Analysis of monosomy‐3 in immunomagnetically isolated circulating melanoma cells in uveal melanoma patients

Monosomy‐3 in primary uveal melanoma (UM) is associated with a high risk of metastasis and mortality. Although circulating melanoma cells (CMC) can be found in most UM patients, only approximately 50% of the patients develop metastases. We utilized a novel immuno‐FISH assay to detect chromosome‐3 in intact CMC isolated by dual immunomagnetic enrichment. Circulating melanoma cells were detected in 91% of the patients (n = 44) with primary non‐metastatic UM, of which 58% were positive for monosomy‐3. The monosomy‐3 status of CMC corresponded to the monosomy‐3 status of the primary tumor in 10 of the 11 patients where this could be tested. Monosomy‐3 in the CMC was associated with an advanced tumor stage (P = 0.046) and was detected in all four patients who developed metastasis within the follow‐up period of 4 yr. This non‐invasive technique may enable the identification of UM patients at risk for metastasis particularly when a primary tumor specimen is unavailable.     

On the Effect of Prevalent Carbazole Homocoupling Defects on the Photovoltaic Performance of PCDTBT:PC71BM Solar Cells

The photophysical properties and solar cell performance of the classical donor–acceptor copolymer PCDTBT (poly(N‐9′‐heptadecanyl‐2,7‐carbazole‐alt ‐5,5‐(4′,7′‐di‐2‐thienyl‐2′,1′,3′‐benzothiadiazole))) in relation to unintentionally formed main chain defects are investigated. Carbazole–carbazole homocouplings (Cbz hc) are found to significant extent in PCDTBT made with a variety of Suzuki polycondensation conditions. Cbz hc vary between 0 and 8 mol% depending on the synthetic protocol used, and are quantified by detailed nuclear magnetic resonance spectroscopy including model compounds, which allows to establish a calibration curve from optical spectroscopy. The results are corroborated by extended time‐dependent density functional theory investigations on the structural, electronic, and optical properties of regularly alternating and homocoupled chains. The photovoltaic properties of PCDTBT:fullerene blend solar cells significantly depend on the Cbz hc content for constant molecular weight, whereby an increasing amount of Cbz hc leads to strongly decreased short circuit currents J_SC. With increasing Cbz hc content, J_SC decreases more strongly than the intensity of the low energy absorption band, suggesting that small losses in absorption cannot explain the decrease in J_SC alone, rather than combined effects of a more localized LUMO level on the TBT unit and lower hole mobilities found in highly defective samples. Homocoupling‐free PCDTBT with optimized molecular weight yields the highest efficiency up to 7.2% without extensive optimization.     

Binding of FAD and tryptophan to the tryptophan 6‐halogenase Thal is negatively coupled

Flavin‐dependent halogenases require reduced flavin adenine dinucleotide (FADH₂), O₂, and halide salts to halogenate their substrates. We describe the crystal structures of the tryptophan 6‐halogenase Thal in complex with FAD or with both tryptophan and FAD. If tryptophan and FAD were soaked simultaneously, both ligands showed impaired binding and in some cases only the adenosine monophosphate or the adenosine moiety of FAD was resolved, suggesting that tryptophan binding increases the mobility mainly of the flavin mononucleotide moiety. This confirms a negative cooperativity between the binding of substrate and cofactor that was previously described for other tryptophan halogenases. Binding of substrate to tryptophan halogenases reduces the affinity for the oxidized cofactor FAD presumably to facilitate the regeneration of FADH₂ by flavin reductases.     

Combined transgene immortalized urothelial cells capable of reprogramming and hepatic differentiation

Human primary cells, including urine-derived cells (UCs), are an excellent source for generation of pluripotent stem cells (iPSCs) to model disease. However, replicative senescence starts early and shortens the time window for generation of iPSCs. We addressed the question whether combinations of transgenes allows efficient immortalization of UCs, iPSC generation, and differentiation into hepatocyte-like cells (HLCs). Retroviral transfer of three gene cassettes HPVE6E7 (H), hTERT/p53DD (T), cyclinD1/CDK4R24C (C) encoding five genes was established in primary UCs. Long-term cell proliferation was observed in cells carrying transgenes H, HT, HC, and HCT, whereas cells carrying transgenes C, T and CT showed early senescence similar to UCs. iPSCs could be exclusively generated from immortalized UCs transduced with transgenes HCT and HC. iPSC colonies appeared however later and in smaller number as compared to UCs. Using an established hepatic differentiation protocol, HLCs were obtained with high efficacy. Of note, a high expression of individual transgenes was observed in immortalized UCs, which was down-regulated after reprogramming in four out of five genes. One transgene was re-expressed in HLCs as compared to iPSCs. Our data suggest that individual transgene combinations result in advanced growth rates of immortalized cells and do not prevent iPSC formation and HLC differentiation. Retroviral transgene expression is mostly silenced in iPSCs but can be rarely re-expressed after hepatic differentiation. An extended time window for iPSC establishment can be proposed that allows straightforward functional analyses of differentiated cells.     

Backbone and sidechain 1H, 13C and 15N resonance assignments of the RGS domain from human RGS14

We have assigned ¹H, ¹⁵N and ¹³C resonances of the RGS domain from the human RGS14 protein, a multi-domain member of the RGS (Regulators of G-protein signalling) family of proteins, important in the down-regulation of specific G-protein signalling pathways.     

Phosphorylation of the tumor necrosis factor receptor CD120a (p55) recruits Bcl-2 and protects against apoptosis

Ligation of the tumor necrosis factor alpha receptor CD120a initiates responses as diverse as apoptosis and the expression of NF-kappaB-dependent pro-survival genes. How these opposing responses are controlled remains poorly understood. Here we demonstrate that phosphorylation by p42(mapk/erk2) inhibits the apoptotic activity of CD120a while preserving its ability to activate NF-kappaB. Phosphorylated CD120a is re-localized from the Golgi complex to tubular structures of the endoplasmic reticulum wherein it recruits Bcl-2. Antisense-mediated down-regulation of Bcl-2 antagonized the localization of CD120a to tubular structures and reversed the protection from apoptosis conferred by receptor phosphorylation. We propose that phosphorylation of CD120a represents a novel, Bcl-2-dependent mechanism by which the apoptotic activity of the receptor may be regulated. Thus, oncogenic activation of p42(mapk/erk2) may serve to inhibit the apoptotic activity of this death receptor while preserving NF-kappaB-dependent responses and may thus indirectly contribute to a failure to eliminate cells bearing oncogenes of the Ras-Raf-MEK-p42(mapk/erk2) pathway.     

Measurement of steady-state values of respiration rate and oxidation levels of respiratory pigments at low oxygen tensions. A new technique

1. An apparatus was developed for the simultaneous measurement of steady-state values of respiration rate and oxidation level of respiratory pigments at low oxygen tensions. An open reaction system is utilized. The liquid sample is in contact with a gas mixture whose oxygen tension can be increased linearly with time at a rate so slow that the system is always practically at a steady state.

2. Assuming Michaelis-Menten kinetics in the respiration, theoretical curves for oxygen tension in the liquid and oxidation level of the terminal oxidase during a linear increase of the oxygen tension in the gas were calculated.

3. Measurements were performed on rat liver mitochondria. Steady-state curves for oxygen tension in the liquid and oxidation level of the terminal oxidase, cytochrome a₃, obtained with coupled mitochondria resembled the theoretical curves. For uncoupled mitochondria the cytochrome a₃ curve was signmoidal, deviating strongly from the theoretical curve.

4. The apparent K_m for oxygen uptake of coupled mitochondria in the presence of pyruvate and malate, in the absence of phosphate was found to be 0.5 μM. In the case of uncoupled mitochondria the oxygen tension in the liquid could not be measured with sufficient accuracy to allow comparison with Michaelis-Menten kinetics. The apparent K_m for oxygen uptake was less than 0.05 μM.