Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, antigen presentation, modulation of apoptosis, and shedding of obsolete protein.Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are significantly enriched for innate immune proteins that include antimicrobial proteins and peptides and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm-Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans. Thus, exosomes are innate immune effectors that contribute to host defense within the urinary tract.
The potential for a variety of environmental contaminants to disturb endocrine function in wildlife and humans has been of recent concern. While much effort is being focused on the assessment of effects mediated through steroid hormone receptor-based mechanisms, there are potentially several other mechanisms that could lead to endocrine disruption. Recent studies have demonstrated that a variety of xenobiotics can alter the gene expression or activity of enzymes involved in steroidogenesis. By altering the production or catalytic activity of steroidogenic or steroid-catabolizing enzymes, these chemicals have the potential to alter the steroid balance in organisms. To assess the potential of chemicals to alter steroidogenesis, an assay system was developed using a human adrenocortical carcinoma cell line, the H295R cell line, which retains the ability to synthesize most of the important steroidogenic enzymes. Methods were developed, optimized, and validated to measure the expression of 10 genes involved in steroidogenesis by the use of real-time quantitative reverse transcriptase PCR. The effects of several model chemicals known to alter steroid metabolism, both inducers and inhibitors, were assessed. Similar expression patterns were observed for chemicals acting through common mechanisms of action. Time-course studies demonstrated distinct time-dependent expression profiles for chemicals able to modulate steroid metabolism. The assay, which allows simultaneous analysis of the expression of numerous steroidogenic enzymes, would be useful as a sensitive and integrative screen for the many effects of chemicals on steroidogenesis.
Gene expression profiles show considerable promise for the evaluation of the toxic potential of environmental contaminants. For example, any alterations in the pathways of steroid synthesis or breakdown have the potential to Cause endocrine disruption. Therefore monitoring these pathways can provide information relative to a chemical's ability to impact endocrine function. One approach to monitoring these pathways has been to use a human adrenocortical carcinoma cell line (H295R) that expresses all the key enzymes necessary for steroidogenesis. In this study we have further developed these methods using accurate and specific quantification methods utilizing molecular beacon-based quantitative RT-PCR (Q-RT-PCR). The assay system was used to analyze the expression patterns of 11 steroidogenic genes in H295R cells. The expression of gene transcripts was measured using a real-time PCR system and quantified based on both a standard curve method using a dilution series of RNA standards and a comparative Ct method. To validate the optimized method, cells were exposed to specific and nonspecific model compounds (inducers and inhibitors of various steroidogenic enzymes) for gene expression profiling. Similar gene expression profiles were exhibited by cells treated with chemicals acting through common mechanisms of action. Overall, our findings demonstrated that the present assay can facilitate the development of compound-specific response profiles, and will provide a sensitive and integrative screen for the effects of chemicals on steroidogenesis.
Nuclear receptor binding protein 1 regulates intestinal progenitor cell homeostasis and tumour formationArising from a ras-interaction screen in C. elegans, nuclear receptor binding protein 1 (NRBP1) is shown to impose a crypt progenitor phenotype in mice and is proposed as a novel tumour suppressor in human cancer.
Exosomes derived from all nephron segments are present in human urine, where their functionality is incompletely understood. Most studies have focused on biomarker discovery rather than exosome function. Through sequencing we identified the miRNA repertoire of urinary exosomes from healthy volunteers; 276 mature miRNAs and 345 pre-miRNAs were identified (43%/7% of reads). Among the most abundant were members of the miR-10, miR-30 and let-7 families. Targets for the identified miRNAs were predicted using five different databases; genes encoding membrane transporters and their regulators were enriched, highlighting the possibility that these miRNAs could modulate key renal tubular functions in a paracrine manner. As proof of concept, cultured renal epithelial cells were exposed to urinary exosomes and cellular exosomal uptake was confirmed; thereafter, reduced levels of the potassium channel ROMK and kinases SGK1 and WNK1 were observed in a human collecting duct cell line, while SPAK was unaltered. In proximal tubular cells, mRNA levels of the amino acid transporter gene SLC38A2 were diminished and reflected in a significant decrement of its encoded protein SNAT2. Protein levels of the kinase SGK1 did not change. Thus we demonstrated a novel potential function for miRNA in urinary exosomes.Urinary exosomes are lipid membrane-bound nanovesicles released from intracellular multivesicular bodies (MVBs) 1,2 and derived from all cells in the urinary tract [3][4][5] . During the inward budding of endosomes that give origin to exosomes, proteins 2 , mRNAs 6 , microRNAs (miRNAs) 7 , noncoding RNA (ncRNA) 8 , transcription factors 9 and other biomolecules present in the cytosol can be incorporated. The lipid bi-layer of these nanovesicles provides the cargo with stable storage conditions and protects it from degradation by extracellular proteases and ribonucleases 10. Studies in other tissues have shown that once exosomes and other microvesicles are released into the extracellular environment, interactions with cells can occur by direct ligand-receptor signalling, by exosomal fusion to the target cell membrane and discharge of exosomal content directly into the cytoplasm, or via phagocytosis/macropinocytosis 11,12 . Exosomes are known to deliver biologic cargo not only to neighbouring cells but also long distance 13 . The majority of studies concerning urinary exosomes have focused on their potential as biomarkers of disease pathology and progression, including prostate and bladder cancers [14][15][16][17] , but their functional significance is now being addressed. Inter-cellular signalling by exosomes in cultured murine renal epithelial cells was demonstrated for the first time by Street et al. 18 , who suggested that collecting duct cell-derived exosomes can transfer the ability to express AQP2. Our previous studies revealed that urinary exosomes inhibit bacterial growth of both commensal and uropathogenic E. coli by inducing bacterial lysis 19 . Bruschi and colleagues demonstrated that urinary exosomes can consume ...
We report a high quality and system-wide proteome catalogue covering 71% (3,542 proteins) of the predicted genes of fission yeast, Schizosaccharomyces pombe, presenting the largest protein dataset to date for this important model organism. We obtained this high proteome and peptide (11.4 peptides/protein) coverage by a combination of extensive sample fractionation, high resolution Orbitrap mass spectrometry, and combined database searching using the iProphet software as part of the Trans-Proteomics Pipeline. All raw and processed data are made accessible in the S. pombe PeptideAtlas. The identified proteins showed no biases in functional properties and allowed global estimation of protein abundances. The high coverage of the PeptideAtlas allowed correlation with transcriptomic data in a system-wide manner indicating that post-transcriptional processes control the levels of at least half of all identified proteins. Interestingly, the correlation was not equally tight for all functional categories ranging from rs >0.80 for proteins involved in translation to rs <0.45 for signal transduction proteins. Moreover, many proteins involved in DNA damage repair could not be detected in the PeptideAtlas despite their high mRNA levels, strengthening the translation-on-demand hypothesis for members of this protein class. In summary, the extensive and publicly available S. pombe PeptideAtlas together with the generated proteotypic peptide spectral library will be a useful resource for future targeted, in-depth, and quantitative proteomic studies on this microorganism.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.