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      Uremic Toxin Lanthionine Interferes with the Transsulfuration Pathway, Angiogenetic Signaling and Increases Intracellular Calcium

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          Abstract

          (1) The beneficial effects of hydrogen sulfide (H 2S) on the cardiovascular and nervous system have recently been re-evaluated. It has been shown that lanthionine, a side product of H 2S biosynthesis, previously used as a marker for H 2S production, is dramatically increased in circulation in uremia, while H 2S release is impaired. Thus, lanthionine could be classified as a novel uremic toxin. Our research was aimed at defining the mechanism(s) for lanthionine toxicity. (2) The effect of lanthionine on H 2S release was tested by a novel lead acetate strip test (LAST) in EA.hy926 cell cultures. Effects of glutathione, as a redox agent, were assayed. Levels of sulfane sulfur were evaluated using the SSP4 probe and flow cytometry. Protein content and glutathionylation were analyzed by Western Blotting and immunoprecipitation, respectively. Gene expression and miRNA levels were assessed by qPCR. (3) We demonstrated that, in endothelial cells, lanthionine hampers H 2S release; reduces protein content and glutathionylation of transsulfuration enzyme cystathionine-β-synthase; modifies the expression of miR-200c and miR-423; lowers expression of vascular endothelial growth factor VEGF; increases Ca 2+ levels. (4) Lanthionine-induced alterations in cell cultures, which involve both sulfur amino acid metabolism and calcium homeostasis, are consistent with uremic dysfunctional characteristics and further support the uremic toxin role of this amino acid.

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          H2S signals through protein S-sulfhydration.

          Hydrogen sulfide (H2S), a messenger molecule generated by cystathionine gamma-lyase, acts as a physiologic vasorelaxant. Mechanisms whereby H2S signals have been elusive. We now show that H2S physiologically modifies cysteines in a large number of proteins by S-sulfhydration. About 10 to 25% of many liver proteins, including actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are sulfhydrated under physiological conditions. Sulfhydration augments GAPDH activity and enhances actin polymerization. Sulfhydration thus appears to be a physiologic posttranslational modification for proteins.
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            The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA.

            Vascular endothelial growth factor (VEGF) was recently identified as a secreted, direct-acting mitogen specific for vascular endothelial cells and capable of stimulating angiogenesis in vivo. Molecular cloning revealed multiple forms of VEGF, apparently arising from alternative splicing of its RNA transcript. We have examined various human cDNA libraries by the polymerase chain reaction technique and discovered a fourth molecular form, VEGF206. This form contains a 41-amino acid insertion relative to the most abundant form, VEGF165, and includes the highly basic 24-amino acid insertion found in VEGF189. Southern blot analysis revealed that a single gene encoded these various forms, and nucleic acid sequence analysis of a portion of the VEGF gene revealed an intron/exon structure compatible with alternative splicing of RNA as a mechanism for their generation. Transient transfection of human embryonic kidney 293 cells showed that, like VEGF189, VEGF206 was predominately cell-associated and only very poorly secreted despite the presence of the signal peptide identical to that found in VEGF121 and VEGF165, both of which are efficiently exported from the cell. Vascular permeability activity was detected in the medium of 293 cells transfected with all four forms of VEGF; however, endothelial cell mitogenic activity was apparent only with VEGF121 and VEGF165. Thus, alternative splicing of VEGF RNA can produce four polypeptides with strikingly different secretion patterns, which suggests multiple physiological roles for this family of proteins.
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              Isolation and Quantification of MicroRNAs from Urinary Exosomes/Microvesicles for Biomarker Discovery

              Recent studies indicate that microRNA (miRNA) is contained within exosome. Here we sought to optimize the methodologies for the isolation and quantification of urinary exosomal microRNA as a prelude to biomarker discovery studies. Exosomes were isolated through ultracentrifugation and characterized by immunoelectron microscopy. To determine the RNA was confined inside exosomes, the pellet was treated with RNase before RNA isolation. The minimum urine volume, storage conditions for exosomes and exosomal miRNA was evaluated. The presence of miRNAs in patients with various kidney diseases was validated with real-time PCR. The result shows that miRNAs extracted from the exosomal fraction were resistant to RNase digestion and with high quality confirmed by agarose electrophoresis. 16ml of urine was sufficient for miRNA isolation by absolute quantification with 4.15×105 copies/ul for miR-200c. Exosomes was stable at 4℃ 24h for shipping before stored at -80℃ and was stable in urine when stored at -80°C for 12months. Exosomal miRNA was detectable despite 5 repeat freeze-thaw cycles. The detection of miRNA by quantitative PCR showed high reproducibility (>94% for intra-assay and >76% for inter-assay), high sensitivity (positive call 100% for CKD patients), broad dynamic range (8-log wide) and good linearity for quantification (R2>0.99). miR-29c and miR-200c showed different expression in different types of kidney disease. In summary, the presence of urinary exosomal miRNA was confirmed for patients with a diversity of chronic kidney disease. The conditions of urine collection, storage and miRNA detection determined in this study may be useful for future biomarker discovery efforts.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                08 May 2019
                May 2019
                : 20
                : 9
                : 2269
                Affiliations
                [1 ]Department of Translational Medical Sciences, University of Campania “Luigi Vanvitelli,” 80131 Naples, Italy; ca.vigorito86@ 123456libero.it (C.V.); anishchenkoea@ 123456gmail.com (E.A.); giovi.capolongo@ 123456gmail.com (G.C.); francesco.trepiccione@ 123456unicampania.it (F.T.); miriam.zacchia@ 123456unicampania.it (M.Z.); patrizia.lombari@ 123456gmail.com (P.L.)
                [2 ]Department of Precision Medicine, University of Campania “Luigi Vanvitelli,” 80138 Naples, Italy; r.capasso74@ 123456icloud.com (R.C.); diego.ingrosso@ 123456unicampania.it (D.I.)
                [3 ]Department of Experimental Medicine, University of Campania “Luigi Vanvitelli,” 80138 Naples, Italy; luigi.mele@ 123456unicampania.it
                [4 ]Biogem A. C. S. R. L. Contrada Camporeale, 83031 Ariano Irpino AV, Italy
                Author notes
                [* ]Correspondence: alessandra.perna@ 123456unicampania.it ; Tel.: +39-081-566-6822
                [†]

                These authors contributed equally to this work.

                Author information
                https://orcid.org/0000-0003-4048-9392
                Article
                ijms-20-02269
                10.3390/ijms20092269
                6539355
                31071929
                817e1378-2f53-4f06-819d-d28b3f9744f4
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 29 March 2019
                : 06 May 2019
                Categories
                Article

                Molecular biology
                lanthionine,glutathione,uremic toxin,chronic kidney disease,h2s,sulfane sulfur,cbs,cse,vegfa,calcium homeostasis

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