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Bastian Welz,* Rolf Bikker,* Leonie Hoffmeister, Mareike Diekmann, Martin Christmann, Korbinian Brand,* René Huber* Institute of Clinical Chemistry, Hannover Medical School, Hannover, 30625, Germany*These authors contributed equally to this workCorrespondence: Korbinian BrandInstitute of Clinical Chemistry, Hannover Medical School, Carl-Neuberg Str. 1, Hannover, 30625, GermanyTel +49 511 532 6614Fax +49 511 532 8614Email brand.korbinian@mh-hannover.deBackground: Termination of TNF-induced signaling plays a key role in the resolution of inflammation with dysregulations leading to severe pathophysiological conditions (sepsis, chronic inflammatory disease, cancer). Since a recent phospho-proteome analysis in human monocytes suggested GSK3 as a relevant kinase during signal termination, we aimed at further elucidating its role in this context.Materials and Methods: For the analyses, THP-1 monocytic cells and primary human monocytes were used. Staurosporine (Stauro) was applied to activate GSK3 by inhibiting kinases that mediate inhibitory GSK3α/β-Ser21/9 phosphorylation (eg, PKC). For GSK3 inhibition, Kenpaulone (Ken) was used. GSK3- and PKC-siRNAs were applied for knockdown experiments. Protein expression and phosphorylation were assessed by Western blot or ELISA and mRNA expression by qPCR. NF-κB activation was addressed using reporter gene assays.Results: Constitutive GSK3β and PKCβ expression and GSK3α/β-Ser21/9 and PKCα/βII-Thr638/641 phosphorylation were not altered during TNF long-term incubation. Stauro-induced GSK3 activation (demonstrated by Bcl3 reduction) prevented termination of TNF-induced signaling as reflected by strongly elevated IL-8 expression (used as an indicator) following TNF long-term incubation. A similar increase was observed in TNF short-term-exposed cells, and this effect was inhibited by Ken. PKCα/β-knockdown modestly increased, whereas GSK3α/β-knockdown inhibited TNF-induced IL-8 expression. TNF-dependent activation of two NF-κB-dependent indicator plasmids was enhanced by Stauro, demonstrating transcriptional effects. A TNF-induced increase in p65-Ser536 phosphorylation was further enhanced by Stauro, whereas IκBα proteolysis and IKKα/β-Ser176/180 phosphorylation were not affected. Moreover, PKCβ-knockdown reduced levels of Bcl3. A20 and IκBα mRNA, both coding for signaling inhibitors, were dramatically less affected under our conditions when compared to IL-8, suggesting differential transcriptional effects.Conclusion: Our results suggest that GSK3 activation is involved in preventing the termination of TNF-induced signaling. Our data demonstrate that activation of GSK3 – either pathophysiologically or pharmacologically induced – may destroy the finely balanced condition necessary for the termination of inflammation-associated signaling.Keywords: TNF, GSK3, PKC, staurosporine, IL-8, NF-κB, termination of TNF-induced signaling, termination of inflammation

The results of an investigation of the three-dimensional distribution of radiant intensity in the probing radiation beam produced in a conventional atomic absorption spectrometer by hollow cathode lamps and electrodeless discharge lamps are presented. The investigation is based on the use of a photodiode array-based digital imaging system. The results obtained revealed that the probing radiation beam is highly non-uniform, both longitudinally and radially. The character of the non-uniformities is greatly dependent on the type of the primary source. An interpretation of the results is given and the possible consequences of the radiation beam non-uniformities for the application of Beer-Lambert's law are discussed.

The spatial distribution of radiant intensity from electrodeless discharge lamps (EDLs) used as radiation sources in atomic absorption spectrometry is investigated with a digital photodiode array imaging system. Intensity distribution over the radial and longitudinal sections of Pb and Hg lamps is measured for both atomic and ionic lines of the analyte and the filler gas. The plasma in the EDLs is highly structured, with metal and filler gas excited species being distributed nonuniformly but in different ways. The clouds of emitting metal and Ar atoms are spatially separated in the volume of the Pb EDL. The excited Pb atoms detected from both the resonance and nonresonance lines have the form of a thin layer concentric to the bulb walls located near the surface of the bulb ("optical skin effect"). In contrast, the emission distribution for Ar atomic lines is bell-shaped with a maximum at the center of the plasma. The spatial distribution of emitting Ar ions is more complex - there is a bulk maximum coinciding with Ar atomic emission maximum and another maximum concentric to the walls coinciding with the maximum of metal atom emission. In the Hg EDL the difference between the spatial intensity profiles of metal and filler gas (Ar) lines is less pronounced because of the use of an increased filler gas pressure in the lamp. Emitting species of both Ar and metal are primarily located in the bulk of the plasma with, however, a small depletion in the vicinity of the lamp axis. Evolution of the spatial intensity profiles during warm-up of the lamps is investigated as well. In both lamps the radial and longitudinal intensity distributions of metal lines are established during the first minutes after lamp ignition, after which there is a slow and monotonic increase of the established intensity profiles. This result implies thermal vaporization as a mechanism of analyte supply to the plasma. The spatial intensity profiles for Ar lines are established in the first seconds after lamp ignition, after which only the absolute values of the established distributions change. The approach to the steady-state intensity of Ar atomic and ionic lines is nonmonotonic; there is a clearly pronounced initial overshoot in intensity of Ar atomic lines that coincides with a decline in the intensity of Ar ion lines. An interpretation for the observed spatial intensity profiles is given on the basis of radial cataphoresis theory.

Spatial non-uniformities in analyte, atomizer gas phase temperature and radiant intensity distributions characteristic for graphite furnace atomic absorption spectrometry are briefly summarized and their effect on the analyte detection is analysed. It is shown that conventional detection of analyte based on the use of photomultiplier tubes provides excellent temporal resolution, sufficient wavelength isolation but totally ignores the spatial aspects of the interaction of the probing radiation beam with the analyte in the atomizer. A new, spatially resolved, method of analyte detection based on the use of a solid state detector located along the monochromator slit is presented. The approach is illustrated by the temporally and spatially resolved detection of Cd atomization and NaCl vaporization. It is shown that severe non-uniformities in atomic and/or background absorbance may be a potential source of analytical error. Advantages of spatially resolved detection as compared with conventional detection are discussed.