Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.

Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBEs) in human hematopoietic stem and progenitor cells (HSPC), potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBEs were analyzed in human CD34+ cells cultured in distinct molecular size fractions of medium, conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double-strand breaks) and chromosomal instability were evaluated in CD34+ cells grown in approximate (I) < 10 kDa, (II) 10–100 kDa and (III) > 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, significantly increased numbers of γH2AX foci (p = 0.0286) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells grown in the (II) 10–100 kDa fraction (0.67 ± 0.10 γH2AX foci per CD34+ cell ∨ 3.8 ± 0.3 aberrant metaphases per CD34+ cell sample; mean ± SEM) when compared to (I) < 10 kDa (0.19 ± 0.01 ∨ 0.3 ± 0.2) or (III) > 100 kDa fractions (0.23 ± 0.04 ∨ 0.4 ± 0.4) or un-/fractionated control medium (0.12 ± 0.01 ∨ 0.1 ± 0.1). Furthermore, RIBEs disappeared after heat inactivation of medium at 75 °C. Taken together, our data suggest that RIBEs are mainly mediated by the heat-sensitive (II) 10–100 kDa fraction of MSC conditioned medium. We postulate proteins as RIBE mediators and in-depth proteome analyses to identify key bystander signals, which define targets for the development of next-generation anti-leukemic drugs.

Introduction: Within the past decade, it has been demonstrated that the outcome of multiple cardiovascular diseases can be modulated via the proteasome, which is already a target of cancer chemotherapeutics. However, the proteasome is a heterogenous group of multi-protein complexes and their precise assembly in vascular cells remains unknown. Therefrore, aim of the study was to map the functional subproteome of 20S proteasome complexes in vascular cells in detail. Methods and Results: Proteasome complexes of cell lysates as well as intact cells were analyzed based on the covalent binding of fluorescent inhibitors, which target only active proteolytic subunits in fully assembled 20S proteasomes. Application of both approaches revealed the existence of an unanticipated proteasome heterogeneity in human vascular and blood-derived cells with a mixed assembly of constitutive and inducible proteasomal subunits even under baseline conditions. Remarkably, the ratio of constitutive to inducible proteasomal subunits allowed to differentiate between the cells. The results obtained via living cell targeting showed that the highest abundance of functional, inducible subunits exists in lymphocytes (LC) and monocytes (MC) followed by platelets (Plt) and endothelial cells (EC), and was minimal in smooth muscle cells (SMC) (e.g. subunit β5i +50% in MC vs. EC, +90% in MC vs. SMC, p<0.01, n=4-8). On the other hand, the functional constitutive counterparts of the inducible proteolytic subunits were most abundant in EC and least abundant in Plt (e.g. subunit β5 +75% in EC vs. MC, +85% in EC vs. LC, + 90% in EC vs. Plt, p<0.01, n=4-8). Notably, the overall abundance of functional proteasome complexes was cell-type dependent as well. Thus, incorporation of proteolytic subunits in active 20S proteasomes was a function of subunits targeted by the fluorescent inhibitors and proteasome concentration in the cell. To validate the results obtained for EC and SMC under cell culture conditions, murine aortae were perfused with the fluorescent inhibitors. Fluorescence microscopy confirmed tissue penetration of the fluorescent inhibitors into the intima, media and adventitia, thus enabling a highly specific staining of active proteolytic subunits of the proteasome, whereby the proteasome assembly in the tissue matched that of cultured cells. Conclusion: Proteasome assembly is characteristic among vascular cells predicting an opportunity for a cell-directed proteasome targeting not only in the pathology of blood-borne cancer, but also in cardiovascular disorders.

Untersuchung der Pilzflora mittels molekulargenetischer Methoden basierend auf der 18S rDNA. Vergleich der Pilzflora von Patienten mit Morbus Crohn, Colitis ulcerosa sowie Normalkontrollen.

Background: Epidemiologic studies reveal a dramatic increase in allergies in the last decades. Air pollution is considered to be one of the factors responsible for this augmentation. The aim of this study was to analyze the impact of urbanization on birch pollen. The birch pollen proteome was investigated in order to identify differences in protein abundance between pollen from rural and urban areas. The allergenicity of birch pollen from both areas was evaluated by assessing its chemotactic potency as well as its protein and allergen contents. Methods: Difference gel electrophoresis (DIGE) was used to analyze the pollen proteome. The chemotactic activity of aqueous pollen extracts was determined by migration assays of human neutrophils. Results: DIGE revealed 26 differences in protein spot intensity between pollen from urban and rural areas. One of these proteins was identified by de novo sequencing as the 14-3-3 protein, which resembles a stress-induced factor in other plant species. Furthermore, extracts from pollen collected in urban areas had higher chemotactic activity on human neutrophils compared to pollen from rural sites. Conclusions: The present study points to an impact of air pollution on allergen carrier proteome and release of chemotactic substances. The increment in proinflammatory substances such as pollen-associated lipid mediators might contribute to the described urban-rural gradient of allergy prevalence. Furthermore, our study suggests that allergenicity is determined by more than the sole allergen content.

The ubiquitin proteasome system (UPS) represents a major pathway for intracellular protein degradation. Proteasome dependent protein quality control participates in cell cycle, immune response and apoptosis. Therefore, the UPS is in focus of therapeutic investigations and the development of pharmaceutical agents. Detailed analyses on proteasome structure and function are the foundation for drug development and clinical studies. Proteomic approaches contributed significantly to our current knowledge in proteasome research. In particular, 2‐DE has been essential in facilitating the development of current models on molecular composition and assembly of proteasome complexes. Furthermore, developments in MS enabled identification of UPS proteins and their PTMs at high accuracy and high‐throughput. First results on global characterization of the UPS are also available. Although the UPS has been intensively investigated within the last two decades, its functional significance and contribution to the regulation of cell and tissue phenotypes remain to be explored. This review recapitulates a variety of applied proteomic approaches in proteasome exploration, and presents an overview of current technologies and their potential in driving further investigations.