC41061 and OPC31260 partially rescue the membrane localization and basal function of these V2R mutants, whereas they inhibit the basal activity of the wild-type V2R. These results indicate that a partial loss of function of Ser-333del and Y128S mutant V2Rs results from defective membrane trafficking. These findings further indicate that V2R antagonists can act as protean agonists, serving as pharmacological chaperones for inactivating V2R mutants and also as inverse agonists of wild-type receptors. We speculate that this protean agonism could underlie the possible dual beneficial effects of the V2R antagonist: improvement of hyponatremia with heart failure or polycystic kidney disease and potential rescue of NDI. PMID:22144672

  12. Estrogen receptor-α, progesterone receptor, and c-erbB/HER-family receptor mRNA detection and phenotype analysis in spontaneous canine models of breast cancer

    PubMed Central

    Kabir, Farruk M. Lutful; DeInnocentes, Patricia; Agarwal, Payal; Mill, Christopher P.; Riese, David J.

    2017-01-01

    Well characterized, stable, p16-defective canine mammary cancer (CMT) cell lines and normal canine mammary epithelial cells were used to investigate expression of the major breast cancer-specific hormone receptors estrogen receptor alpha (ER1) and progesterone receptor (PR) as well as luminal epithelial-specific proto-oncogenes encoding c-erbB-1 (epidermal growth factor receptor/EGFr), c-erbB-2/HER2, c-erbB-3, and c-erbB-4 receptors. The investigation developed and validated quantitative reverse transcriptase polymerase chain reaction assays for each transcript to provide rapid assessment of breast cancer phenotypes for canine cancers, based on ER1, PR, and c-erbB-2/HER2 expressions, similar to those in human disease. Roles for relatively underexplored c-erbB-3 and c-erbB-4 receptor expressions in each of these breast cancer phenotypes were also evaluated. Each quantitative assay was validated by assessment of amplicon size and DNA sequencing following amplification. Differential expression of ER1, PR, and c-erbB-2 in CMT cell lines clearly defined distinct human-like breast cancer phenotypes for a selection of CMT-derived cell lines. Expression profiles for EGFr family genes c-erbB-3 and c-erbB-4 in CMT models also provided an enriched classification of canine breast cancer identifying new extended phenotypes beyond the conventional luminal-basal characterization used in human breast cancer. PMID:27515268

  13. 7 CFR 1b.2 - Policy.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 1 2013-01-01 2013-01-01 false Policy. 1b.2 Section 1b.2 Agriculture Office of the Secretary of Agriculture NATIONAL ENVIRONMENTAL POLICY ACT § 1b.2 Policy. (a) All policies and programs of... environment for present and future generations. (b) Each USDA agency is responsible for compliance with this...

  14. 7 CFR 1b.2 - Policy.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Policy. 1b.2 Section 1b.2 Agriculture Office of the Secretary of Agriculture NATIONAL ENVIRONMENTAL POLICY ACT § 1b.2 Policy. (a) All policies and programs of... environment for present and future generations. (b) Each USDA agency is responsible for compliance with this...

  15. 2-Aminoethyl Methylphosphonate, a Potent and Rapidly Acting Antagonist of GABA A-ρ1 Receptors

    DOE PAGES

    Xie, A.; Yan, J.; Yue, L.; ...

    2011-08-02

    All three classes of receptors for the inhibitory neurotransmitter GABA (GABAR) are expressed in the retina. This study investigated roles of GABAR, especially GABA(C)R (GABA(A)-rho), in retinal signaling in vivo by studying effects on the mouse electroretinogram (ERG) of genetic deletion of GABA(C)R versus pharmacological blockade using receptor antagonists. Brief full-field flash ERGs were recorded from anesthetized GABA(C)R(-/-) mice, and WT C57BL/6 (B6) mice, before and after intravitreal injection of GABA(C)R antagonists, TPMPA, 3-APMPA, or the more recently developed 2-AEMP; GABA(A)R antagonist, SR95531; GABA(B)R antagonist, CGP, and agonist, baclofen. Intravitreal injections of TPMPA and SR95531 were also made in Brownmore » Norway rats. The effect of 2-AEMP on GABA-induced current was tested directly in isolated rat rod bipolar cells, and 2-AEMP was found to preferentially block GABA(C)R in those cells. Maximum amplitudes of dark (DA) and light-adapted (LA) ERG b-waves were reduced in GABA(C)R(-/-) mice, compared to B6 mice, by 30-60%; a-waves were unaltered and oscillatory potential amplitudes were increased. In B6 mice, after injection of TPMPA (also in rats), 3-APMPA or 2-AEMP, ERGs became similar to ERGs of GABA(C)R(-/-) mice. Blockade of GABA(A)Rs and GABA(B)Rs, or agonism of GABA(B)Rs did not alter B6 DA b-wave amplitude. The negative scotopic threshold response (nSTR) was slightly less sensitive in GABA(C)R(-/-) than in B6 mice, and unaltered by 2-AEMP. However, amplitudes of nSTR and photopic negative response (PhNR), both of which originate from inner retina, were enhanced by TPMPA and 3-APMPA, each of which has GABA(B) agonist properties, and further increased by baclofen. The finding that genetic deletion of GABA(C)R, the GABA(C)R antagonist 2-AEMP, and other antagonists all reduced ERG b-wave amplitude, supports a role for CABA(C)R in determining the maximum response amplitude of bipolar cells contributing to the b-wave. GABA(C)R antagonists

  16. Amygdala Infusions of an NR2B-Selective or an NR2A-Preferring NMDA Receptor Antagonist Differentially Influence Fear Conditioning and Expression in the Fear-Potentiated Startle Test

    ERIC Educational Resources Information Center

    Walker, David L.; Davis, Michael

    2008-01-01

    Within the amygdala, most N-methyl-D-aspartic acid (NMDA) receptors consist of NR1 subunits in combination with either NR2A or NR2B subunits. Because the particular subunit composition greatly influences the receptors' properties, we investigated the contribution of both subtypes to fear conditioning and expression. To do so, we infused the…

  17. R- and S-terbutaline activate large conductance and Ca2+ dependent K+ (BKCa) channel through interacting with β2 and M receptor respectively.

    PubMed

    Fan, Zhuo; Lin, Wei; Lv, Nanying; Ye, Yanrui; Tan, Wen

    2016-11-01

    This study investigated the effect of the β 2 receptor agonist terbutaline on the single channel activity of BK Ca channel. The effects of racemate and two isomers of terbutaline were all assessed. β 2 adrenoceptors were stably overexpressed on HEK293 cells by lentiviral transduction method and chicken BK Ca channels were transiently expressed on normal HEK293 cell line or HEK293 cells overexpressing β 2 receptors. Data showed that terbutaline significantly increased the single channel open probability of BK Ca channel within 10min. The channel activating effects of terbutaline are stereoselective and mainly stay with the R-enantiomers. The opening probability of BK Ca channel at 10min after drug application normalized to that just before drug application (Po10/Po0s) for R- and S-terbutaline were 7.85±3.20 and 1.06±0.45 respectively at 1μM concentration, corresponding to 28.37±9.96 and 2.68±1.09 at the higher concentration of 10μM. ICI 118551 blocked the effect of R- but not S-terbutaline (10μM), whereas atropine blocked the channel activating effects of S-terbutaline of higher concentration. In addition, the muscarinic receptor agonist carbachol increased the BK Ca channel activity in an atropine-sensitive manner as an positive control experiment, which indicate the involvement of M receptor in the channel activating effect of S-terbutaline. Copyright © 2016. Published by Elsevier B.V.

  18. Conventional light chains inhibit the autonomous signaling capacity of the B cell receptor.

    PubMed

    Meixlsperger, Sonja; Köhler, Fabian; Wossning, Thomas; Reppel, Michael; Müschen, Markus; Jumaa, Hassan

    2007-03-01

    Signals from the B cell antigen receptor (BCR), consisting of mu heavy chain (muHC) and conventional light chain (LC), and its precursor the pre-BCR, consisting of muHC and surrogate light chain (SLC), via the adaptor protein SLP-65 regulate the development and function of B cells. Here, we compare the effect of SLC and conventional LC expression on receptor-induced Ca(2+) flux in B cells expressing an inducible form of SLP-65. We found that SLC expression strongly enhanced an autonomous ability of muHC to induce Ca(2+) flux irrespective of additional receptor crosslinking. In contrast, LC expression reduced this autonomous muHC ability and resulted in antigen-dependent Ca(2+) flux. These data indicate that autonomous ligand-independent signaling can be induced by receptor forms other than the pre-BCR. In addition, our data suggest that conventional LCs play an important role in the inhibition of autonomous receptor signaling, thereby allowing further B cell differentiation.

  19. CCR2 antagonist CCX140-B provides renal and glycemic benefits in diabetic transgenic human CCR2 knockin mice

    PubMed Central

    Sullivan, Timothy; Miao, Zhenhua; Dairaghi, Daniel J.; Krasinski, Antoni; Wang, Yu; Zhao, Bin N.; Baumgart, Trageen; Ertl, Linda S.; Pennell, Andrew; Seitz, Lisa; Powers, Jay; Zhao, Ruiping; Ungashe, Solomon; Wei, Zheng; Boring, Landin; Tsou, Chia-Lin; Charo, Israel; Schall, Thomas J.; Jaen, Juan C.

    2013-01-01

    Chemokine (C-C motif) receptor 2 (CCR2) is central for the migration of monocytes into inflamed tissues. The novel CCR2 antagonist CCX140-B, which is currently in two separate phase 2 clinical trials in diabetic nephropathy, has recently been shown to reduce hemoglobin A1c and fasting blood glucose levels in type 2 diabetics. In this report, we describe the effects of this compound on glycemic and renal function parameters in diabetic mice. Since CCX140-B has a low affinity for mouse CCR2, transgenic human CCR2 knockin mice were generated and rendered diabetic with either a high-fat diet (diet-induced obesity) or by deletion of the leptin receptor gene (db/db). CCX140-B treatment in both models resulted in decreased albuminuria, which was associated with decreased glomerular hypertrophy and increased podocyte density. Moreover, treatment of diet-induced obese mice with CCX140-B resulted in decreased levels of fasting blood glucose and insulin, normalization of homeostatic model assessment of insulin resistance values, and decreased numbers of adipose tissue inflammatory macrophages. Unlike other CCR2 antagonists, CCX140-B had no effect on plasma levels of the CCR2 ligand CCL2 or on the numbers of blood monocytes. These results support the ongoing evaluation of this molecule in diabetic subjects with impaired renal function. PMID:23986513

  20. Pirfenidone attenuates IL-1β-induced COX-2 and PGE2 production in orbital fibroblasts through suppression of NF-κB activity.

    PubMed

    Choi, Youn-Hee; Back, Keum Ok; Kim, Hee Ja; Lee, Sang Yeul; Kook, Koung Hoon

    2013-08-01

    The aim of this study was to determine the effect of pirfenidone on interleukin (IL)-1β-induced cyclooxygenase (COX)-2 and prostaglandin (PG)E2 expression in orbital fibroblasts from patients with thyroid-associated ophthalmopathy (TAO). Primary cultures of orbital fibroblasts from patients with TAO (n = 4) and non-TAO subjects (n = 4) were prepared. The level of PGE2 in orbital fibroblasts treated with IL-1β in the presence or absence of pirfenidone was measured using an enzyme-linked immunosorbent assay. The effect of pirfenidone on IL-1β-induced COX-2 expression in orbital fibroblasts from patients with TAO was evaluated by reverse transcription-polymerase chain reaction (PCR) and quantitative real-time PCR analyses, and verified by Western blot. Activation of nuclear factor-κB (NF-κB) was evaluated by immunoblotting for inhibitor of κB (IκB)α and phosphorylated IκBα, and DNA-binding activity of p50/p65 NF-κB was analyzed by electrophoretic mobility shift assay. In addition, IL-1 receptor type 1 (IL-1R1) expression was assessed by RT-PCR in IL-1β-treated cells with or without pirfenidone. Pirfenidone significantly attenuated IL-1β-induced PGE2 release in both TAO and non-TAO cells. IL-1β-induced COX-2 mRNA and protein expression decreased significantly following co-treatment with pirfenidone. IL-1β-induced IκBα phosphorylation and degradation decreased in the presence of pirfenidone and led to decreased nuclear translocation and DNA binding of the active NF-κB complex. In our system, neither IL-1β nor pirfenidone co-treatment influenced IL-1R1 expression. Our results suggest that pirfenidone attenuates the IL-1β-induced PGE2/COX-2 production in TAO orbital fibroblasts, which is related with suppression of the NF-κB activation. Copyright © 2013 Elsevier Ltd. All rights reserved.