Journal: PLoS ONE

Article Title: Increased Migration of Monocytes in Essential Hypertension Is Associated with Increased Transient Receptor Potential Channel Canonical Type 3 Channels

doi: 10.1371/journal.pone.0032628

Figure Lengend Snippet: A ; Immunoblot showing specificity of antibodies against TRPC3 in monocytes from normotensive control subjects (NT) and patients with essential hypertension (HT) in the absence or presence of TRPC3 antigens (TRPC3+Ag). The predicted molecular weight of TRPC3 is 97 kDa. B ; Immunoblot showing specificity of antibodies against TRPC3 in monocytes from normotensive control subjects (NT, n = 8), patients with type 2 diabetes mellitus (DM, n = 9), patients with essential hypertension (HT, n = 8) or hypertensive patients with type 2 diabetes mellitus (HT+DM, n = 10). Summary data of the TRPC3 expression (normalized to GAPDH). *p<0.05, compared to NT. Data are mean ± SEM. C ; Representative in-cell western assay and summary data of the TRPC3 expression (normalized to CD14 expression used as an internal reference) in monocytes from normotensive control subjects (Normotensive, and opened bars, n = 3) and patients with essential hypertension (Hypertensive, filled bars, n = 3) under control conditions and after transfection with scrambled siRNA or specific siRNA against TRPC3 for 48 h. In-cell western assay was performed using specific antibodies and fluorescence-labeled secondary antibodies. TRPC3 (visible in green) normalized to CD14 (used as an internal reference). Measurements were performed in duplicate for each sample. *p<0.05 or **p<0.01 for the comparison with their controls; and ## p<0.01 for the comparison Hypertensive (filled bars) vs. Normotensive (open bars). D ; Representative in-cell western assay and summary data of the TRPC3 and TRPC6 expression in monocytes from normotensive control subjects under control conditions and after transfection with specific siRNA against TRPC3 for 48 h. In-cell western assay was performed using specific antibodies and fluorescence-labeled secondary antibodies. TRPC3 and TRPC6 expression (visible in green) normalized to CD14 (visible in red used as an internal reference). Measurements were performed in duplicate for each sample. **p<0.01 compared to control conditions. Data are mean ± SEM of three independent experiments. E ; Summary data of the fMLP-induced monocyte migration from hypertensive patients (HT, filled bars) and normotensive control subjects (NT, opened bars) quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. Monocytes chemotaxis was expressed as the mean number of migrated cells per high-power fields from duplicate wells. Experiments were performed under control conditions, after transfection with scrambled siRNA or specific siRNA against TRPC3. *p<0.05; **p<0.01 compared to normotensive control subjects under control conditions. Data are mean ± SEM of eight independent experiments. F ; Spontaneous migrations of monocytes from normotensive control subjects (NT; open bars) and hypertensive patients (HT, filled bars) were tested using medium or after transfection with scrambled siRNA or specific siRNA against TRPC3. The data was quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. P>0.05 compared to NT. Data are percent of medium as mean ± SEM of three independent experiments.

Article Snippet: After that incubated with rabbit anti-TRPC3 antibodies (1∶1000, Alomone Laboratories, Jerusalem, Israel) as the primary antibodies for 2 h, washed, incubated with IRDye 800 CW infrared fluorescent dye conjugated goat anti-rabbit antibodies (1∶1000, Biomol, Hamburg, Germany) as the secondary antibody overnight and washed, and quantitative imaging was performed at 810 nm emission with an excitation wavelength of 780 nm.

Techniques: Western Blot, Molecular Weight, Expressing, In-Cell ELISA, Transfection, Fluorescence, Labeling, Migration, Chemotaxis Assay

Journal: PLoS ONE

Article Title: Increased Migration of Monocytes in Essential Hypertension Is Associated with Increased Transient Receptor Potential Channel Canonical Type 3 Channels

doi: 10.1371/journal.pone.0032628

Figure Lengend Snippet: A , B ; fMLP activates ERK or phosphorylation of ERK ( A ) and Akt or phosphorylation of Akt ( B ) in a dose- and time-dependent manner in monocytes from normotensive control subjects. 10 nmol/L open bars, 100 nmol/L filled bars. Data are mean ± SEM, n = 3. *p<0.05 compared to lower concentration conditions. C , D ; Increased fMLP-induced phosphorylation of ERK ( C ) and Akt ( D ) in monocytes from patients with essential hypertension. The proteins were measured using immunoblotting with specific antibodies. Data are mean ± SEM from three independent experiments. *p<0.05 compared to normotensive control subjects. E ; fMLP activates monocytes by an ERK-dependent and Akt-dependent pathway. Akt, ERK, or pERK and pAkt were measured using immunoblotting with specific antibodies. In the presence of 2-APB or after administration of specific siRNA against TRPC3, the fMLP-induced ERK, pERK; Akt and pAkt were significantly reduced when compared with control conditions. Data are mean ± SEM from six independent experiments. *p<0.05; **p<0.01 compared to control.

Article Snippet: After that incubated with rabbit anti-TRPC3 antibodies (1∶1000, Alomone Laboratories, Jerusalem, Israel) as the primary antibodies for 2 h, washed, incubated with IRDye 800 CW infrared fluorescent dye conjugated goat anti-rabbit antibodies (1∶1000, Biomol, Hamburg, Germany) as the secondary antibody overnight and washed, and quantitative imaging was performed at 810 nm emission with an excitation wavelength of 780 nm.

Techniques: Concentration Assay, Western Blot

Journal:

Article Title: Antibodies to the desmoglein 1 precursor proprotein but not to the mature cell surface protein cloned from individuals without pemphigus

doi: 10.4049/jimmunol.0901691

Figure Lengend Snippet: Furin, which processes preDsg1 to matDsg1, causes decreased binding of anti-Dsg1 antibodies cloned from individuals without pemphigus. A, Furin treatment of Dsg1 ELISA plates causes decreased binding of K2D14-4, a mAb from a normal individual, but increased binding of F24-9, a pathogenic cell surface mAb from patient PF2. B, Immunoblot with anti-E-tag. Dsg1Ehis recombinant protein without furin treatment shows a high and lower molecular weight band (lane 7). Treatment with furin shows decreased intensity of the higher molecular weight band from processing of preDsg1 to matDsg1 (lane 6). T2D14-6 immunoprecipitates preDsg1 (lane 2) that is not longer detectable after furin treatment (lane 1). However, F24-9 precipitates only matDsg1 with or without furin treatment (lanes 3,4).

Article Snippet: In some experiments, to increase the ratio of the mature form of Dsg1 on ELISA plates, we pretreated the plates with 10U/well of furin (New England Biolabs) in 20 mM Tris, 500 mM sodium chloride, pH7.5 [TBS], with 1mM CaCl 2 at room temperature overnight.

Techniques: Binding Assay, Clone Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant, Molecular Weight

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Molecular events for CRBP1 gene in cervical epithelium samples. A: In order to know the gain of copy number of the CRBP1 gene, DNA of healthy cervix and CC samples, were subjected to real time PCR with specific Taqman probes. White bar (healthy cervix samples) represents the mean of the normal cervices (n = 26) without extra copies of CRBP1 gene. Black bars show CC samples with gain of copy number (2-20X); while gray dotted line bars are showing CC samples that do not change in the copies number. Values above the cut-off line (as 1), being assigned as increased gene copy number compared with normal cervical epithelium. CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [26]. In X-axis represents cervical samples, Y-axis relative copies fold change of CRBP1 gene. B: CRBP1 expression was observed as positive immunostaining result on tissue microarray as mentioned in Methods section The DNAs used for gain of copy number (panel A) were also used for the methylation assay. Methylation result represents the methylation of the CRBP1 promoter. In this case, each healthy or CC sample, correspond to each column for CRBP1 expression and methylation status. Interestingly, in most of the cases, there was an association between the lack of expression of the CRBP1 gene and its methylation status.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunostaining, Microarray, Methylation

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: CRBP1 immunodetection in the uterine cervix samples. A: (1) Cytoplasmic CRBP1 expression is present in cells of the basal layer of normal cervical epithelium (healthy tissue); (2) the immunodetection in the transformed cells of a cervical cancer (CC03) tissue harboring gain of CRBP1 gene. (3) CC samples without gain CRBP1 gene showing negative immunostaining (CC16 sample). A kidney tissue section (4) was used as positive control, while a heart tissue section for negative control (5). B: cervical progression spectrum. The tissue section shows a brownish reaction (positive reaction) in the basal cell layer of the “normal” region, in the high-grade lesion, and also in the invasive region. All tissue sections were hematoxylin counterstained, 200X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunodetection, Expressing, Transformation Assay, Immunostaining, Positive Control, Negative Control, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Association between CRBP1 gene gain copy number and its expression in cervical cancer samples

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Immunodetection

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Correlation between CRBP1 expression and clinic pathological variables in cervical cancer

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Activity Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Immunolocalization of CRBP1 by immunofluorescence in cervical cells. Nuclei were Dapi stained in blue color (A-C). The immunodetection of CRBP1 was observed in green color (D-F). Cytoplasmic immunodetection of CRBP1 in the merge imaging (G-I). 100X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunofluorescence, Staining, Immunodetection, Imaging, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Methylation promoter of CRBP1 gene in cervical cancer samples. Example of CRBP1 gene promoter methylation analysis. Lanes: Healthy cervix sample, CC03 and CC06 samples with un-methylated status; lanes CC 10 and CC 16 with methylated status; HeLa cells as un-methylated control (109 bp), or MCF-7 cells as methylated control (99 bp). MW: molecular weight marker of 100 bp.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Methylation, Molecular Weight, Marker

Journal: Vaccines

Article Title: Antigen Targeting of Porcine Skin DEC205 + Dendritic Cells.

doi: 10.3390/vaccines10050684

Figure Lengend Snippet: Figure 1. Design, expression, and characterization of rAb ZH9F7-Cap and isotype control 7DEU-Cap. (a) Schematic illustration of the recombinant mouse × pig chimeric rAb ZH9F7-Cap and isotype control rAb 7DEU-Cap. (b) 10% SDS–PAGE followed by Coomassie blue staining of purified rAb ZH9F7-Cap and rAb 7DEU-Cap. Lane 1: molecular weight marker; lane 2: rAb ZH9F7 without Cap; lane 3: purified rAb ZH9F7-Cap; lane 4: purified rAb 7DEU-Cap. (c) Western blot of the chimeric mouse × pig rAb ZH9F7-Cap and rAb 7DEU-Cap. Lane 1: molecular weight marker; lane 2: rAb ZH9F7 without Cap; lane 3: purified rAb ZH9F7-Cap; lane 4: purified rAb 7DEU-Cap. HRP- conjugated goat anti-porcine IgG (H + L) was used to confirm the expression of both mouse × pig recombinant antibodies.

Article Snippet: For extracellular staining, IgG2a mouse anti-pig CD8α (Bio-Rad, Watford, UK) and IgG2b mouse anti-pig CD4α (Bio-Rad, Watford, UK) were added and incubated for 15 min at room temperature.

Techniques: Expressing, Control, Recombinant, SDS Page, Staining, Molecular Weight, Marker, Western Blot

Journal: Vaccines

Article Title: Antigen Targeting of Porcine Skin DEC205 + Dendritic Cells.

doi: 10.3390/vaccines10050684

Figure Lengend Snippet: Figure 7. Immune response promoted by intradermal application of rAb ZH9F7-Cap in swine. (a) Immunization schedule. (b) Indirect ELISA for the detection of PCV2a anti-Cap antibodies. Serum samples were tested for anti-PCV2a Cap IgG detection at weeks 0 and 4 postimmunization. (c) Dot plots represent the intracellular IFN-γ in cells stimulated with PHA, rAb ZH9F7-Cap (d), and rAb ZH9F7 without PCV2 Cap as a control (e). In (b–d), graphs represent the average percentage of IFN-γ expression by the CD4+CD8−, CD4−CD8+, and CD4+CD8+ populations. Black bars-dots represent cells from the control group (n = 2), and blue bars-dots represent cells from the rAb ZH9F7-Cap vaccinated group (n = 3). Two-way ANOVA and Tukey–Kramer tests for multiple comparisons of means were performed (p < 0.05). p-values denote statistically significant differences in the means of the different treatments. In (b–d), the basal expression of IFN-γ from unstimulated cells was subtracted.

Article Snippet: For extracellular staining, IgG2a mouse anti-pig CD8α (Bio-Rad, Watford, UK) and IgG2b mouse anti-pig CD4α (Bio-Rad, Watford, UK) were added and incubated for 15 min at room temperature.

Techniques: Indirect ELISA, Control, Expressing

Journal: Clinical and Experimental Immunology

Article Title: Neutralization of mitogenic lectins by intravenous immunoglobulin (IVIg) prevents T cell activation: does IVIg really have a direct effect on T cells?

doi: 10.1111/j.1365-2249.2011.04476.x

Figure Lengend Snippet: Depletion of phytohaemagglutinin (PHA)-reactive IgG from intravenous immunoglobulin (IVIg) abolishes its inhibitory activity. (a) The presence of PHA-reactive IgG in IVIg before and after passage on a PHA-Sepharose column was evaluated by enzyme-linked immunosorbent assay (ELISA) using PHA as capture antigen and 50 µg/ml of each fraction. ***P < 0·001 (unpaired t-test, Welch corrected). (b) Jurkat T cells were incubated in the presence of PHA (0·5 µg/ml) and 5 mg/ml of human serum albumin (HSA), IVIg or IVIg–PHA for 24 h, prior to interleukin (IL)-2 determination by ELISA. ***P < 0·001 (Kruskal–Wallis with Dunn's post-test). Results [± standard deviation (s.d.)] are representative of at least four independent experiments.

Article Snippet: Undigested IgG and Fc fragments were removed by chromatography on protein A-Sepharose (GE Healthcare) followed by chromatography on an anti-human IgG-Sepharose column prepared using the mouse monoclonal C5-1 anti-human IgG antibody [ 18 ] conjugated to NHS-activated Sepharose (GE Healthcare).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Incubation, Standard Deviation

Journal: Clinical and Experimental Immunology

Article Title: Neutralization of mitogenic lectins by intravenous immunoglobulin (IVIg) prevents T cell activation: does IVIg really have a direct effect on T cells?

doi: 10.1111/j.1365-2249.2011.04476.x

Figure Lengend Snippet: Intravenous immunoglobulin (IVIg) contains IgG reactive with lectins other than phytohaemagglutinin (PHA) and neutralizes their stimulatory activity. (a) Molecular weight analysis of IVIg and peptide:N-glycosidase F (PNGase F)-treated IVIg by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% gel. (b) The reactivity of IVIg or Fc-deglycosylated IVIg (20 µg/ml) with PHA, concanavalin A (Con A) and pokeweed mitogen (PWM) was assessed in enzyme-linked immunosorbent assay (ELISA). Results [± standard deviation (s.d.)] are representative of at least two independent experiments. *P < 0·05; ***P < 0·001; n.s.: not significant (unpaired t-test). (c) The presence of Con A-reactive IgG in IVIg before and after passage on a Con A-sepharose column was evaluated by ELISA. Results (mean ± s.d.) are representative of three independent experiments. ***P < 0·001 (unpaired t-test). (d) Jurkat T cells were incubated in the presence of Con A (20 µg/ml) and 5 mg/ml of IVIg or IVIg–Con A for 24 h, prior to interleukin (IL)-2 determination by ELISA. Mean (± s.d.) of two independent experiments. ***P < 0·001 (Kruskal–Wallis with Dunn's post test).

Article Snippet: Undigested IgG and Fc fragments were removed by chromatography on protein A-Sepharose (GE Healthcare) followed by chromatography on an anti-human IgG-Sepharose column prepared using the mouse monoclonal C5-1 anti-human IgG antibody [ 18 ] conjugated to NHS-activated Sepharose (GE Healthcare).

Techniques: Activity Assay, Molecular Weight, Polyacrylamide Gel Electrophoresis, SDS Page, Enzyme-linked Immunosorbent Assay, Standard Deviation, Incubation

Journal: EMBO Molecular Medicine

Article Title: Protein-based tools for the detection and characterisation of Oropouche virus infection

doi: 10.1038/s44321-025-00291-7

Figure Lengend Snippet: ( A ) Amino acid sequence alignment of selected nanobodies that recognise OROV N (NpXX) or Gc spike (SpXX). Complementarity Determining Regions (CDRs) as defined by IGMT (Giudicelli et al, ) are highlighted. ( B ) Coomassie-stained SDS-PAGE of nanobodies that had been biotinylated in vitro. Upper bands represent the Avi-tagged, biotinylated nanobodies while lower bands represent nanobodies where the tag had been lost, presumably by proteolysis during the purification procedure. ( C ) Electrophoretic mobility shift assay to confirm biotinylation. After boiling of the nanobody in SDS-PAGE loading buffer, streptavidin was added at a 2:1, 1:1 or 1:2 molar ratio (nanobody:streptavidin) and samples were subjected to SDS-PAGE. Appearance of a high apparent molecular weight band, and disappearance of the low molecular weight band, confirms biotinylation of the nanobody. Asterisk (*) denotes streptavidin that was boiled before SDS-PAGE, rather than being added to the sample buffer after boiling. ( D ) Coomassie-stained SDS-PAGE of His 8 -tagged nanobodies. ( E ) OROV N competition BLI sensorgrams. Streptavidin biosensors loaded with bNbs were sequentially incubated with 1 µM OROV N then with 1 µM OROV N plus 25 µM of each competitive nanobody, with biosensor regeneration between incubation (association) cycles. After the final competition step, the biosensor was incubated with 1 µM OROV N to confirm that the bNbs remained active. For each association in the presence of competitive nanobodies, the response for each sensor at 180 s (dotted grey line) was divided by the response of the same sensor in the presence of OROV N alone at 180 s to generate the heatmap shown in Fig. . .

Article Snippet: Plates were incubated with biotinylated nanobodies (2 μg/mL in blocking buffer) for 1 h, HRP-conjugated streptavidin (1:10,000 dilution, Thermo Fisher Scientific N100) for 15 min, and then developed.

Techniques: Sequencing, Staining, SDS Page, In Vitro, Purification, Electrophoretic Mobility Shift Assay, Molecular Weight, Incubation

Journal: EMBO Molecular Medicine

Article Title: Protein-based tools for the detection and characterisation of Oropouche virus infection

doi: 10.1038/s44321-025-00291-7

Figure Lengend Snippet: ( A ) Nanobody detection of purified OROV antigens. Antigens were immobilised on the capture surface via passive adsorption (2 µg/mL) and detected via incubation with specified biotinylated nanobodies (bNbs) plus streptavidin-HRP. CRIV Gc spike was included as a negative control. The data represent one experiment. ( B ) Sandwich ELISA using purified OROV antigens to determine nanobody competition groups. Surfaces were coated with nanobodies (2 µg/mL) before being incubated with antigen then detection bNbs and streptavidin-HRP. For non-competing nanobodies, data are representative of two independent experiments. For competing Gc spike nanobodies the experiment was performed once. ( C ) Competition biolayer interferometry (BLI) to map nanobody competition for OROV N epitopes. Streptavidin biosensors were loaded with bNbs then incubated with 1 µM OROV N plus 25 µM competitor nanobody. Heatmap shows BLI responses after 180 s of association, normalised relative to the response obtained for each bNb-loaded biosensor incubated with 1 µM OROV N alone. BLI traces are shown in Fig. . Data are representative of two independent experiments. ( D ) Limit of detection (LOD) of purified Gc spike (left) or N (right) in sandwich ELISA using optimised nanobody pairings. LOD is calculated as (3.3 * standard deviation [background])/slope. Data are representative of two independent experiments. ( E ) Detection of Gc spike (left) and N (right) in laboratory stocks of OROV strains TRVL (Anderson et al, ) and BeAn19991 (Acrani et al, ; Aquino et al, ) by sandwich ELISA. Data shown are from one experiment performed in technical triplicate. .

Article Snippet: Plates were incubated with biotinylated nanobodies (2 μg/mL in blocking buffer) for 1 h, HRP-conjugated streptavidin (1:10,000 dilution, Thermo Fisher Scientific N100) for 15 min, and then developed.

Techniques: Purification, Adsorption, Incubation, Negative Control, Sandwich ELISA, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Protein-based tools for the detection and characterisation of Oropouche virus infection

doi: 10.1038/s44321-025-00291-7

Figure Lengend Snippet: ( A , B ) Coomassie-stained SDS-PAGE of ( A ) untagged nanobody-Fc fusions, and ( B ) biotinylated nanobody-Fc fusions. ( C ) Electrophoretic mobility shift assay to confirm biotinylation. After boiling of the nanobody-Fc fusion in SDS-PAGE loading buffer, streptavidin was added at a 8:1, 4:1, 2:1, 1:1 or 1:2 molar ratio (nanobody-Fc fusion:streptavidin) and samples were subjected to SDS-PAGE. Appearance of a high apparent molecular weight bands, and reduction of the lower molecular weight band, confirms biotinylation of the nanobody-Fc fusion.

Article Snippet: Plates were incubated with biotinylated nanobodies (2 μg/mL in blocking buffer) for 1 h, HRP-conjugated streptavidin (1:10,000 dilution, Thermo Fisher Scientific N100) for 15 min, and then developed.

Techniques: Staining, SDS Page, Electrophoretic Mobility Shift Assay, Molecular Weight

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Plasminogen activator inhibitor-1 (PAI-1) mechanism and approaches to modulating PAI-1 activity in vivo. The PAI-1 reaction includes inhibitory (ki), substrate (ks), and latent (klat) branches, which result in a loss of activity and formation of a stable inhibitory complex (E-PAI-1), cleaved serpin (PAI-1*), and latent serpin (PAI-1lat), respectively. PAI-1 in an active conformation (PAI-1) complexed with endogenous vitronectin (Vn) in the pleural space interacts with the plasminogen activator [wild-type urokinase (wt-uPA) or urokinase with 179RHRGGS184→179AAAAAA184 substitutions (ΔDS-uPA); enzyme, E] and forms a transient Michaelis complex (E·PAI-1·Vn). Once the enzyme cleaves PAI-1 and forms an acyl-enzyme, conformational changes in the serpin result in its stabilization in an inhibitory complex (E-PAI-1). Under physiological conditions, >90% of the PAI-1 reaction follows the inhibitory branch (ki), resulting in mutual, stoichiometric inhibition of the enzyme and PAI-1. Active PAI-1 can also slowly, spontaneously transform (klat) into an inactive, latent conformation (PAI-1lat), losing the ability to bind Vn. The substrate branch (ks) yields an inactive, cleaved serpin (PAI-1*) and an active enzyme. The PAI-1 reaction was modulated by three distinct mechanisms (mechanisms I–III). In mechanism I, alanine mutations of positively charged residues in the 37-loop of uPA result in ΔDS-uPA, which interacts with PAI-1 via the inhibitory branch (ki) considerably more slowly than the wild-type enzyme. In mechanism II, ligands (L), such as inactive uPA with Ser195Ala substitution (S195A-uPA) or monoclonal antibody MA-56A7C10, compete with uPA and bind PAI-1/Vn with nanomolar affinity [Kd = koff/kon, where Kd is the dissociation constant and koff and kon are the first-order rate constants of dissociation and association, respectively, of PAI-1/S195A-two-chain uPA (tcuPA) or MA-56A7C10] forming nonproductive “molecular sandwich”-type complexes (L·PAI-1·Vn), which compete with the Michaelis complex for uPA (Kd << Km). L·PAI-1·Vn also stabilizes the active conformation of PAI-1, inhibiting the latent branch (klat). The rate of enzyme inactivation by L·PAI-1·Vn becomes limited by a low koff. In mechanism III, monoclonal antibody MA-33B8 accelerates the transition of active PAI-1 or its complex with Vn to inactive PAI-1 (PAI-1lat).

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Activity Assay, In Vivo, Inhibition

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Effects of a 179RHRGGS184→179AAAAAA184 urokinase mutant (ΔDS-uPA) on the rate of interaction with plasminogen activator inhibitor-1 (PAI-1) and activation of Glu-plasminogen. A: dependence of the observed first-order rate constants (kobs) for the interaction of PAI-1 with wild-type urokinase (wt-uPA; ○) and ΔDS-uPA (△) on enzyme concentration. The data for wt- and ΔDS-uPA are shown in different scales. The values of kobs were determined as previously described (45, 49, 51). Linear equations were fit (solid lines; r2 > 0.95) to the data, and values of the second-order association rate constant (kass) were determined from the slopes. The ratio of kass for wt-uPA over ΔDS-uPA was 63.0 for inhibition by PAI-1. B: dependence of the rates of accumulation of plasmin due to activation of Glu-plasminogen by wt-uPA (○) and ΔDS-uPA (△) on enzyme concentration. The rates of plasmin accumulation were determined from the slopes of linear equations, which were fit (solid lines; r2 > 0.99) to the data as described previously (49). The ratio of slopes for wt-uPA over ΔDS-uPA for activation of Glu-plasminogen was 1.5. AU, arbitrary units.

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Mutagenesis, Activation Assay, Concentration Assay, Inhibition

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Accumulation of intrapleural α-macroglobulin (αM)/urokinase with 179RHRGGS184→179AAAAAA184 substitutions (ΔDS-uPA) “molecular cage” complexes during intrapleural fibrinolytic therapy (IPFT). A: time dependence of the formation of intrapleural αM/ΔDS-uPA during IPFT with ΔDS-scuPA (0.0625 mg/kg). ΔDS-uPA amidolytic activity was measured after samples of pleural fluid withdrawn at 0–40 min were supplemented with an excess (100–200 nM) of exogenous recombinant human plasminogen activator inhibitor-1 (PAI-1) to inhibit free enzyme. PAI-1-resistant ΔDS-uPA amidolytic activity, which represents intrapleural ΔDS-uPA in “molecular cage” complexes with αM (46, 50), was converted to concentrations (nM) and plotted against time. A single exponential equation was fit to the dependence of the concentration of αM/ΔDS-uPA (A) or αM/uPA (not shown) on time to obtain the values of the apparent first-order rate constants (kapp; B) as previously described (46).

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Activity Assay, Recombinant, Concentration Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Intrapleural plasminogen activator inhibitor-1 (PAI-1)-independent inactivation of free urokinase with 179RHRGGS184→179AAAAAA184 substitutions (ΔDS-uPA) is twofold faster than wild-type urokinase (wt-uPA). A: time dependence of the amidolytic activity of intrapleural free uPA (△) during intrapleural fibrinolytic therapy (IPFT) with ΔDS-prourokinase (ΔDS-scuPA; 0.0625 mg/kg). Briefly, the total ΔDS-uPA amidolytic activity was measured in samples of pleural fluid withdrawn at 10–40 min after IPFT. ΔDS-uPA activity that is resistant to an excess of exogenous human recombinant PAI-1 [represents α-macroglobulin (αM)/ΔDS-uPA complexes] was subtracted from total amidolytic activity to estimate the level of free ΔDS-uPA in the sample ([free ΔDS-uPA] = [total ΔDS-uPA] − [αM/ΔDS-uPA]). B: time dependence of free (not complexed) intrapleural uPA amidolytic activity (○) during IPFT with wt-scuPA (0.0625 mg/kg). Amidolytic activity of free uPA was the difference between total uPA activity and activity of αM/uPA complexes. C: observed first-order rate constants (kobs) for the intrapleural inactivation of free ΔDS- and wt-uPA. Values of kobs are given for loss of intrapleural amidolytic (▽) and plasminogen-activating (△) activities of ΔDS-uPA, as well as the plasminogen-activating activity of wt-uPA (○) during IPFT with 0.0625 mg/kg (n = 6). Values of kobs were estimated from the changes in activity with respect to time, as described previously (42, 49). A single exponential equation was fit to the dependence of [free enzyme] on time to obtain kobs of PAI-1-independent inactivation of ΔDS- and wt-uPA as previously described (46). The rate of intrapleural inactivation of ΔDS-uPA was statistically (P < 0.05) higher than that for wt-uPA. There was no statistically significant difference (P > 0.05) between the kobs of ΔDS-uPA inactivation estimated from measurements of amidolytic and Glu-plasminogen-activating activities.

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Activity Assay, Recombinant

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Two-chain urokinase with Ser195Ala substitution (S195A-tcuPA; 0.5 mg/kg) does not affect the rate of intrapleural inactivation of urokinase (uPA). A: time dependence of the amidolytic activity of intrapleural, free uPA during intrapleural fibrinolytic therapy (IPFT) with 0.5 mg/kg of S195A-tcuPA with (●; n = 5) and without (■; n = 3) 0.25 mg/kg of scuPA. The amidolytic activity of free uPA was determined in samples of pleural fluid withdrawn at 10–40 min after IPFT. The amidolytic activity of free uPA was calculated as the difference between total uPA activity and activity attributed to α-macroglobulin (αM)/uPA complexes, as previously described (46). There was a statistically significant difference (P < 0.05) between free uPA activity in animals treated with (○) and without (□) scuPA (0.25 mg/kg). B: time dependence of the amidolytic activity of free uPA during IPFT with scuPA (0.25 mg/kg, ○; n = 5) and vehicle control (PBS, □; n = 3). The amidolytic activity of free uPA was the difference between total uPA activity and activity of αM/uPA complexes. There was a statistically significant difference (P < 0.05) between free uPA activity in animals treated with (○) and without (□) scuPA (0.25 mg/kg). C: observed first-order rate constants (kobs) for the intrapleural inactivation of uPA with (●; n = 5) and without (○; n = 5) S195A-tcuPA (0.5 mg/kg). A single exponential equation was fit to the dependence of [free uPA] with respect to time to obtain the kobs of PAI-1-independent inactivation of uPA as previously described (46). There was a statistically significant difference (P < 0.05) between kobs in animals treated with (○) and without (□) scuPA (0.25 mg/kg). There was no statistically significant difference (P > 0.05) between the kobs observed for IPFT with scuPA (0.25 mg/kg) with (●) or without (○) S195A-tcuPA (0.5 mg/kg).

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Activity Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Effect of two-chain urokinase with Ser195Ala substitution (S195A-tcuPA; 0.5 mg/kg) on the accumulation of α-macroglobulin (αM)/urokinase (uPA) complexes during intrapleural fibrinolytic therapy (IPFT) with 0.25 mg/kg prourokinase (scuPA). A: time dependence of intrapleural levels of αM/uPA during IPFT with 0.5 mg/kg of S195A-tcuPA, with (●) and without (■) scuPA 0.25 mg/kg. Samples of pleural fluid withdrawn at 0–40 min after IPFT were supplemented with 100–200 nM of exogenous recombinant human plasminogen activator inhibitor-1 (PAI-1), and the amidolytic activity of uPA was measured as previously described (46). There was a statistically significant difference (P < 0.05) between αM/uPA in animals treated with S195A-tcuPA/scuPA and with S195A-tcuPA alone at 10, 20, and 40 min. B: values of the apparent first-order rate constants (kapp) for intrapleural accumulation of αM/uPA with (solid symbols) or without (open symbols) 0.5 mg/kg of S195A-tcuPA. A single exponential equation was fit to the dependence of [αM/uPA] for treatments with (A) or without (not shown) S195A-tcuPA on time as previously described (46). There was no statistically significant difference between the rates of accumulation of αM/uPA during IPFT with 0.25 mg/kg scuPA alone or in the presence of S195A-tcuPA (P > 0.05). C: levels of intrapleural αM/uPA “molecular cage”-type complexes at 24 h after IPFT with 0.25 mg/kg scuPA with (●) or without (○) S195A-tcuPA (0.5 mg/kg). Briefly, the amidolytic activity of uPA was measured after supplementation of samples of pleural fluid withdrawn at 24 h after IPFT with an excess (20–40 nM) of exogenous recombinant human PAI-1. There was no statistically significant difference between [αM/uPA] observed for treatment with 0.25 mg/kg scuPA with (●) or without (○) S195A-tcuPA (0.5 mg/kg; P > 0.05).

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Recombinant, Activity Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Accumulation of active plasminogen activator inhibitor-1 (PAI-1) in pleural fluids 24 h after intrapleural fibrinolytic therapy (IPFT). A: changes in the activity of exogenous two-chain urokinase (tcuPA; 0.5 nM) added to the samples of pleural fluid collected 24 h after IPFT with 0.25 mg/kg prourokinase (scuPA) and MA-56A7C10 (dashed line) or MA-33B8 (dotted line). Thin, solid lines represent the best fit of a single exponential [first-order rate constant of intrapleural uPA inactivation (kobs) = 4.5 × 10−3 min−1] and linear equation to the data, respectively. The levels of PAI-1 activity in the pleural fluid are shown in the Table 1. B: Western blot analysis of PAI-1 complexed with MA-56A7C10 and tcuPA with Ser195Ala substitution (S195A-tcuPA) isolated from pleural fluids of animals by immunoprecipitation. Complexes of endogenous active PAI-1 were precipitated with magnetic beads (Dynabeads M-280 with sheep anti-mouse IgG; Invitrogen by Thermo Fisher Scientific) per the manufacturer’s protocol, as described in experimental procedures. Western blot analysis detected rabbit PAI-1 in the precipitates obtained from pleural fluids of animals treated with MA-56A7C10 and scuPA (0.5 and 0.25 mg/kg, respectively; lane 4) or S195A-tcuPA (0.5 mg/kg; lane 2), but not in pleural fluids of animals treated with mouse IgG (0.5 mg/kg) or vehicle control [Dulbecco’s phosphate-buffered saline (DPBS); lanes 3 and 1, respectively]. Two pairs of lanes (lanes 1–4) represent parts of the same gel. The positioning of molecular weight markers is shown at right. Treatments are described in the table above the Western blot image. Bands on the Western blot other than rabbit PAI-1, which are present in every lane, represent nonspecific binding of secondary antibodies. *Pleural fluids of rabbits treated with DPBS and S195A-tcuPA (lanes 1 and 2, respectively) were supplemented with anti-human uPA monoclonal antibody (4–8 µg), 10 min before addition of magnetic beads. C: PAI-1 activity was precipitated by sheep anti-mouse IgG magnetic beads from pleural fluids of animals treated with MA-56A7C10 in combination with scuPA (0.5 and 0.25 mg/kg, respectively) and with S195A-tcuPA (0.5 mg/kg), but not from pleural fluids of animals treated with mouse IgG alone (0.5 mg/kg) or vehicle control. PAI-1 activity was measured by incubating an aliquot (5–10 µl) of the bead slurry with 0.2 nM uPA and fluorogenic substrate in DPBS with BSA (1 mg/ml). Amidolytic activity of uPA was measured using fluorogenic substrate as described in experimental procedures and elsewhere (42). Relative levels of active PAI-1 bound to the resin were estimated from decreases in the uPA activity. The levels of active PAI-1 (on average, 2 independent experiments) in pleural fluids were expressed in arbitrary units (AU).

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Activity Assay, Western Blot, Isolation, Immunoprecipitation, Magnetic Beads, Molecular Weight, Binding Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00579.2016

Figure Lengend Snippet: Changing plasminogen activator inhibitor-1 (PAI-1) activity affects intrapleural fibrinolytic therapy (IPFT) outcomes under conditions of slow fibrinolysis in the pleural space. Successful IPFT in tetracycline (TCN)-induced pleural injury in rabbits requires maintaining plasminogen-activating activity for 4–8 h (48). The minimal time necessary for effective fibrinolysis (4–8 h) is shown as a yellow zone between effective (green zone; >8 h) and ineffective (red zone; <4 h) IPFT outcomes. The rate of PAI-1-independent inactivation of uPA remains the same with different doses of prourokinase (scuPA; 46), shown as parallel solid lines, with the minimal effective dose in the middle. Fibrinolysis stops as soon as endogenous PAI-1 (black dashed line) inhibits the plasminogen activator(s) present (intercept of solid and dashed lines), which in turn determines outcomes for effective (yellow and green zones) and ineffective (red zone) IPFT. Neutralizing PAI-1 (blue arrow) decreases PAI-1 activity (blue dotted line). Consequently, an otherwise ineffective dose of scuPA (the lowest solid line) provides positive plasminogen-activating activity for >8 h (the intercept with the blue dotted line in the green zone), representing the increased efficacy of IPFT (decreasing the minimal effective dose). On the other hand, increasing the PAI-1 activity in the pleural space (red arrow) results in faster inhibition of intrapleural plasminogen activator (the intercept with the red dotted line in the red zone) and in ineffective IPFT with doses of scuPA that are normally effective. The efficacy of IPFT in tetracycline (TCN)-induced pleural injury was increased when PAI-1 was neutralized with MA-33B8 [Table 1; gross lung injury score (GLIS) = 3] or with monoclonal antibodies that redirect the PAI-1 mechanism toward the substrate branch (Fig. 1, ks; 25). The adverse effects of increased PAI-1 were observed during IPFT in the presence of MA-56A7C10 (Table 1; GLIS = 50), in animals subjected to serial computed chest tomography (48) and in rabbits with infectious pleural injury (empyema; 47). AU, arbitrary units; FT, fibrinolytic therapy.

Article Snippet: Levels of active rabbit PAI-1 in the pleural fluids were determined either by titrating active inhibitor with solutions of uPA of a known concentration, as previously described ( 50 ), or by using a commercially available ELISA (Molecular Innovations) following the manufacturer’s protocol.

Techniques: Activity Assay, Inhibition, Tomography