Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: 2-O, 3-O Desulfated Heparin Blocks High Mobility Group Box 1 Release by Inhibition of p300 Acetyltransferase Activity

doi: 10.1165/rcmb.2016-0069OC

Figure Lengend Snippet: 2-O, 3-O desulfated heparin (ODSH) inhibited neutrophil elastase (NE)– and LPS-induced high mobility group box 1 (HMGB1) release. RAW264.7 cells in serum-free media were pretreated with ODSH (0.5, 2, or 7.5 μM) for 30 minutes, and then treated with NE (0.5 μM) for 4 hours. (A) Conditioned media were collected and tested for HMGB1 expression using Western analysis. Densitometric analysis of autoradiographs from three experiments (mean ± SEM; n = 3) was summarized graphically and is expressed as relative values compared with control treatment (Ctrl). (B) +HMGB1 levels after NE treatment were significantly greater than Ctrl or HMGB1 levels after 7.5 μM ODSH treatment alone (P < 0.05); *HMGB1 levels after NE + 0.5 or NE + 2 μM ODSH were significantly lower than HMGB1 levels after NE treatment alone (P < 0.05); **HMGB1 levels after NE + 7.5 μM ODSH were significantly lower than HMGB1 levels after NE treatment alone (P < 0.01). (C) RAW264.7 cells were treated with LPS ± ODSH (0.5, 2, or 5 μM) in OPTI-MEM media for 24 hours. Conditioned media were collected, and Western analysis for HMGB1 was performed as described in Materials and Methods. Molecular weight markers are shown (KD). (D) Densitometric analysis of autoradiographs from three experiments (mean ± SEM; n = 3) was summarized graphically and is expressed as relative values compared with Ctrl. ++HMGB1 levels after LPS treatment were significantly greater than Ctrl or HMGB1 levels after 5 μM ODSH treatment alone (P < 0.01); *HMGB1 levels after LPS + 2 μM ODSH treatment were significantly lower than HMGB1 levels after LPS treatment alone (P < 0.05); **HMGB1 levels after LPS + 5 μM ODSH were significantly lower than HMGB1 levels after LPS treatment alone (P < 0.01).

Article Snippet: CoA, coenzyme A. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 4. caption a7 ODSH inhibited p300 histone acetyltransferase (HAT) activity and p300-induced HMGB1 acetylation in vitro in a concentration-dependent manner. ( A ) To test whether ODSH inhibited p300 HAT activity, recombinant p300 HAT activity (50 ng/50 μl total reaction volume; Active Motif) was assayed in the presence or absence of ODSH (0–850 μM) using histone H3 as a substrate.

Techniques: Expressing, Western Blot, Control, Molecular Weight

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: 2-O, 3-O Desulfated Heparin Blocks High Mobility Group Box 1 Release by Inhibition of p300 Acetyltransferase Activity

doi: 10.1165/rcmb.2016-0069OC

Figure Lengend Snippet: ODSH inhibited HMGB1 acetylation induced by RAW264.7 nuclear lysates and by human blood monocyte–derived macrophage (hBMDM) nuclear lysates in a concentration-dependent manner. (A) RAW264.7 cell or (B) hBMDM nuclear extract was incubated with recombinant HMGB1-his-tag, acetyl-CoA (Active Motif, Carlsbad, CA), and ODSH (8.5–850 μM), at room temperature for 2 hours. The reaction mixture was collected using His-tagged Dynabeads and was resuspended in loading buffer. An aliquot (20 μl) was separated by 4–20% polyacrylamide gel electrophoresis for Western analyses for acetyllysine and recombinant HMGB1. Western blots are representative of three separate experiments for RAW264.7 cells and hBMDM. Molecular weight markers are shown. (C and D) Graphic summaries of densitometric analyses of autoradiographs were expressed as the ratio of acetyllysine to HMGB1 normalized to the no-ODSH control treatment condition (mean ± SEM; n = 3). ***ODSH significantly inhibited RAW264.7 nuclear extract–induced HMGB1 acetylation (P < 0.001); *ODSH significantly inhibited hBMDM nuclear extract–induced HMGB1 acetylation (P < 0.05). CoA, coenzyme A.

Article Snippet: CoA, coenzyme A. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 4. caption a7 ODSH inhibited p300 histone acetyltransferase (HAT) activity and p300-induced HMGB1 acetylation in vitro in a concentration-dependent manner. ( A ) To test whether ODSH inhibited p300 HAT activity, recombinant p300 HAT activity (50 ng/50 μl total reaction volume; Active Motif) was assayed in the presence or absence of ODSH (0–850 μM) using histone H3 as a substrate.

Techniques: Derivative Assay, Concentration Assay, Incubation, Recombinant, Polyacrylamide Gel Electrophoresis, Western Blot, Molecular Weight, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: 2-O, 3-O Desulfated Heparin Blocks High Mobility Group Box 1 Release by Inhibition of p300 Acetyltransferase Activity

doi: 10.1165/rcmb.2016-0069OC

Figure Lengend Snippet: ODSH inhibited p300 histone acetyltransferase (HAT) activity and p300-induced HMGB1 acetylation in vitro in a concentration-dependent manner. (A) To test whether ODSH inhibited p300 HAT activity, recombinant p300 HAT activity (50 ng/50 μl total reaction volume; Active Motif) was assayed in the presence or absence of ODSH (0–850 μM) using histone H3 as a substrate. Relative HAT activity was determined using a HAT activity assay kit (Active Motif) and was expressed as relative fluorescent units and normalized to the incubation control with no ODSH (0). Data summarize three experiments (mean ± SEM; n = 3). ***ODSH 85 and 850 μM significantly inhibited p300 HAT activity (P < 0.001). (B) To evaluate whether ODSH inhibited p300-catalyzed lysine acetylation of HMGB1, recombinant p300 (50 ng/50 μl total reaction volume; Active Motif) was incubated with recombinant HMGB1 (2 μg), acetyl-CoA (Active Motif), and a concentration range of ODSH (8.5–850 μM). An aliquot of reaction mixture (5 μl) was separated by 4–20% polyacrylamide gel electrophoresis, and Western analyses were performed for acetyllysine and HMGB1. Graphic summary of densitometric analyses of Western autoradiographs are expressed as the ratio of acetyllysine/HMGB1 normalized to a no-ODSH control treatment condition (mean ± SEM; n = 3 experiments). (C) ***All ODSH treatments significantly decreased acetyllysine/HMGB1 levels compared with no ODSH treatment (P < 0.001).

Article Snippet: CoA, coenzyme A. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 4. caption a7 ODSH inhibited p300 histone acetyltransferase (HAT) activity and p300-induced HMGB1 acetylation in vitro in a concentration-dependent manner. ( A ) To test whether ODSH inhibited p300 HAT activity, recombinant p300 HAT activity (50 ng/50 μl total reaction volume; Active Motif) was assayed in the presence or absence of ODSH (0–850 μM) using histone H3 as a substrate.

Techniques: Activity Assay, In Vitro, Concentration Assay, Recombinant, HAT Activity Assay, Incubation, Control, Polyacrylamide Gel Electrophoresis, Western Blot

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: 2-O, 3-O Desulfated Heparin Blocks High Mobility Group Box 1 Release by Inhibition of p300 Acetyltransferase Activity

doi: 10.1165/rcmb.2016-0069OC

Figure Lengend Snippet: Hypothetical schema of ODSH mechanisms to inhibit release of HMGB1. HMGB1 acetylation and cellular release require p300 catalytic activity. ODSH inhibits p300 activity by binding to the catalytic domain via an allosteric mechanism. ODSH also inhibits NE protease activity, which is required for NE-induced HMGB1 release. Ac-HMGB1, Acetyl-HMGB1.

Article Snippet: CoA, coenzyme A. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 4. caption a7 ODSH inhibited p300 histone acetyltransferase (HAT) activity and p300-induced HMGB1 acetylation in vitro in a concentration-dependent manner. ( A ) To test whether ODSH inhibited p300 HAT activity, recombinant p300 HAT activity (50 ng/50 μl total reaction volume; Active Motif) was assayed in the presence or absence of ODSH (0–850 μM) using histone H3 as a substrate.

Techniques: Activity Assay, Binding Assay

Journal: Journal of Immunology Research

Article Title: Reduced Activity of HDAC3 and Increased Acetylation of Histones H3 in Peripheral Blood Mononuclear Cells of Patients with Rheumatoid Arthritis

doi: 10.1155/2018/7313515

Figure Lengend Snippet: HAT and HDAC activities in the nuclear extracts of PBMCs in RA patients and HCs. HAT activity was significantly increased in RA patients compared with HCs. HDAC activity was significantly decreased in RA patients compared to that in HCs.

Article Snippet: We next used the EpiQuik™ total HDAC activity and HDAC (1–3, 8) activity assay colorimetric kit (EpiGentek, Farmingdale, NY) and the HAT activity assay kit (Enzo Life Sciences, Koropi, Greece) to evaluate HAT and HDAC activities in PBMC nuclear extracts.

Techniques: Activity Assay