PMC:1472690 / 32969-40917 JSON TXT

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pmc-enju-pas

bionlp-st-ge-2016-test-proteins

bionlp-st-ge-2016-uniprot

UBERON-AE

{"project":"UBERON-AE","denotations":[{"id":"T17617","span":{"begin":1008,"end":1013},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T17616","span":{"begin":484,"end":489},"obj":"http://purl.obolibrary.org/obo/UBERON_0000178"}],"text":"Methods\n\nMaterials\nWater soluble phosphorylated β-1→3-D-glucan (GP) was prepared as done before [20]. The physicochemical characteristics of GP were determined as reported previously [20,26]. GP was dissolved in aqueous media, filter-sterilized (0.2 μm) and screened for endotoxin contamination with the Endospecy assay (Seigakaku, Tokyo, Japan), which is specific for endotoxin but does not respond to β-1→3-D-glucans.\n\nIsolation of human PBMC\nBuffy coats were obtained from healthy blood donors in compliance with the Helsinki declaration and with the approval of the ethics committee of the University of Luebeck. The buffy coats were separated over a Ficoll-Hypaque (Biochrom, Berlin, Germany) gradient [54,55]. Following isolation, PBMC were cultured at a final concentration of 3 × 106 cells/ml (for ELISA measurements) or 5 × 106/ml (for gel shifts) or 1 × 106 cells/ml (for flow cytometry) in RPMI 1640 medium (BioWhittaker, Heidelberg, Germany, LPS-free), containing 10% heat-inactivated fetal calf serum (FCS, low-LPS, Invitrogen, Karlsruhe, Germany), 1% penicillin (10000 U/ml), 1% streptomycin (10000 μg/ml), and 1% 200 mM L-glutamine (BioWhittaker).\n\nStimulation conditions\nFor induction of transcription factors and cytokines, PBMC were cultured in a volume of 4 ml in sterile pyrogen-free 6-well culture plates (Falcon, Heidelberg, Germany). Cultures were stimulated for 1 h (EMSA) or from 0 h to 48 h (ELISA) with 1 or 100 μg per 106 cells of GP, with 250 ng/106 cells TSST-1 (Toxin Technologies, Sarasota, FL, USA) or with 250 ng/106 cells wild-type LPS from Escherichia coli serotype 0111:B4 (Sigma, Munich, Germany). In costimulatory experiments, PBMC were supplemented simultaneously with a combination of GP and TSST-1 or with a combination of GP and LPS.\n\nOligonucleotides (Oligos) and 5'-32-P-Labeling\nThe complementary double-stranded (ds) oligonucleotides (oligos) from the TNFα, IL-8, IFNγ and IL-6 promoters were synthesised from single stranded (ss) oligos (illustrated in Table 1), (TIB Molbiol, Berlin, Germany) and 32P-labeled with 5'γ-P-ATP (3,000 Ci/mmol, Amersham, Braunschweig, Germany) using the Ready-To-Go-Polynucleotide-Kinase Kit (PNK Kit, Pharmacia LKB, Freiburg, Germany) according to the manufacturer's instructions. To test the binding specificity of the oligos, mutated oligos were used as an additional control. Mutations in the P2 site of the IFNγ promoter [29] and in the κ consensus sequence of the TNFα promoter [55] have been reported to interfere with sequences, which seem to be crucial for the binding of NFAT and NFκB proteins, respectively. Afterwards, the ds oligos were purified via gel filtration using Probe Quant G-50 Micro Columns (Pharmacia) according to the manufacturer's instructions.\n\nPreparation of nuclear extracts and electrophoretic mobility shift assays (EMSA)\nNuclear extracts of purified PBMCs were prepared according to a technique described by Trede et al. [27] and modified for our experimental setup [56]. The intensity of shifted bands was normalized to bands of unstimulated controls. Competition experiments with labeled and unlabeled mutated oligos of the cytokine promoter binding sites (see above) were carried out in order to prevent non-specific binding to nuclear proteins. For such experiments, an excess (5–50×) of unlabeled oligos was added to the nuclear extracts. For specificity of binding, supershift assays for NFκB with a combined p65/50 antibody were performed (data not shown). To exclude non-specific reactions, a 30-fold molar excess of Oct-1 DNA (unrelated DNA, Table 1) was used, which did not compete with specific binding.\n\nImmuno (Dot) blot\nTo determine whether the DNA binding proteins were related to NFκB and NFAT, dot blots using a SRC96D SNS minifold I dot blotter (Schleicher \u0026 Schüll, Dassel, Germany) were performed with the following positive controls and antibodies (all from Santa Cruz Biotechnology, Heidelberg, Germany): positive controls (10 ng/dot): NFκB p52 (80 kD) sc-4095WB, c-Rel (61 kD) sc-4030WB; Jurkat nuclear extract, PMA-stimulated; primary antibodies (1 μg/dot): anti-NFκB p50 (NLS) sc-114, anti-NFκB p65 (A) sc-109, anti-NFκB p52 (447) sc-848, a rabbit polyclonal IgG1 antiserum, anti-c-rel (N466) sc-272, anti-NFATc2 (M-20) sc-1151, anti-NFATc1 (K-18) sc-1149, two goat polyclonal IgG1 antisera; secondary antibodies (0.8 μg/dot): goat anti-rabbit IgG AP-conjugate sc-2007, and donkey anti-goat IgG AP-conjugate sc-2022. The nuclear proteins were blotted onto a nitrocellulose membrane (Bio-Rad). After blocking with PBS/3% BSA (Fluka, Deisenhofen, Germany), the blot was incubated overnight with the primary antibodies diluted 1:2000 in PBS/1% BSA, washed again and incubated with the alkaline phosphatase-conjugated secondary antibody (1:500 in PBS/1% BSA). The blot was developed using the Vectastain® staining kit (Vector Laboratories Inc. Burlingame, CA, USA).\n\nCytokine measurements\nSupernatants of cell cultures were harvested 3, 6, 9, 12, 18, 24, 30, 36 and 48 hours after stimulation and stored at -80 °C until used and thawed only once. IFNγ, TNFα, IL-1 and IL-6 were determined using an ELISA from Bender Systems (Vienna, Austria), IL-1RA and IL-8 by an ELISA from R\u0026D Systems (Wiesbaden-Nordenstadt, Germany), and TGFβ 1 using an ELISA as previously described [57]. Quantification of IL-2 as an indicator for the bioactivity of IL-1 was done as previously described [46]. Samples were diluted in the same buffer as that used for the standards. All cytokines were quantified using an ELISA plate reader (Anthos Labotec, Salzburg, Austria or Microplate Reader, BioRad). Cytokine amounts were all within the range of the standard curve. Only stimulation of PBMC observed in donors showing no spontaneous cytokine release was included in the statistics.\n\nInhibition of transcription factors\nTo inhibit translocation and binding of transcription factors to the IL-1RA promoter, PBMC were incubated for 1 h with each of the following substances (concentrations were based upon 50% inhibition): 50 μg/ml of CAPE (caffeic-3,4-dihydroxycinnamic-acid-phenyl-ester), (Biomol, Hamburg, Germany); 400 ng/ml cyclosporin A (Calbiochem-Merck Biosciences, Bad Soden, Germany); 10 μg/ml cycloheximide (Santa Cruz Biotechnology) prior to incubation with GP. Band shifts were determined after 1 h incubation with GP as described before, including mutated oligos for NFIL-6 (TTA CAA CAG TGG ATT GCG ACA CTT AGT GGG) and NFATP2/3 (GGC GCA GAA AAG GTA AAA TAT TTA CTA TCT) binding sites within the IL-1RA promoter. PBMC RNA was isolated with an RNeasy kit (Qiagen, Germany) and messenger RNA was transcribed into cDNA with Reactin Ready First Strand kit (Biomol) and analysed for IL-1RA and GAPDH transcripts by PCR with a HotStart \"sweet\" PCR mastermix (Biomol) (95°C, 30 sec, 55°C, 30 sec, 72°C, 30 sec, 25 cycles for IL-1RA and 94°C for 30 sec, 50°C for 30 sec and 72°C for 45 sec, 25 cycles for GAPDH) following 18 h incubation with GP. IL-1RA protein was measured in the supernatant by ELISA after 24 h incubation with GP.\n\nStatistical analysis\nThe Kolmogorow-Smirnov test was used to evaluate, whether cytokine amounts and binding activities to transcription factors were normally distributed. Correlation coefficients and corresponding significances were analyzed by the Pearson test (normal distribution) or the Spearman test (non-normal distribution). To compare experimental data to a theoretical value (for example GP + TSST-1 vs GP/TSST-1 calc.) we calculated the sum of the individual effects and added these values into Fig. 1B, 3, 4, 5, and 6. Significances of differences between experimental data (for example GP vs LPS) as well as between experimental data and theoretical values (for example GP + TSST-1 vs GP/TSST-1 calc.) were analysed using the Student's t-test (normal distribution) or the Wilcoxon Signed-Rank test (non-normal distribution), (SPSS for Windows; SPSS Science Software, Erkrath, Germany)."}

GO-BP

GO-MF

GO-CC

sentences

ICD10

{"project":"ICD10","denotations":[{"id":"T17621","span":{"begin":484,"end":496},"obj":"http://purl.bioontology.org/ontology/ICD10/Z52.0"},{"id":"T17465","span":{"begin":234,"end":244},"obj":"http://purl.bioontology.org/ontology/ICD10/Z30.2"}],"text":"Methods\n\nMaterials\nWater soluble phosphorylated β-1→3-D-glucan (GP) was prepared as done before [20]. The physicochemical characteristics of GP were determined as reported previously [20,26]. GP was dissolved in aqueous media, filter-sterilized (0.2 μm) and screened for endotoxin contamination with the Endospecy assay (Seigakaku, Tokyo, Japan), which is specific for endotoxin but does not respond to β-1→3-D-glucans.\n\nIsolation of human PBMC\nBuffy coats were obtained from healthy blood donors in compliance with the Helsinki declaration and with the approval of the ethics committee of the University of Luebeck. The buffy coats were separated over a Ficoll-Hypaque (Biochrom, Berlin, Germany) gradient [54,55]. Following isolation, PBMC were cultured at a final concentration of 3 × 106 cells/ml (for ELISA measurements) or 5 × 106/ml (for gel shifts) or 1 × 106 cells/ml (for flow cytometry) in RPMI 1640 medium (BioWhittaker, Heidelberg, Germany, LPS-free), containing 10% heat-inactivated fetal calf serum (FCS, low-LPS, Invitrogen, Karlsruhe, Germany), 1% penicillin (10000 U/ml), 1% streptomycin (10000 μg/ml), and 1% 200 mM L-glutamine (BioWhittaker).\n\nStimulation conditions\nFor induction of transcription factors and cytokines, PBMC were cultured in a volume of 4 ml in sterile pyrogen-free 6-well culture plates (Falcon, Heidelberg, Germany). Cultures were stimulated for 1 h (EMSA) or from 0 h to 48 h (ELISA) with 1 or 100 μg per 106 cells of GP, with 250 ng/106 cells TSST-1 (Toxin Technologies, Sarasota, FL, USA) or with 250 ng/106 cells wild-type LPS from Escherichia coli serotype 0111:B4 (Sigma, Munich, Germany). In costimulatory experiments, PBMC were supplemented simultaneously with a combination of GP and TSST-1 or with a combination of GP and LPS.\n\nOligonucleotides (Oligos) and 5'-32-P-Labeling\nThe complementary double-stranded (ds) oligonucleotides (oligos) from the TNFα, IL-8, IFNγ and IL-6 promoters were synthesised from single stranded (ss) oligos (illustrated in Table 1), (TIB Molbiol, Berlin, Germany) and 32P-labeled with 5'γ-P-ATP (3,000 Ci/mmol, Amersham, Braunschweig, Germany) using the Ready-To-Go-Polynucleotide-Kinase Kit (PNK Kit, Pharmacia LKB, Freiburg, Germany) according to the manufacturer's instructions. To test the binding specificity of the oligos, mutated oligos were used as an additional control. Mutations in the P2 site of the IFNγ promoter [29] and in the κ consensus sequence of the TNFα promoter [55] have been reported to interfere with sequences, which seem to be crucial for the binding of NFAT and NFκB proteins, respectively. Afterwards, the ds oligos were purified via gel filtration using Probe Quant G-50 Micro Columns (Pharmacia) according to the manufacturer's instructions.\n\nPreparation of nuclear extracts and electrophoretic mobility shift assays (EMSA)\nNuclear extracts of purified PBMCs were prepared according to a technique described by Trede et al. [27] and modified for our experimental setup [56]. The intensity of shifted bands was normalized to bands of unstimulated controls. Competition experiments with labeled and unlabeled mutated oligos of the cytokine promoter binding sites (see above) were carried out in order to prevent non-specific binding to nuclear proteins. For such experiments, an excess (5–50×) of unlabeled oligos was added to the nuclear extracts. For specificity of binding, supershift assays for NFκB with a combined p65/50 antibody were performed (data not shown). To exclude non-specific reactions, a 30-fold molar excess of Oct-1 DNA (unrelated DNA, Table 1) was used, which did not compete with specific binding.\n\nImmuno (Dot) blot\nTo determine whether the DNA binding proteins were related to NFκB and NFAT, dot blots using a SRC96D SNS minifold I dot blotter (Schleicher \u0026 Schüll, Dassel, Germany) were performed with the following positive controls and antibodies (all from Santa Cruz Biotechnology, Heidelberg, Germany): positive controls (10 ng/dot): NFκB p52 (80 kD) sc-4095WB, c-Rel (61 kD) sc-4030WB; Jurkat nuclear extract, PMA-stimulated; primary antibodies (1 μg/dot): anti-NFκB p50 (NLS) sc-114, anti-NFκB p65 (A) sc-109, anti-NFκB p52 (447) sc-848, a rabbit polyclonal IgG1 antiserum, anti-c-rel (N466) sc-272, anti-NFATc2 (M-20) sc-1151, anti-NFATc1 (K-18) sc-1149, two goat polyclonal IgG1 antisera; secondary antibodies (0.8 μg/dot): goat anti-rabbit IgG AP-conjugate sc-2007, and donkey anti-goat IgG AP-conjugate sc-2022. The nuclear proteins were blotted onto a nitrocellulose membrane (Bio-Rad). After blocking with PBS/3% BSA (Fluka, Deisenhofen, Germany), the blot was incubated overnight with the primary antibodies diluted 1:2000 in PBS/1% BSA, washed again and incubated with the alkaline phosphatase-conjugated secondary antibody (1:500 in PBS/1% BSA). The blot was developed using the Vectastain® staining kit (Vector Laboratories Inc. Burlingame, CA, USA).\n\nCytokine measurements\nSupernatants of cell cultures were harvested 3, 6, 9, 12, 18, 24, 30, 36 and 48 hours after stimulation and stored at -80 °C until used and thawed only once. IFNγ, TNFα, IL-1 and IL-6 were determined using an ELISA from Bender Systems (Vienna, Austria), IL-1RA and IL-8 by an ELISA from R\u0026D Systems (Wiesbaden-Nordenstadt, Germany), and TGFβ 1 using an ELISA as previously described [57]. Quantification of IL-2 as an indicator for the bioactivity of IL-1 was done as previously described [46]. Samples were diluted in the same buffer as that used for the standards. All cytokines were quantified using an ELISA plate reader (Anthos Labotec, Salzburg, Austria or Microplate Reader, BioRad). Cytokine amounts were all within the range of the standard curve. Only stimulation of PBMC observed in donors showing no spontaneous cytokine release was included in the statistics.\n\nInhibition of transcription factors\nTo inhibit translocation and binding of transcription factors to the IL-1RA promoter, PBMC were incubated for 1 h with each of the following substances (concentrations were based upon 50% inhibition): 50 μg/ml of CAPE (caffeic-3,4-dihydroxycinnamic-acid-phenyl-ester), (Biomol, Hamburg, Germany); 400 ng/ml cyclosporin A (Calbiochem-Merck Biosciences, Bad Soden, Germany); 10 μg/ml cycloheximide (Santa Cruz Biotechnology) prior to incubation with GP. Band shifts were determined after 1 h incubation with GP as described before, including mutated oligos for NFIL-6 (TTA CAA CAG TGG ATT GCG ACA CTT AGT GGG) and NFATP2/3 (GGC GCA GAA AAG GTA AAA TAT TTA CTA TCT) binding sites within the IL-1RA promoter. PBMC RNA was isolated with an RNeasy kit (Qiagen, Germany) and messenger RNA was transcribed into cDNA with Reactin Ready First Strand kit (Biomol) and analysed for IL-1RA and GAPDH transcripts by PCR with a HotStart \"sweet\" PCR mastermix (Biomol) (95°C, 30 sec, 55°C, 30 sec, 72°C, 30 sec, 25 cycles for IL-1RA and 94°C for 30 sec, 50°C for 30 sec and 72°C for 45 sec, 25 cycles for GAPDH) following 18 h incubation with GP. IL-1RA protein was measured in the supernatant by ELISA after 24 h incubation with GP.\n\nStatistical analysis\nThe Kolmogorow-Smirnov test was used to evaluate, whether cytokine amounts and binding activities to transcription factors were normally distributed. Correlation coefficients and corresponding significances were analyzed by the Pearson test (normal distribution) or the Spearman test (non-normal distribution). To compare experimental data to a theoretical value (for example GP + TSST-1 vs GP/TSST-1 calc.) we calculated the sum of the individual effects and added these values into Fig. 1B, 3, 4, 5, and 6. Significances of differences between experimental data (for example GP vs LPS) as well as between experimental data and theoretical values (for example GP + TSST-1 vs GP/TSST-1 calc.) were analysed using the Student's t-test (normal distribution) or the Wilcoxon Signed-Rank test (non-normal distribution), (SPSS for Windows; SPSS Science Software, Erkrath, Germany)."}

simple1

BioNLP16_DUT

BioNLP16_Messiy

DLUT931

bionlp-st-ge-2016-test-ihmc

bionlp-st-ge-2016-spacy-parsed

bionlp-st-ge-2016-test-tees

testone

test3

PMC:1472690 / 32969-40917 JSONTXT

Annnotations TAB JSON ListView MergeView

PMC:1472690 / 32969-40917 JSON TXT