Purification and mass spectrometry based characterization of a pediocin produced by Pediococcus acidilactici 13.

Bacteriocins are antimicrobial peptides produced by several bacterial species. Among the bacteriocins pediocin-like bacteriocins have a significant inhibitory activity on the foodborne pathogens especially on Listeria monocytogenes. This study aims to select a simple and usable purification method to purify/concentrate the antimicrobial peptide and characterization of the bacteriocin produced by Pediococcus acidilactici 13 by using proteomic approaches which is a recent omic technology. For purification dialysis, ultrafiltration method was used, and as a result of this study the bacteriocin activity reached 819,200 AU/mL from 102,400 AU/mL initially. Two dimensional gel electrophoresis and then matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) analysis were carried out to identify the current bacteriocin and related proteins. Obtained data revealed similarity to pediocin PA-1 transport/processing ATP-binding protein PedD (accession number: P36497), pediocin operon PedC (accession number: Q68GC4) and bacteriocin pediocin PA-1 (accession number: P29430) from UniProtKB/Swiss-Prot databank, thus the bacteriocin produced by P. acidilactici 13 is considered similar to pediocin PA-1.

Proteomic identification of erythrocyte membrane protein deficiency in hereditary spherocytosis.

Hereditary spherocytosis (HS) is the most common congenital hemolytic anemia in Caucasians, with an estimated prevalence ranging from 1:2000 to 1:5000. The molecular defect in one of the erythrocytes (RBC) membrane proteins underlying HS like; spectrin-α, spectrin-ß, ankyrin, band 3 and protein 4.2 that lead to membrane destabilization and vesiculation, may change the RBCs into denser and more rigid cells (spherocytes), which are removed by the spleen, leading to the development of hemolytic anemia. It is classified as mild, moderate and severe, according to the degree of the hemolytic anemia and the associated symptoms. Two-dimensional gel electrophoresis (2-DE) is potentially valuable method for studying heritable disorders as HS that involve membrane proteins. This separation technique of proteins based upon two biophysically unrelated parameters; molecular weight and charge, is a good option in clinical proteomics in terms of ability to separate complex mixtures, display post-translational modifications and changes after phosphorylation. In this study, we have used contemporary methods with some modifications for the solubilisation, separation and identification of erythrocyte membrane proteins in normal and in HS RBCs. Spectrin alpha and beta chain, ankyrin and band 3 proteins expression differences were found with PDQuest software 8.0.1. and peptide mass fingerprinting (PMF) analysis performed for identification of proteins in this study.

Depletion of eIF2·GTP·Met-tRNAi translation initiation complex up-regulates BRCA1 expression in vitro and in vivo.

Most sporadic breast and ovarian cancers express low levels of the breast cancer susceptibility gene, BRCA1. The BRCA1 gene produces two transcripts, mRNAa and mRNAb. mRNAb, present in breast cancer but not in normal mammary epithelial cells, contains three upstream open reading frames (uORFs) in its 5'UTR and is translationally repressed. Comparable tandem uORFs are characteristically seen in mRNAs whose translational efficiency paradoxically increases when the overall translation rate is decreased due to phosphorylation of eukaryotic translation initiation factor 2 α (eIF2α). Here we show fish oil derived eicosopanthenoic acid (EPA) that induces eIF2α phosphorylation translationally up-regulates the expression of BRCA1 in human breast cancer cells. We demonstrate further that a diet rich in EPA strongly induces expression of BRCA1 in human breast cancer xenografts.

Comprehensive identification of erythrocyte membrane protein deficiency by 2D gel electrophoresis based proteomic analysis in hereditary elliptocytosis and spherocytosis.

PURPOSE: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1DE) may reveal qualitative or quantitative defects in red blood cell (RBC) membrane proteins, two-dimensional gel electrophoresis (2DE) can be used for determination of the protein changes caused by the disease process in a relatively high-throughput manner, because it permits an analysis of thousands of modified or unmodified proteins simultaneously. The principal aim of this study was to compare hereditary elliptocytosis (HE), hereditary spherocytosis (HS), and control RBC membrane protein profiles and identify proteins as a clinical marker by the sensitive methods. EXPERIMENTAL DESIGN: RBC membrane proteins of HE, HS, and control groups were compared by 2DE and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis was obtained using peptide mass fingerprint for protein identification. RESULTS: Twenty proteins were identified with peptide mass fingerprint analysis and different expression levels were found in HE, HS, and controls for some proteins that includes three biomarker proteins (ankyrin, spectrin, band 3) that may have prognostic or predictive importance. CONCLUSIONS AND CLINICAL RELEVANCE: 2DE of RBC proteins is a potentially valuable method for studying heritable disorders such as HE and HS. By identifying a deficiency in membrane proteins associated with the RBC cytoskeleton, the diagnosis of HE and HS can be confirmed.