Although proteomics analyses identified many storage-associated protein changes, these varied significantly by method suggesting that a combination of protein-centric (2D gel or DIGE) and peptide-centric (iTRAQ or ICAT) approaches are essential to acquire adequate data. The use of one proteomics method to study changes in stored blood products may give insufficient information.
A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1. The highest dehalogenase activity was observed with the biomass cultured at 22°C, compared to 30 and 37°C, where the cell suspensions were 2.2 and 9.6 times less active, respectively. The reductive dehalogenase was purified 12.7-fold to apparent homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa. Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor. Several polychlorophenols were dechlorinated at the meta and para positions. The apparent K m for 3,5-dicholorophenol was 49.3 ؎ 3.1 M at a methyl viologen concentration of 2 mM. Six internal tryptic peptides were sequenced by mass spectrometry. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences. This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase. The corresponding ORF (named cprA5) in D. frappieri PCP-1 was cloned and sequenced. The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion. The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases. This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions.Several strictly anaerobic bacteria are able to reductively dehalogenate a large variety of chlorinated compounds and use them as terminal electron acceptors (8). Desulfomonile tiedjei DCB-1, Dehalobacter restrictus PER-K23, Sulfurospirillum (formerly Dehalospirillum) multivorans, and many members of the genus Desulfitobacterium have been the most studied for their dechlorinating activity. Three types of reductive dehalogenases have been isolated from dehalorespiring bacteria. The most frequently reported dehalogenases consist of a single polypeptide containing one corrinoid cofactor and two iron-sulfur clusters: tetrachloroethene (PCE) reductive dehalogenases of S. multivorans (18), Desulfitobacterium sp. strain PCE-S (16), and Desulfitobacterium frappieri TCE-1 (27), ortho-chlorophenol reductive dehalogenases of Desulfitobacterium hafniense (5), Desulfitobacterium dehalogenans (28), and Desulfitobacterium chlororespirans (10), and PCE-and trichloroethene (TCE)-reductive dehalogenases of Dehalococcoides ethenogenes (14). Two reductive dehalogenases with one corrinoid cofactor but without an iron-sulfur cluster have also been reported: the ortho-chlorophenol reductive dehalogenase from D. frappieri PCP-1 (3) and the PCE reductive dehalogenase from Clostridium bifermentans DPH-1 (22). These two proteins are d...
Desulfitobacterium are Gram positive, spore-forming, strictly anaerobic bacteria, that belong to the Firmicutes, Clostridia, Clostridiales, and Peptococcaceae. Most known members of the genus Desulfitobacterium have the ability to dechlorinate several halogenated compounds by a mechanism of reductive dehalogenation and use them as electron acceptors to generate energy (halorespiration). Desulfitobacteria are therefore perfect candidates to be used in bioremediation treatments of environment polluted with halogenated compounds. Understanding the physiology and the molecular mechanisms of these bacteria will help to develop better bioremediation systems. This report summarizes works that have been done in our laboratories with D. frappieri PCP-1 on reductive dehalogenases, genes encoding these dehalogenases and their expression, and the development of lab-scale PCP-degrading reactors using this bacterium.
Platelet transfusion is a very common live-saving medical procedure for patients with platelet-deficient diseases like leukemia. In contrast to other blood components, the availability of platelets is restricted since they have a limited shelf-life of 5 days for transfusion purposes. This is due to storage-related deterioration in product quality resulting in the clearance from circulation. To overcome this problem, it is important to understand the molecular mechanisms leading to blood platelet lesion during storage. We were using two different proteomic approaches combined with functional biochemistry to investigate time-dependent changes in the blood platelet proteome. One type of analysis consisted of the separation of the platelet proteome at two different time points of storage, day 1 and day 8, using 2-dimensional (2D) gel electrophoresis for qualitative and DIGE technology for quantitative analysis. The identification of proteins changing in spot intensity was carried out by liquid chromatography and tandem mass spectrometry. The second method was based on stable isotope labeling with ITRAQ/ICAT reagents in combination with protease treatment, equivalent mixing, separation of the resulting peptides and quantitative analysis by mass spectrometry. Taken together, for the 2D/DIGE approach we analyzed 977 spots corresponding to 103 different proteins and for the ITRAQ approach 1428 peptides corresponding to 355 proteins, resulting in 37 proteins significantly changing both quantitatively due to protein synthesis or degradation and qualitatively due to post-translational modification and enzymatic activity. The high degree of correlation between the two approaches validates the experimental set-up and confirmed the requirement for complementary tools to enhance proteome coverage. Among others, increased amounts of integrins and other proteins known to form receptor signaling complexes with these integrins as well as proteins observed in platelet activation were detected. This proves, for the first time, that there is an apparent link between blood platelet storage lesion and cell signaling.
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