egta  (Dojindo Labs)

Journal: Scientific Reports

Article Title: ATP Exhibits Antimicrobial Action by Inhibiting Bacterial Utilization of Ferric Ions

doi: 10.1038/srep08610

Figure Lengend Snippet: ATP's antimicrobial effect is attributable to its iron-chelating activity. (a) Blocking effects of MgCl 2 and FeCl 3 on ATP's anti- M. intracellulare antimicrobial activity. (b) Anti- M. intracellulare activities of ATP and various metal-chelating agents (pyrophosphate (PPi), EDTA, EGTA) and blocking effects of MgCl 2 . (c) Anti- S. aureus activities of ATP and various metal-chelating agents. (d) Ferric ion-chelating activities of ATP and EDTA measured by CAS assay. (e) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against M. intracellulare . (f) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against P. aeruginosa . (g) Siderophore production by ATP-resistant E. coli during the course of cultivation. (h) Siderophore production by two ATP-resistant K. pneumoniae strains during 48-h cultivation in the presence of ATP. (i) Siderophore production by ATP-susceptible S. aureus and ATP-resistant E. coli during 24-h cultivation in the presence of ATP (5 mM). (j) Siderophore production by ATP-susceptible and ATP-resistant strains of P. aeruginosa during 24-h cultivation.

Article Snippet: Special agents The following agents were used: ATP (Sigma Aldrich Co., St. Louis, MO; MP Biomedicals, Solon, OH, USA, Calbiochem Co., La Jolla, CA, and Roche Diagnostic Co., Indianapolis, IN), ADP (Sigma), AMP (Sigma), adenosine (Sigma), benzoylbenzoyl ATP (Sigma), oxidized ATP (Sigma), suramin (Wako, Tokyo, Japan), MIA (methyl isobutyl amiloride, Wako), DIDS (4,4′- Diisothiocyanatostilbene-2,2′-disulfonic acid, Sigma), EDTA (Dojindo, Tokyo, Japan), EGTA (Dojindo), dipyridyl (Sigma), CAS (Chrome Azurol S, Sigma), E. coli S17-1 and pK18mobSacB (kindly provided by Dr. F. Taguchi, Okayama University), Instagene matrix (Bio-Rad, Hercules, CA), KOD-Plus (Toyobo, Osaka, Japan), Wizard SV Gel and PCR cleanup system (Promeg, Madison, WI), Ligation High (Toyobo), Protein Assay Rapid Kit (Wako), [14 C] isoleucine (Moravek Biochemicals, Inc, Brea, CA), [14 C]uracil (Moravek Biochemicals, Inc.), α-defensin-1 (Peptide institute, Inc, Osaka, Japan.), cathepsin G (Sigma), vancomycin (Wako), clarithromycin (Taisho-Toyama Pharmaceutical Co., Tokyo), rifampin (Daiichi Sankyo Co., Tokyo), and ethambutol (Sigma), gatifloxacin (Wako), FLUOS (Sigma), LB medium (Invitrogen, San Diego, CA), M9 medium (prepared by our laboratory), Heart infusion agar (Eiken Chemical Co., Tokyo, Japan), Middlebrook 7H9 medium (Becton Dickinson, Cockeysville, MD), and Middlebrook 7H11 medium (Becton Dickinson).

Techniques: Activity Assay, Blocking Assay

Journal: Biology Open

Article Title: Microtubule stabilizer reveals requirement of Ca2+-dependent conformational changes of microtubules for rapid coiling of haptonema in haptophyte algae

doi: 10.1242/bio.036590

Figure Lengend Snippet: Effects of Ca 2+ on taxol-induced haptonematal bending. (A) A phase contrast image of a haptophyte treated with taxol (20 μM) in the absence of Ca 2+ (CFSW+EGTA+BAPTA-AM) showing a helical configuration of the haptonema (left). The plot to the right shows the curvature along the haptonema (see Fig. S5 ). The curvature of the haptonema was calculated from the microscopic image, which is a two-dimensionally projected image. The curvature of a helical structure calculated in this way shows oscillation with a repeat distance equivalent to that of the helix, and a shift in the pattern represents propagation of the wave form. Scale bar: 10 µm. (B) Ca 2+ -dependent propagation of a taxol-induced haptonematal helix. The helix is also converted to planar bending with the maximum curvature of bend propagating to the proximal region. Trace images of haptophytes are shown as insets.

Article Snippet: To examine the effects of Ca2+ , 100 µl of concentrated cells were mixed with 900 µl of Ca2+ -free ASW (462.01 mM NaCl, 9.39 mM KCl, 59.08 mM MgCl2 , 10 mM HEPES, pH 8.0), 10 mM EGTA in Ca2+ -free ASW, or 10 µM EGTA and 50 µM BAPTA-AM (Dojindo, 50 mM stock solution in DMSO) in Ca2+ -free ASW.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Specific Activation of the G Protein-coupled Receptor BNGR-A21 by the Neuropeptide Corazonin from the Silkworm, Bombyx mori, Dually Couples to the Gq and Gs Signaling Cascades *

doi: 10.1074/jbc.M112.441675

Figure Lengend Snippet: Intracellular Ca 2+ influx. HEK293 ( A ) and Sf21 ( B ) cells, respectively, transfected with FLAG-A21 and BmFlag-A21 were measured in response to different concentrations of corazonin peptide using the fluorescent Ca 2+ indicator fura-2. C , effect of pretreatment of the G q inhibitor on corazonin (1 μ m )-mediated Ca 2+ influx in HEK293 cells. D , effects of pretreatment of calcium chelators (EGTA, 5 m m , and BAPTA-AM, 50 μ m ) and l -calcium channel (nefidipine, 10 μ m ) inhibitors on corazonin-mediated Ca 2+ influx in HEK293 cells. Effect of pretreatment of PLC inhibitor (U73122, 10 μ m ) on corazonin-mediated Ca 2+ influx in HEK293 ( E ) and Sf21 ( F ) cells. The figures are representative of more than three independent experiments.

Article Snippet: The cells were detached by a Nonenzymatic Cell Dissociation Solution (M & C Gene Technology, China) or 0.02% EGTA and then loaded with 3 μm Fura 2-AM (Dojindo Laboratories, Japan) for 30 min at 37 °C.

Techniques: Transfection, Planar Chromatography