Journal: Frontiers in Immunology

Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)

doi: 10.3389/fimmu.2019.00012

Figure Lengend Snippet: Extracellular NETotic DNA is rich in MPO. Representative fluorescence images of NETs induced by CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) activated in supplement-free RPMI/HEPES. The images show a clear colocalization of MPO (red) with the extracellular DNA-fibers (blue) of released NETs. Scale = 10 μm.

Article Snippet: Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively.

Techniques: Fluorescence

Journal: Frontiers in Immunology

Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)

doi: 10.3389/fimmu.2019.00012

Figure Lengend Snippet: Influence of serum and serum albumin supplements on NET formation of murine neutrophils. (A) Representative fluorescence images of nuclei of murine neutrophils (Hoechst) after stimulation with CaI (4 μM), PMA (100 nM) or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI/ HEPES without supplements and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS, as indicated. Chromatin decondensation is only inducible in RPMI/ HEPES (white arrow heads). Scale = 50 μm. (B) Percentage of decondensed nuclei/ NETs after stimulation with PMA (100 nM), CaI (4 μM), or LPS (10, 25, or 100 μg/ml) for 180 min. Murine neutrophils were studied in RPMI/ HEPES and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS. Addition of 0.5% BSA or 2% hiFCS to RPMI/ HEPES, inhibits chromatin decondensation induced by PMA, CaI and LPS completely. Error bars = mean ± SEM. ns = not significant. **** p

Article Snippet: Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively.

Techniques: Fluorescence

Journal: Frontiers in Immunology

Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)

doi: 10.3389/fimmu.2019.00012

Figure Lengend Snippet: Influence of serum and serum albumin supplements on NET formation of human neutrophils. (A) Representative fluorescence images of human neutrophils (chromatin stained by Hoechst) after stimulation with CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI 1,640 with 10 mM HEPES (RPMI/HEPES), RPMI/ HEPES + 0.5% human serum albumin (HSA) or + 1% heat inactivated (56°C) fetal calf serum (hiFCS). Chromatin decondensation induced by PMA is clearly visible with all three culture conditions, while LPS or CaI only cause NET formation in BSA- and HSA-free RPMI/HEPES. Scale = 50 μm. (B) Neutrophils were stimulated to undergo NET formation with CaI (4 μM), PMA (100 nM) or LPS (10, 25, or 100 μg/ml), respectively. Both, HSA and BSA inhibit CaI and LPS-induced formation of NETs (determined as percentage of decondensed nuclei/NETs of total neutrophils). NETosis stimulated by PMA is independent of serum albumin addition. Error bars = mean ± SEM. ns, not significant. * p

Article Snippet: Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively.

Techniques: Fluorescence, Staining, Albumin Addition

Journal: Biochemistry

Article Title: The Interface between Escherichia coli Elongation Factor Tu and Aminoacyl-tRNA

doi: 10.1021/bi500533x

Figure Lengend Snippet: Comparison of yeast Phe-tRNA Phe binding to mutations made in E. coli EF-Tu (Table 1 ) with identical mutations made at orthologous sites in T. thermophilus EF-Tu. 49 E. coli numbering is used. Experiments performed in 50 mM HEPES pH 7, 30 mM KCl, 70 mM NH 4 Cl, 10 mM MgCl 2 , 10 μM GTP, 3 mM phosphoenolpyruvate, 50 μg/mL of pyruvate kinase, and 1 mM DTT at 0 °C for E. coli EF-Tu. ΔΔ G o values for each mutation were calculated relative to those of corresponding wild-type proteins (Δ G o = 10.1 ± 0.3 for E. coli and 9.8 ± 0.2 for T. thermophilus ). Since the complex was not stable enough, ΔΔ G o value displays the lower limit for T. thermophilus E259A, and the actual value is larger than 1.8. 49

Article Snippet: GTP Hydrolysis Rate Measurement Ternary complex was formed by mixing 0.4 μM EF-Tu with 1.2 μM Tyr-tRNATyr or 1.2 μM Tyr-T1 Tyr-tRNATyr , and 50 μM γ-32 P-GTP in 50 mM HEPES pH 7, 30 mM KCl, 70 mM NH4 Cl, 10 mM MgCl2 , 10 μM GTP, 3 mM phosphoenolpyruvate, 50 μg/mL of pyruvate kinase, and 1 mM DTT according to published protocols., , , Heat activated ternary complex solution was passed through two Bio-Spin 6 columns (BIO-RAD) to remove excess [γ-32 P]-GTP.

Techniques: Binding Assay, Mutagenesis

Journal: Biochemistry

Article Title: The Interface between Escherichia coli Elongation Factor Tu and Aminoacyl-tRNA

doi: 10.1021/bi500533x

Figure Lengend Snippet: Time courses of ribosome-catalyzed GTP hydrolysis of ternary complexes containing Tyr-tRNA Tyr and the indicated EF-Tu mutations. Experiments were performed in 50 mM HEPES pH 7, 30 mM KCl, 70 mM NH 4 Cl, 10 mM MgCl 2 , 10 μM GTP, 3 mM phosphoenolpyruvate, 50 μg/mL of pyruvate kinase, and 1 mM DTT at 20 °C. Lines for each mutant indicate the best fit to a single binding equilibrium adjusted to an extent and a k obs. Values for all mutants are given in Table 2

Article Snippet: GTP Hydrolysis Rate Measurement Ternary complex was formed by mixing 0.4 μM EF-Tu with 1.2 μM Tyr-tRNATyr or 1.2 μM Tyr-T1 Tyr-tRNATyr , and 50 μM γ-32 P-GTP in 50 mM HEPES pH 7, 30 mM KCl, 70 mM NH4 Cl, 10 mM MgCl2 , 10 μM GTP, 3 mM phosphoenolpyruvate, 50 μg/mL of pyruvate kinase, and 1 mM DTT according to published protocols., , , Heat activated ternary complex solution was passed through two Bio-Spin 6 columns (BIO-RAD) to remove excess [γ-32 P]-GTP.

Techniques: Mutagenesis, Binding Assay