Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 7. Validation of compound-target interactions through molecular docking and protein-small molecule binding assays. (A) Chemical structure of EGCG and molecular docking models showing its interactions with NOX2 and p47phox. (B) Chemical structure of quercetin and molecular docking models illustrating its interactions with NOX2 and p47phox. (C) An SPR sensorgram displaying the binding affinity of the NOX2-EGCG interaction. (D) An SPR sensorgram showing the binding affinity of the NOX2-quercetin interaction. (E) A BLI sensorgram illustrating the binding affinity of the p47phox-EGCG interaction. (F)A BLI sensorgram depicting the binding affinity of the p47phox-quercetin interaction. Abbreviations: BLI, Bio-layer interferometry; EGCG, epigallocatechin-3-gallate; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; Que, quercetin; SPR, surface plasmon resonance.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques: Biomarker Discovery, Binding Assay, SPR Assay

Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 8. QKPC and its components, EGCG and quercetin, reduce NOX2-p47phox/ROS signaling in vivo and in vitro. (A) Protein levels of NOX2 and p47phox in human neutrophils were assessed by Western blotting analysis, with relative density quantification shown as the ratio of NOX2 or p47phox to GAPDH (n = 3). (B) Protein levels of NOX2 and p47phox in lung tissues of mice were determined by Western blotting analysis and presented as the ratio of NOX2 or p47phox to GAPDH (n = 3). (C) Cellular ROS levels were measured using the oxidant-sensing probe DCFH-DA, and fluorescence intensity was recorded for each group. EGCG, quercetin, and the positive drugs NAC (20 μM, a ROS scavenger), DPI (50 μM, a general NADPH oxidase inhibitor), and GSK (50 μM, a NOX2-specific inhibitor) effectively suppressed ROS levels elevated by the combined CSE and LPS exposure (n = 6–8). (D) MDA levels in lung tissues were measured with an MDA assay kit. Data are presented as mean ± SD, with statistical significance set at p < 0.05. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviations: CS, cigarette smoke; CSE, cigarette smoke extract; DPI, Diphenyleneiodonium chloride; EGCG, epigallocatechin-3-gallate; GSK, GSK2795039; LPS, lipopolysaccharide; MDA, malondialdehyde; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; QKPC, Qingke Pingchuan; Que, quercetin; ROS, reactive oxygen species.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques: In Vivo, In Vitro, Western Blot, Fluorescence, Multiple Displacement Amplification

Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 9. Proposed mechanisms of QKPC action against ECOPD. Abbreviations: COPD, chronic obstructive pulmonary disease; EGCG, epigallocatechin-3-gallate; LPS, lipopolysaccharide; MPO, myeloperoxidase; NE, neutrophil Elastase; NET, neutrophil extracellular trap; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; QKPC, Qingke Pingchuan; ROS, reactive oxygen species.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques:

Journal:

Article Title: Isolation and Characterization of an Arabidopsis Mutant That Overaccumulates O -Acetyl- l -Ser 1 [w]

doi: 10.1104/pp.104.047068

Figure Lengend Snippet: Positional cloning of the OSH1. A, Physical map of a region of chromosome V where OSH1 was mapped. BAC clones, markers used for fine mapping using 3,600 chromatids, and the number of recombinants detected are indicated. OSH1 was mapped between the markers F7A7.2 and F7A7.3. B, Four overlapping probes (a–d) used for Southern hybridization to detect methylation of HpaII site. The thin vertical lines indicate the positions of CCGG sequence. The thick horizontal bar represents the 2.3-kb HpaII fragment (221,355–223,688 bp of chromosome V) that was detected in osh1-1 but not in wild type. Asterisk indicates the HpaII site that was methylated in wild type but was demethylated in osh1-1 (see below). C, Detection of difference in methylation status by PCR. Genomic DNA from wild type and osh1-1 was cleaved with MspI or HpaII and amplified with two primer sets covering each end of the 2.3-kb HpaII fragment in B. Results of three replicates with independent DNA extraction are shown. Intact DNA (uncut) was also amplified. D, Predicted genes within the mapped region and their orientations. E, Detection of difference in methylation status by bisulfite sequencing. Genomic DNA corresponding to the top strand of 223,234 to 224,340 was examined and extent of methylation at each C residue was calculated in wild type (upper) and osh1-1 (lower). The middle part shows the exons (thick bar) and introns (thin bar) of At5g01580 and At5g01590. F, DNA sequence of the hypermethylated region. Hypermethylated (>10%) C residues in wild type are underlined. Asteriskes in E and F indicate the C residue at 223689 that were demethylated in osh1-1.

Article Snippet: Recombinant At5g01580 protein or E. coli thioredoxin (Wako Pure Chemicals, Osaka) or egg white lysozyme (Wako Pure Chemicals) was preactivated with dithiothreitol (final concentration, 25 μ m ) for 10 min at 37°C, and added to the reaction mixture (20 μ L) at 300 n m . Each reaction contained 4 ng of cy3-conjugated F(ab′) 2 .

Techniques: Clone Assay, Hybridization, Methylation, Sequencing, Amplification, DNA Extraction, Methylation Sequencing

Journal:

Article Title: Isolation and Characterization of an Arabidopsis Mutant That Overaccumulates O -Acetyl- l -Ser 1 [w]

doi: 10.1104/pp.104.047068

Figure Lengend Snippet: A, Accumulation of At5g01580 mRNAs in osh1-1. Plants were grown as in Figure 1A. Total RNA extracted from shoots was reverse transcribed and subjected to real-time PCR analysis to monitor amplification of At5g01580 cDNAs in wild type (white bars) and osh1-1 (black bars). Accumulation of these mRNAs relative to that of ubiquitin5 was determined. Values are normalized with the means of wild-type plants (+S). Means and sd of seven plants are shown. Asterisks indicate significant difference between wild type and osh1-1 plants (P < 0.05, Student's t test). B, Accumulation of At5g01580 mRNAs in various tissues of nontransgenic Col-0 plants. Total RNA was extracted from rosette leaves (Lf) and roots (Rt) of 2-week-old plants (2w), rosette leaves, stems (St), flowers (Fl), and siliques (Sq) of 5-week-old plants (5w). Accumulation of the mRNAs relative to that of ubiquitin5 was determined as in A, and values were normalized with the means of 2-week-old rosette leaves. Means and sd of three plants are shown.

Article Snippet: Recombinant At5g01580 protein or E. coli thioredoxin (Wako Pure Chemicals, Osaka) or egg white lysozyme (Wako Pure Chemicals) was preactivated with dithiothreitol (final concentration, 25 μ m ) for 10 min at 37°C, and added to the reaction mixture (20 μ L) at 300 n m . Each reaction contained 4 ng of cy3-conjugated F(ab′) 2 .

Techniques: Real-time Polymerase Chain Reaction, Amplification

Journal:

Article Title: Isolation and Characterization of an Arabidopsis Mutant That Overaccumulates O -Acetyl- l -Ser 1 [w]

doi: 10.1104/pp.104.047068

Figure Lengend Snippet: Relative mRNA accumulations of genes within the mapped region in shoots of osh1-1

Article Snippet: Recombinant At5g01580 protein or E. coli thioredoxin (Wako Pure Chemicals, Osaka) or egg white lysozyme (Wako Pure Chemicals) was preactivated with dithiothreitol (final concentration, 25 μ m ) for 10 min at 37°C, and added to the reaction mixture (20 μ L) at 300 n m . Each reaction contained 4 ng of cy3-conjugated F(ab′) 2 .

Techniques:

Journal:

Article Title: Isolation and Characterization of an Arabidopsis Mutant That Overaccumulates O -Acetyl- l -Ser 1 [w]

doi: 10.1104/pp.104.047068

Figure Lengend Snippet: Relative mRNA accumulations of At5g01580, APR1 and Sultr2;2 in a line of transgenic Arabidopsis carrying 35S::At5g01580

Article Snippet: Recombinant At5g01580 protein or E. coli thioredoxin (Wako Pure Chemicals, Osaka) or egg white lysozyme (Wako Pure Chemicals) was preactivated with dithiothreitol (final concentration, 25 μ m ) for 10 min at 37°C, and added to the reaction mixture (20 μ L) at 300 n m . Each reaction contained 4 ng of cy3-conjugated F(ab′) 2 .

Techniques: Transgenic Assay

Journal:

Article Title: Isolation and Characterization of an Arabidopsis Mutant That Overaccumulates O -Acetyl- l -Ser 1 [w]

doi: 10.1104/pp.104.047068

Figure Lengend Snippet: Sequence comparison between At5g01580 and related proteins in database. A, Phylogenetic relationships. Translated amino acid sequence of At5g01580 was compared using the BLASTP search program. Protein sequences from animals showing E-values under e−16 and translated sequences from plants with full-length coding sequences showing E-values under e−35 were compared using the CLASTALW program. GenBank accession number is preceded by a species identifier. At, Arabidopsis; Os, O. sativa; Rn, R. norvegicus; Mm, M. musculus; Hs, H. sapiens; Ce, C. elegans; Dm, D. melanogaster. Scale bar, 0.1 substitutions/site. B, Alignment of translated sequence of At5g01580 and protein sequence of human GILT, HsP13284. The signal peptide and propeptide of HsP13284 (Arunachalam et al., 2000) are italicized and underlined, respectively. The predicted signal peptide of At5g01580 by the iPSORT program was also italicized. The active site (CXXC) of HsP13284 (Arunachalam et al., 2000) is enclosed by a rectangle.

Article Snippet: Recombinant At5g01580 protein or E. coli thioredoxin (Wako Pure Chemicals, Osaka) or egg white lysozyme (Wako Pure Chemicals) was preactivated with dithiothreitol (final concentration, 25 μ m ) for 10 min at 37°C, and added to the reaction mixture (20 μ L) at 300 n m . Each reaction contained 4 ng of cy3-conjugated F(ab′) 2 .

Techniques: Sequencing

Journal:

Article Title: Isolation and Characterization of an Arabidopsis Mutant That Overaccumulates O -Acetyl- l -Ser 1 [w]

doi: 10.1104/pp.104.047068

Figure Lengend Snippet: In vitro thiol reductase assay. A, Lysozyme (negative control), recombinant At5g01580, and thioredoxon were incubated with F(ab′)2 conjugated with cy3 under different pH conditions. Samples were separated by nonreducing SDS-PAGE and fluorescence from cy3 was scanned. Positions of F(ab′)2, Fab′, and H′+L chains are marked. B, The signal intensities of F(ab′)2, Fab′, and H′+L chains were quantified and the intensities of F(ab′)2 and H′+L chains relative to that of total were plotted against pH.

Article Snippet: Recombinant At5g01580 protein or E. coli thioredoxin (Wako Pure Chemicals, Osaka) or egg white lysozyme (Wako Pure Chemicals) was preactivated with dithiothreitol (final concentration, 25 μ m ) for 10 min at 37°C, and added to the reaction mixture (20 μ L) at 300 n m . Each reaction contained 4 ng of cy3-conjugated F(ab′) 2 .

Techniques: In Vitro, Reductase Assay, Negative Control, Recombinant, Incubation, SDS Page, Fluorescence

Journal:

Article Title: Isolation and Characterization of an Arabidopsis Mutant That Overaccumulates O -Acetyl- l -Ser 1 [w]

doi: 10.1104/pp.104.047068

Figure Lengend Snippet: Confocal laser scanning micrographs of Arabidopsis epidermal cells (A) or tobacco Bright Yellow-2 cells (B) expressing GFP or At5g01580 tagged with GFP. Projections of 10 × 1 μm confocal optical sections through the cell are shown. Bars = 20 μm.

Article Snippet: Recombinant At5g01580 protein or E. coli thioredoxin (Wako Pure Chemicals, Osaka) or egg white lysozyme (Wako Pure Chemicals) was preactivated with dithiothreitol (final concentration, 25 μ m ) for 10 min at 37°C, and added to the reaction mixture (20 μ L) at 300 n m . Each reaction contained 4 ng of cy3-conjugated F(ab′) 2 .

Techniques: Expressing

Journal:

Article Title: Lysostaphin Disrupts Staphylococcus aureus and Staphylococcus epidermidis Biofilms on Artificial Surfaces

doi: 10.1128/AAC.47.11.3407-3414.2003

Figure Lengend Snippet: Strains of staphylococci used in these studies

Article Snippet: Recombinant homogenous lysostaphin was purchased from Nutrition 21, Inc. (Purchase, N.Y.), or produced by Biosynexus Incorporated.

Techniques: Knock-Out, Variant Assay, Isolation, In Vitro

Journal:

Article Title: Lysostaphin Disrupts Staphylococcus aureus and Staphylococcus epidermidis Biofilms on Artificial Surfaces

doi: 10.1128/AAC.47.11.3407-3414.2003

Figure Lengend Snippet: Oxacillin or vancomycin had no visible effect on S. aureus biofilms in PBS or bacterial media after incubation for 24 h. Polystyrene 96-well tissue culture wells were inoculated with, per ml, ∼108 CFU of S. aureus ATCC 35556 (MIC of lysostaphin, 0.004 μg/ml; MIC of oxacillin, 0.125 μg/ml; and MIC of vancomycin, 1 μg/ml). Following 24 h of biofilm formation, wells were washed twice with PBS and then treated for 24 h in either PBS (A) (this panel shows one of the plates from the experiment reported in Fig. ​Fig.4)4) or TSB plus 0.25% glucose bacterial medium (B) with either no added antibiotic (first column of wells in each plate) or serial twofold dilutions of lysostaphin (first two rows; 0.8 to 200 μg/ml), oxacillin (third and fourth rows; 1.6 μg/ml to 400 μg/ml), or vancomycin (bottom two rows; 3.2 to 800 μg/ml) as indicated on the figure. Following treatment, the wells were washed with PBS and then stained with safranin. The darkly staining wells indicate the presence of biofilm following treatment. Lysostaphin in PBS cleared the biofilm at 0.8 μg/ml (A), while lysostaphin in TSB plus 0.25% glucose cleared the biofilm at 12.5 μg/ml (B).

Article Snippet: Recombinant homogenous lysostaphin was purchased from Nutrition 21, Inc. (Purchase, N.Y.), or produced by Biosynexus Incorporated.

Techniques: Incubation, Staining

Journal:

Article Title: Lysostaphin Disrupts Staphylococcus aureus and Staphylococcus epidermidis Biofilms on Artificial Surfaces

doi: 10.1128/AAC.47.11.3407-3414.2003

Figure Lengend Snippet: Lysostaphin caused an immediate and continuous drop in the absorbance of S. aureus biofilms, which continued over time. Polystyrene 96-well tissue culture wells were inoculated, per ml, with ∼108 CFU of S. aureus ATCC 35556 (MIC of lysostaphin, 0.004 μg/ml; MIC of oxacillin, 0.125 μg/ml; and MIC of vancomycin, 1 μg/ml). Following 24 h of biofilm formation, wells were washed twice with PBS and then treated with either lysostaphin (6.25 μg/ml), oxacillin (400 μg/ml), or vancomycin (800 μg/ml) in PBS. The absorbance at 650 nm following treatment was monitored by a microtiter plate reader every 20 min for 3 h and then again at 24 h. The data are the means of results for six samples from three separate experiments ± the standard deviations.

Article Snippet: Recombinant homogenous lysostaphin was purchased from Nutrition 21, Inc. (Purchase, N.Y.), or produced by Biosynexus Incorporated.

Techniques:

Journal:

Article Title: Lysostaphin Disrupts Staphylococcus aureus and Staphylococcus epidermidis Biofilms on Artificial Surfaces

doi: 10.1128/AAC.47.11.3407-3414.2003

Figure Lengend Snippet: Lysostaphin-disrupted biofilms of MRSA strains Col and MBT 5040. Ninety-six-well polystyrene tissue culture wells (four for each sample) were inoculated with ∼108 CFU of S. aureus strain Col or MBT 5040 (as indicated). After we allowed 48 h for biofilm formation, the wells were washed twice and then incubated with (+) or without (−) 50 μg of lysostaphin/ml in PBS for 3 h. Following incubation, the wells were washed again and stained with safranin to visualize biofilms.

Article Snippet: Recombinant homogenous lysostaphin was purchased from Nutrition 21, Inc. (Purchase, N.Y.), or produced by Biosynexus Incorporated.

Techniques: Incubation, Staining

Journal:

Article Title: Lysostaphin Disrupts Staphylococcus aureus and Staphylococcus epidermidis Biofilms on Artificial Surfaces

doi: 10.1128/AAC.47.11.3407-3414.2003

Figure Lengend Snippet: Lysostaphin did not disrupt biofilms formed by lysostaphin-resistant S. aureus variants. Tissue culture wells (three for each sample) of a microtiter plate were inoculated with ∼108 CFU of S. aureus strain SA113, SA5 LysoR, MBT 5040, or MBT 5040 LysoR (as indicated). After we allowed 48 h for biofilm formation, the wells were washed twice and then incubated with (+) or without (−) 50 μg of lysostaphin/ml in PBS for 3 h. Following incubation, the wells were washed again and stained with safranin to visualize biofilms.

Article Snippet: Recombinant homogenous lysostaphin was purchased from Nutrition 21, Inc. (Purchase, N.Y.), or produced by Biosynexus Incorporated.

Techniques: Incubation, Staining

Journal:

Article Title: Lysostaphin Disrupts Staphylococcus aureus and Staphylococcus epidermidis Biofilms on Artificial Surfaces

doi: 10.1128/AAC.47.11.3407-3414.2003

Figure Lengend Snippet: Lysostaphin-disrupted S. epidermidis biofilms. Glass chamber slide wells were inoculated with ∼5 × 107 CFU of either S. aureus strain SA113 as a control (A), S. epidermidis strain Hay (B), S. epidermidis strain ATCC 35984 (C), or S. epidermidis strain SE1175 (D) per ml. Biofilms were allowed to form for 24 h, and then wells were washed twice with PBS. Established biofilms were treated with PBS alone or with lysostaphin in PBS (200 μg/ml) for 3 h. Following treatment, the wells were washed with PBS followed by ddH2O and then Gram stained. The biofilms in the PBS-treated row (−) stained gram positive, while no gram-positive cells were seen in lysostaphin-treated (+) S. epidermidis wells. Only the residual extracellular glycocalyx in the corners of the wells stained gram negative in the lysostaphin-treated wells. The two enlarged sections reveal the multilayered biofilm of S. epidermidis strain ATCC 35984 (top) and the residual glycocalyx of the same strain with no intact staphylococci following lysostaphin treatment (bottom).

Article Snippet: Recombinant homogenous lysostaphin was purchased from Nutrition 21, Inc. (Purchase, N.Y.), or produced by Biosynexus Incorporated.

Techniques: Staining

Journal:

Article Title: Lysostaphin Disrupts Staphylococcus aureus and Staphylococcus epidermidis Biofilms on Artificial Surfaces

doi: 10.1128/AAC.47.11.3407-3414.2003

Figure Lengend Snippet: SEM revealed that lysostaphin eradicates S. aureus biofilms, removing both the sessile cells and the extracellular matrix. Polycarbonate transwells were inoculated with 5 × 108 CFU of S. aureus ATCC 49521 per ml. Following 48 h of biofilm formation, transwells were washed and then treated with either PBS (A and B) or 100 μg of lysostaphin/ml in PBS (C and D) for 3 h. The treated wells were washed again and then fixed with gluteraldehyde prior to SEM. The results shown are representative of the entire well. Magnifications, ×1,800 (A and C) and ×5,940 (B and D).

Article Snippet: Recombinant homogenous lysostaphin was purchased from Nutrition 21, Inc. (Purchase, N.Y.), or produced by Biosynexus Incorporated.

Techniques: