CFP and YFP photostabilities are differentially affected by common mounting fluids

The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking and tracing of specific proteins or cells or to determine protein interactions. In the later case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is visualized by specific microscopy techniques. When we applied a commonly used FRET microscopy technique - the increase in CFP-fluorescence after bleaching of YFP, we noticed that it worked well for live cells, but that most of the FRET-signal was lost in fixed cells mounted in commercial microscopy mounting fluids. Subsequently, we could show that CFP bleached much faster in the mounting medium than in live cells, while the opposite effect was observed for YFP. This change in photostability was not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increased in live cells after illumination at the YFP-excitation wavelength – a phenomenon, which might cause a false-positive signal in the FRET-microscopy technique that is based on bleaching of YFP. All together our results show that it is problematic to use commercially available mounting fluids for fluorescent proteins due to their differential effects on the bleaching kinetics and that the FRET microscopy technique based on bleaching of the acceptor is prone to artefacts at least for the CFP/YFP pair

Interaction of the TNFR-receptor associated factor TRAF1 with I-kappa B kinase 2 (IKK2, IKK-beta, IKBKB) and TRAF2 indicating a dose dependent regulatory function of TRAF1 for NF-kappa B signaling

IKK2 is one of the most crucial signaling kinases for activation of the transcription factor NF-kappa B. Since many NF-kappa B activating pathways converge at the level of IKK2, we searched for interaction partners of this kinase using the C-terminal part (aa 466-756) as bait in a yeast two-hybrid system. We identified the N-terminal part (aa 1-228) of the TNF-receptor associated factor TRAF1 as putative interaction partner, which was subsequently confirmed in mammalian cells by coimmunoprecipitation experiments. However, this interaction seemed weaker than the interaction between TRAF1 and TRAF2, an important activating adapter molecule of NF-kappa B signaling indicating that relative levels of IKK2, TRAF1 and TRAF2 might be important for the final biological readout. Reporter gene and kinase assays using ectopic expression of TRAF1 indicated that it can have both activating and inhibiting functions for IKK2 and NF-kappa B. Co-expression of fluorescently tagged TRAF1 and TRAF2 at different ratios implied that TRAF1 can affect clustering and presumably the activating function of TRAF2 in a dose dependent manner

Sorption and activation of hydrocarbons by molecular sieves

Substantial progress has been made recently in the understanding of sorption and activation of alkanes. This progress reflects the emergence of new theoretical and experimental results, leading to a more quantitative picture of the elementary steps involved in the ordering of alkanes in molecular sieves and their chemical interaction with the acid site. Conversion of n-alkanes over various zeolites is now well understood to depend mainly upon the concentration of reactants sorbed. The sorption enthalpy and entropy of these molecules are linearly related and this relationship is characteristic of a particular molecular sieve (compensation effect). The interfacial chemistry that alkanes and alkenes undergo involves ionic species only in their transition state, whereas the stable intermediates are covalently bound. This leads to a description of the chemical transformations that resemble nucleophilic and electrophilic substitutions