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The isolation of valinomycin was carried out by semi-preparative HPLC (Phenomenex Luna SemiPrep RP18e 10 x 250 mm) using H2O (A) and CH3OH (B) as the solvents and the following gradient: flow 4.5 mL/min; 0-5 min 90% B, 11-15 min 100% B yielding 8.4 mg of the compound (Rt = 13.085 min).
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The isolation of valinomycin was carried out by semi-preparative HPLC (Phenomenex Luna SemiPrep RP18e 10 x 250 mm) using H2O (A) and CH3OH (B) as the solvents and the following gradient: flow 4.5 mL/min; 0-5 min 90% B, 11-15 min 100% B yielding 8.4 mg of the compound (Rt = 13.085 min).
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84856100665
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Leishmania major promastigotes were seeded at a cell density of 1 x 107 cells/mL into 96-well plates in complete medium (RPMI with NaHCO3, 10% FCS, 2 mM glutamine, 10 mM Hepes pH 7.2, 100 U/mL penicillin, 50 μg/mL gentamicin, 50 mM 2-mercaptoethanol) without phenol red (200 mL, in the absence or presence of different concentrations of the compounds. These were then incubated for 24 h at 26 ° C, 5% CO2 and 95% humidity. Following the addition of 20 mL of Alamar Blue, the plates were incubated again and the optical densities (ODs) measured 24 h and 48 h later with an enzyme-linked immunosorbent assay (ELISA) reader (Multiskan Ascent, Germany) using a test wavelength of 540 nm and a reference wavelength of 630 nm. Absorbance in the absence of compounds was set as 100% of growth. Amphotericin B was used as a reference compound and positive control. The effects of cell density, incubation time and the concentration of DMSO were examined in con
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2 and 95% humidity. Following the addition of 20 mL of Alamar Blue, the plates were incubated again and the optical densities (ODs) measured 24 h and 48 h later with an enzyme-linked immunosorbent assay (ELISA) reader (Multiskan Ascent, Germany) using a test wavelength of 540 nm and a reference wavelength of 630 nm. Absorbance in the absence of compounds was set as 100% of growth. Amphotericin B was used as a reference compound and positive control. The effects of cell density, incubation time and the concentration of DMSO were examined in control experiments. The final concentration of DMSO in the medium never exceeded 1% vol/vol and had no effect on the proliferation of extracellular or intracellular parasites. For each experiment, each drug concentration was assayed in duplicate wells [31].
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22
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84856100666
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Trypomastigote forms of Trypanosoma brucei brucei laboratory strain TC 221 were cultured in complete Baltz medium [80 mL Baltz medium basic solution, 0.8 mL 2 mercaptoethanol stock solution (20 mM, 0.8 mL penicillin/streptomycin (10,000 U/mL, 16 mL FCS (inactivated for 30 min at 56 ° C, Baltz medium basic solution is composed of the following: 500 mL MEM with Earle's salts and L-glutamine, 3 g Hepes, 0.5 g monohydrate glucose, 0.110 g sodium pyruvate, 0.007 g hypoxanthine, 0.002 g thymidine, 0.0107 g adenosine, 0.0141 g bathocuproine disulfonic acid disodium salt, 0.146 g glutamine, 5 mL sterile non-essential amino acid concentrate (100x, pH 7.5, A defined number of parasites (104 trypanosomes per mL) were exposed in test chambers of 96-well plates to various concentrations of the test substances (previously dissolved in DMSO) to make a final of 200 μL in duplicates. Positive (trypanosomes in culture medium) and negative controls test substance without trypano
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2 for a total time period of 72 h. After 24 h, 20 μL of Alamar Blue was added. The activity of the test substances was measured by light absorption using MR 700 Microplate Reader at a wavelength of 550 nm with a reference wavelength of 630 nm. The first reading was done at 48 h and subsequently at 72 h. The effect of the test substances was quantified in IC50 values by linear interpolation of three independent measurements [32].
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Production of valinomycin, an insecticidal antibiotic, by Streptomyces griseus var. flexipartum var. nov
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Heisey, R.; Huang, J.; Mishka, S.K.; Keller, J.E.; Miller, J.R.; Putnam, A.R.; D'Silva, T.D. Production of valinomycin, an insecticidal antibiotic, by Streptomyces griseus var. flexipartum var. nov. J. Agric. Food Chem. 1988, 36, 1283-1286.
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D'Silva, T.D.7
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24
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77649198100
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The isolation of staurosporine was carried out by semi-preparative HPLC (Phenomenex Luna SemiPrep RP18e 10 x 250 mm) using H2O + 0.1% TFA (A) and CH3OH (B) as the solvents and the following gradient: flow 4.5 mL/min; 0-5 min 70% B, 10 min 80% B, 20-25 min 100% B to yield 1.4 mg of the compound (Rt = 4.162 min).
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The isolation of staurosporine was carried out by semi-preparative HPLC (Phenomenex Luna SemiPrep RP18e 10 x 250 mm) using H2O + 0.1% TFA (A) and CH3OH (B) as the solvents and the following gradient: flow 4.5 mL/min; 0-5 min 70% B, 10 min 80% B, 20-25 min 100% B to yield 1.4 mg of the compound (Rt = 4.162 min).
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J774.1 macrophages were cultured in complete medium (RPMI with NaHCO3, 10% FCS, 2 mM glutamine, 10 mM Hepes pH 7.2, 100 U/mL penicillin, 50 μg/mL gentamicin, 50 μM 2-mercaptoethanol) without phenol red in the absence or presence of increasing concentrations of the compounds at a cell density of 1 x 105 cells/mL (200 μL) for 24 h at 37 ° C, 5% CO2 and 95% humidity. Following the addition of 20 ?L of Alamar Blue, the plates were incubated and the ODs measured at 24 h, 48 h and 72 h. The same Alamar blue assay previously described for Leishmania was followed [21, Kidney epithelial 293T cells were also tested in the same manner as the macrophages but using complete DMEM medium (4.5 g/L solution of DMEM high glucose solution with sodium pyruvate but without L-glutamine, FBS superior at final concentration of 20, 200 mM L-glutamine 100x) and cell density 2 x 104 cells/mL
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4 cells/mL).
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