Project description:Lymphocytes are the standard cell of a majority of biodosimetry approaches because of their availability and radiosensitivity. But progresses remain to be performed to bring down the minimum threshold of dose sensitivity. To find a lymphocyte subtype able to respond to low doses of ionizing radiation, we examined gene expression variations in different lymphocyte subpopulations using the microarray technology. Blood samples were independently exposed to 0, 0.05 and 0.5 Gray of ionizing radiations. Three and 24 hours after exposure, CD56+, CD4+ and CD8+ cells were negatively isolated. RNA from each condition was competitively hybridized on oligonucleotide microarrays. Keywords: Dose response, stress response, cell type comparison We characterized genes whose expression level was affected following exposure to γ rays by carrying out genome-wide expression profiling on each lymphocyte subpopulation. Total blood samples were collected from 5 healthy male donors. Each sample was splitted into three parts which were exposed respectively to 0 Gy (sham-irradiated), 0.05 Gy and 0.5 Gy of ionizing radiations. Lymphocytes subpopulations were sorted 3 and 24 hours after irradiation. RNA was prepared from each cell type and amplified. The amino-allyl amplified RNAs were labeled and independently hybridized to the array. RNA from MNC obtained from 20 independent healthy donors constituted the reference group for competitive oligonucleotide microarray hybridization. According to our criteria of quality, we considered a total of 114 microarrays
Project description:Lymphocytes are the standard cell of a majority of biodosimetry approaches because of their availability and radiosensitivity. But progresses remain to be performed to bring down the minimum threshold of dose sensitivity. To find a lymphocyte subtype able to respond to low doses of ionizing radiation, we examined gene expression variations in different lymphocyte subpopulations using the microarray technology. Blood samples were independently exposed to 0, 0.05 and 0.5 Gray of ionizing radiations. Three and 24 hours after exposure, CD56+, CD4+ and CD8+ cells were negatively isolated. RNA from each condition was competitively hybridized on oligonucleotide microarrays. Keywords: Dose response, stress response, cell type comparison
Project description:Ionizing radiations are categorized by linear energy transfer (LET) into low-LET and high-LET. High-LET is considered to have a higher relative biological effectiveness (RBE) than low-LET radiations. However, the details of the effects have not been clearly determined. The aim of this study was to characterize the difference between high-LET and low LET radiations. The global effects of the three types of high-LET radiations (fast neutron, heavy ion (C) and thermal neutron) were compared with the low-LET radiation (gamma ray) using yeast DNA microarrays. Highly induced genes by the three types of high-LET radiations were those genes related to oxidative stress. Oxidative stress was one of the common factors associated with the four types of radiations. Oxidative stress induced by high-LET radiations may be more serious than that induced by gamma rays. Additionally, genes related to protein synthesis and the ubiquitin and proteasome system were detected. This suggests that more protein damages can be induced by high-LET radiation that denatures the proteins in yeast cells. The genes specifically altered by each type of high-LET radiation were also studied. Keywords: stress response
Project description:The growing number of particle treatment facilities worldwide and patients treated with particles instead of X-rays marks the upcoming rearrangement of modern radiotherapy. Especially for tumors being difficult to access and for tumors that are resistant to conventional X-ray treatment particle radiotherapy is a beneficial technology. At the Heidelberg Ion Beam Therapy Center (HIT) patients are treated with this technology since 2009 as it offers clear benefits. Contrary to X-rays, which show an exponential dose decrease (after reaching electron equilibrium) with increasing tissue depth, charged particles deposit most of their energy to a small region within the tissue with a sharp dose fall-off after the so-called Bragg peak. This precise dose localization enables further dose escalation within the tumor while sparing healthy tissue. Besides physical advantages, particle radiotherapy offers additional biological advantages. In radiobiology, the term relative-biological effectiveness (RBE) is defined as the ratio of X-rays dose to an alternative irradiation modality dose which produce the same biological effect (e.g. survival or number of DSBs). While protons have a relative biological effectiveness (RBE) comparable to X-rays, carbon ions are more effective in inducing DNA damage(1, 2) and are therefore especially useful for radioresistant tumors. This is due to the fact, that carbon ions induce clustered and direct DNA damage, which is considered to be less dependent on cell cycle stage, oxygen level, genetic background and hinders DNA repair mechanisms(3–6). Nevertheless, their exact mode of action and cellular mechanisms are largely unknown. We show the first comprehensive proteomic and phosphoproteomic study elucidating the cellular response to treatment with protons, carbon ions and X-rays. We found that 2h after treatment with these radiations negligible regulation occurred at protein expression level. But 181 phosphorylation sites were deregulated by ionizing radiation contributing mainly to DNA damage response functionalities. Interestingly we found 55 phosphorylation sites being differentially regulated between the radiations. Here, we observed protons and carbon ions producing equal cellular response whereas X-rays show altered regulation for certain phosphorylation sites. A subset of 28 phosphorylation sites being involved in the DNA damage response or differentially regulated between the ionizing radiations was selected for result confirmation.
Project description:To elucidate the epithelial cell diversity within the nasal inferior turbinates, a comprehensive investigation was conducted comparing control subjects to individuals with house dust mite-induced allergic rhinitis. This study aimed to delineate the differential expression profiles and phenotypic variations of epithelial cells in response to allergic rhinitis. This research elucidated distinct subpopulations and rare cell types of epithelial cells within the nasal turbinates, discerning alterations induced by allergic rhinitis. Furthermore, by interrogating transcriptomic signatures, the investigation provided novel insights into the cellular dynamics and immune responses underlying allergic rhinitis pathogenesis
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.