Proteomic analysis of the effect of Antrodia camphorata extract on human lung cancer A549 cell

Antrodia camphorata (niu-chang-chih) is a fungus native to Taiwan that is believed to be effective in preventing diseases. This study demonstrates that 0.2-2% v/v ethanol extracts of A. camphorata cultivated by solid-state fermentation (SACE) can effectively impede the proliferation of human non-small cell lung carcinoma A549 cells but not primary human fetal lung fibroblast MRC-5. The results of apoptotic analyses implicate that SACE might trigger the apoptosis in the A549 cells by inducing endoplasmic reticulum stress. Two-dimensional gel maps of non-treated and treated A549 cells were compared using PDQUEST analytical software to discover five statistically significant twofold or above-twofold differentially-expressed protein spots. The five protein spots that were significantly de-regulated were chosen for subsequent identification by high performance liquid chromatography electro-spray tandem mass spectrometry. The five proteins were later identified as human galectin-1, human eukaryotic translation initiation factor 5A, human Rho GDP dissociation inhibitor alpha, human calcium-dependent protease small subunit and human annexin V. All five proteins were confirmed to be down-regulated by Western blotting. The analytical results of this study help to provide insight into the effect of SACE on the gene expression of the tumor cells.     

Anti-inflammatory effects of moxifloxacin and human beta-defensin 2 association in human lung epithelial cell line (A549) stimulated with lipopolysaccharide

Epithelia in the human airways, from the nasal aperture to the alveoli, are covered in a protective film of fluid containing a number of antimicrobial proteins. Defensins are single-chain, strongly cationic peptides and are one of the most extensively studied classes of antimicrobial peptides. Moxifloxacin (MXF) is a fluoroquinolone that acts against both Gram positive and Gram negative bacteria. In this study, we evaluated the effects of HBD2, MXF and the association MXF/HBD2 on some cytokines and on the ICAM-1 expression in LPS-stimulated A549 cells. Our results suggest that by lowering the epithelial cell-derived IL-1β, IL-6, IL-8 and ICAM-1 expression, the MXF/HBD2 association interferes with the multifunctional cytokine network evolving during inflammatory processes of the respiratory tract; this anti-inflammatory potential could be of great value in the treatment of inflammatory disorders.     

Proteomic analysis of Bacillus cereus growing in liquid soil organic matter

Bacillus cereus is believed to be a soil bacterium, but studied solely in laboratory culture media. The aim of this study was to assess the physiology of B. cereus growing on soil organic matter by a proteomic approach. Cells were cultured to mid-exponential phase in soil extracted solubilized organic matter (SESOM), which mimics the nutrient composition of soil, and in Luria-Bertani broth as control. Silver staining of the two-dimensional gels revealed 234 proteins spots up-regulated when cells were growing in SESOM, with 201 protein spots down-regulated. Forty-three of these differentially expressed proteins were detected by Colloidal Coomassie staining and identified by matrix assisted laser desorption ionization-time of flight MS of tryptic digests. These differentially expressed proteins covered a range of functions, primarily amino acid, lipid, carbohydrate and nucleic acid metabolism. These results suggested growth on soil-associated carbohydrates, fatty acids and/or amino acids, concomitant with shifts in cellular structure.     

Reproducibility,sensitivity and compatibility of the ProteoExtract® subcellular fractionation kit with saturation labeling of laser microdissected tissues

Laser microdissection (LMD), a method of isolating specific microscopic regions of interest from a tissue that has been sectioned, is increasingly being applied to study proteomics. LMD generally requires tissues to be fixed and histologically stained, which can interfere with protein recovery and subsequent analysis. We evaluated the compatibility and reproducibility of protein extractions from laser microdissected human colon mucosa using a subcellular fractionation kit (ProteoExtract^®, Calbiochem). Four protein fractions corresponding to cytosol (fraction 1), membrane/organelle (fraction 2), nucleus (fraction 3) and cytoskeleton (fraction 4) were extracted, saturation labeled with Cy5 and 5 μg separated by both acidic (pH 4–7) and basic (pH 6–11) 2‐DE. The histological stains and fixation required for LMD did not interfere with the accurate subcellular fractionation of proteins into their predicted fraction. The combination of subcellular fractionation and saturation CyDye labeling produced very well resolved, distinct protein spot maps by 2‐DE for each of the subcellular fractions, and the total number of protein spots consistently resolved between three independent extractions for each fraction was 893, 1128, 1245 and 1577 for fractions 1, 2, 3 and 4, respectively. Although significant carryover of protein did occur between fractions, this carryover was consistent between experiments, and very low inter‐experimental variation was observed. In summary, subcellular fractionation kits are very compatible with saturation labeling DIGE of LMD tissues and provide greater coverage of proteins from very small amounts of microdissected material.