Journal: PLoS Pathogens

Article Title: Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

doi: 10.1371/journal.ppat.1000857

Figure Lengend Snippet: ( A ) Sequence of prothrombin and overlapping peptides (indicated by numbers). In addition to the regular overlapping peptides, peptide regions of high net charge, and/or content of predicted helical regions (Agadir; http://www.embl-heidelberg.de/Services/serrano/agadir/agadir-start.html ) were selected. Peptides described in subsequent experiments are also indicated; GKY25, VFR17, and the major ∼11 kDa peptide (amino acids 527–622). ( B ) Overlapping peptides of prothrombin were analyzed for antimicrobial activities against E. coli . The inhibitory zones, relative hydrophobic moment (μHrel) as well as net charge of respective peptides (only active peptides are numbered) are indicated in the 3-D graph. Peptides showing antimicrobial activity are also indicated by red color in ( A ). For determination of antibacterial activities, E. coli (4×10 6 cfu) was inoculated in 0.1% TSB agarose gel. Each 4 mm-diameter well was loaded with 6 µl of peptide (at 100 µM). The zones of clearance correspond to the inhibitory effect of each peptide after incubation at 37°C for 18–24 h (mean values are presented, n = 3). ( C ) Helical wheel representation of the C-terminal peptide VFR17. The amino acids are indicated. ( D ) LPS-binding activity of the prothrombin-derived peptide sequences. Peptides (5 µg) were applied to nitrocellulose membranes followed by incubation in PBS (containing 2% bovine serum albumin) with iodinated ( 125 I)-LPS. Only peptides from the C-terminal part of prothrombin demonstrated significant binding to LPS. ( E ) Molecular model of thrombin. The peptides GKY25 (green-orange, indicated in ) and VFR17 (orange, peptide 48 in ) are indicated in the crystal structure of human thrombin (PDB code 1C5L). ( F ) Activities of prothrombin (PT), thrombin (T), GKY25 and VFR17 on E. coli ATCC 25922. In viable count assays GKY25 and VFR17 displayed significant antibacterial activities. 2×10 6 cfu/ml of bacteria were incubated in 50 µl with proteins and peptides at a concentration of 3 and 6 µM, respectively.

Article Snippet: In order to deplete acute wound fluids (AWF) of thrombin and fragments containing C-terminal peptides, AWF was diluted with an equal volume of PBS, and passaged 5 times through an affinity column (0.3 ml, Thermo Scientific) having coupled IgG antibodies specific for VFR17 (Innovagen AB).

Techniques: Sequencing, Activity Assay, Agarose Gel Electrophoresis, Incubation, Binding Assay, Derivative Assay, Concentration Assay

Journal: PLoS Pathogens

Article Title: Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

doi: 10.1371/journal.ppat.1000857

Figure Lengend Snippet: ( A ) RP-HPLC separation of thrombin digested with neutrophil elastase. The bars indicate the antibacterial activity of the fractions in low (gray) as well as high salt conditions (black). Fraction 30 (lower left) contained two peaks of masses 2034.78 and 2270.88, perfectly matching the indicated sequences obtained after ESI-MS/MS analysis. Fraction 38 was analyzed by MALDI-MS, and subsequently by ESI-MS. The ESI-MS analysis identified a dominant mass of 11041 corresponding to the 96-amino acid long peptide N527-E622 (indicated in ) with two intact disulphide bridges. A minor mass corresponding to V528-E622 was also detected by ESI-MS/MS. N- and C-terminal sequencing yelded NLPI and EGFQ, respectively. The rightmost insets illustrate the ∼11 kDa peptide analysed by SDS-PAGE and stained for protein (stain), or after immunoblot (blot), and below, the peptide (F38) was analyzed by gel-overlay for detection of antibacterial activity. The activity of F38 was identical to the major clearing zone generated by elastase-digested thrombin (T+NE). Right arrow indicates the position of clearing zone generated by the peptide GKY25. The gel was run top to bottom. Finally, peptides of fractions 20–21 were predicted using the FINDPEPT tool ( www.expasy.org/tools/findpept.html ) . ( B ) Degradation of thrombin by neutrophil supernatants generates antibacterial activity in RDA (upper inset). RDA was performed in low-salt conditions. E. coli (4×10 6 cfu) was used as test organism. Each 4 mm-diameter well was loaded with 6 µl of material (C, supernatant only; T, thrombin only; T+NS; thrombin incubated for 30 and 180 min respectively, with neutrophil supernatants). The digests were analysed by SDS-PAGE (16.5% Tris-Tricine gels) and immunoblotting with antibodies against VFR17 (lower panel). ( C ) Prothrombin was digested with the enzymes as indicated for 3 h, and analyzed by SDS-PAGE (16.5% Tris-Tricine gels) and immunoblotting using antibodies against VFR17 (NE, neutrophil elastase; CG, cathepsin G; PAE, P. aeruginosa elastase). ( D ) RDA results of prothrombin digested with cathepsin G (CG) and P. aeruginosa elastase (PAE) for different time periods. VFR17 and LL-37 (10 µM) are shown for comparison.

Article Snippet: In order to deplete acute wound fluids (AWF) of thrombin and fragments containing C-terminal peptides, AWF was diluted with an equal volume of PBS, and passaged 5 times through an affinity column (0.3 ml, Thermo Scientific) having coupled IgG antibodies specific for VFR17 (Innovagen AB).

Techniques: Activity Assay, Tandem Mass Spectroscopy, Sequencing, SDS Page, Staining, Western Blot, Generated, Incubation

Journal: PLoS Pathogens

Article Title: Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

doi: 10.1371/journal.ppat.1000857

Figure Lengend Snippet: ( A ) Fibrin clots were produced from human plasma and incubated with neutrophil elastase for the indicated time periods (Fibrin), or obtained from a patient with a venous, non-infected, chronic ulcer (PF), extracted, and analyzed by immunoblotting using polyclonal antibodies against the thrombin C-terminal peptide VFR17. ( B ) Human plasma, incubated with neutrophil elastase for the indicated time periods (Plasma, left panel), acute wound fluid (patients 1–2, AWF, middle panel), or wound fluid from patients with chronic ulcers (patients 1–6, CWF, right panel) was analysed by Western blot using polyclonal antibodies against the thrombin C-terminal peptide VFR17. ( C ) Flow cytometry analysis of binding of C-terminal thrombin epitopes to P. aeruginosa bacteria. Bacteria were incubated for 4 h with control plasma (P), human plasma depleted of prothrombin (DP), depleted plasma supplemented with the peptide GKY25, or, acute wound fluid (AWF). Binding of C-terminal epitopes to the bacteria was detected using primary antibodies against the C-terminal epitope VFR17 followed by addition of FITC-labeled secondary antibodies. ( D ) Visualization of binding and membrane damage by TCPs. P. aeruginosa bacteria were incubated ex vivo with human plasma (P), acute wound fluid (AWF), or wound fluid from a chronic leg ulcer (CWF), or visualized in vivo in fibrin slough (CWS) derived from a patient with a chronic ulcer infected by S. aureus . Arrows in P, AWF, and CWF point to damaged bacterial membranes. Coccoid bacteria (indicated by an arrow in CWS) show extensive binding of antibodies directed against the C-terminal peptide VFR17 (negative and positive bacterial controls, and additional material are found in and ). ( E ) TCPs inhibit bacterial growth in human plasma. Control plasma (P), plasma depleted of prothrombin (DP), depleted plasma supplemented with either prothrombin (DP+PT), or GKY25 (DP+GKY25) (PT and GKY25 at 1.5 µM), or control AWF or depleted AWF (D-AWF), were inoculated with P. aeruginosa bacteria under similar conditions as in ( C–D ). The multiplication factors at various time points are given. After incubation, CFUs were determined by plating. Experiments were repeated three times and a representative experiment is shown. ( F ) The thrombin C-terminal peptide GKY25 significantly increases survival. Mice were injected i.p. with P. aeruginosa bacteria, followed by subcutaneous injection of GKY25 or buffer only, after 1 h. The injections were repeated after 24 hours. Treatment with the peptide significantly increased survival (n = 10 for controls and treated, p = 0.002). ( G ) GKY25 suppresses bacterial dissemination to the spleen and kidney. Mice were infected as above, GKY25 was administrated subcutaneously after 1 h, and the cfu of P. aeruginosa in spleen and kidney was determined after a time period of 8 h (n = 10 for controls and treated, P<0.05 for spleen and kidney. Horizontal line indicates median value).

Article Snippet: In order to deplete acute wound fluids (AWF) of thrombin and fragments containing C-terminal peptides, AWF was diluted with an equal volume of PBS, and passaged 5 times through an affinity column (0.3 ml, Thermo Scientific) having coupled IgG antibodies specific for VFR17 (Innovagen AB).

Techniques: Produced, Incubation, Infection, Western Blot, Flow Cytometry, Binding Assay, Labeling, Ex Vivo, In Vivo, Derivative Assay, Injection

Journal: PLoS Pathogens

Article Title: Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

doi: 10.1371/journal.ppat.1000857

Figure Lengend Snippet: ( A ) Electron microscopy analysis. P. aeruginosa and S. aureus bacteria was incubated for 2 h at 37°C with 30 µM of GKY25 and LL-37 and analyzed with electron microscopy. Scale bar represents 1 µm. Control; Buffer control. ( B ) Permeabilizing effects of peptides on E. coli . Bacteria were incubated with the indicated peptides at 30 µM and permeabilization was assessed using the impermeant probe FITC. ( C ) Helical content of the thrombin-derived C-terminal peptides GKY25 and VFR17 in presence of negatively charged liposomes (PA). The two peptides showed a marked helix induction upon addition of the liposomes. ( D ) CD spectra of GKY25 and VFR17 in Tris-buffer and in presence of LPS. For control, CD spectra for buffer and LPS alone are also presented. ( E ) Effects of the indicated peptides on liposome leakage. The membrane permeabilizing effect was recorded by measuring fluorescence release of carboxyfluorescein from PA (negatively charged) liposomes. The experiments were performed in 10 mM Tris-buffer, in absence and presence of 0.15 M NaCl. Values represents mean of triplicate samples. ( F ) Activities of corresponding C-terminal peptides of the indicated coagulation factors. Peptides were tested in RDA against the indicated bacteria. Bacteria (4×10 6 cfu) were inoculated in 0.1% TSB agarose gels. Each 4 mm-diameter well was loaded with 6 µl of peptide at 100 µM. The zones of clearance correspond to the inhibitory effect of each peptide after incubation at 37°C for 18–24 h. ( G ) Overlay 3D-model showing the three coagulation factors thrombin, and factor X and IX. The C-terminal parts are indicated.

Article Snippet: In order to deplete acute wound fluids (AWF) of thrombin and fragments containing C-terminal peptides, AWF was diluted with an equal volume of PBS, and passaged 5 times through an affinity column (0.3 ml, Thermo Scientific) having coupled IgG antibodies specific for VFR17 (Innovagen AB).

Techniques: Electron Microscopy, Incubation, Derivative Assay, Fluorescence, Coagulation

Journal: Endocrinology

Article Title: Evocalcet Rescues Secondary Hyperparathyroidism-driven Cortical Porosity in CKD Male Rats

doi: 10.1210/endocr/bqad022

Figure Lengend Snippet: List of primary/secondary antibodies used in this study

Article Snippet: Rabbit polyclonal anti-SLC20A1 (Pit1) , GTX105062 , GeneTex., Inc., Irvine, CA , 1: 100 , AB_1951921.

Techniques:

Journal: Endocrinology

Article Title: Evocalcet Rescues Secondary Hyperparathyroidism-driven Cortical Porosity in CKD Male Rats

doi: 10.1210/endocr/bqad022

Figure Lengend Snippet: Osteocytic apoptosis, empty lacunae, and Pit1/Pit2, PTHR, and FGFR1 localization in the osteocytes of CKD-SHPT rats. (A, B) Osteocytic death at the endpoint (after 6 weeks of drug administration; scale bars, 10 μm). (A) Apoptotic osteocytes by TUNEL staining. (B) Empty lacunae by von Kossa staining. TUNEL-positive osteocytes and empty lacunae were observed in the vehicle groups (ii, v) but hardly detected in the Evo groups (iii, iv, vi). (C-F) Immunolocalization of (C) PTHR, (D) FGFR1, (E) Pit1, and (F) Pit2. Osteocytes of the vehicle groups (ii, v) exhibit PTHR and FGFR1 positivity (brown, arrows). Although the osteocytes in all groups show Pit1 immunopositivity (brown), Pit2 reactivity is relatively intense in the vehicle groups (ii, v). (G) Percentage of TUNEL-positive osteocytes (n = 10 per group). (H) Percentage of empty lacunae (n = 10 per group). The percentages of TUNEL-positive osteocytes and empty lacunae in the vehicle group significantly increased. (I, J) mRNA expression levels of (I) Pthr and (J) Fgfr1 in the whole femora at the endpoint (n = 10 per group; scale bars, 10 μm). Pthr and Fgfr1 expression levels significantly increased in the vehicle groups (ii, v) compared with those in the sham group (i). (K-N) Expression levels of (K, M) Pit1 and (L, N) Pit2 in the femoral cortical bone at the midpoint of the experimental period (n = 5 per group) and in the whole femora at the endpoint (n = 10 per group). Pit1 and Pit2 expression levels in the vehicle groups at the midpoint significantly increased compared with those in the sham group but decreased in the Evo groups. At the endpoint, Pit2 expression levels were increased in the vehicle groups and decreased in the Evo groups. Data are shown as the mean ± SE; differences with P < 0.05 are considered significant (see Methods). ***, P < 0.001; **, P < 0.01; *, P < 0.05. Abbreviations: FGFR1, fibroblast growth factor receptor 1; Pit1, sodium-dependent phosphate transporter 1; Pit2, sodium-dependent phosphate transporter 2; PTHR, PTH receptor; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Article Snippet: Rabbit polyclonal anti-SLC20A1 (Pit1) , GTX105062 , GeneTex., Inc., Irvine, CA , 1: 100 , AB_1951921.

Techniques: TUNEL Assay, Staining, Expressing, End Labeling

Journal: Endocrinology

Article Title: Evocalcet Rescues Secondary Hyperparathyroidism-driven Cortical Porosity in CKD Male Rats

doi: 10.1210/endocr/bqad022

Figure Lengend Snippet: Effect of PTH and Pi on MLO-Y4 cell death. (A–C) Cell death levels in MLO-Y4 cells cultured with (A) Pi or the mixture of Pi and PTH (Pi + PTH); (B) PTH, and (C) FGF23 for 1 to 3 days (n = 4). Cell death levels in cells treated with Pi were much higher than those in controls in a dose and time-dependent manner. Cell death levels in cells treated with Pi + PTH were higher than those in the cells treated with Pi. Conversely, cell death levels in cells treated with PTH or FGF23 were similar to those in the control cells. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (compared with control). (D) Localization of Pit1 and Pit2 (green) in MLO-Y4 cells after 6 and 12 hours of Pi treatment (n = 3; nuclei stained with DAPI). Pit1 and Pit2 immunoreactivities were localized around the cell membrane in the MLO-Y4 cells without Pi treatment. In Pi-treated MLO-Y4 cells, Pit1 and Pit2 immunoreactivities were observed in the granular structure inside the cells. (E-J) Expression levels of Pit1 and Pit2 in MLO-Y4 cells treated with indicated compounds for 3, 6, 12, and 24 hours (n = 3). Pit1 expression levels in MLO-Y4 cells increased significantly after 3 hours in all Pi, PTH, and FGF23 treatments; thereafter, the expression levels decreased with increased duration of treatment. Pit1 expression levels were elevated more substantially by Pi or PTH treatment than by FGF23 treatment. Pit2 expression in MLO-Y4 cells slightly increased when the cells were treated with Pi and PTH. The Pit2 expression did not markedly change by FGF23 treatment. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (compared with the 3-hour controls). ###, P < 0.001; ##, P < 0.01; #, P < 0.05 (compared with the 6-hour controls). †††, P < 0.001; ††, P < 0.01; †, P < 0.05 (compared with the 12-hour controls). §§§, P < 0.001; §§, P < 0.01; §, P < 0.05 (compared with the 24-hour controls). Data are shown as the mean ± SE; differences with P < 0.05 were considered significant. Abbreviations: FGF23, fibroblast growth factor 23; Pi, inorganic phosphate.

Article Snippet: Rabbit polyclonal anti-SLC20A1 (Pit1) , GTX105062 , GeneTex., Inc., Irvine, CA , 1: 100 , AB_1951921.

Techniques: Cell Culture, Control, Staining, Membrane, Expressing

Journal: Endocrinology

Article Title: Evocalcet Rescues Secondary Hyperparathyroidism-driven Cortical Porosity in CKD Male Rats

doi: 10.1210/endocr/bqad022

Figure Lengend Snippet: Schematic illustration of the cellular mechanism of cortical porosity and the inhibitory effect of evocalcet on SHPT model rats. Elevated serum PTH and Pi in CKD-SHPT induced osteocytic osteolysis, Phex/SIBLING-mediated inhibition of mineralization, and osteocytic death, which were the first steps of cortical porosity. PTH enhanced extracellular Pi sensing by stimulating Pit1 expression; PTH and Pi exacerbated cortical porosity in SHPT by enhancing their action toward each other. However, evocalcet, as a calcimimetic agent, prevented the PTH–Pi interaction and inhibited the enlargement of osteocytic lacunae and cortical porosity by decreasing the serum PTH concentration. Abbreviations: Pi, inorganic phosphate; SHPT, secondary hyperparathyroidism.

Article Snippet: Rabbit polyclonal anti-SLC20A1 (Pit1) , GTX105062 , GeneTex., Inc., Irvine, CA , 1: 100 , AB_1951921.

Techniques: Inhibition, Expressing, Concentration Assay