Journal: The Journal of Trauma and Acute Care Surgery

Article Title: PAD2 disturbs cardiomyocyte calcium homeostasis by citrullinating SERCA2a protein in hemorrhagic shock induced arrhythmia

doi: 10.1097/TA.0000000000004644

Figure Lengend Snippet: PAD2 protein is increased in trauma patients with hemorrhagic shock and knockout of Pad2 improves hemorrhagic ventricular arrhythmias. ( A ) Peripheral blood plasma PAD2 concentration (ELISA) in patients with traumatic hemorrhagic shock (n = 22) and healthy volunteers (n = 30),**** p < 0.0001; ( B ) Correlation analysis between PAD2 concentration and lactate concentration in peripheral blood plasma of patients with traumatic hemorrhagic shock (n = 22), r = 0.5174, *** p = 0.0002; ( C ) Expression of PAD2 in RNA Seq of cardiac tissue in GEO datasets of patients with arrhythmia compared to healthy volunteers; ( D ) Construction strategy of Pad2 knockout ( Pad2 −/− ) mice (upper), Pad2 expression level in heart tissues of Pad2 −/− mice and WT mice by western blot (bottom); ( E ) Schematic diagram of hemorrhagic shock model; ( F ) After hemorrhagic shock, the Kaplan-Meier survival rate curve of WT mice (n = 7) and Pad2 −/− mice (n = 7), **** p < 0.0001; ( G ) Representative electrocardiogram monitoring images of WT mice and Pad2 −/− mice in hemorrhagic shock model; ( H ) The statistical data of heart rate of WT mice before and after hemorrhagic shock (n = 6, *** p = 0.0008), and the statistical data of heart rate of Pad2 −/− mice before and after hemorrhagic shock (n = 6 vs. n = 6, *** p = 0.0002); ( I ) The statistical data of PP interval of WT mice before and after hemorrhagic shock (n = 6, ** p = 0.0053), and the statistical data of PP interval of Pad2 −/− mice before and after hemorrhagic shock (n = 6 vs. n = 6, *** p = 0.0002); ( J ) The statistical data of QT interval of WT mice before and after hemorrhagic shock (n = 6, ** p = 0.009), and the statistical data of PP interval of Pad2 −/− mice before and after hemorrhagic shock (n = 6 vs. n = 6); ( K ) After hemorrhagic shock, Total duration of ventricular tachycardia (left) (n = 6 vs. n = 6, **** p < 0.0001) and Incidence of ventricular fibrillation (right) of WT mice and Pad2 −/− mice.

Article Snippet: PAD2 cDNA (ORIGENE, RC223889) and Pad2-Ca2-mutant (Tsingke Biotech) plasmids were obtained commercially.

Techniques: Knock-Out, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, RNA Sequencing, Western Blot

Journal: The Journal of Trauma and Acute Care Surgery

Article Title: PAD2 disturbs cardiomyocyte calcium homeostasis by citrullinating SERCA2a protein in hemorrhagic shock induced arrhythmia

doi: 10.1097/TA.0000000000004644

Figure Lengend Snippet: Cardiac overexpression of PAD2 increases mortality and susceptibility to ventricular arrhythmias following hemorrhagic shock. Construct cardiomyocyte PAD2 overexpression in WT mice by inject AAV9-PAD2 via tail vein of WT for 4 weeks, AAV9-MCS injection as control; Construct cardiomyocyte PAD2 reintroduction in Pad2 −/− mice by inject AAV9-PAD2 via tail vein of Pad2 −/− mice for 4 weeks, AAV9-MCS injection as control; ( A ) Construction strategy of cardiomyocyte PAD2 overexpression in WT mice, cardiomyocytes PAD2 reintroduction in Pad2 −/− mice; ( B ) Representative images of confocal microscopy to verify the overexpression (top)/reintroduction (bottom) efficiency of Pad2 in WT/ Pad2 −/− mouse hearts; ( C ) Representative protein immunoblotting images to validate the overexpression (top)/reintroduction (bottom) efficiency of Pad2 in WT/ Pad2 −/− mouse hearts; ( D ) Kaplan-Meier survival rate curve of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice after hemorrhagic shock, n = 6 vs. n = 5, ** p = 0.0044; ( E ) Representative electrocardiogram monitoring images of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice after hemorrhagic shock in hemorrhagic shock model; ( F ) Kaplan-Meier survival rate curve of cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice after hemorrhagic shock, n = 6 for each group, ** p = 0.003; ( G ) Representative electrocardiogram monitoring images of cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice after hemorrhagic shock; ( H ) Before and after hemorrhagic shock, heart beats statistical value of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice (left), n = 6 vs. n = 5, * p = 0.0363, *** p = 0.0004; cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and control mice (right), n = 6 for each group, *** p = 0.0001, *** p = 0.0008; ( I ) Before and after hemorrhagic shock, P amplitude statistical value of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice (left), n = 6 vs. n = 5, * p = 0.0467, * p = 0.0302; cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice (right), n = 6 for each group, **** p < 0.0001, * p = 0.0158; ( J ) Before and after hemorrhagic shock, PP interval statistical value of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice (left), n = 6 vs. n = 5, *** p = 0.0002; cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice (right), n = 6 for each group, **** p < 0.0001, *** p = 0.0009; ( K ) Before and after hemorrhagic shock, QT interval statistical value of cardiomyocytes PAD2 overexpression in WT mice and AAV9-MCS infected WT mice (left), n = 6 vs. n = 5, ** p = 0.0077, ** p = 0.0015; cardiomyocytes PAD2 reintroduction in Pad2 −/− mice and AAV9-MCS infected Pad2 −/− mice(right), n = 6 for each group, *** p = 0.0003, *** p = 0.0003.

Article Snippet: PAD2 cDNA (ORIGENE, RC223889) and Pad2-Ca2-mutant (Tsingke Biotech) plasmids were obtained commercially.

Techniques: Over Expression, Construct, Injection, Control, Confocal Microscopy, Western Blot, Infection

Journal: The Journal of Trauma and Acute Care Surgery

Article Title: PAD2 disturbs cardiomyocyte calcium homeostasis by citrullinating SERCA2a protein in hemorrhagic shock induced arrhythmia

doi: 10.1097/TA.0000000000004644

Figure Lengend Snippet: PAD2 deficiency protects cardiomyocytes from arrhythmia by maintaining Ca 2+ homeostasis. ( A ) Representative diagram of cytosolic Ca 2+ recordings of adult mouse cardiomyocytes in response to 10-V, 0.5-Hz to 7.0-Hz electronic pacing; n = 20 from 5 mice in WT group, n = 18 from 5 mice in Pad2 KO group. ( B ) Average Ca 2+ decay Tau of adult mouse cardiomyocytes in response to 10-V, 0.5-Hz (n = 6 vs. n = 6, ** p = 0.0025), 2.0-Hz (n = 6 vs. n = 6, ** p = 0.0015), and 3.0 Hz (n = 4 vs. n = 6, *** p = 0.0005) separately. ( C ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal in response to 10 mmol/L caffeine stimulation in adult mouse cardiomyocytes, ( D ) representative recordings, represented by a curve showing the variation of fluorescence intensity value (F/F 0 ) over time. ( E ) Sarcoplasmic reticulum calcium content quantifications of the peak amplitude of cytosolic Ca 2+ as determined by the Fluo-4 AM fluorescence signal, the calcium storage content is defined as the maximum value of F/F 0 , and n = 10 cells from 6 mice in each group. ** p < 0.0001. ( F ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal in response to 10 mmol/L caffeine stimulation in adult rat cardiomyocytes infected with Ad-Scramble or Ad-sh-PAD2, ( G ) representative recordings, represented by a curve showing the variation of fluorescence intensity value (F/F 0 ) over time, and ( H ) Sarcoplasmic reticulum calcium content quantifications of the peak amplitude of cytosolic Ca 2+ as determined by the Fluo-4 AM fluorescence signal, the calcium storage content is defined as the maximum value of F/F 0 . n = 6 cells from 6 rats and n = 10 cells from 6 rats, *** p = 0.0003. ( I ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal in response to 10 mmol/L caffeine stimulation in adult rat cardiomyocytes infected with Ad-LacZ or Ad-PAD2, ( J ) representative recordings, represented by a curve showing the variation of fluorescence intensity value (F/F 0 ) over time, and ( K ) Sarcoplasmic reticulum calcium content quantifications of the peak amplitude of cytosolic Ca 2+ as determined by the Fluo-4 AM fluorescence signal, the calcium storage content is defined as the maximum value of F/F0. n = 28 cells from 10 rats and n = 25 cells from 10 rats, **** p < 0.0001. ( L ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal of spontaneous calcium waves in adult mouse cardiomyocytes from WT and Pad2 −/− mice. ( M ) Percentage of cells with spontaneous calcium waves. n = 15 cells from 5 mice in each group. ( N ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal of spontaneous calcium sparks in adult mouse cardiomyocytes from WT and Pad2 −/− mice.

Article Snippet: PAD2 cDNA (ORIGENE, RC223889) and Pad2-Ca2-mutant (Tsingke Biotech) plasmids were obtained commercially.

Techniques: Fluorescence, Infection

Journal: The Journal of Trauma and Acute Care Surgery

Article Title: PAD2 disturbs cardiomyocyte calcium homeostasis by citrullinating SERCA2a protein in hemorrhagic shock induced arrhythmia

doi: 10.1097/TA.0000000000004644

Figure Lengend Snippet: Hypoxia induces PAD2 translocation and increases its co-localization with the SR. ( A ) Representative Western blot (left) and average data (right) illustrating Pad2 protein levels in cardiomyocytes under normoxic or hypoxic conditions for 1 hour. Each group consists of n = 6 vs. n = 6 samples. ( B ) Immunofluorescence staining (top) showing the translocation of Pad2 out of the nucleus under hypoxic stimulation. Scale bar = 5 μm (upper)Scale bar = 10 μm (bottom). The quantification of Pad2 fluorescence intensity in the nucleus and cytoplasm is shown below, with values expressed as the ratio of average fluorescence intensity to pixels. After hypoxia stimulation, the average fluorescence intensity of Pad2 in the nucleus decreased significantly (n = 7 vs. n = 7, * p = 0.013), while the average fluorescence intensity of Pad2 in the cytoplasm increased significantly (n = 7 vs. n = 7, ** p = 0.0078). H9c2 cells were exposed to either normoxia or hypoxia, and cytoplasmic and nuclear proteins were isolated. ( C ) Quantification of Pad2 protein levels in the cytoplasm and nucleus was performed using Western blotting (WB). Representative Western blot (top) and average data (bottom) demonstrate a significant decrease in nuclear Pad2 and a significant increase in cytoplasmic Pad2 after hypoxia exposure (n = 6 per group, **** p < 0.0001). ( D ) Immunofluorescence staining (top) showing the co-localization of Pad2 with PDI in cardiomyocytes with and without hypoxic stimulation. Scale bar = 10 μm (upper) Scale bar = 20 μm (bottom). Pearson’s correlation coefficient was calculated to quantify the degree of co-localization between Pad2 and PDI (bottom). **** p < 0.0001. ( E ) Sarcoplasmic reticulum proteins were separated and extracted, and Pad2 protein levels were quantified using Western blotting. Representative Western blot (top) and average data (bottom) indicate the levels of Pad2 in the sarcoplasmic reticulum. n = 4 independent experiments, * p = 0.014.

Article Snippet: PAD2 cDNA (ORIGENE, RC223889) and Pad2-Ca2-mutant (Tsingke Biotech) plasmids were obtained commercially.

Techniques: Translocation Assay, Western Blot, Immunofluorescence, Staining, Fluorescence, Isolation

Journal: The Journal of Trauma and Acute Care Surgery

Article Title: PAD2 disturbs cardiomyocyte calcium homeostasis by citrullinating SERCA2a protein in hemorrhagic shock induced arrhythmia

doi: 10.1097/TA.0000000000004644

Figure Lengend Snippet: Hypoxia inhibits SERCA2a activity through PAD2-mediated citrullination. Neonatal rat cardiomyocytes were infected with Ad-PAD2, and CoIP-MS (co-immunoprecipitation mass spectrometry) was performed using Pad2 and IgG controls separately. ( A ) Heatmap of CoIP-MS. ( B ) Gene Ontology (GO) enrichment analysis of CoIP-MS. ( C ) KEGG pathway enrichment analysis of CoIP-MS. ( D ) ER Proteins interacting with Pad2. ( E ) The results of protein immunoprecipitation (CoIP) showed that endogenous Pad2 protein interacts with SERCA2a protein in neonatal rat cardiomyocytes. ( F ) Western blot analysis (left) and average data (right) showing SERCA2a protein levels in neonatal rat cardiomyocytes under normoxic or hypoxic conditions (n = 4 vs. n = 4 per group). ( G ) Measurement of Pad2 enzymatic activity in neonatal rat cardiomyocytes followed by normoxia or hypoxia stimulation for 1 hour. (n = 6 for each group). ( H ) Measurement of SERCA2a enzymatic activity in neonatal rat cardiomyocytes infected with Ad-LacZ or Ad-PAD2 for 48 hours, followed by normoxia or hypoxia stimulation for 1 hour (n = 7 vs. n = 7 per group, * p = 0.004, * p = 0.0478, **** p < 0.0001). ( I ) Measurement of SERCA2a enzymatic activity in neonatal rat cardiomyocytes infected with Ad-sh-Scramble or Ad-sh-PAD2 for 48 hours, followed by normoxia or hypoxia stimulation for 1 hour (n = 6 vs. n = 6 per group, **** p < 0.0001, *** p = 0.0002). ( J ) CoIP (co-immunoprecipitation) analysis showing the interaction of Serca2a protein with a citrullination antibody in neonatal cardiomyocytes under normoxic or hypoxic conditions for 1 hour. ( K ) CoIP (co-immunoprecipitation) analysis showing the interaction of SERCA2a protein with a citrullination antibody in heart tissue under hemorrhagic shock. ( L ) Cardiomyocytes transfected with the pCMV-PAD2-Ca2 mutation plasmid exhibited significantly reduced Pad2 enzyme activity compared to those transfected with the WT pCMV-PAD2 plasmid (n = 5 vs. n = 5 per group, **** p < 0.0001). ( M ) Cardiomyocytes transfected with the pCMV-PAD2-Ca2 mutation plasmid reversed hypoxia-induced reductions in SERCA2a activity compared to those transfected with the WT pCMV-PAD2 plasmid (n = 3 vs. n = 3, ** p = 0.0016, *** p = 0.0005).

Article Snippet: PAD2 cDNA (ORIGENE, RC223889) and Pad2-Ca2-mutant (Tsingke Biotech) plasmids were obtained commercially.

Techniques: Activity Assay, Infection, Immunoprecipitation, Mass Spectrometry, Western Blot, Transfection, Mutagenesis, Plasmid Preparation

Journal: The Journal of Trauma and Acute Care Surgery

Article Title: PAD2 disturbs cardiomyocyte calcium homeostasis by citrullinating SERCA2a protein in hemorrhagic shock induced arrhythmia

doi: 10.1097/TA.0000000000004644

Figure Lengend Snippet: PAD2 inhibitor prevents hemorrhagic shock-induced cardiac arrhythmia. Thirty minutes before the hemorrhagic shock model, mice were treated with different concentrations of the Pad2 selective inhibitor AFM-30a (10 mg/kg, 20 mg/kg, and 40 mg/kg) via intraperitoneal injection, with DMSO as the vehicle control. ( A ) Survival rates of hemorrhagic shock mice treated with various concentrations of AFM-30a. n = 12 for each treated group and n = 16 for the vehicle group. * p = 0.0338, ** p = 0.0084. ( B ) Serum lactate concentrations from mice treated with various concentrations of AFM-30a. n = 6 for each treated group and n = 10 for the vehicle group. * p = 0.0251, * p = 0.0323. ( C ) Representative electrocardiogram (ECG) monitoring images of mice treated with vehicle (DMSO), or 40 mg/kg of AFM-30a for 30 minutes before the hemorrhagic shock model. In hemorrhagic shock, ( D ) Statistical values of heart rate from ECG recordings (top). Statistical values of QT interval from ECG recordings (bottom). n = 6 for each group. ( E ) Representative diagram of cytosolic Ca 2+ recordings of adult mouse cardiomyocytes pretreated with 25 μM of AFM-30a or DMSO for 24 hours in response to 10-V, 0.5-Hz to 7.0-Hz electronic pacing; n = 20 from 5 mice in control group, n = 18 from 5 mice in AFM-30a treated group. ( F ) Average Ca 2+ decay Tau of adult mouse cardiomyocytes in response to 10-V, 0.5-Hz (n = 6 vs. n = 6, **** p < 0.0001), 2.0-Hz (n = 6 vs. n = 6, * p = 0.0138), and 3.0 Hz (n = 6 vs. n = 6, ** p = 0.0028) separately. ( G ) Representative confocal images of cytosolic Ca 2+ via the Fluo-4 AM fluorescence signal in response to 10 mmol/L caffeine stimulation in adult mouse cardiomyocytes treated with 25 μM of AFM-30a or DMSO for 24 hours, ( H ) representative recordings, represented by a curve showing the variation of fluorescence intensity value (F/F 0 ) over time. ( I ) Sarcoplasmic reticulum calcium content quantifications of the peak amplitude of cytosolic Ca 2+ as determined by the Fluo-4 AM fluorescence signal, the calcium storage content is defined as the maximum value of F/F 0 , and n = 10 cells from 6 mice in each group. ** p < 0.0001.

Article Snippet: PAD2 cDNA (ORIGENE, RC223889) and Pad2-Ca2-mutant (Tsingke Biotech) plasmids were obtained commercially.

Techniques: Injection, Control, Fluorescence

Journal: Arthritis Research & Therapy

Article Title: Effect of Porphyromonas gingivalis infection on gut dysbiosis and resultant arthritis exacerbation in mouse model

doi: 10.1186/s13075-020-02348-z

Figure Lengend Snippet: Effect of PgPAD on the onset of joint arthritis. The effect of PgPAD on the onset of arthritis was determined. In this experiment, PgPAD deletion mutant 33277 strain (⊿ pad ) and Pg 33277 wild-type (WT) strain were used, unless Pg W83 strain was specifically described in caption of Figures. ABL of the upper jaw in each group was measured ( a ). Ctrl, PBS inoculation; WT, Pg wild-type inoculation; ⊿ pad , Pg pad knockout strain inoculation. To evaluate inflammation of the gingival tissue 6 weeks after Pg inoculation, IL-6 production in gingival tissue of SKG mice was measured by ELISA ( b ). AS was measured 3 and 6 weeks after Pg inoculation with LA i.p. injection ( c ). To assess CP synthesis, ELISA was performed ( d gingival tissue, e joint tissue, f small intestine, g large intestine). The titer of serum ACPA 6 weeks after Pg inoculation with LA i.p. injection in mice from each group was measured by ELISA ( h ). The production of endogenous PADI2 and PADI4 in joint tissue was determined by Western blotting, and then the density of each band was measured by NIH Image-J ( i and j ). Data represent mean ± SD ( b – j ) of 5 mice per group. Statistical analyses were performed using the Tukey-Kramer test and Bonferroni-corrected Mann-Whitney U test for multiple comparisons (* P < 0.05, ** P < 0.01)

Article Snippet: As a control, the amount of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected by anti-GAPDH antibody (10 μg/ml, HRP-60004, PROTEINTECH JAPAN, Japan) The density of target PADI2 and PADI4 bands was measured by NIH Image-J software.

Techniques: Mutagenesis, Knock-Out, Enzyme-linked Immunosorbent Assay, Injection, Western Blot, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

doi: 10.1186/s13075-014-0498-9

Figure Lengend Snippet: In situ citrullination of human fibrinogen. (A) Fibrinogen (Fib) or citrullinated fibrinogen (cFib) was immobilized to enzyme-linked immunosorbent assay (ELISA) plates at a concentration of 1 μg/ml. Increasing concentrations of the anti-cFib monoclonal antibody (mAb; clone 20B2) were added, followed by addition of enzyme-conjugated rabbit anti-mouse immunoglobulin antibodies and o -phenylenediamine substrate. All data points represent means and ranges of duplicate measurements of optical density at 490 nm. (B) ELISA plates were coated with 1.0 μg/ml human fibrinogen and incubated with citrullination buffer including recombinant human peptidylarginine deiminase (rhPAD) enzyme, ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), MQPAD4 (49 mU/ml) or rhPAD4 (1,500 ng/ml). Shown is the activity as a percentage of the maximal activity obtained for each enzyme, corresponding to the activity at 4 hours. Anti-cFib mAb was used at a concentration of 0.5 μg/ml, as determined from the graph shown in (A). Symbols and error bars represent means and ranges of duplicate measurements.

Article Snippet: Enzyme-linked immunosorbent assay plates were coated with 1.0 μg/ml human fibrinogen and incubated with four different recombinant human peptidylarginine deiminase (rhPAD) preparations: ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), rhPAD4 (1,500 ng/ml) or MQPAD4 (49 mU/ml).

Techniques: In Situ, Enzyme-linked Immunosorbent Assay, Concentration Assay, Incubation, Recombinant, Activity Assay

Journal: Arthritis Research & Therapy

Article Title: Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

doi: 10.1186/s13075-014-0498-9

Figure Lengend Snippet: Comparison between enzymatic activities of peptidylarginine deiminases 2 and 4. Enzyme-linked immunosorbent assay plates were coated with 1.0 μg/ml human fibrinogen. (A) Recombinant human peptidylarginine deiminase 2 (rhPAD2) or rhPAD4 raised in-house were used for citrullination at mass concentrations ranging from 0.1 ng/ml to 15 μg/ml. (B) Commercially available ModiQuest (MQ) PAD2 or MQPAD4 enzymes were used in units ranging from 0.1 mU/ml to 100 mU/ml. Following a 3-hour citrullination period, the anti-cFib mAb (0.5 μg/ml) was used for detection of citrullinated fibrinogen. Duplicate measurements of optical density (OD) at 490 nm are shown as mean and range.

Article Snippet: Enzyme-linked immunosorbent assay plates were coated with 1.0 μg/ml human fibrinogen and incubated with four different recombinant human peptidylarginine deiminase (rhPAD) preparations: ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), rhPAD4 (1,500 ng/ml) or MQPAD4 (49 mU/ml).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Arthritis Research & Therapy

Article Title: Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

doi: 10.1186/s13075-014-0498-9

Figure Lengend Snippet: Calcium dependency of peptidylarginine deiminases. Enzyme-linked immunosorbent assay plates were coated with 1.0 μg/ml human fibrinogen and incubated with four different recombinant human peptidylarginine deiminase (rhPAD) preparations: ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), rhPAD4 (1,500 ng/ml) or MQPAD4 (49 mU/ml). Dithiothreitol was added at a concentration of 1.0 mM, and CaCl 2 was added at concentrations ranging from 50 μM to 10 mM. Following 3 hours of incubation, citrullination was measured using mouse anti-cFib monoclonal antibody (0.5 μg/ml). Shown is the activity as a percentage of maximal activity for each enzyme. Symbols and error bars represent means and ranges of duplicate measurements.

Article Snippet: Enzyme-linked immunosorbent assay plates were coated with 1.0 μg/ml human fibrinogen and incubated with four different recombinant human peptidylarginine deiminase (rhPAD) preparations: ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), rhPAD4 (1,500 ng/ml) or MQPAD4 (49 mU/ml).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, Concentration Assay, Activity Assay

Journal: Arthritis Research & Therapy

Article Title: Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

doi: 10.1186/s13075-014-0498-9

Figure Lengend Snippet: Peptidylarginine deiminases activity in synovial fluid samples from rheumatoid arthritis patients. (A) Synovial fluid (SF) samples from five rheumatoid arthritis patients were applied on enzyme-linked immunosorbent assay plates coated with fibrinogen (1 μg/ml) after 1:3 dilution into Tris buffer with 1 mM dithiothreitol (DTT; black columns), Tris buffer containing 1 mM DTT and 10 mM CaCl 2 (grey columns) or Tris buffer containing 1 mM DTT and 10 mM ethylenediaminetetraacetic acid (EDTA; white columns). Following a 3-hour incubation period, the monoclonal mouse anti-citrullinated fibrinogen (anti-cFib) (0.5 μg/ml) was used for detection of citrullinated fibrinogen. Peptidylarginine deiminase (PAD) activity is presented as optical density (OD) at 490 nm of duplicated measurements. (B) Association between PAD2 concentration and PAD activity in the five SF samples diluted 1:3 in citrullination buffer containing 10 mM CaCl 2 (grey circles, solid line) or no additive calcium (black circles, dotted line). Pearson’s correlation coefficients ( r ) and levels of significance are shown.

Article Snippet: Enzyme-linked immunosorbent assay plates were coated with 1.0 μg/ml human fibrinogen and incubated with four different recombinant human peptidylarginine deiminase (rhPAD) preparations: ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), rhPAD4 (1,500 ng/ml) or MQPAD4 (49 mU/ml).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: PAD activity in NAWM of MS patients. (A) Quantification of PAD2 protein in white matter from normal and MS brain by immunoslot blot ( n =5, P <0.0001). (B) Citrullinated protein in white matter from normal and MS brains by immunoslot blot as pixel density ( n =4, P <0.01). (C) PAD enzyme activity in normal and MS tissue, with or without pre-incubation with 2CA ( n =5, P <0.05). Each dot represents one patient analysed three times. The means (horizontal bar) for all the MS patients were compared with the normal.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Activity Assay, Incubation

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: Interaction of PAD with 2CA. (A) PAD2 and PAD4 inhibition curves in the presence of increasing 2CA concentrations. Insert: PAD1-4 enzymes contain a common C-terminal active-site cysteine residue (Cys656) bound by 2CA, confirmed by ESI mass spectrometry of tryptic digests of PAD2-acetamidine adducts. (B) Schematic of the nucleophilic reaction between 2CA and the Cys656 residue in the active site of PAD2 . (C) Tabular summary of peptide fragment atomic masses for 2CA-modified and native PAD2.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Inhibition, Residue, Mass Spectrometry, Modification

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: mAb4E12 (anti-2CA adduct) immunogold labeled optic nerve cryosections from control and 2CA-treated PAD2 transgenic mice. Minimal labeling in untreated mice (black arrows). Numerous gold particles (white arrows) in nuclei (N) and cytoplasm of oligodendrocytes, myelin and axonoplasm (Ax) of 2CA-treated mice. Scale bars: 500 nm.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Labeling, Control, Transgenic Assay

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: 2CA attenuates demyelinating disease in ND4 mice. (A) ND4 mice treated with PBS, 2CA (5 mg/kg), or 2CA+B12 (5 mg/kg and 10 mg/kg) starting at 2 months before disease onset ( n =5, P <0.0001). (B) ND4 mice treated at disease onset ( n =4, P <0.0001). (C) Stopping 2CA, but continuing B12 at 3.5 months in ND4 mice ( n =5, P <0.0001) demonstrates that B12 alone does not attenuate disease. (D) PAD activity in brains from animals shown in ( n =5, P <0.05). The first four bars represent results from animals at 6 months of age, whereas the post-treatment animals were 8 months of age. (E) PAD2 RT PCR in white matter extracts from normal, PBS-, 2CA- and 2CA+B12-treated ND4 mice ( n =9, P <0.05). (F) LFB and hematoxylin stain of cerebella from normal, PBS-, 2CA- and 2CA+B12-treated ND4 mice (40×). WM, white matter; GM, grey matter.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: 2CA attenuates PAD2 overexpressor. (A) Demyelinating disease in PAD2 transgenic mice treated with PBS, 2CA or 2CA+B12 starting at 6 months of age ( n =5, P <0.0001). (B) PAD activity in brain extracts of PAD2 transgenic mice treated with PBS, 2CA or 2CA+B12, and non-transgenic littermates ( n =4, P <0.05). PAD activity is reduced to normal levels by treatment.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Transgenic Assay, Activity Assay