Bacterial Ammonia Causes Significant Plant Growth Inhibition

<div><p>Many and complex plant-bacteria inter-relationships are found in the rhizosphere, since plants release a variety of photosynthetic exudates from their roots and rhizobacteria produce multifaceted specialized compounds including rich mixtures of volatiles, e.g., the bouquet of <i>Serratia odorifera</i> 4Rx13 is composed of up to 100 volatile organic and inorganic compounds. Here we show that when growing on peptone-rich nutrient medium <i>S. odorifera</i> 4Rx13 and six other rhizobacteria emit high levels of ammonia, which during co-cultivation in compartmented Petri dishes caused alkalization of the neighboring plant medium and subsequently reduced the growth of <i>A. thaliana</i>. It is argued that in nature high-protein resource degradations (carcasses, whey, manure and compost) are also accompanied by bacterial ammonia emission which alters the pH of the rhizosphere and thereby influences organismal diversity and plant-microbe interactions. Consequently, bacterial ammonia emission may be more relevant for plant colonization and growth development than previously thought.</p></div

Metabolic Profiling Reveals Sphingosine-1-Phosphate Kinase 2 and Lyase as Key Targets of (Phyto-) Estrogen Action in the Breast Cancer Cell Line MCF-7 and Not in MCF-12A

<div><p>To search for new targets of anticancer therapies using phytoestrogens we performed comparative metabolic profiling of the breast cancer cell line MCF-7 and the non-tumorigenic breast cell line MCF-12A. Application of gas chromatography-mass spectrometry (GC-MS) revealed significant differences in the metabolic levels after exposure with 17ß-estradiol, genistein or a composition of phytoestrogens within a native root flax extract. We observed the metabolites 3-(4-hydroxyphenyl)-lactic acid, cis-aconitic acid, 11-beta-hydroxy-progesterone, chenodeoxycholic acid and triacontanoic acid with elevated levels due to estrogen action. Particularly highlighted were metabolites of the sphingolipid metabolism. Sphingosine and its dihydro derivate as well as ethanolaminephosphate were significantly altered after exposure with 1 nM 17ß-estradiol in the cell line MCF-7, while MCF-12A was not affected. Treatment with genistein and the flax extract normalized the sphingosine concentrations to the basic levels found in MCF-12A cells. We could further demonstrate that the expression levels of the sphingosine metabolizing enzymes: sphingosine-1-phosphate kinase (Sphk) and lyase (S1P lyase) were significantly influenced by estrogens as well as phytoestrogens. The isoform Sphk2 was overexpressed in the tumorigenic cell line MCF-7, while S1P lyase was predominantly expressed in the non-tumorigenic cell line MCF-12A. Importantly, in MCF-7 the weak S1P lyase expression could be significantly increased after exposure with 10 µM genistein and 1 µg/ml root flax extract. Here, we present, for the first time, an analysis of metabolic response of phytoestrogens to breast cancer cell lines. The contrasting regulation of sphingolipid enzymes in MCF-7 and MCF-12A render them as preferred targets for future anticancer strategies.</p> </div

Alterations in sphingolipid metabolites.

<p>Boxplots of absolute metabolite levels for sphingosine, dihydrosphingosine and ethanolaminephosphate under the treatment of 17ß-estradiol (E), genistein (G) and flax extract (L) in the cell lines MCF-12A and MCF-7. Each box plot shows the median, and upper and lower quartiles. Outlier values are indicated by individual data points.</p

Expression regulation of Sphk1 and Sphk2.

<p>Western blotting (<b>A</b>, <b>D</b>), quantification of western blotting results (<b>B</b>, <b>E</b>) and immunofluorescence staining (<b>C</b>, <b>F</b>) of the sphingosine-1-phosphate kinase isoform 1 and 2 (Sphk1; Sphk2) expression level after 48 h exposure to 17ß-estradiol (E), genistein (G) and the root flax extract (L) at different concentration in the cell lines MCF-7 and MCF-12A. Western blotting and immunofluorescence staining were carried out with same primary antibody and were repeated at least three times with individual passaged cells. Single representative western blot and fluorescence images were displayed. SPHK expression in the immunofluorescence pictures was taken with a constant exposure time of 5.6 s for Sphk1 and 2.0 s Sphk2 (green); nucleus (blue). Mean ± SD values (n = 3−5). * = <i>p</i><0.01; ** = <i>p</i><0.001 as compared to EtOH control (unpaired <i>t</i> test).</p

Heat map of metabolites comparing between MCF-12A and MCF-7.

<p>Principal component analysis (PCA) and heat map presentation of metabolite profiles from MCF-7 cells (squares) and MCF-12A (circles) treated with 0.1% EtOH (C, white), 1 nM 17β-estradiol (E, red), 1 µM genistein (G1, light green), 10 µM genistein (G2, dark green), 0.01 µg/ml <i>Linum usitatissimum</i> (L1, light blue) and 1 µg/ml <i>Linum usitatissimum</i> (L2, dark blue) for 48 hours. <b>A</b>: For log10-transformed values the main sources of variance are cell type (PC1, 49%) and treatment (PC2, 21%). <b>B</b>: For data expressed relative to the respective control samples the main sources of variance are treatment (PC1, 38%) and treatment within cell type (PC2, 16%). <b>C</b>: Heat map presentation of all identified metabolites by GC-MS based profiling under the treatment with 17ß-estradiol (E), genistein (G) and flax extract (L) in the cell lines MCF-12A and MCF-7 (see color bar for scale; blue color indicates down regulated metabolites; red color indicates up regulated metabolites). The data were normalized to the untreated control of MCF-12A to identify significantly altered metabolites in the tumorigenic cell line MCF-7. Metabolites were grouped according to cell line and each treatment condition (indicated with specific colors in the upper panel) and hierarchically clustered. Correlation coefficients were calculated by applying the Pearson algorithm using R software.</p

Expression regulation of Sphingosine lyase.

<p>Western blotting (<b>A</b>), quantification of western blotting results (<b>B</b>) and immunofluorescence staining (<b>C</b>) of the sphingosine-1-phosphate lyase (S1P lyase) expression level after 48 h exposure to 17ß-estradiol (E), genistein (G) and the root flax extract (L) at different concentration in the cell lines MCF-7 and MCF-12A. Western blotting and immunofluorescence staining were carried out with same primary antibody and were repeated at least three times with individual passaged cells. Single representative western blot and fluorescence images were displayed. S1P lyase expression in the immunofluorescence pictures was taken with a constant exposure time of 2.3 s. S1P lyase (green); nucleus (blue).Mean ± SD values (n = 3−5). * = <i>p</i><0.01; ** = <i>p</i><0.001 as compared to EtOH control (unpaired <i>t</i> test).</p

Online monitoring of cell metabolism.

<p>Online monitoring of cell metabolism in the cancerous cell line MCF-7 after exposure with 1 µM sphingosine-1-phosphate and 1 µM D-sphingosine. Displayed were the percentage of the standardized and normalized rates of cell adhesion (impedance), respiration (O₂ consumption) and extracellular acidification in a period of 24 h. Displayed were three individual replicates. Grey shadowed area marks the adaption phase (4 h) of the cells to the new conditions.</p

List of the ranked metabolites.

<p>Ranking of the 29 metabolites affected stronger by 17ß-estradiol in the tumorigenic cell line MCF-7 compared to the non-tumorigenic cell line MCF-12A. Related pathways and the KEGG and/or PubChem number are given.</p