Journal: iScience

Article Title: Monitoring GPCR conformation with GFP-inspired dyes

doi: 10.1016/j.isci.2024.110466

Figure Lengend Snippet: Structural and spectral changes of A 2A AR L225C -DyeC induced by various ligands (A) Schematic representation of structural changes caused by agonists and the allosteric modulator HMA in A 2A AR L225C -DyeC. Structures of the active and inactive A 2A AR are sketched from PDB IDs 6GDG and 3RFM , respectively. , Agonist binding results in an outward shift of the intracellular part of the TM6. The structural effects of the allosteric modulator HMA remain unknown. (B) Emission spectra of A 2A AR L225C -DyeC bound to antagonists (ZM241385 and SCH58261), agonists (NECA and adenosine), allosteric modulator (HMA), and in the apo state. (C) Variation of the integrated intensity of A 2A AR L225C -DyeC bound to different ligands. The integrated intensity is quantified as the area under the fluorescence emission spectrum from 430 to 700 nm. (D) Variation of the intensity ratio I 520 /I 460 for different ligands. Each condition in C and D was measured at least 9 times with protein from at least three independent purifications; each protein sample was mixed with the ligand independently. The data represent the mean ± SD. The protein concentration was maintained at 10 μM; all ligands were added at a saturating concentration of 100 μM. The significance level is given according to the ordinary one-way ANOVA with the post hoc Tukey HSD test: ∗∗ p < 0.005, ∗ p < 0.05, ns, not significant..

Article Snippet: Figure 4 Molecular dynamics simulations of A 2A AR L225C -DyeC (A and B) Isosurfaces (yellow) delineate the low-free-energy regions (at +25 kJ/mol level relative to the global free energy minimum) explored by the proximal carbon atom of the dimethoxybenzene ring of DyeC label in the active (A) and inactive (B) states in metadynamics simulations.

Techniques: Binding Assay, Fluorescence, Protein Concentration, Concentration Assay

Journal: iScience

Article Title: Monitoring GPCR conformation with GFP-inspired dyes

doi: 10.1016/j.isci.2024.110466

Figure Lengend Snippet: Molecular dynamics simulations of A 2A AR L225C -DyeC (A and B) Isosurfaces (yellow) delineate the low-free-energy regions (at +25 kJ/mol level relative to the global free energy minimum) explored by the proximal carbon atom of the dimethoxybenzene ring of DyeC label in the active (A) and inactive (B) states in metadynamics simulations. The A 2A AR L225C helices are labeled from TM1 to H8 (TM6 is colored in red), the G-protein binding site is labeled in the active state. (C and D) Positions of the dimethoxybenzene ring of the DyeC label in the active (C) and inactive (D) complexes throughout the unbiased (i.e., without any external forces applied) MD simulations shown every 0.1 ns as orange/yellow dots, respectively. Each system was simulated for 1,000 ns in two replicates. The positions of the lipid head groups are schematically indicated by the gray dotted line. (E) Autocorrelation functions (ACFs) calculated for a vector describing the DyeC label position in the unbiased simulations shown in (C) and (D). Higher values of ACF suggest slower reorientational dynamics of the label. Results for two replicates in the active/inactive states are shown in orange/yellow. (F) Correlation between fluorescence emission maximum of DyeC in different solvents and their partition coefficient, logP. The logP values were obtained from the PubChem/Chemeo databases , and provided in Table S1 . The inverse correlation implies that the translocation of DyeC into the region of the polar head groups of lipids, as observed in the simulations of the active state, leads to a red shift in the fluorescence emission maximum.

Article Snippet: Figure 4 Molecular dynamics simulations of A 2A AR L225C -DyeC (A and B) Isosurfaces (yellow) delineate the low-free-energy regions (at +25 kJ/mol level relative to the global free energy minimum) explored by the proximal carbon atom of the dimethoxybenzene ring of DyeC label in the active (A) and inactive (B) states in metadynamics simulations.

Techniques: Labeling, Protein Binding, Plasmid Preparation, Fluorescence, Translocation Assay

Journal: Inflammatory bowel diseases

Article Title: Potential for Developing Purinergic Drugs for Gastrointestinal Diseases

doi: 10.1097/MIB.0000000000000047

Figure Lengend Snippet: Drugs in clinical trials targeting purinergic receptors

Article Snippet: N/A , STW5/II , A 2A AR Antag , IBS , Steigerwald Arzneimittelwerke GmbH , II , Completed 189 , Yes.

Techniques: Functional Assay, Inhibition

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dexmedetomidine Attenuates Orthotopic Liver Transplantation-Induced Acute Gut Injury via α 2 -Adrenergic Receptor-Dependent Suppression of Oxidative Stress

doi: 10.1155/2019/9426368

Figure Lengend Snippet: Blocking α 2A -AR siRNA reversed the protective role of Dex on OLT-induced intestinal injury in rats. (a) Representative images showing the microscopic findings of intestines stained with hematoxylin and eosin in seven groups. Bar scale, 10 μ m. (b) Chiu's scoring quantitating the intestinal mucosal damage. (c–d) Immunohistochemistry staining of CD3 (c) and CD4 (d) in intestinal issues. Red arrow: positive cells. Data are represented as the mean ± standard deviation, n = 8 for each group. S: sham-operated group; M: the model group with OLT; D1: rats that were pretreated with 10 μ g/kg before OLT; D2: rats that were pretreated with 50 μ g/kg before OLT; B1: rats that received 500 g/kg atipamezole at 40 min before receiving 50 μ g/kg Dex prior to OLT; B2: rats that received 50 g/kg ARC239 at 40 min before receiving 50 μ g/kg Dex prior to OLT; B3: rats that received 1.5 mg/kg BRL-44408 at 40 min before receiving 50 μ g/kg Dex prior to OLT. ∗ P < 0.05, compared to Group S; # P < 0.05, compared to Group M; $ P < 0.05, compared to Group D2.

Article Snippet: The α 2A -AR siRNA duplexes (5′-CGAGCUGCAAGAUUAACGA-3′) targeting the rat α 2A -AR siRNA gene (gene ID: 25083; nucleotide accession number: NM_012739.3) were commercially obtained from Biomics (Jiangsu, China).

Techniques: Blocking Assay, Staining, Immunohistochemistry, Standard Deviation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dexmedetomidine Attenuates Orthotopic Liver Transplantation-Induced Acute Gut Injury via α 2 -Adrenergic Receptor-Dependent Suppression of Oxidative Stress

doi: 10.1155/2019/9426368

Figure Lengend Snippet: Blocking α 2A -AR siRNA reversed the protective role of Dex on OLT-induced intestinal mucosal barrier injury in rats. (a–b) Western blotting results showing the levels of occludin (a) and ZO-1 (b) in rat intestines. (c–f) ELISA results showing the levels of serum biomarkers of intestinal mucosal barrier function, including DAO (c), LPS (d), I-FABP2 (e), and D-LA (f). Data are represented as the mean ± standard deviation, n = 8 for each group. S: sham-operated group; M: the model group with OLT; D1: rats that were pretreated with 10 μ g/kg before OLT; D2: rats that were pretreated with 50 μ g/kg before OLT; B1: rats that received 500 g/kg atipamezole at 40 min before receiving 50 μ g/kg Dex prior to OLT; B2: rats that received 50 g/kg ARC239 at 40 min before receiving 50 μ g/kg Dex prior to OLT; B3: rats that received 1.5 mg/kg BRL-44408 at 40 min before receiving 50 μ g/kg Dex prior to OLT. ∗ P < 0.05, compared to Group S; # P < 0.05, compared to Group M; $ P < 0.05, compared to Group D2.

Article Snippet: The α 2A -AR siRNA duplexes (5′-CGAGCUGCAAGAUUAACGA-3′) targeting the rat α 2A -AR siRNA gene (gene ID: 25083; nucleotide accession number: NM_012739.3) were commercially obtained from Biomics (Jiangsu, China).

Techniques: Blocking Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dexmedetomidine Attenuates Orthotopic Liver Transplantation-Induced Acute Gut Injury via α 2 -Adrenergic Receptor-Dependent Suppression of Oxidative Stress

doi: 10.1155/2019/9426368

Figure Lengend Snippet: Blocking α 2A -AR siRNA counteracted the alleviation of oxidative stress by Dex in intestines of rats with OLT. The levels of oxidant ROS (a) and antioxidants including GST α 1 (b), SOD (c), and GSH (d) were measured in rat intestines. Data are represented as the mean ± standard deviation, n = 8 for each group. S: sham-operated group; M: the model group with OLT; D1: rats that were pretreated with 10 μ g/kg before OLT; D2: rats that were pretreated with 50 μ g/kg before OLT; B1: rats that received 500 g/kg atipamezole at 40 min before receiving 50 μ g/kg Dex prior to OLT; B2: rats that received 50 g/kg ARC239 at 40 min before receiving 50 μ g/kg Dex prior to OLT; B3: rats that received 1.5 mg/kg BRL-44408 at 40 min before receiving 50 μ g/kg Dex prior to OLT. ∗ P < 0.05, compared to Group S; # P < 0.05, compared to Group M; $ P < 0.05, compared to Group D2.

Article Snippet: The α 2A -AR siRNA duplexes (5′-CGAGCUGCAAGAUUAACGA-3′) targeting the rat α 2A -AR siRNA gene (gene ID: 25083; nucleotide accession number: NM_012739.3) were commercially obtained from Biomics (Jiangsu, China).

Techniques: Blocking Assay, Standard Deviation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dexmedetomidine Attenuates Orthotopic Liver Transplantation-Induced Acute Gut Injury via α 2 -Adrenergic Receptor-Dependent Suppression of Oxidative Stress

doi: 10.1155/2019/9426368

Figure Lengend Snippet: Silencing of α 2A -AR siRNA reversed the protective role of Dex on cell apoptosis in IEC-6 cells with simulated H/R stimulation. (a) qRT-PCR data showing effective knockdown of α 2A -AR by siRNA transfection in IEC-6 cells. Control: untreated IEC-6 cells; NC-siRNA: IEC-6 cells transfected with nonspecific control siRNA; α 2A -AR siRNA: IEC-6 cells transfected with α 2A -AR-specific siRNA. (b) Results of the cell proliferation assay by Cell Counting Kit-8 in IEC-6 cells with different stimulations. (c) Summary of cell apoptosis data of IEC-6 cells with different stimulations. (d) Representative flow cytometry profiles showing the cell apoptosis assay by annexin V and propidium iodide staining, n = 6 for each group. A: control IEC-6 cells; B: IEC-6 cells with H/R treatment; C: IEC-6 cells that were pretreated with 1 nM Dex for 1 h before inducing H/R injury; D: IEC-6 cells with silencing of α 2A -AR siRNA; E: IEC-6 cells with silencing of α 2A -AR siRNA and H/R treatment; F: IEC-6 cells with silencing of α 2A -AR siRNA that were pretreated with Dex prior to H/R injury. ∗ P < 0.05, compared to Group A; # P < 0.05, compared to Group B; $ P < 0.05, compared to Group C.

Article Snippet: The α 2A -AR siRNA duplexes (5′-CGAGCUGCAAGAUUAACGA-3′) targeting the rat α 2A -AR siRNA gene (gene ID: 25083; nucleotide accession number: NM_012739.3) were commercially obtained from Biomics (Jiangsu, China).

Techniques: Quantitative RT-PCR, Knockdown, Transfection, Control, Proliferation Assay, Cell Counting, Flow Cytometry, Apoptosis Assay, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dexmedetomidine Attenuates Orthotopic Liver Transplantation-Induced Acute Gut Injury via α 2 -Adrenergic Receptor-Dependent Suppression of Oxidative Stress

doi: 10.1155/2019/9426368

Figure Lengend Snippet: Silencing of α 2A -AR siRNA erased the protective role of Dex on antioxidation in IEC-6 cells with simulated H/R stimulation. (a) Summary of ROS levels in IEC-6 cells with different treatments. (b) Representative fluorescence immunostaining images of Nrf2 in IEC-6 cells with different treatments. Scale bar, 100 μ m. (c–d) Expression of Nrf2, NQO1, and Keap1 proteins and mRNA levels in intestinal tissues. (e–g) Summary of the relative expression levels of the transcripts for the genes HMOX1 (e), SOD1 (f), and CAT (g) in IEC-6 cells with different treatments, n = 6 for each group. A: control IEC-6 cells; B: IEC-6 cells with H/R treatment; C: IEC-6 cells that were pretreated with 1 nM Dex for 1 h before inducing H/R injury; D: IEC-6 cells with silencing of α 2A -AR siRNA; E: IEC-6 cells with silencing of α 2A -AR siRNA and H/R treatment; F: IEC-6 cells with silencing of α 2A -AR siRNA that were pretreated with Dex prior to H/R injury. ∗ P < 0.05, compared to Group A; # P < 0.05, compared to Group B; $ P < 0.05, compared to Group C.

Article Snippet: The α 2A -AR siRNA duplexes (5′-CGAGCUGCAAGAUUAACGA-3′) targeting the rat α 2A -AR siRNA gene (gene ID: 25083; nucleotide accession number: NM_012739.3) were commercially obtained from Biomics (Jiangsu, China).

Techniques: Fluorescence, Immunostaining, Expressing, Control