Osmotic stress-dependent serine phosphorylation of the histidine kinase homologue DokA

BACKGROUND: Two-component systems consisting of histidine kinases and their corresponding receivers are widespread in bacterial signal transduction. In the past few years, genes coding for homologues of two-component systems were also discovered in eukaryotic organisms. DokA, a homologue of bacterial histidine kinases, is an element of the osmoregulatory pathway in the amoeba Dictyostelium. The work described here addresses the question whether DokA is phosphorylated in vivo in response to osmotic stress. RESULTS: We have endogenously overexpressed individual domains of DokA to investigate post-translational modification of the protein in response to osmotic shock in vivo. Dictyostelium cells were labeled with [(32)P]-orthophosphate, exposed to osmotic stress and DokA fragments were subsequently isolated by immunoprecipitation. Thus, a stress-dependent phosphorylation could be demonstrated, with the site of phosphorylation being located in the kinase domain. We demonstrate biochemically that the phosphorylated amino acid is serine, and by mutational analysis that the phosphorylation reaction is not due to an autophosphorylation of DokA. Furthermore, mutation of the conserved histidine did not affect the osmostress-dependent phosphorylation reaction. CONCLUSIONS: A stimulus-dependent serine phosphorylation of a eukaryotic histidine kinase homologue was demonstrated for the first time in vivo. That implies that DokA, although showing typical structural features of a bacterial two-component system, might be part of a eukaryotic signal transduction pathway that involves serine/threonine kinases

Laminar-Turbulent Transition Localization in Thermographic Flow Visualization by Means of Principal Component Analysis

Thermographic flow visualization is a contactless, non-invasive technique to visualize the boundary layer flow on wind turbine rotor blades, to assess the aerodynamic condition and consequently the efficiency of the entire wind turbine. In applications on wind turbines in operation, the distinguishability between the laminar and turbulent flow regime cannot be easily increased artificially and solely depends on the energy input from the sun. State-of-the-art image processing methods are able to increase the contrast slightly but are not able to reduce systematic gradients in the image or need excessive a priori knowledge. In order to cope with a low-contrast measurement condition and to increase the distinguishability between the flow regimes, an enhanced image processing by means of the feature extraction method, principal component analysis, is introduced. The image processing is applied to an image series of thermographic flow visualizations of a steady flow situation in a wind tunnel experiment on a cylinder and DU96W180 airfoil measurement object without artificially increasing the thermal contrast between the flow regimes. The resulting feature images, based on the temporal temperature fluctuations in the images, are evaluated with regard to the global distinguishability between the laminar and turbulent flow regime as well as the achievable measurement error of an automatic localization of the local flow transition between the flow regimes. By applying the principal component analysis, systematic temperature gradients within the flow regimes as well as image artefacts such as reflections are reduced, leading to an increased contrast-to-noise ratio by a factor of 7.5. Additionally, the gradient between the laminar and turbulent flow regime is increased, leading to a minimal measurement error of the laminar-turbulent transition localization. The systematic error was reduced by 4% and the random error by 5.3% of the chord length. As a result, the principal component analysis is proven to be a valuable complementary tool to the classical image processing method in flow visualizations. After noise-reducing methods such as the temporal averaging and subsequent assessment of the spatial expansion of the boundary layer flow surface, the PCA is able to increase the laminar-turbulent flow regime distinguishability and reduce the systematic and random error of the flow transition localization in applications where no artificial increase in the contrast is possible. The enhancement of contrast increases the independence from the amount of solar energy input required for a flow evaluation, and the reduced errors of the flow transition localization enables a more precise assessment of the aerodynamic condition of the rotor blade

Osmotic stress response in Dictyostelium is mediated by cAMP

DokA, a homolog of bacterial hybrid histidine kinases, is essential for hyperosmotic stress resistance in Dictyostelium. We show that a transient intracellular cAMP signal, dependent on the presence of DokA, is generated in response to an osmotic shock. This variation of cAMP levels contributes to survival under hypertonic conditions. In contrast to the low cAMP levels observed in dokA(–) strains, overexpression of the receiver domain of DokA causes an increase in cAMP levels, resulting in a rapidly developing phenotype. We present biochemical and cell biological data indicating that the DokA receiver domain is a dominant-negative regulator of a phosphorelay, which controls the intracellular cAMP phosphodiesterase RegA. The activity of the DokA receiver domain depends on a conserved aspartate, mutation of which reverses the developmental phenotype, as well as the deregulation of cAMP metabolism

Mimicking hypoxia to treat anemia: HIF-stabilizer BAY 85-3934 (Molidustat) stimulates erythropoietin production without hypertensive effects.

Oxygen sensing by hypoxia-inducible factor prolyl hydroxylases (HIF-PHs) is the dominant regulatory mechanism of erythropoietin (EPO) expression. In chronic kidney disease (CKD), impaired EPO expression causes anemia, which can be treated by supplementation with recombinant human EPO (rhEPO). However, treatment can result in rhEPO levels greatly exceeding the normal physiological range for endogenous EPO, and there is evidence that this contributes to hypertension in patients with CKD. Mimicking hypoxia by inhibiting HIF-PHs, thereby stabilizing HIF, is a novel treatment concept for restoring endogenous EPO production. HIF stabilization by oral administration of the HIF-PH inhibitor BAY 85-3934 (molidustat) resulted in dose-dependent production of EPO in healthy Wistar rats and cynomolgus monkeys. In repeat oral dosing of BAY 85-3934, hemoglobin levels were increased compared with animals that received vehicle, while endogenous EPO remained within the normal physiological range. BAY 85-3934 therapy was also effective in the treatment of renal anemia in rats with impaired kidney function and, unlike treatment with rhEPO, resulted in normalization of hypertensive blood pressure in a rat model of CKD. Notably, unlike treatment with the antihypertensive enalapril, the blood pressure normalization was achieved without a compensatory activation of the renin-angiotensin system. Thus, BAY 85-3934 may provide an approach to the treatment of anemia in patients with CKD, without the increased risk of adverse cardiovascular effects seen for patients treated with rhEPO. Clinical studies are ongoing to investigate the effects of BAY 85-3934 therapy in patients with renal anemia

Experimental <it>in vivo</it> and <it>in vitro</it> treatment with a new histone deacetylase inhibitor belinostat inhibits the growth of pancreatic cancer

Abstract Background Treatment options for pancreatic ductal adenocarcinoma (PDAC) are limited. Histone deacetylase inhibitors are a new and promising drug family with strong anticancer activity. The aim of this study was to examine the efficacy of in vitro and in vivo treatment with the novel pan-HDAC inhibitor belinostat on the growth of human PDAC cells. Methods The proliferation of tumour cell lines (T3M4, AsPC-1 and Panc-1) was determined using an MTT assay. Apoptosis was analysed using flow cytometry. Furthermore, p21Cip1/Waf1 and acetylated histone H4 (acH4) expression were confirmed by immunoblot analysis. The in vivo effect of belinostat was studied in a chimeric mouse model. Antitumoural activity was assessed by immunohistochemistry for Ki-67. Results Treatment with belinostat resulted in significant in vitro and in vivo growth inhibition of PDAC cells. This was associated with a dose-dependent induction of tumour cell apoptosis. The apoptotic effect of gemcitabine was further enhanced by belinostat. Moreover, treatment with belinostat increased expression of the cell cycle regulator p21Cip1/Waf1 in Panc-1, and of acH4 in all cell lines tested. The reductions in xenograft tumour volumes were associated with inhibition of cell proliferation. Conclusion Experimental treatment of human PDAC cells with belinostat is effective in vitro and in vivo and may enhance the efficacy of gemcitabine. A consecutive study of belinostat in pancreatic cancer patients alone, and in combination with gemcitabine, could further clarify these effects in the clinical setting.</p

Effects of BAY 85-3934 administration in male Wistar rats treated with gentamicin to induce renal anemia.

<p>Data are presented as means ± SEM. (A) Plasma EPO levels, and (B) kidney and (C) liver relative expression of erythropoietin (EPO) mRNA 4 h after oral administration of BAY 85-3934. Before administration, rats had been treated with vehicle or gentamicin. (<i>n</i> = 5 animals per group). *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001 compared with vehicle group,^§<i>p</i><0.05 and ^§§<i>p</i><0.001 compared with control group, t-test. (D) Kidney and (E) liver mRNA expression levels of hypoxia-inducible factor target genes relative to mean of vehicle treated animals after oral administration of BAY 85-3934 (<i>n</i> = 4 to 5 animals per group, error bars not shown for clarity of presentation). For definition of gene symbols, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111838#pone-0111838-t001" target="_blank">Table 1</a>. (F) Time-course of changes in packed cell volume (PCV) following treatment with BAY 85-3934 or vehicle (once daily, five times per week, number of animals as indicated). (G) Hemoglobin levels 7 days after start of treatment with BAY 85-3934 or vehicle at day 22 of experiment shown in (F). *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001 compared with vehicle group; ^#<i>p</i><0.05 compared with sham group; t-test.</p

Characterization of the <i>in vivo</i> activity of BAY 85-3934 (in male Wistar rats).

<p>Data are presented as means ± SEM. (A) Increase in plasma erythropoietin (EPO) at 4 h and (B) reticulocytes (as a proportion of red blood cells [RBCs]) at 72 h following single oral dosing of BAY 85-3934. Data were pooled from two sequential experiments (<i>n</i> = 2×5 animals per group). *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001; unpaired t-test, sequentially pairwise-applied to dose groups and corresponding vehicle group. (C) Change in packed cell volume (PCV) during once-daily dosing with BAY 85-3934 (<i>n</i> = 12 animals per group). **<i>p</i><0.01 and ***<i>p</i><0.001; two-way ANOVA with Dunnett’s multiple comparison test versus vehicle group. (D) Induction of erythropoiesis after subcutaneous administration of recombinant human EPO (rhEPO) twice weekly or BAY 85-3934 (2.5 mg/kg) once daily (<i>n</i> = 10 animals per group). *<i>p</i><0.001 compared with control (t-test) at day 30. (E) BAY 85-3934 plasma levels, kidney EPO relative mRNA expression, and plasma EPO levels after oral administration of BAY 85-3934 (5 mg/kg) (<i>n</i> = 5 animals per group). (F) Relative mRNA expression levels of HIF target genes in rat kidney after administration of BAY 85-3934 (5 mg/kg). Baseline expression was set at 1 (<i>n</i> = 5 animals per group; error bars not show for clarity of presentation). For definition of gene symbols, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111838#pone-0111838-t001" target="_blank">Table 1</a>.</p

Oligonucleotide primers and probes used for quantitative RT-PCR analysis of samples from rat tissues, and human cell lines (in italics).

<p>Oligonucleotide primers and probes used for quantitative RT-PCR analysis of samples from rat tissues, and human cell lines (in italics).</p