Abstract: To assess responses to low-dose ionizing radiation (LD-IR) exposures potentially encountered during medical diagnostic procedures, nuclear accidents or terrorist acts, a quantitative proteomic approach was used to identify changes in protein abundance in a reconstituted human skin tissue model treated with 0.1 Gy of ionizing radiation. To improve the dynamic range of the assay, subcellular fractionation was employed to remove highly abundant structural proteins and to provide insight into radiation-induced alterations in protein localization. Relative peptide quantification across cellular fractions, control and irradiated samples was performing using 8-plex iTRAQ labeling followed by online two-dimensional nano-scale liquid chromatography and high resolution MS/MS analysis. A total of 107 proteins were detected with statistically significant radiation-induced change in abundance (>1.5 fold) and/or subcellular localization compared to controls. The top biological pathways identified using bioinformatics include organ development, anatomical structure formation and the regulation of actin cytoskeleton. From the proteomic data, a change in proteolytic processing and subcellular localization of the skin barrier protein, filaggrin, was identified, and the results were confirmed by western blotting. This data indicate post-transcriptional regulation of protein abundance, localization and proteolytic processing playing an important role in regulating radiation response in human tissues.
Abstract: Imatinib mesylate is the leading compound to treat chronic myeloid leukemia (CML) and other cancers, through its inhibition of Bcr-Abl tyrosine kinases. However, resistance to imatinib develops frequently, particularly in late-stage disease and has necessitated the development of new Bcr-Abl inhibitors. The synthesis of a new series of phenylaminopyrimidines, structurally related to imatinib, showed large interest since the introduction of nilotinib. Here, we compare the protein levels in K562 cells treated with either imatinib or with novel imatinib derivates. Our results revealed that among the 986 quantified proteins, 35 had significantly altered levels of expression by imatinib or its derivates. In a second series of experiments, we directly compared the proteomes of imatinib treated K562 cells with those K562 cells treated with any of the four imatinib derivates. More than 1029 protein were quantified, 80 of which had altered levels of expression. Both experiments pointed to changes in the expression of the ATP-dependent RNA helicase DDX3X and of two mitochondrial coiled-coil-helix-coiled-coil-helix domain-containing proteins.
Abstract: The risks of non-cancerous diseases associated with exposure to low doses of radiation are at present not validated by epidemiological data, and pose a great challenge to the scientific community of radiation protection research. Here, we show that premature senescence is induced in human fibroblasts when exposed to chronic low dose rate (LDR) exposure (5 or 15 mGy/h) of gamma rays from a 137Cs source. Using a proteomic approach we determined differentially expressed proteins in cells after chronic LDR radiation compared to control cells. We identified numerous proteins involved in protection against oxidative stress, suggesting that these pathways protect against premature senescence. In order to further study the role of oxidative stress for radiation induced premature senescence, we also used human fibroblasts, isolated from a patient with a congenital deficiency in glutathione synthetase (GS). We found that these GS deficient cells entered premature senescence after a significantly shorter time of chronic LDR exposure as compared to the GS proficient cells. In conclusion, we show that chronic LDR exposure induces premature senescence in human fibroblasts, and propose that a stress induced increase in reactive oxygen species (ROS) is mechanistically involved.
Abstract: The present study was performed to unravel the mechanisms of systemic acquired resistance (SAR) establishment and resistance signaling pathways against the canker-rot fungus (Inonotus obliquus strain IO-U1) infection in Japanese birch plantlet No.8. Modulation of protein-profile induced by salicylic acid (SA)-administration was analyzed, and SA-responsive proteins were identified. In total, 5 specifically expressed, 3 significantly increased, and 3 significantly decreased protein spots were identified using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the sequence tag method. These proteins were malate dehydrogenase, succinate dehydrogenase, phosphoglycerate kinase, diaminopimalate decarboxylase, arginase, chorismate mutase, cyclophilin, aminopeptidase, and unknown function proteins. These proteins are considered to be involved in SAR-establishment mechanisms in the Japanese birch plantlet No 8.
Abstract: Due to changing climate, flooding (waterlogged soils and submergence) becomes a major problem in agriculture and crop production. In the present study, the effect of waterlogging was investigated on peroxidases of maize (Zea mays L.) leaves. The plants showed typical adaptations to flooding stress, i.e., alterations in chlorophyll a/b ratios and increased basal shoot diameter. Seven peroxidase bands could be detected by first dimension modified SDS-PAGE and 10 bands by first dimension high resolution Clear Native Electrophoresis that altered in dependence on plant development and time of waterlogging. Native isoelectric focusing revealed three acidic to neutral and four alkaline guaiacol peroxidases that could be further separated by high resolution Clear Native Electrophorese in the second dimension. One neutral peroxidase (pI 7.0) appeared to be down-regulated within four hours after flooding, whereas alkaline peroxidases (pI 9.2, 8.0 and 7.8) were up-regulated after 28 or 52 h. Second dimensions revealed molecular masses of 133 kDa and 85 kDa for peroxidases at pI 8.0 and 7.8, respectively. Size exclusion chromatography revealed native molecular masses of 30–58 kDa for peroxidases identified as class III peroxidases and ascorbate peroxidases by mass spectrometry. Possible functions of these peroxidases in flooding stress will be discussed.
Abstract: The bone marrow is one of the most radio-sensitive tissues. Accidental ionizing radiation exposure can damage mature blood cells and hematopoietic progenitor/stem cells, and mortality can result from hematopoietic insufficiency and infection. Ionizing radiation induces alterations in gene and protein expression in hematopoietic tissue. Here we investigated radiation effects on protein carbonylation, a primary marker for protein oxidative damage. C57BL/6 mice were either sham irradiated or exposed to 7.5 Gy 60Co (0.6 Gy/min) total body irradiation. Bone marrow was obtained 24 h post-irradiation. Two dimensional (2-D) gel electrophoresis and Oxyblot immunodetection were used to discover carbonylated proteins, and peptide mass fingerprinting was performed for identification. 2D gels allowed the detection of 13 carbonylated proteins in the bone marrow; seven of these were identified, with two pairs of the same protein. Baseline levels of carbonylation were found in 78 kDa glucose-related protein, heat shock protein cognate 71 KDa, actin, chitinase-like protein 3 (CHI3L1), and carbonic anhydrase 2 (CAII). Radiation increased carbonylation in four proteins, including CHI3L1 and CAII, and induced carbonylation of one additional protein (not identified). Our findings indicate that the profile of specific protein carbonylation in bone marrow is substantially altered by ionizing radiation. Accordingly, protein oxidation may be a mechanism for reduced cell viability.