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lengths of 544 and 620 nm, respectively, in a Fluostar Galaxy microplate fluorimeter (BMG Labtechnologies, Offenburg, Germany). The correlation between PI and CFUs as a measure for candidacidal activity has been previously described (van der Kraan et al. 2004; Veerman et al. 2004). In the present experiments, the maximum PI fluorescence is assayed to verify 100% killing. To verify killing, the number of CFUs in representative wells was determined after 1 h by diluting the cells in buffer and plating them on appropriate growth media. Following an incubation period of 48 h, the number of CFUs was counted. LC50-values are the concentrations of peptide resulting in 50% killing. All experiments were repeated at least twice in duplicate. The candidacidal activity of the fluorescently labeled peptides was confirmed to be comparable with that of unlabeled peptide
Reference Reading Report
Biochem. Cell Biol(2012) 1-9
Chimerization of lactoferricin and lactoferrampin peptides strongly potentiates the killing activity against Candida albicans1 Jan Bolscher, Kamran Nazmi, Jan van Marle, Wim van ‘t Hof, and Enno Veerman
Methods and Materials
Peptide synthesis, FITC-labeling, purification, and characterization
BLF peptides, LFampin265–284 (N-DLIWKLLSKAQEKFGKNKSR-C) and LFcin17– 30 (N-FKCRRWQWRMKKLG-C), and LFchimera (N-FKCRRWQWRMKKLG-K3RSKNKGFKEQAKSLLKWILD-N) were manufactured using Fmoc-protected amino acids with a MilliGen 9050 synthesizeras described previously. On-resin labeling of an aliquotof each of the 3 peptides was carried out with a 20-fold excess of fluorescein-5-isothiocyanate on peptides N-terminally elongated with the linker Fmoc-L-g-aminobutyric acid. The aminobutyric acid linker prevented Edmann-type rearrangements during the cleavage reaction. Peptides were purified to a purity of at least 95% by semipreparative reversed phase – high-performance liquid chromatographyon a Vydac C18-columnand the authenticity of the peptides was confirmed by MALDI-TOF mass spectrometry on a Microflex LRF mass spectrometer equipped with an additional gridless reflectron.
孟庆森 2012-04-24
2012年 2.653
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Abstract Methods and Materials Results Discussion
Abstract
Bovine lactoferrin harbors 2 antimicrobial sequences (LFcin and LFampin), situated in close proximity in the N1• domain. To mimic their semi parallel configuration we have synthesized a chimeric peptide (LFchimera) in which these sequences are linked in a head-to-head fashion to the aand 3-amino group, respectively, of a single lysine. In line
Candidacidal activity of peptides
• • • • • • • • • • • • • The peptide-mediated membrane permeabilization, monitored by the fluorescence enhancement of propidium iodide (PI; Invitrogen), was used as a measure for the relative candidacidal activity of peptides. This PI-assay is a quick assay to compare relative candidacidal activities of a series of peptides under various conditions and after various incubation times (van der Kraan et al. 2004; Veerman et al. 2004). In brief, 50 µL aliquots from a mid-log phase culture of C. albicans (∼2 × 107 cells/mL) were supplemented with PI (final concentration 6 µmol/L) and incubated at 30 °C with equal volumes of peptide solutions at final concentrations ranging from 0.2 to 50 µmol/L. PI fluorescence was measured at 5 min intervals for 1 h at excitation and emission wave-
Fluorescence microscopy The ability of peptides to penetrate into C. albicans was determined by fluorescence microscopy. Candida albicans cells, either in yeast or pseudohyphal morphology (2 × 107 cells/mL), were incubated with 2–20 µmol/L FITC-labeled peptides for 5 and 30 min, and cells were examined with a fluorescence microscope (Leica DMRA, Heidelberg, Germany) and photographed with a Leica DMDL digital camera
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with previously described bactericidal effects, this peptide was also a stronger candidacidal agent than the antimicrobial peptides LFcin17–30 and LFampin265–284, or a combination of these 2. Conditions that strongly reduced the candidacidal activities of LFcin17–30 and LFampin265–284, such as high ionic strength and energy depletion, had little influence on the activityof LFchimera.
Growth conditions of C. albicans
Candida albicans 315 (ATCC 10231), in yeast morphology, was cultured aerobically at 30 °C on Sabouraud dextrose agar plates (SDA; Oxoid, Hampshire, UK) or in Sabouraud dextrose broth (Difco, Becton Dickinson Microbiology Systems,Sparks, Md.). To obtain a mid-log phase culture at thetime of harvesting, a single colony of the yeast was culturedovernight and subcultured for 1–2 h in 20 mL of Sabourauddextrose broth in a 100 mL Erlenmeyer flask. Cells werewashed twice in 5 mmol/L potassium phosphate buffer(PPB) and resuspended to a cell density of ∼2 × 107 cells/mL.Candida albicans, in hyphal morphology, was culturedaerobically at 37 °C with 5% CO2 in Sabouraud dextrosebroth containing 5% fetal calf serum