Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 1. Ntrk2 is up-regulated in mouse lungs after 24 hours of hypoxia exposure (pO2 0.01). Total RNA from mice lung homogenates was isolated (n 18 per group), direct labeled, and hybridized to 44K 60mer oligonucleotide microarrays (MWG). A: Volcano plot: log2-fold regulation compared to probability of regulation. Genes with log odds values 5 were considered to be regulated; black spots, exemplary genes. B: Functional annotation of regulated genes was performed according to Gene Ontology (GO). Ntrk2 (TrkB) belongs to MAPK signaling pathway. C: A time course of TrkB expression was performed using real-time PCR of lung homogenates harvested 1, 7, or 21 days after hypoxia exposure (n 7 to 9, each). D: Immunohistochemical staining of TrkB and its ligand, BDNF in mouse lung sections. The arrow indicates positive staining. E and F: Real-time PCR analysis of TrkB and BDNF expression in (E) isolated major pulmonary arteries (n 6), and (F) laser-microdissected intrapulmonary vessels (40 vessels per animal, n 4), from mice exposed to 21 days of hypoxia or normoxia. G: Expression levels of TrkB and BDNF in mouse PASMC as indicated by real-time PCR; lower CT values represent a more abundant transcript level. H: Immunofluorescence staining of TrkB and BDNF in mouse PASMC. The arrows indicate TrkB localization. Inset, negative control staining. *P 0.05.

Article Snippet: Predesigned, commercially available siRNA against human Egr-1 (siGenomeSMART pool) was purchased from Dharmacon (Chicago, IL), TrkB (siGenomeSMART pool) from Dharmacon and Santa Cruz Biotechnology, siRNA against BDNF (Santa Cruz Biotechnology), control siRNA from Ambion.

Techniques: Isolation, Labeling, Functional Assay, Expressing, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining, Immunofluorescence, Negative Control

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 2. Analysis of TrkB and BDNF expression in rat PAH models. A: Real-time PCR analysis of TrkB and BDNF mRNA expression 14 and 28 days after monocrotaline administration (n 4 per group) in lung homogenates. B: Immunohistochemical staining of TrkB and BDNF in monocrotaline- treated lungs and lungs from control animals. C: Real-time PCR analysis of TrkB and BDNF mRNA expression in lung homogenates in the Su5416 rat model (n 4 controls, n 7 Su5416). n.s., not significant. *P 0.05.

Article Snippet: Predesigned, commercially available siRNA against human Egr-1 (siGenomeSMART pool) was purchased from Dharmacon (Chicago, IL), TrkB (siGenomeSMART pool) from Dharmacon and Santa Cruz Biotechnology, siRNA against BDNF (Santa Cruz Biotechnology), control siRNA from Ambion.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining, Control

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 3. Up-regulation of TrkB and BDNF in human lung tissue from IPAH. A: Colocalization of BDNF and TrkB with SMA as indicated by immunofluorescence staining in donor and IPAH patients. B: Real-time PCR analysis of TrkB and BDNF in laser-microdissected intrapulmonary vessels from donor and IPAH patients. Expression is given in comparison to samples from donor lungs (n 5 per group). C: Real-time PCR analysis of expression levels of TrkB and BDNF in human PASMC; lower CT values represent a more abundant transcript level. D: Immunofluorescence staining of TrkB and BDNF in human PASMC derived from donor and IPAH patients. E: Colocalization of BDNF and TrkB with SMA in PASMC as indicated by immunofluorescence staining, IgG, isotype control staining. *P 0.05.

Article Snippet: Predesigned, commercially available siRNA against human Egr-1 (siGenomeSMART pool) was purchased from Dharmacon (Chicago, IL), TrkB (siGenomeSMART pool) from Dharmacon and Santa Cruz Biotechnology, siRNA against BDNF (Santa Cruz Biotechnology), control siRNA from Ambion.

Techniques: Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Expressing, Comparison, Derivative Assay, Control

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 5. BDNF stimulation leads to MAP kinase activation. A: Time-dependent phosphorylation in response to BDNF of the ERK1/2 kinase with densitometric analysis. B and C: Effects on BDNF signaling by pretreatment with the inhibitors (B) K252a (TrkB) and (C) U0126 (ERK1/2) with densitometric analysis. Equal protein loading was confirmed by immunoblotting against -actin. D: Effects of blocking BDNF antibody on ERK1/2 phosphorylation with densitometric analysis. M(kDa), molecular weight in kilodaltons; n.s., not significant. *P 0.05.

Article Snippet: Predesigned, commercially available siRNA against human Egr-1 (siGenomeSMART pool) was purchased from Dharmacon (Chicago, IL), TrkB (siGenomeSMART pool) from Dharmacon and Santa Cruz Biotechnology, siRNA against BDNF (Santa Cruz Biotechnology), control siRNA from Ambion.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Blocking Assay, Molecular Weight

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 4. Functional role of TrkB and its ligand BDNF in remodeling. Proliferation after BDNF stimulation was assessed by direct [3H] thymidine incorporation (A) (n 6), or immunofluorescence staining of Ki-67 proliferation marker (B). The effect of BDNF on cell apoptosis was measured by annexin V staining (C) and caspase-3 cleavage (D). 5% FCS and staurosporine were used as positive controls for proliferation and apoptosis studies, respectively. *P 0.05.

Article Snippet: Predesigned, commercially available siRNA against human Egr-1 (siGenomeSMART pool) was purchased from Dharmacon (Chicago, IL), TrkB (siGenomeSMART pool) from Dharmacon and Santa Cruz Biotechnology, siRNA against BDNF (Santa Cruz Biotechnology), control siRNA from Ambion.

Techniques: Functional Assay, Immunofluorescence, Staining, Marker

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 6. BDNF regulates and activates Egr-1. A: Induction of early growth-response factor 1 (Egr-1) protein in human PASMC as analyzed by Western blotting. B: BDNF-induced Egr-1 protein localization in PASMC as assessed by immunofluorescence analysis. C: Electrophoretic mobility shift assay for Egr-1 induction and DNA–protein interactions from nuclear extracts prepared from BDNF (10 ng/mL)-treated PASMC, incubated with radiolabeled oligonucleotide containing Egr-1 consensus sequence. Incubation with an excess (10-fold) of an unlabeled probe served as a control for specific binding. PDGF (10 ng/mL) treated cells served as a positive control. D: Egr-1 expression in lung parenchyma of donor and IPAH patients. An Egr-1/ mouse (KO-Egr-1) served as negative control. E: Real-time PCR analysis for Egr-1 expression in IPAH patient and donor laser-microdissected intrapulmonary vessels (n 5). n.s., not significant. *P 0.05.

Article Snippet: Predesigned, commercially available siRNA against human Egr-1 (siGenomeSMART pool) was purchased from Dharmacon (Chicago, IL), TrkB (siGenomeSMART pool) from Dharmacon and Santa Cruz Biotechnology, siRNA against BDNF (Santa Cruz Biotechnology), control siRNA from Ambion.

Techniques: Western Blot, Immunofluorescence, Electrophoretic Mobility Shift Assay, Incubation, Sequencing, Control, Binding Assay, Positive Control, Expressing, Negative Control, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: Moesin domain organization, conformational states, and binding sites. Correspondence between regions of sequence and the domain architecture of full-length moesin. Colors correspond to distinct functional parts: lobe A of FERM domain (green), lobe B (orange), lobe C (yellow), α-helical region (blue), FLAP (red), and C-terminal tail (gray). A, domains of moesin within the primary sequence. Residue numbers in human moesin are shown for boundaries and for the C-terminal phosphorylation site. B, schematic of full-length inactive and active conformations. Also shown in light blue and labeled in the figure are four known binding sites of the ERM protein that include two for PIP2 (1 and 2) and two for protein ligands (3 and 4). The two PIP-2 binding sites are shaded dark green. The two protein-binding sites are enclosed with dashed regions. a, schematic of inactive conformation (based on structure 2I1K) in which three binding sites are masked (by α-helical region, FLAP, and tail) but the PIP2-binding lysine PATCH (1) remains exposed. (The connecting peptide between the FLAP and C-terminal tail is poorly visible because it runs behind lobe C in this perspective.) b, schematic of active conformation in which three of the binding sites are unmasked. The helical region is shown with dashes in the middle to indicate it is longer than shown.

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Binding Assay, Sequencing, Functional Assay, Labeling, Protein Binding

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: FLAP, sequence and structure analysis. A, electrostatic surface potential of full-length insect ERM protein (PDB 2I1K) and B, of unmasked moesin FERM domain with color charge scale on the left. The molecules are oriented to show the PIP2 binding POCKET, which is covered by the FLAP in the closed conformation of moesin (A) and uncovered in the FERM domain (B). Labels indicate locations of the A, B, and C lobes. Location of PIP2 binding POCKET is indicated by arrows. C, ribbon representation of the closed ERM structure. FLAP is red (N-terminal and C-terminal parts) or magenta (reconstructed tip, amino acids 473–485). β1 to β4 strands of the PH-like domain are green; β5-β7 strands are yellow; α-helix is blue. C-terminal tail is light blue. Light Blue circles indicate locations of the two critical residues, Lys-63 and Lys-278, in the POCKET, which are masked by the FLAP when moesin is closed. D, multiple sequence analysis of the FLAP of human ezrin, radixin, and moesin and flanking regions. Red dashed rectangle highlights the FLAP. Above the sequence is a row of symbols that scores the extent of sequence conservation scored by ClustalX (* = identity; : = all residues belong to a strong conservation group; · = all residues belong to a weak conservation group). Arrowheads indicate residues in moesin that are acidic (red) or short side chains (black). Statistical significance compared with Moesin WT membrane enrichment is shown as: *, p < 0.1 and **, p < 0.01.

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: The PIP2 binding POCKET defined by crystallography is necessary for ERM activation. A, the complex of radixin FERM domain with IP3 (PDB 1GC6) represented as ribbon drawing. The dashed box highlights the basic cleft between lobes A and C. The IP3 molecule is shown in a ball and stick model. The positively charged residues proposed to interact with the phosphate groups of IP3 are shown in space-filled and colored blue. The side chains of IP3 are shown in ball-and-stick models. Inset shows expanded view of this cleft. B, membrane enrichment of GFP-tagged constructs of WT or mutant moesin (and GFP-only as control) was assessed in representative midplane confocal images of transfected Jurkat cells. The large nucleus of Jurkat is indicated with “N.” Scale bar is 5 μm. C, quantitation of membrane enrichment of moesin constructs in Jurkat cells having the indicated mutations of lysines in the PIP2-binding POCKET. The enrichment characteristic of the WT protein is indicated by the upper dashed line. Proteins not enriched at the plasma membrane typically have ratios of membrane/cytoplasmic localization of ∼1.0 (lower dashed line). Statistical significance compared with Moesin WT membrane enrichment is shown as: *, p < 0.1; **, p < 0.01; ***, p < 0.001.

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Binding Assay, Activation Assay, Construct, Mutagenesis, Transfection, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: Mutation of the PIP2 POCKET abolishes PIP2 binding. A, binding of WT and K63N/K278N moesin to PIP2 in LUV was measured by a cosedimentation assay over the indicated range of PIP2 concentrations. Dissociation constants (mean of two independent experiments ± S.D.) are presented in the lower panel. B and C, binding of moesin to the cytoplasmic tail of CD44 (B) and the tail of NHERF1 (C) were assessed by pulldown assays. Recombinant proteins WT and K63/278N moesin were tested in the presence of lipid control PS (phosphatidylserine) (upper panels) or PIP2 (lower panels). Regions shown are from the pellet using anti-His to detect moesin pulled down and Coomassie stain to validate equal loading of GST-CD44 or GST-NHERF1.

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Mutagenesis, Binding Assay, Recombinant, Staining

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: Binding of PATCH mutant constructs to PIP2. A, binding of WT and mutant moesin to PIP2 in LUV was measured by a cosedimentation assay over the indicated range of PIP2 concentrations. Moesin constructs tested included WT, each of the single lysine mutations (K253N, K254N, K262N, K263N), and double mutations of the two pairs (K253N/K254N and K262N/K263N) that strongly impaired membrane localization in cells and the combined mutation of the 4 lysines K253N/K254N/K262N/K263N (4N). B, tabulation of dissociation constant (mean of two independent experiments ± S.D.).

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Binding Assay, Mutagenesis, Construct

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: Single lysine mutations in the PATCH impair PIP2-induced activation of moesin binding to CD44 and NHERF1. A, binding of moesin to CD44 was assessed using pulldown of moesin-His by CD44 tail-GST immobilized on beads. Moesin constructs tested included WT, each of the single lysine mutations that strongly impaired membrane binding in cells, and combined mutations of lysines in a pair. Gel regions shown are from the pellet using anti-His to detect moesin pulled down and Coomassie stain to validate equal loading of GST-CD44. B and C, similar analysis as A but using NHERF1 tail-GST (B) or NHERF1 full-length-GST (C) instead of CD44-GST.

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Activation Assay, Binding Assay, Construct, Staining

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: FLAP removal increases PIP2-induced protein ligands binding to moesin. A, SDS-PAGE analysis of the pellet from pulldown assays using soluble moesin-His and the cytoplasmic tail of CD44-GST (or NHERF1-GST) immobilized on beads. Moesin in the pellet is detected by anti-His antibody. CD44-GST (or NHERF1-GST) is detected by Coomassie Blue to verify equal loading. B, quantitation of the relative amounts of bound moesin determined by densitometry.

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Binding Assay, SDS Page, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: Analysis of stoichiometry and cooperativity of moesin binding to PIP2. A, cooperativity. Binding of moesin-Alexa 488 to LUVs composed of varying percentages (0–15%) of PIP2 assessed by co-sedimentation assays followed by spectrofluorimetric analysis. LUVs having fixed total lipid concentration (0.28 mm accessible lipid) but vary in their mole fractions of PIP2 were added to a fixed concentration of moesin-Alexa 488 (0.4 μm). The percent of moesin bound is plotted as a function of the percentage of PIP2 in the LUVs where each point is the average of two experiments. The solid line is the nonlinear least squares best fit of all the data. B, stoichiometry. A standard protein to membrane FRET assay was employed to quantitate membrane-bound moesin. The FRET measured occurs between intrinsic tryptophan donors in moesin and the dansyl-PE acceptors in the LUVs. LUVs containing 10 mol % PIP2 in a lipid mixture mimicking the plasma membrane inner leaflet (PE/PC/PS/l-α-phosphatidylinositol/sphingomyelin/cholesterol/dansyl-PE/PIP2 (23.8:9.1:18.1:4.5:4.5:25:5:10)) were titrated into a fixed concentration of nonlabeled moesin (3.6 μm). These conditions were chosen to drive high affinity PIP2 binding such that the titration yields a linear increase in membrane-associated moesin until all proteins had been bound and a plateau achieved. High variability (a time dependent signal loss we ascribe to vesicle aggregation) was observed when LUV concentrations exceeded 6.0 μm PIP2 (data point shown in parentheses). The intersection of the best-fit straight lines for the linear increase and plateau regions represent the saturation point, yielding 3.1 ± 0.25 μm PIP2 molecules per 3.6 μm moesin molecules equivalent to a PIP2/moesin ratio of about 0.86 ± 0.07 (average of 5 experiments ± S.E.).

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Binding Assay, Sedimentation, Concentration Assay, Titration

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: Representation of a proposed additional mechanism for conformational change when PIP2 binds to the PATCH. Panel A shows a model of the PATCH of intact ERM interacting with a lipid bilayer including a PIP2 molecule. Lobe C, the PH-like domain, has been colored to facilitate visualization: β1–β4 strands in green, β5–β7 strands in yellow, and the α-helix in blue. The FLAP is colored red. The headgroup of PIP2 is accommodated between the two pairs of lysines (Lys-253/Lys-254 and Lys-262/Lys-263) whose side chains are shown in ball and stick representation. Panel B is a magnified view of the binding site.

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Binding Assay

Journal: Asia-Pacific Journal of Sports Medicine, Arthroscopy, Rehabilitation and Technology

Article Title: Neural structural alterations correlates of quadriceps muscle strength deficits in patients after anterior cruciate ligament reconstruction

doi: 10.1016/j.asmart.2024.11.001

Figure Lengend Snippet: The associations between CST structure and injured quadriceps strength and masks of corticospinal tracts (in red, yellow, green, and purple) on the images of DTI outcomes. (A) The position of quadriceps strength measurement. (B) The picture of the hand-held dynamometer. (C, D) The hot plots of relationships between DTI outcomes of CST structure and quadriceps strength of injured limb before (C) and after (D) controlling age, BMI, Tegner activity score, and graft type in patients with ACLR and subgroup analyses of synthetics and hamstring autografts before (C) and after (D) controlling age, BMI, Tegner activity score. (E) the scatter plots of significant correlations between the DTI outcomes of CST structure and quadriceps strength of injured limb (E). DTI, diffusion tensor imaging; CST, corticospinal tract; LSI, limb symmetry index; BMI, body mass index. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The mean values of the bilateral/left/right sides of the CSTs were extracted from the voxel within the individual masks registered from Johns Hopkins University International Consortium for Brain Mapping DTI tracts.

Techniques: Activity Assay, Diffusion-based Assay, Imaging

Journal: Asia-Pacific Journal of Sports Medicine, Arthroscopy, Rehabilitation and Technology

Article Title: Neural structural alterations correlates of quadriceps muscle strength deficits in patients after anterior cruciate ligament reconstruction

doi: 10.1016/j.asmart.2024.11.001

Figure Lengend Snippet: The associations between the ratio of DTI outcomes in corticospinal tracts of the injured hemisphere to those of the non-injured hemisphere and LSI of quadriceps strength, and masks of corticospinal tracts (in red, yellow, green, and purple) on the images of the ratio of DTI outcomes. (A, B) The hot plots of relationships between the ratio of DTI outcomes in corticospinal tracts of the injured hemisphere to those of the non-injured hemisphere and LSI of quadriceps strength before (A) and after (B) controlling age, BMI, Tegner activity score, and graft type in patients with ACLR and subgroup analyses of synthetics and hamstring autografts before (A) and after (B) controlling age, BMI, Tegner activity score. (C, D, E, F) the scatter plots of significant correlations between the ratio of DTI outcomes in corticospinal tracts of the injured hemisphere to those of the non-injured hemisphere and LSI of quadriceps strength (C, D, E, F). DTI, diffusion tensor imaging; CST, corticospinal tract; LSI, limb symmetry index; BMI, body mass index. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The mean values of the bilateral/left/right sides of the CSTs were extracted from the voxel within the individual masks registered from Johns Hopkins University International Consortium for Brain Mapping DTI tracts.

Techniques: Activity Assay, Diffusion-based Assay, Imaging

Journal: Antioxidants

Article Title: PTEN/PKM2/ERα-Driven Glyoxalase 1 Overexpression Sustains PC3 Prostate Cancer Cell Growth Through MG-H1/RAGE Pathway Desensitization Leading to H 2 O 2 -Dependent KRIT1 Downregulation

doi: 10.3390/antiox14091120

Figure Lengend Snippet: Glyoxalase 1 (Glo1) is overexpressed in aggressive prostate cancer (PCa) tissues and cell lines. Representative images of Glo1 protein expression, evaluated by immunohistochemistry, in PCa tissues from ( a ) low-grade (LG) and ( b ) high-grade (HG) patients with PCa. Magnification 200×. ( c ) Glo1 protein expression, measured by ELISA; ( d ) Glo1 specific enzyme activity, evaluated by a specific spectrophotometric method; ( e ) cell proliferation, measured by the CCK-8 assay and the clonogenic assay; ( f ) 20 µM etoposide and 1 µM staurosporine-driven apoptosis, evaluated by the increased expression of active caspase-3, using ELISA; and ( g ) MG-H1 intracellular amounts, measured by a specific ELISA kit, in moderately aggressive DU145 and highly aggressive PC3 PCa cells. The histograms indicate the mean ± SD of three different cultures. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human prostate cancer cell lines DU145 (derived from brain metastasis) and PC3 (derived from bone metastasis) were acquired from the American Type Culture Collection (ATCC) and maintained, following the supplier’s instructions, at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Activity Assay, CCK-8 Assay, Clonogenic Assay

Journal: Antioxidants

Article Title: PTEN/PKM2/ERα-Driven Glyoxalase 1 Overexpression Sustains PC3 Prostate Cancer Cell Growth Through MG-H1/RAGE Pathway Desensitization Leading to H 2 O 2 -Dependent KRIT1 Downregulation

doi: 10.3390/antiox14091120

Figure Lengend Snippet: Glyoxalase 1 (Glo1) sustains prostate cancer (PCa) aggressiveness to control methylglyoxal (MG)-derived hydroimidazolone 1 (MG-H1). ( a ) MG-H1 intracellular amounts, measured by a specific ELISA kit, and ( b ) cell proliferation, measured by the CCK-8 assay, of PC3 cells upon Glo1 transient silencing (siGlo1) ( a , b ) and of DU145 cells upon Glo1 ectopic expression (pCMV-Glo1) ( a , b ). The histograms indicate the mean ± SD of three different cultures. siCtr: control (non-specific siRNA); pCMV-Ctr: control containing plasmid DNA. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human prostate cancer cell lines DU145 (derived from brain metastasis) and PC3 (derived from bone metastasis) were acquired from the American Type Culture Collection (ATCC) and maintained, following the supplier’s instructions, at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Control, Derivative Assay, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Expressing, Plasmid Preparation

Journal: Antioxidants

Article Title: PTEN/PKM2/ERα-Driven Glyoxalase 1 Overexpression Sustains PC3 Prostate Cancer Cell Growth Through MG-H1/RAGE Pathway Desensitization Leading to H 2 O 2 -Dependent KRIT1 Downregulation

doi: 10.3390/antiox14091120

Figure Lengend Snippet: Glyoxalase 1 (Glo1) upregulation is driven by the PTEN-dependent pathway. ( a ) PTEN expression, measured by a specific ELISA kit, in PC3 cells upon PTEN ectopic expression (pCMV-PTEN) decreased ( b ) Glo1 mRNA expression and specific enzyme activity, evaluated by qRT-PCR and a specific spectrophotometric method, respectively; ( c ) PTEN expression, measured by a specific ELISA kit, in transiently PTEN-knocked-down (siPTEN) DU145 cells increased ( d ) Glo1 transcript levels and specific enzyme activity. The histograms indicate the mean ± SD of three different cultures. pCMV-Ctr: control containing plasmid DNA; siCtr: control (non-specific siRNA). *** p < 0.001, **** p < 0.0001.

Article Snippet: Human prostate cancer cell lines DU145 (derived from brain metastasis) and PC3 (derived from bone metastasis) were acquired from the American Type Culture Collection (ATCC) and maintained, following the supplier’s instructions, at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, Quantitative RT-PCR, Control, Plasmid Preparation

Journal: Antioxidants

Article Title: PTEN/PKM2/ERα-Driven Glyoxalase 1 Overexpression Sustains PC3 Prostate Cancer Cell Growth Through MG-H1/RAGE Pathway Desensitization Leading to H 2 O 2 -Dependent KRIT1 Downregulation

doi: 10.3390/antiox14091120

Figure Lengend Snippet: Glyoxalase 1 (Glo1) upregulation is driven by the PI3/AKT/mTOR pathway. ( a , c ) Ectopic expression of PTEN (pCMV-PTEN) in PC3 cells or ( b , d ) PTEN knockdown (siPTEN) in DU145 cells led to desensitization or activation of the PI3K/AKT/mTOR pathway, respectively. Treatment of PC3 cells with the PI3K inhibitor LY294002 (LY), the AKT inhibitor MK2206 (MK), and the mTOR inhibitor rapamycin (Rapam) further confirmed the involvement of the PI3K/AKT/mTOR signaling cascade in regulating ( e ) Glo1 at both the transcript and the functional level. The PI3/AKT/mTOR pathway was evaluated by p-AKT and p-mTOR expression measured by both specific ELISA kits and Western blotting. mRNA expression was evaluated by qRT-PCR, while specific enzyme activity was measured by a specific spectrophotometric method. β-actin served as the internal control. The histograms indicate the mean ± SD of three different cultures. pCMV-Ctr: control containing plasmid DNA; siCtr: control (non-specific siRNA). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human prostate cancer cell lines DU145 (derived from brain metastasis) and PC3 (derived from bone metastasis) were acquired from the American Type Culture Collection (ATCC) and maintained, following the supplier’s instructions, at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Knockdown, Activation Assay, Functional Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR, Activity Assay, Control, Plasmid Preparation

Journal: Antioxidants

Article Title: PTEN/PKM2/ERα-Driven Glyoxalase 1 Overexpression Sustains PC3 Prostate Cancer Cell Growth Through MG-H1/RAGE Pathway Desensitization Leading to H 2 O 2 -Dependent KRIT1 Downregulation

doi: 10.3390/antiox14091120

Figure Lengend Snippet: Expression of the receptor for AGEs (RAGE), levels of hydrogen peroxide (H 2 O 2 ), and KRIT1 expression in DU145 and PC3 cells. ( a ) RAGE transcript levels, measured by qRT-PCR, and RAGE protein expression, evaluated by Western blotting. ( b ) H 2 O 2 levels were measured by a specific kit, and ( c ) KRIT1 expression was studied by using a specific ELISA kit. β-actin served as the internal control. The histograms represent the mean ± SD from three independent experiments. ** p < 0.01, *** p < 0.001.

Article Snippet: Human prostate cancer cell lines DU145 (derived from brain metastasis) and PC3 (derived from bone metastasis) were acquired from the American Type Culture Collection (ATCC) and maintained, following the supplier’s instructions, at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Aging Neuroscience

Article Title: Increase in Blood Levels of Growth Factors Involved in the Neuroplasticity Process by Using an Extremely Low Frequency Electromagnetic Field in Post-stroke Patients

doi: 10.3389/fnagi.2018.00294

Figure Lengend Snippet: Comparison of the BDNF level obtained from the ELF-EMF group vs. the non-ELF-EMF group. (A) BDNF plasma concentration. Statistical significance between ELF-EMF and non-ELF-EMF groups: b vs. d: p < 0.0001. (B) BDNF mRNA expression. Statistical significance between ELF-EMF and non-ELF-EMF groups: b vs. d: p < 0.0001.

Article Snippet: Expression levels of the studied genes were obtained using the following TaqMan probes: Hs02718934_s1 for human BDNF gene, and Hs02786624_g1 as an endogenous control, which was a human GAPDH gene (Life Technologies, Carlsbad, CA, United States).

Techniques: Comparison, Clinical Proteomics, Concentration Assay, Expressing

Journal: Frontiers in Aging Neuroscience

Article Title: Increase in Blood Levels of Growth Factors Involved in the Neuroplasticity Process by Using an Extremely Low Frequency Electromagnetic Field in Post-stroke Patients

doi: 10.3389/fnagi.2018.00294

Figure Lengend Snippet: Plasma levels (in pg/ml) of BDNF and VEGF measured before and after treatment in ELF-EMF and non-ELF-EMF groups.

Article Snippet: Expression levels of the studied genes were obtained using the following TaqMan probes: Hs02718934_s1 for human BDNF gene, and Hs02786624_g1 as an endogenous control, which was a human GAPDH gene (Life Technologies, Carlsbad, CA, United States).

Techniques: Clinical Proteomics

Journal: Frontiers in Aging Neuroscience

Article Title: Increase in Blood Levels of Growth Factors Involved in the Neuroplasticity Process by Using an Extremely Low Frequency Electromagnetic Field in Post-stroke Patients

doi: 10.3389/fnagi.2018.00294

Figure Lengend Snippet: Scatterplots presenting correlation between changes of BDNF plasma level (in pg/ml) in ELF-EMF group and (A) changes of ADL; (B) changes of MMSE; (C) changes of GDS.

Article Snippet: Expression levels of the studied genes were obtained using the following TaqMan probes: Hs02718934_s1 for human BDNF gene, and Hs02786624_g1 as an endogenous control, which was a human GAPDH gene (Life Technologies, Carlsbad, CA, United States).

Techniques: Clinical Proteomics

Journal: Frontiers in Aging Neuroscience

Article Title: Increase in Blood Levels of Growth Factors Involved in the Neuroplasticity Process by Using an Extremely Low Frequency Electromagnetic Field in Post-stroke Patients

doi: 10.3389/fnagi.2018.00294

Figure Lengend Snippet: Correlation coefficient values obtained for the change in BDNF plasma level (ΔBDNF) and parameters of functional status (ADL, MMSE, and GDS) after ELF-EMF treatment.

Article Snippet: Expression levels of the studied genes were obtained using the following TaqMan probes: Hs02718934_s1 for human BDNF gene, and Hs02786624_g1 as an endogenous control, which was a human GAPDH gene (Life Technologies, Carlsbad, CA, United States).

Techniques: Clinical Proteomics, Functional Assay

Journal: bioRxiv

Article Title: Light-driven repair: Photobiomodulation restores blood–brain barrier function following hypoxic injury

doi: 10.64898/2026.02.15.706027

Figure Lengend Snippet: A) Experimental workflow illustrating the transwell-based BBB model, hypoxic exposure (6 h, 1% O₂), PBM treatment schedule, and downstream functional and molecular analyses (created with BioRender). (B–E) TEER expressed as relative change (%) from baseline. (B) Normoxic controls at 48 h. (C)TEER immediately following hypoxia. (D)TEER at 24 h post-hypoxia. (E)TEER at 48 h post-hypoxia. PBM significantly restored endothelial barrier resistance under hypoxic conditions. Statistical comparisons were performed using multiple unpaired t-tests with Holm–Šídák correction; N=4 independent biological replicates; *p<0.05. (F)ZO-1 mRNA expression in HBMECs at 48 h, normalised to RPL13A. (G)Quantification of ZO-1 protein levels relative to control, measured as ZO-1-positive area normalised to Hoechst nuclear area. (H)Representative ICC images showing ZO-1 (magenta) and nuclei (Hoechst, blue) in normoxic and hypoxic HBMECs with and without PBM. For mRNA and protein analyses, statistical significance was determined using one-way ANOVA with Šídák’s post hoc correction; N=3–4 independent biological replicates. Data are presented as mean±SEM.

Article Snippet: The cell lines used were purchased from Innoprot: human brain microvascular endothelial cells (HBMECs) (Innoprot; P10361-IM), human astrocytes (HAs) (Innoprot; P10251-IM), and human brain vascular pericytes (HVPCs) (Innoprot; P10363-IM).

Techniques: Functional Assay, Expressing, Control

Journal: bioRxiv

Article Title: Light-driven repair: Photobiomodulation restores blood–brain barrier function following hypoxic injury

doi: 10.64898/2026.02.15.706027

Figure Lengend Snippet: (A)vWF mRNA expression in HBMECs 48 h after normoxia or hypoxia (6 h, 1% O₂), with or without PBM treatment, normalised to RPL13A. (B)Representative ICC images showing vWF (yellow) and nuclei (Hoechst, blue) in normoxic and hypoxic endothelial cells ±PBM. (C)Quantification of vWF protein levels, expressed as mean nuclear-normalised fluorescence intensity relative to normoxia −PBM controls. (D)Validation of vWF knockdown efficiency in siRNA-transfected endothelial cells, shown as relative vWF mRNA expression normalised to RPL13A. (E-F) Relative TEER changes (%) in endothelial monocultures and BBB tri-cultures at 24 h and 48 h under normoxic (E) and hypoxic (F) conditions following vWF silencing. Statistical comparisons for mRNA and protein expression were performed using one-way ANOVA with Šidák’s post hoc test (N=3–4 biological replicates). siRNA validation was analysed using an unpaired two-tailed t-test (N=8 biological replicates). TEER data were analysed using multiple unpaired t-tests with Holm–Šidák correction (N=4 biological replicates). Data are presented as mean±SEM.

Article Snippet: The cell lines used were purchased from Innoprot: human brain microvascular endothelial cells (HBMECs) (Innoprot; P10361-IM), human astrocytes (HAs) (Innoprot; P10251-IM), and human brain vascular pericytes (HVPCs) (Innoprot; P10363-IM).

Techniques: Expressing, Fluorescence, Biomarker Discovery, Knockdown, Transfection, Two Tailed Test