Membrane proteins and membrane lipids are frequently organized in submicron-sized domains within cellular membranes. Factors thought to be responsible for domain formation include lipid-lipid interactions, lipid-protein interactions and protein-protein interactions. However, it is unclear whether the domain structure is regulated by other factors such as divalent cations. Here, we have examined in native plasma membranes and intact cells the role of the second messenger Ca 2 þ in membrane protein organization. We find that Ca 2 þ at low micromolar concentrations directly redistributes a structurally diverse array of membrane proteins via electrostatic effects. Redistribution results in a more clustered pattern, can be rapid and triggered by Ca 2 þ influx through voltage-gated calcium channels and is reversible. In summary, the data demonstrate that the second messenger Ca 2 þ strongly influences the organization of membrane proteins, thus adding a novel and unexpected factor that may control the domain structure of biological membranes.

Nuclear factor kappaB (NF-κB) is a central transcription factor in the immune system and modulates cell survival in response to radiotherapy. Activation of NF-κB was shown to be an early step in the cellular response to ultraviolet A (UVA) and ionizing radiation exposure in human cells. NF-κB activation by the genotoxic stress-dependent sub-pathway after exposure to different radiation qualities had been evaluated to a very limited extent. In addition, the resulting gene expression profile, which shapes the cellular and tissue response, is unknown. Therefore, in this study the activation of NF-κB after exposure to low- and high-linear energy transfer (LET) radiation and the expression of its target genes were analyzed in human embryonic kidney (HEK) cells. The activation of NF-κB via canonical and genotoxic stress-induced pathways was visualized by the cell line HEK-pNF-κB-d2EGFP/Neo L2 carrying the destabilized enhanced green fluorescent protein (d2EGFP) as reporter. The NF-κB-dependent d2EGFP expression after irradiation with X rays and heavy ions was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after irradiation with X rays (significant NF-κB activation for doses >4 Gy) and heavy ions (significant NF-κB activation at doses as low as 1 Gy), it was expected that radiation quality (LET) played an important role in the cellular radiation response. In addition, the relative biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival were compared for heavy ions having a broad LET range (∼0.3-9,674 keV/μm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real-time reverse transcriptase quantitative PCR (RT-qPCR). The maximal RBE for NF-κB activation and cell killing occurred at an LET value of 80 and 175 keV/μm, respectively. There was a dose-dependent increase in expression of NF-κB target genes NF-κB1A and CXCL8. A qPCR array of 84 NF-κB target genes revealed that TNF and a set of CXCL genes (CXCL1, CXCL2, CXCL8, CXCL10), CCL2, VCAM1, CD83, NF-κB1, NF-κB2 and NF-κBIA were strongly upregulated after exposure to X rays and neon ions (LET 92 keV/μm). After heavy-ion irradiations, it was noted that the expression of NF-κB target genes such as chemokines and CD83 was highest at an LET value that coincided with the LET resulting in maximal NF-κB activation, whereas expression of the NF-κB inhibitory gene NFKBIA was induced transiently by all radiation qualities investigated. Taken together, these findings clearly demonstrate that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ∼50-200 keV/μm. The upregulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, CXCL8/IL-8 and TNF) could be important for cell-cell communication among hit as well as nonhit cells (bystander effect).

Charged particles, such as carbon ions, bear the promise of a more effective cancer therapy. In human spaceflight, exposure to charged particles represents an important risk factor for chronic and late effects such as cancer. Biological effects elicited by charged particle exposure depend on their characteristics, e.g., on linear energy transfer (LET). For diverse outcomes (cell death, mutation, transformation, and cell-cycle arrest), an LET dependency of the effect size was observed. These outcomes result from activation of a complex network of signaling pathways in the DNA damage response, which result in cell-protective (DNA repair and cell-cycle arrest) or cell-destructive (cell death) reactions. Triggering of these pathways converges among others in the activation of transcription factors, such as p53, nuclear factor κB (NF-κB), activated protein 1 (AP-1), nuclear erythroid-derived 2-related factor 2 (Nrf2), and cAMP responsive element binding protein (CREB). Depending on dose, radiation quality, and tissue, p53 induces apoptosis or cell-cycle arrest. In low LET radiation therapy, p53 mutations are often associated with therapy resistance, while the outcome of carbon ion therapy seems to be independent of the tumor’s p53 status. NF-κB is a central transcription factor in the immune system and exhibits pro-survival effects. Both p53 and NF-κB are activated after ionizing radiation exposure in an ataxia telangiectasia mutated (ATM)-dependent manner. The NF-κB activation was shown to strongly depend on charged particles’ LET, with a maximal activation in the LET range of 90–300 keV/μm. AP-1 controls proliferation, senescence, differentiation, and apoptosis. Nrf2 can induce cellular antioxidant defense systems, CREB might also be involved in survival responses. The extent of activation of these transcription factors by charged particles and their interaction in the cellular radiation response greatly influences the destiny of the irradiated and also neighboring cells in the bystander effect.

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This article introduces 'Tox-Box', a joint research project designed to develop a holistic approach towards a harmonized testing strategy for exposure-and hazard-based risk management of anthropogenic trace substances in drinking water to secure a long-term drinking water supply. The main task of the Tox-Box consortium is to enhance the existing health-related indicator value concept (German: GOW-Konzept -Gesundheitlicher Orientierungswert) through development and prioritization of additional end point-related testing strategies for genotoxicity, neurotoxicity, germ cell damage, and endocrine effects. In this context, substance-specific modes of action will be identified and characterized. Toxicological data collected by the 12 Tox-Box subprojects will be evaluated and weighted to structure a hierarchical testing strategy for an improved risk assessment. A technical guidance document for exposure and hazard-based risk management of anthropogenic trace substances in drinking water will eventually be prepared.

To understand the mechanisms of disturbed differentiation and development by radiation, murine CGR8 embryonic stem cells (mESCs) were exposed to ionizing radiation and differentiated by forming embryoid bodies (EBs). The colony forming ability test was applied for survival and the MTT test for viability determination after X-irradiation. Cell cycle progression was determined by flow cytometry of propidium iodide-stained cells, and DNA double strand break (DSB) induction and repair by γH2AX immunofluorescence. The radiosensitivity of mESCs was slightly higher compared to the murine osteoblast cell line OCT-1. The viability 72 h after X-irradiation decreased dose-dependently and was higher in the presence of leukemia inhibitory factor (LIF). Cells exposed to 2 or 7 Gy underwent a transient G2 arrest. X-irradiation induced γH2AX foci and they disappeared within 72 h. After 72 h of X-ray exposure, RNA was isolated and analyzed using genome-wide microarrays. The gene expression analysis revealed amongst others a regulation of developmental genes (Ada, Baz1a, Calcoco2, Htra1, Nefh, S100a6 and Rassf6), downregulation of genes involved in glycolysis and pyruvate metabolism whereas upregulation of genes related to the p53 signaling pathway. X-irradiated mESCs formed EBs and differentiated toward cardiomyocytes but their beating frequencies were lower compared to EBs from unirradiated cells. These results suggest that X-irradiation of mESCs deregulate genes related to the developmental process. The most significant biological processes found to be altered by X-irradiation in mESCs were the development of cardiovascular, nervous, circulatory and renal system. These results may explain the X-irradiation induced-embryonic lethality and malformations observed in animal studies.

Astronauts are exposed to considerable doses of space radiation during long-term space missions. As complete shielding of the highly energetic particles is impracticable, the cellular response to space-relevant radiation qualities has to be understood in order to develop countermeasures and to reduce radiation risk uncertainties. The transcription factor Nuclear Factor κB (NF-κB) plays a fundamental role in the immune response and in the pathogenesis of many diseases. We have previously shown that heavy ions with a linear energy transfer (LET) of 100–300 keV/µm have a nine times higher potential to activate NF-κB compared to low-LET X-rays. Here, chemical inhibitor studies using human embryonic kidney cells (HEK) showed that the DNA damage sensor Ataxia telangiectasia mutated (ATM) and the proteasome were essential for NF-κB activation in response to X-rays and heavy ions. NF-κB’s role in cellular radiation response was determined by stable knock-down of the NF-κB subunit RelA. Transfection of a RelA short-hairpin RNA plasmid resulted in higher sensitivity towards X-rays, but not towards heavy ions. Reverse Transcriptase real-time quantitative PCR (RT-qPCR) showed that after exposure to X-rays and heavy ions, NF-κB predominantly upregulates genes involved in intercellular communication processes. This process is strictly NF-κB dependent as the response is completely absent in RelA knock-down cells. NF-κB’s role in the cellular radiation response depends on the radiation quality.

Non-vesicular lipid transport steps play a crucial role in lipid trafficking and potentially include spontaneous exchange. Since membrane contact facilitates this lipid transfer, it is most likely to occur at membrane contact sites (MCS). However, to date it is unknown whether closely attached biological membranes exchange lipids spontaneously. We have set up a system for studying the exchange of lipids at MCS formed between the endoplasmic reticulum (ER) and the plasma membrane. Contact sites were stably anchored and the lipids cholesterol and phosphatidylcholine (PC) were not capable of transferring spontaneously into the opposed bilayer. We conclude that physical contact between two associated biological membranes is not sufficient for transfer of the lipids PC and cholesterol.