human β3 receptor  (R&D Systems)

Journal: PLoS ONE

Article Title: Chemokine (C-C Motif) Ligand 20, a Potential Biomarker for Graves' Disease, Is Regulated by Osteopontin

doi: 10.1371/journal.pone.0064277

Figure Lengend Snippet: (A) Induction of CCL20 level in plasma from uGD patients and healthy controls. Medium: RPMI 1640; NP, normal plasma; NP+ OPN: normal plasma added with 0.1 µg/ml OPN; PP, patient plasma; PP+OPNmAb : patient plasma pretreated with 5 µg/ml OPN-neutralizing antibody for 30 min. *, P <0.05 versus medium control; #, P <0.05 versus Ctrl mAb. (B, C) OPN induced CCL20 mRNA expressions (B) and protein levels (C) in a time and dose-dependent manner. For time course, the PBMCs were cultured in the presence of human recombinant OPN (1 µg/ml) and analyzed at the indicated time points. To determine the dose dependent manner of CCL20 expression, the PBMCs were cultured for 12 hours with OPN at the indicated concentrations. CD4+T cells and culture medium were then purified and analyzed. Shown are representative results from 3 independent experiments with separate specimens. All data were presented as mean±SEM. *, P <0.05; **, P <0.01, versus medium control.

Article Snippet: For blocking experiments, purified antibodies against human OPN (Human OPN Affinity Purified Polyclonal Ab, AF1433, 5µg/ml), human β3 receptor (Human Integrin alpha V beta 3 MAb, MAB3050, 5µg/ml), human IL-17 (Human IL-17 Affinity Purified Polyclonal Ab, AF-317-NA, 1µg/ml) and isotype-matched control antibodies (all purchased from R&D Systems) were added.

Techniques: Cell Culture, Recombinant, Expressing, Purification

Journal: PLoS ONE

Article Title: Chemokine (C-C Motif) Ligand 20, a Potential Biomarker for Graves' Disease, Is Regulated by Osteopontin

doi: 10.1371/journal.pone.0064277

Figure Lengend Snippet: (A) CCL20 gene expressions in isolated CD4+T cells from 8 hCD and 8 uGD in the presence or absence of 1 µg/ml rOPN for 12 h. (B) Expressions of OPN receptors on CD4+ T cells from uGD patients, eGD patients, nGD patients and hCD. (C) β3 integrin receptor antibody blocked induction of CCL20 mRNA and protein levels by OPN. The freshly isolated PBMCs were cultured in the presence or absence of 1 µg/ml rOPN for 12 h. Blocking Abs to integrin β3, or its isotype control mAb (IgG) were added at 5 µg/ml. Supernatants from cultures and CD4+T cells separated from PBMCs were used to analyze the expression levels of CCL20. Shown are representative results from 3 independent experiments with separate specimens. All data were presented as mean±SEM. *, P <0.05; ***, P <0.001, versus medium control; #, P <0.05 versus Ctrl mAb.

Article Snippet: For blocking experiments, purified antibodies against human OPN (Human OPN Affinity Purified Polyclonal Ab, AF1433, 5µg/ml), human β3 receptor (Human Integrin alpha V beta 3 MAb, MAB3050, 5µg/ml), human IL-17 (Human IL-17 Affinity Purified Polyclonal Ab, AF-317-NA, 1µg/ml) and isotype-matched control antibodies (all purchased from R&D Systems) were added.

Techniques: Isolation, Cell Culture, Blocking Assay, Expressing

Journal: PLoS ONE

Article Title: Chemokine (C-C Motif) Ligand 20, a Potential Biomarker for Graves' Disease, Is Regulated by Osteopontin

doi: 10.1371/journal.pone.0064277

Figure Lengend Snippet: (A, B) PBMCs were isolated from normal subjects and treated with rOPN (1 µg/ml) at different time point. The IL-17 mRNA expression in purified CD4+T cells (A) and protein level in culture medium (B) were analyzed. Induction of CCL20 mRNA in CD4+T cells (C) and protein levels in culture medium (D) by OPN was blocked by antibody against IL-17, and inhibitors of IKK and MAPKs. Shown are representative results from 3 independent experiments with separate specimens. All data were presented as mean±SEM. *, P <0.05 versus OPN; # P <0.05 versus medium control; ns>0.05 versus OPN.

Article Snippet: For blocking experiments, purified antibodies against human OPN (Human OPN Affinity Purified Polyclonal Ab, AF1433, 5µg/ml), human β3 receptor (Human Integrin alpha V beta 3 MAb, MAB3050, 5µg/ml), human IL-17 (Human IL-17 Affinity Purified Polyclonal Ab, AF-317-NA, 1µg/ml) and isotype-matched control antibodies (all purchased from R&D Systems) were added.

Techniques: Isolation, Expressing, Purification

Journal: Anesthesiology

Article Title: Alphaxalone Binds in Inner Transmembrane β + –α − Interfaces of α1β3γ2 γ-Aminobutyric Acid Type A Receptors

doi: 10.1097/ALN.0000000000001978

Figure Lengend Snippet: The transmembrane domain of a α1β3γ2L structural homology model based on GluCl (pdb 4COF) is depicted 25. Subunit peptide backbones are shown as ribbons (α1 = yellow; β3 = blue; γ2L = green), with sidechains of interest (see Table 1) shown in space-filling mode and labeled. Amino acid sidechains on β3-M3 and α1-M1 that are directly photolabeled by analogs of one or more study anesthetics are colored orange-red. Anesthetic contact sidechains that have previously been identified using substituted cysteine modification-protection are colored purple. Other β3-M2 and β3-M3 sidechains that line the β+–α– cleft and three sidechains predicted to face the β3 intra-subunit helix bundle pocket (Y284, G287, and E298), are colored gray. The location of α1Q242 (pink) is also shown. Inserts display the molecular space-filling structures of propofol, etomidate, and alphaxalone, approximately scaled to the receptor model. Hydrogens have been hidden for clarity.

Article Snippet: Complementary DNAs encoding human α1, β3, and γ2L GABA A receptor subunits in pCDNA3.1 expression vectors (Thermo Fisher Scientific, Waltham, MA, USA) were used.

Techniques: Labeling, Modification

Journal: British Journal of Pharmacology

Article Title: 2?-Methoxy-6-methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA A receptors

doi: 10.1111/j.1476-5381.2011.01604.x

Figure Lengend Snippet: 2′MeO6MF is a positive modulator of α1-containing GABAA receptors expressed in Xenopus oocytes. (A) Representative current trace showing the potentiation of GABA (EC10: 10 µM) by various concentrations of 2′MeO6MF (1, 10, 30, 100 and 300 µM) at human recombinant α1β2γ2L GABAA receptors. Horizontal bars show the duration of drug application. In the absence of GABA, 2′MeO6MF (300 µM) produced ≍2% of the current produced by the maximum GABA concentration. Note the rebound current following removal of 2′MeO6MF (indicated by the arrows) (B) 2′MeO6MF potentiates the response to GABA (EC10) at recombinant α1β2, α1β1γ2, α1β2γ2L and α1β3γ2L receptors expressed in oocytes. Control GABA concentration at each receptor subtype was 10 µM. Data are mean ± SEM (n = 3–6 oocytes). (C) Representative current trace from an oocyte showing potentiation of GABA (10 µM) by 2′MeO6MF (100 µM) at recombinant α1β2γ2L GABAA receptors. As shown, the enhancement of GABA (10 µM) by 2′MeO6MF (100 µM) was not inhibited by flumazenil (1 and 10 µM) whereas potentiation of GABA (10 µM) by diazepam (1 µM) was inhibited by flumazenil (1 and 10 µM). Horizontal bars show the duration of drug application.

Article Snippet: GABA receptor subunit constructs Human α1, α2, β2 and γ2L DNA in pcDM8 were provided by Dr Paul Whiting (Merck, Sharpe and Dohme Research Labs, Harlow, UK), α3 and β3 in pGEMHE, α5 in pcDNA3 and β1 in PCDM8 were a gift from Dr Bjarke Ebert (H. Lundbeck A/S, Valby, Denmark).

Techniques: Recombinant, Produced, Concentration Assay, Control

Journal: British Journal of Pharmacology

Article Title: 2?-Methoxy-6-methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA A receptors

doi: 10.1111/j.1476-5381.2011.01604.x

Figure Lengend Snippet: The effects of 2′MeO6MF at the various recombinant ionotropic GABA receptor subtypes expressed in Xenopus oocytes

Article Snippet: GABA receptor subunit constructs Human α1, α2, β2 and γ2L DNA in pcDM8 were provided by Dr Paul Whiting (Merck, Sharpe and Dohme Research Labs, Harlow, UK), α3 and β3 in pGEMHE, α5 in pcDNA3 and β1 in PCDM8 were a gift from Dr Bjarke Ebert (H. Lundbeck A/S, Valby, Denmark).

Techniques: Recombinant

Journal: British Journal of Pharmacology

Article Title: 2?-Methoxy-6-methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA A receptors

doi: 10.1111/j.1476-5381.2011.01604.x

Figure Lengend Snippet: 2′MeO6MF directly activates human recombinant α2β2/3γ2L and α2β2/3 GABAA receptors expressed in oocytes. (A) Current trace showing the effects of increasing concentrations of 2′MeO6MF on recombinant α2β2γ2L GABAA receptors expressed in Xenopus oocytes against the maximum effect of GABA (3000 µM). Horizontal bars indicate duration of drug application. (B) Representative trace showing the potentiation of GABA (EC10: 5 µM) in the presence of diazepam (1 µM) and this effect was attenuated by flumazenil (10 µM) at α2β2γ2L GABAA receptors. In contrast, the direct activation of 2′MeO6MF (30 µM) on the same receptor was not attenuated by flumazenil (10 µM). (C) Representative trace showing that a 3 min pre-incubation with bicuculline (10 µM) attenuates the response produced by 2′MeO6MF (100 µM) by 56 ± 4% at α2β2γ2L GABAA receptors. Gabazine (10 µM and 100 µM) also attenuated the response produced by 2′MeO6MF on α2β2γ2L GABAA receptors by 45 ± 4% and 100% respectively. (D) Concentration–response curves for 2′MeO6MF alone and 2′MeO6MF in the presence of bicuculline (10 µM) and gabazine (1 µM) at α2β2γ2L GABAA receptors expressed in Xenopus oocytes. Data are mean ± SEM (n = 4–6 oocytes). (E) Concentration–response curves for 2′MeO6MF on α2β3γ2L, α2β2γ2L, α2β3 and α2β2 recombinant GABAA receptors expressed in oocytes. Currents are expressed as a percentage of the peak current elicited by 3 mM GABA. Data are mean ± SEM (n = 4–6 oocytes).

Article Snippet: GABA receptor subunit constructs Human α1, α2, β2 and γ2L DNA in pcDM8 were provided by Dr Paul Whiting (Merck, Sharpe and Dohme Research Labs, Harlow, UK), α3 and β3 in pGEMHE, α5 in pcDNA3 and β1 in PCDM8 were a gift from Dr Bjarke Ebert (H. Lundbeck A/S, Valby, Denmark).

Techniques: Recombinant, Activation Assay, Incubation, Produced, Concentration Assay

Journal: British Journal of Pharmacology

Article Title: 2?-Methoxy-6-methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA A receptors

doi: 10.1111/j.1476-5381.2011.01604.x

Figure Lengend Snippet: (A) Representative trace showing the activation of 2′MeO6MF (300 µM) is significantly reduced at the mutant α2β2N265Sγ2L GABAA receptor. In contrast, 2′MeO6MF (300 µM) was able to positively modulate GABA (10 µM) by 454 ± 16%. Horizontal bars show duration of drug application. (B) Concentration–response curves for 2′MeO6MF in the presence of GABA (EC10) at α2β1, α2β1γ2L and α2β2N265Sγ2L. Control GABA dose was 10 µM. Data are mean ± SEM (n = 4 oocytes). (C) Representative trace showing 2′MeO6MF (100 µM) directly activating recombinant α2β1S265Nγ2L mutant GABAA receptors. The current generated by 2′MeO6MF (100 µM) in the presence of GABA (10 µM) is the sum of the individual currents produced by GABA (10 µM) and 2′MeO6MF (100 µM) alone. Horizontal bars show duration of drug application. (D) Concentration–response curve for 2′MeO6MF expressed relative the maximal GABA concentration (3 mM) at α2β1S265Nγ2L GABAA receptors showing that the converse mutation converted 2′MeO6MF from a potentiator to a direct activator. Data are mean ± SEM for three to four oocytes.

Article Snippet: GABA receptor subunit constructs Human α1, α2, β2 and γ2L DNA in pcDM8 were provided by Dr Paul Whiting (Merck, Sharpe and Dohme Research Labs, Harlow, UK), α3 and β3 in pGEMHE, α5 in pcDNA3 and β1 in PCDM8 were a gift from Dr Bjarke Ebert (H. Lundbeck A/S, Valby, Denmark).

Techniques: Activation Assay, Mutagenesis, Concentration Assay, Control, Recombinant, Generated, Produced