Hydrogen Peroxide Stimulates Exosomal Cathepsin B Regulation of the Receptor for Advanced Glycation End-Products (RAGE)

Exosomes are nano-sized vesicles that are secreted into the extracellular environment. These vesicles contain various biological effector molecules that can regulate intracellular signaling pathways in recipient cells. The aim of this study was to examine a correlation between exosomal cathepsin B activity and the receptor for advanced glycation end-products (RAGE). Type 1 alveolar epithelial (R3/1) cells were treated with or without hydrogen peroxide and exosomes isolated from the cell conditioned media were characterized by NanoSight analysis. Lipidomic and proteomic analysis showed exosomes released from R3/1 cells exposed to oxidative stress induced by hydrogen peroxide or vehicle differ in their lipid and protein content, respectively. Cathepsin B activity was detected in exosomes isolated from hydrogen peroxide treated cells. The mRNA and protein expression of RAGE increased in cultured R3/1 cells treated with exosomes containing active cathepsin B while depletion of exosomal cathepsin B attenuated RAGE mRNA and protein expression. These results suggest exosomal cathepsin B regulates RAGE in type 1 alveolar cells under conditions of oxidative stress. J. Cell. Biochem. 119: 599–606, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc

Mammalian mitochondrial ribosomal proteins (2) - Amino acid sequencing, characterization, and identification of corresponding gene sequences

Four different classes of mammalian mitochondrial ribosomal proteins were identified and characterized. Mature proteins were purified from bovine liver and subjected to N-terminal or matrix-assisted laser-desorption mass spectroscopic amino acid sequencing after tryptic in-gel digestion and high pressure liquid chromatography separation of the resulting peptides. Peptide sequences obtained were used to virtually screen expressed sequence tag data bases from human, mouse, and rat. Consensus cDNAs were assembled in silico from various expressed sequence tag sequences identified. Deduced mammalian protein sequences were characterized and compared with ribosomal protein sequences of Escherichia coli and yeast mitochondria. Significant sequence similarities to ribosomal proteins of other sources were detected for three out of four different mammalian protein classes determined. However, the sequence conservation between mitochondrial ribosomal proteins of mammalian and yeast origin is much less than the sequence conservation between cytoplasmic ribosomal proteins of the same species. In particular, this is shown for the mammalian counterparts of the E. coli EcoL2 ribosomal protein (MRP-L14), that do not conserve the specific and functional highly important His(229) residue of E. coli and the corresponding yeast mitochondrial Rml2p

Effect-based tools for monitoring (xeno)estrogens in surface waters: Variability and reproducibility of sample preparation and five different in vitro assays

In vitro bioassays are increasingly used to assess estrogenic activity of environmental water samples and have been suggested as suitable tools for monitoring estrogenic contamination of surface waters. Such assays are of particular use as they measure the overall estrogenic activity of a sample, including the potent steroids 17ß-estradiol (E2) and 17a-ethinyl estradiol (EE2); proposed annual average Environmental Quality Standards (AA-EQS) are 400 and 35 pg/L, respectively. For most routine chemical methods these EQS are below analytical limits of quantification, resulting in difficulties for monitoring E2 and EE2 under the watchlist mechanism of the Water Framework Directive. The use of sensitive in vitro assays could circumvent current detection problems by measuring the total activation potential of estrogenic substances in environmental samples by expressing overall estrogenicity in E2-equivalents (EEQs). It has been shown, however, that different assays lead to different EEQs for the same sample. Reasons for these differences are known, for example sensitivity differences of the assays towards certain substancesbut it remains unclear how to use and interpret bioassay results. Hence the aims of this study are to (1) compare EEQs of reconstituted water samples and extracts assessed by five commonly used bioassays in order to determine the variability and reproducibility of the assays and the sample preparation method (solid phase extraction) and (2) get insights into their validity for environmental monitoring. Initial results for three different bioassays show that inter-day reproducibility of derived EEQs varied between 2.5 and 30%. Comparison of the results from different in vitro assays showed that all assays were able to correctly detect the EEQ of the positive control. Only the ER-CALUX was able to derive EEQs close to the calculated EEQs for the reconstituted samples. The 10x concentration difference between the two reconstituted samples was detected by the ER-CALUX and the T47D-Kbluc. The YES underestimated the estrogenic load of these samples, whereas the T47D-Kbluc overestimated the EEQs of both mixtures. Data from the MELN and GeneBLAzer ER assays are yet to be analyzed. Overall, our findings suggest that in vitro bioassays are comparable to chemical measurements regarding variability and reproducibility of the derived EEQ concentrations