Durchflusszytometrie als Methode zur Quantifizierung der Wirkung ionisierender Strahlen auf dreidimensionale Organkokulturen von humanem Bronchialepithel und einer Bronchialepithel-Tumorzelllinie

Monolayerkulturen von Normal- und Tumorzellen verhalten sich unter Bestrahlung substantiell unterschiedlich. Wir haben ein Ko-Kultursystem (COCs), bestehend aus dreidimensionalen Organkulturen von humanem Bronchialepithels (OCs') und der GFP-transfizierten Lungenkarzinom-Zelllinie EPLC entwickelt, und können in Kombination mit der Durchflusszytometrie den DNA-Gehalt getrennt für Tumor- und Normalzellen analysieren. Damit kann die Wirkung ionisierender Strahlung auf die Proliferation, die Apoptose und die Zellzahl im Verlauf quantifiziert werden. Die humane Bronchialepithel-Zelllinie BEAS-2-B zeigt im Monolayer gesteigerte Proliferation und erhöhte Strahlensenitivität im Vergleich zum normalen Bronchialepithel in der COC. Ebenso ist die Proliferation und Strahlensenitivität der Tumorzellen, wenn als Monolayer kultiviert, erhöht im Vergleich zu Tumorzellen in COCs. Normalepithel-Zellen zeigten mit Tumorzellen in COCs kultiviert, verminderte Proliferation und, nach Bestrahlung keine gesteigerte Apoptose. Diese Daten zeigen, dass mit COCs in Verbindung mit Durchflusszytometrie die Auswirkung ionisierender Strahlung in einem In-vivo-nahen System erfassbar ist und das sowohl die räumliche Organisation als auch die Interaktion von Normal- und Tumorzellen für die Wirksamkeit einer Antitumortherapie entscheidend sind

Mechanisms altering airway smooth muscle cell Ca(2+) homeostasis in two asthma models

Background: Asthma is characterized by airway remodeling, altered mucus production and airway smooth muscle cell (ASMC) contraction causing extensive airway narrowing. In particular, alterations of ASMC contractility seem to be of crucial importance. The elevation of the cytoplasmic Ca(2+) concentration is a key event leading to ASMC contraction and changes in the agonist- induced Ca(2+) increase in ASMC have been reported in asthma. Objective: The aim of this study was to investigate mechanisms underlying these changes. Methods: Murine tracheal smooth muscle cells (MTSMC) from T- bet KO mice and human bronchial smooth muscle cells (HBSMC) incubated with IL-13 and IL-4 served as asthma models. Acetylcholine- induced changes in the cytoplasmic Ca(2+) concentration were recorded using fluorescence microscopy and the expression of Ca(2+) homeostasis regulating proteins was investigated with Western blot analysis. Results: Acetylcholine- induced Ca(2+) transients were elevated in both asthma models. This correlated with an increased Ca(2+) content of the sarcoplasmic reticulum (SR). In MTSMC from T-bet KO mice, the expression of the SR Ca(2+) buffers calreticulin and calsequestrin was higher compared to wild- type mice. In HBSMC incubated with IL-13 or IL-4, the expression of ryanodine receptors, inositol-3-phosphate receptors and sarcoplasmic/ endoplasmic reticulum Ca 2+ ATPases 2 was increased compared to HBSMC without incubation with interleukins. The enlarged acetylcholine- induced Ca(2+) transients could be reversed by blocking inositol-3- phosphate receptors. Conclusions: We conclude that in the murine asthma model the SR Ca(2+) buffer capacity is increased, while in the human asthma model the expression of SR Ca(2+) channels is altered. The investigation of the Ca(2+) homeostasis of ASMC has the potential to provide new therapeutical options in asthma. Copyright (C) 2008 S. Karger AG, Basel

The properties of dynamically ejected runaway and hyper-runaway stars

Runaway stars are stars observed to have large peculiar velocities. Two mechanisms are thought to contribute to the ejection of runaway stars, both involve binarity (or higher multiplicity). In the binary supernova scenario a runaway star receives its velocity when its binary massive companion explodes as a supernova (SN). In the alternative dynamical ejection scenario, runaway stars are formed through gravitational interactions between stars and binaries in dense, compact clusters or cluster cores. Here we study the ejection scenario. We make use of extensive N-body simulations of massive clusters, as well as analytic arguments, in order to to characterize the expected ejection velocity distribution of runaways stars. We find the ejection velocity distribution of the fastest runaways (>~80 km s^-1) depends on the binary distribution in the cluster, consistent with our analytic toy model, whereas the distribution of lower velocity runaways appears independent of the binaries properties. For a realistic log constant distribution of binary separations, we find the velocity distribution to follow a simple power law; Gamma(v) goes like v^(-8/3) for the high velocity runaways and v^(-3/2) for the low velocity ones. We calculate the total expected ejection rates of runaway stars from our simulated massive clusters and explore their mass function and their binarity. The mass function of runaway stars is biased towards high masses, and depends strongly on their velocity. The binarity of runaways is a decreasing function of their ejection velocity, with no binaries expected to be ejected with v>150 km s^-1. We also find that hyper-runaways with velocities of hundreds of km s^-1 can be dynamically ejected from stellar clusters, but only at very low rates, which cannot account for a significant fraction of the observed population of hyper-velocity stars in the Galactic halo.Comment: Now matching published ApJ versio

Lack of MSH2 involvement differentiates V(D)J recombination from other non-homologous end joining events

V(D)J recombination and class switch recombination are the two DNA rearrangement events used to diversify the mouse and human antibody repertoires. While their double strand breaks (DSBs) are initiated by different mechanisms, both processes use non-homologous end joining (NHEJ) in the repair phase. DNA mismatch repair elements (MSH2/MSH6) have been implicated in the repair of class switch junctions as well as other DNA DSBs that proceed through NHEJ. MSH2 has also been implicated in the regulation of factors such as ATM and the MRN (Mre11, Rad50, Nbs1) complex, which are involved in V(D)J recombination. These findings led us to examine the role of MSH2 in V(D)J repair. Using MSH2(−/−) and MSH2(+/+) mice and cell lines, we show here that all pathways involving MSH2 are dispensable for the generation of an intact pre-immune repertoire by V(D)J recombination. In contrast to switch junctions and other DSBs, the usage of terminal homology in V(D)J junctions is not influenced by MSH2. Thus, whether the repair complex for V(D)J recombination is of a canonical NHEJ type or a separate microhomology-mediated-end joining (MMEJ) type, it does not involve MSH2. This highlights a distinction between the repair of V(D)J recombination and other NHEJ reactions

Nucleon Polarizibilities for Virtual Photons

We generalize the sum rules for the nucleon electric plus magnetic polarizability $\Sigma=\alpha+\beta$ and for the nucleon spin-polarizability $\gamma$, to virtual photons with $Q^2>0$. The dominant low energy cross sections are represented in our calculation by one-pion-loop graphs of relativistic baryon chiral perturbation theory and the $\Delta(1232)$-resonance excitation. For the proton we find good agreement of the calculated $\Sigma_p(Q^2)$ with empirical values obtained from integrating up electroproduction data for $Q^2<0.4 GeV^2$. The proton spin-polarizability $\gamma_p(Q^2)$ switches sign around $Q^2= 0.4 GeV^2$ and it joins smoothly the "partonic" curve, extracted from polarized deep-inelastic scattering, around $Q^2=0.7 GeV^2$. For the neutron our predictions of $\Sigma_n(Q^2)$ and $\gamma_n(Q^2)$ agree reasonably well at $Q^2=0$ with existing determinations. Upcoming (polarized) electroproduction experiments will be able to test the generalized polarizability sum rules investigated here.Comment: 12 pages, 5 figures, submittes to Nuclear Physics