Journal: The Journal of Biological Chemistry

Article Title: Autophagy Protects against Colitis by the Maintenance of Normal Gut Microflora and Secretion of Mucus *

doi: 10.1074/jbc.M114.632257

Figure Lengend Snippet: Generation of Atg7 cKO mice. A, expression of Chst4 and Villin1 (Vil1) mRNA in lower gastrointestinal tracts. The relative quantity of each mRNA to that of β-actin was determined by quantitative RT-PCR. Error bars represent the standard deviation (S.D.) of samples within a group. n = 3 per group. B, frozen sections of lower gastrointestinal tracts from GlcNAc6ST-2-Cre+/R26R reporter mice were stained with X-gal solution and Nuclear Fast Red as described previously (26). Bar, 50 μm. C, quantitative RT-PCR analysis of Atg7 mRNA in the colon. Error bars represent the S.D. of samples within a group. An unpaired Student's t test was used to compare WT versus cKO mice. **, p < 0.01. n = 7–9 per group. D, Western blot analysis of whole colonic lysates from WT and cKO mice. The arrowhead in each panel indicates the position of each protein. #, high molecular weight form of p62. E, quantification of the ATG7 protein in the whole colon. Western blot bands (D) were quantified. The error bars represent the S.D. of the samples within a group. An unpaired Student's t test was used to compare WT versus cKO mice. **, p < 0.01. n = 5 per group.

Article Snippet: The cDNA was synthesized with the PrimeScript RT-PCR kit with gDNA Eraser (TaKaRa) and subjected to quantitative RT-PCR using SYBR Premix Ex TaqII (Tli RNase H Plus; TaKaRa).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Staining, Western Blot, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Autophagy Protects against Colitis by the Maintenance of Normal Gut Microflora and Secretion of Mucus *

doi: 10.1074/jbc.M114.632257

Figure Lengend Snippet: Comparison of major microbial populations in fecal samples. A–C, pyrosequencing analysis was used to examine microbial diversity in feces from WT and cKO mice. Sequences were obtained from pooled stool samples of WT (n = 3; 145,188 reads) and cKO mice (n = 3; 111,639 reads). A, comparisons of the diversity at the phylum or class level. Each chart represents the taxonomic composition. B, comparison of the diversity at the family level in Clostridiales. C, comparison of the diversity at the family level in Bacteroidales. D, total bacteria in fecal samples were quantified by quantitative RT-PCR using universal bacterial 16S rRNA genes (WT, n = 4; cKO, n = 6). E, microbial composition of the feces of WT and cKO mice treated with (Abx-cKO) or without (WT and cKO) a combination of metronidazole and ciprofloxacin for 4 weeks was analyzed by quantitative PCR using group- or genus-specific primers of the bacterial 16S rRNA genes. Error bars represent the S.D. of samples within the group (WT, n = 4; cKO, n = 4; Abx-cKO, n = 8). An unpaired Student's t test and a paired Student's t test were used to compare WT versus cKO mice and cKO versus Abx-cKO mice, respectively. **, p < 0.01; *, p < 0.05. n = 4 per group.

Article Snippet: The cDNA was synthesized with the PrimeScript RT-PCR kit with gDNA Eraser (TaKaRa) and subjected to quantitative RT-PCR using SYBR Premix Ex TaqII (Tli RNase H Plus; TaKaRa).

Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Autophagy Protects against Colitis by the Maintenance of Normal Gut Microflora and Secretion of Mucus *

doi: 10.1074/jbc.M114.632257

Figure Lengend Snippet: Quantitative RT-PCR analyses of antimicrobial peptides, antiparasitic peptides, and cytokines in WT and Atg7 cKO mice. Quantitative RT-PCR analyses of antimicrobial peptides, antiparasitic peptides, and cytokines were performed using total RNA samples from the medial colons of WT and cKO mice treated with (Abx-cKO) or without (WT and cKO) a combination of metronidazole and ciprofloxacin for 4 weeks. A, expression of antimicrobial peptides and antiparasitic peptides. B, expression of Th1/Th2 cytokines. C, expression of epithelium-derived cytokines. Error bars represent the S.D. of samples within a group. An unpaired Student's t test and a paired Student's t test were used to compare WT versus cKO mice and cKO versus Abx-cKO mice, respectively. **, p < 0.01; *, p < 0.05. n = 3–4 per group.

Article Snippet: The cDNA was synthesized with the PrimeScript RT-PCR kit with gDNA Eraser (TaKaRa) and subjected to quantitative RT-PCR using SYBR Premix Ex TaqII (Tli RNase H Plus; TaKaRa).

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Autophagy Protects against Colitis by the Maintenance of Normal Gut Microflora and Secretion of Mucus *

doi: 10.1074/jbc.M114.632257

Figure Lengend Snippet: Secretion of colonic mucins in WT and Atg7 cKO mice. Hematoxylin and eosin (A), Alcian blue-PAS (B), and anti-MUC2 (C) staining of the mouse distal colon. D, percentage of mucus-secreting crypts. E, quantification of inner mucus layer thickness. F, detection of fecal MUC2 by ELISA. G, quantitative RT-PCR. The relative quantity of Muc2 mRNA compared with the quantity of β-actin was determined by the ΔΔCt method. Error bars represent the S.D. of samples within a group. Asterisk in A–C, mucus-secreting crypt. Arrows in A–C, mucus-secreting goblet cells. Dashed line in C, mucosal surface. An unpaired Student's t test was used to compare WT versus cKO mice. **, p < 0.01; *, p < 0.05. n = 3 per group.

Article Snippet: The cDNA was synthesized with the PrimeScript RT-PCR kit with gDNA Eraser (TaKaRa) and subjected to quantitative RT-PCR using SYBR Premix Ex TaqII (Tli RNase H Plus; TaKaRa).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

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: 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: Veterinary Immunology and Immunopathology

Article Title: Generation and characterization of anti-α-enolase single-chain antibodies in chicken

doi: 10.1016/j.vetimm.2010.06.001

Figure Lengend Snippet: Characterization of recombinant α-enolase and polyclonal anti-α-enolase IgY antibodies. Samples in each panel are protein markers (lane M), purified GST (lane 1), purified α-enolase (lane 2), and purified GST-α-enolase fusion protein (lane 3). Purified proteins visualized by Coomassie blue staining (panel A) were blotted onto nitrocellulose paper and probed with anti-GST antibodies (panel B) or sera from 4th-immunized chicken (panel C). The molecular weight of recombinant α-enolase protein is about 48 kD.

Article Snippet: All the proteins were transferred onto nitrocellulose membranes (Amersham Biosciences, UK), which were then blocked with 5% skim milk in TBST for 1 h. Polyclonal goat anti-chicken IgY light chain antibodies (Bethyl Laboratories, Montgomery, TX, USA) were added at 1:3000 dilution and incubated for an additional hour.

Techniques: Recombinant, Purification, Staining, Molecular Weight

Journal: Veterinary Immunology and Immunopathology

Article Title: Generation and characterization of anti-α-enolase single-chain antibodies in chicken

doi: 10.1016/j.vetimm.2010.06.001

Figure Lengend Snippet: Binding activity of scFv antibodies to purified α-enolase analyzed by ELISA. Cellular lysates containing scFv antibodies from randomly selected clones from the 4th panning cycle were examined for their binding to purified α-enolase coated onto the plate wells. Binding activity was detected using the goat anti-chicken light chain antibodies at 1:3000 dilution, followed by HRP-conjugated donkey anti-goat IgG and measured at 450 nm. Two anti-SARS-CoV scFv antibodies (SCoS-S8 and SCoS-L22) were used as negative controls. One additional control experiment was carried out as described without adding primary recombinant scFv antibodies. Polyclonal IgY antibodies from chickens immunized with purified α-enolase were used as a positive control. The ELISA data were represented as means of the duplicated experiments.

Article Snippet: All the proteins were transferred onto nitrocellulose membranes (Amersham Biosciences, UK), which were then blocked with 5% skim milk in TBST for 1 h. Polyclonal goat anti-chicken IgY light chain antibodies (Bethyl Laboratories, Montgomery, TX, USA) were added at 1:3000 dilution and incubated for an additional hour.

Techniques: Binding Assay, Activity Assay, Purification, Enzyme-linked Immunosorbent Assay, Clone Assay, Control, Recombinant, Positive Control

Journal: European Journal of Cell Biology

Article Title: Tumor necrosis factor-α-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase (MMP)-1 and MMP-3 ex vivo

doi: 10.1016/j.ejcb.2014.10.001

Figure Lengend Snippet: MMP-1 and MMP-3 expression in control and TNF-α-treated skin analyzed by ELISA (A and D), Western blot (B and E) and β-casein zymography (F), and MMP-1 immunohistochemistry (C) and MMP-3 correlation to ICTP (G) after 8 days of incubation. (A and D) Pooled tissue extracts and media from five explants per donor and group were assayed. Mean ± SEM. ** p < 0.01, *** p < 0.005 versus control. n = number of skin donors. (B and E) The PVDF membrane was first probed with the polyclonal MMP-1 antibody (B), then stripped and reprobed with the polyclonal MMP-3 antibody (E). Lanes 1 and 3, control; 2 and 4, TNF-α; 1 and 2, pooled tissue extract (18 μl/lane) from 30 individual 8-mm skin explants from the 6 donors (1–6) per group; lanes 3 and 4, pooled media (18 μl/lane) from 24 individual 8-mm skin explants from 5 donors (media from donor 1 was lost) per group. Std.: 42.7 kDa rhMMP-1 (14 ng). Glycosylated and nonglycosylated latent and active MMP-1/MMP-3 doublets are indicated. (C) Representative sections of native (a, d and g), or cultured skin explants from control (b, e and h) and TNF-α groups (c, f and i) treated with primary monoclonal MMP-1 antibody that detects latent and active forms (a–f) or with isotype (negative) control (g–i). (a–c, 40×; d–i, 900×). (F) Casein gels were incubated in the absence (Buffer) or presence of 10 μM GM6001. Lane 1, rhMMP-3 (5 ng); 2, pooled media from control-treated explants (15 μl); 3, pooled media from TNF-α-treated explants (15 μl); 4, rhMMP-1 (2 ng). Mark12™ (Life Technologies) molecular weight marker was run in parallel lane. Upper doublets represent latent forms of MMP-3 (upper bands) and MMP-1 (lower bands) and lower doublets active forms of MMP-3 (upper bands) and MMP-1 (lower bands). (G) Tissue MMP-3 contents and corresponding ICTP in media. Each symbol as indicated in represents pooled tissue extracts and media from five explants per donor. The TNF-α-treated skin explants of donor 1 is missing due to lost pooled media sample. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: MMP-1 and MMP-3 were quantified by ELISA kits (Boster Biological Technology, Fremont, CA, USA).

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Zymography, Immunohistochemistry, Incubation, Membrane, Cell Culture, Negative Control, Molecular Weight, Marker

Journal: European Journal of Cell Biology

Article Title: Tumor necrosis factor-α-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase (MMP)-1 and MMP-3 ex vivo

doi: 10.1016/j.ejcb.2014.10.001

Figure Lengend Snippet: MMP-2 (A and B) and TIMP-1 (C) tissue levels. (A) Total and active (lower bars) MMP-2 contents estimated by gelatin zymography. (B) In the zymogram, MMP-2 standard and pooled tissue extracts (1.0 μl) from 30 individual 8-mm skin explants per group were loaded into each lane. Lane 1, rhMMP-2 (50 pg; PF037); 2, native skin; 3, control; 4, TNF-α. (C) TIMP-1 levels determined by ELISA. (A and C) Pooled tissue extracts from five separate native or organ-cultured 8-mm skin explants from each of the donors and group were used for the analyses. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Mean ± SEM ( n = 6).

Article Snippet: MMP-1 and MMP-3 were quantified by ELISA kits (Boster Biological Technology, Fremont, CA, USA).

Techniques: Zymography, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Immunity

Article Title: Caspase-8 Collaborates with Caspase-11 to Drive Tissue Damage and Execution of Endotoxic Shock

doi: 10.1016/j.immuni.2018.06.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For IHC detection, paraffin-embedded sections were prepared and stained as described ( Liang et al., 2014 ) with rabbit anti-cleaved-Casp3 (1:100 dilution) or anti-cleaved-Casp8 (1:100 dilution) at 4C, followed by biotinylated goat anti-rabbit secondary antibody (BA-1000, Vector Laboratories), streptavidine-horseradish peroxidase (HRP, SA-5004, Vector Laboratories) and peroxidase reaction reagents from an ABC kit (PK-4002, Vector Laboratories).

Techniques: Labeling, Plasmid Preparation, Recombinant, In Vivo, Molecular Weight, Cell Culture, Cell Viability Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Software

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