Mass Spectrometry-Based Identification of Urinary Antimicrobial Peptides in Dairy Cows

Background:: Urine is considered one of the biological fluids in which antimicrobial peptides are secreted or expressed. Cow urine has not been investigated for the presence of these peptides using MALDI-TOF-MS. Objective:: The aim of this study is to isolate, identify and assess the antimicrobial activity of urinary antimicrobial peptides from healthy normal cycling cows. Method:: We analyzed the urine sample using diafiltration, ion exchange chromatography, Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), acid urea polyacrylamide gel electrophoresis (AU-PAGE) coupled with identification through Peptide Mass Fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF- MS). The in vitro antimicrobial effects of purified fractions were assessed using Radial Diffusion Assay (RDA) and microtitre broth dilution assay against Gram-positive and Gramnegative bacteria. Results: : Proteins corresponding to the peaks were identified using SWISSPROT protein database. This study revealed constitutive expression of β-Defensin-1 (DEFB1), β-Defensin-4A (DFB4A), Neutrophil Defensin-1 (DEF1), Neutrophil Defensin-3 (DEF3) in cow urine. The identified peptides are cationic antimicrobial peptides of the defensin family. The purified fractions exhibited antimicrobial effects in radial diffusion assay and MIC values in the range of 2.93-29.3 &*#181;M/L. Conclusion:: This study concludes that cow urine, previously unexplored with regard to antimicrobial peptides, would be a promising source of highly potent AMPs and an effective alternative to the resistant antibiotics.

A study of antimicrobial activity of polyphenols derived from wood

Due to the spreading and increasing drug resistance of pathogens, the search for novel antibiotics is becoming ever more important. Plant-derived polyphenols are a vast and promising class of compounds with a potential to fight infectious diseases. Still, they are not routinely used in clinical practice. No reports on the in vivo studies of these compounds have been presented. The aim of our work was to compare the antimicrobial activity of resveratrol (stilbene), dihydroquercetin and dihydromyricetin (flavonols) extracted from the bark and wood of conifers against the dermatophytes Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Using the radial diffusion assay, we established that dihydroquercetin, resveratrol and dihydromyricetin exhibit high activity against S. aureus even at the smallest possible concentrations of 0.22, 0.15, and 0.15 mM, respectively. In contrast, the highest achievable concentrations of these compounds in the solutions (21.5, 15.5 and 15.0 mM for dihydroquercetin, resveratrol and dihydromyricetin, respectively) have no effect on the growth of P. aeruginosa and C. albicans. These findings suggest that polyphenols derived from conifers could have a potential to be used as a medicine for topical application to treat bacterial infections of the skin caused by S. aureus.

Interaction of Moringa oleifera seed lectin with humic acid

The aim of this work was to characterise the affinity of protein preparations from Moringa oleifera seeds, specifically extract (seeds homogenised with 0.15 M NaCl), fraction (extract precipitated with 390 mg mL−1 of ammonium sulphate) and cMoL (coagulant M. oleifera lectin) to bind humic acids using a haemagglutinating activity assay with rabbit erythrocytes and a radial diffusion assay in agarose gel. Specific haemagglutinating activity (SHA) decreased by 94 % for the extract and cMoL and by 50 % for the fraction in the presence of humic acid. Precipitation bands were observed in the diffusion gel. Both results suggested humic acid-cMoL binding. Carbohydrates, potassium, and calcium ions and pH affected the SHA of cMoL. As an example of application, cMoL was immobilised on a column packed with sepharose receiving 20 mg mL−1 of carbon humic acid solution, 30 mg of humic acid per gram of support was removed. This result suggested that protein preparations might be used in water treatment to remove humic acids.

Activity of Protegrins against Yeast-PhaseCandida albicans

ABSTRACT We used a two-stage radial diffusion assay to perform a structure-activity study of the antifungal effects of protegrin-1 (PG-1) on yeast-phase Candida albicans. While doing so, we computed MICs from the radial diffusion assay data by three methods and compared the respective values with results from colony count and broth microdilution assays. This allowed us to identify several technical modifications that improved the sensitivity and accuracy of radial diffusion assays. We found that both PG-1 and enantiomeric PG-1 (composed exclusively of d-amino acids) were potently fungicidal for yeast-phase C. albicans. The protegrins PG-2, -3, and -5, but not PG-4, were as effective as PG-1. At least one intramolecular disulfide bond was required to retain optimal candidacidal activity at physiological NaCl concentrations. Truncated variants of PG-1 that lacked its first four residues showed decreased candidacidal activity, although their activity against bacteria was substantially intact. Altering the β-turn region (residues 9 to 12) of PG-1 or its variants further decreased candidacidal activity. These studies suggest that only 12 residues are needed to endow protegrin molecules with strong antibacterial activity and that at least 4 additional residues are needed to add potent antifungal properties. Thus, the 16-residue protegrin PG-2 likely represents the minimal structure needed for broad-spectrum antimicrobial activity encompassing bacteria and fungi.