12 research outputs found
Uremic Toxin Lanthionine Interferes with the Transsulfuration Pathway, Angiogenetic Signaling and Increases Intracellular Calcium
(1) The beneficial effects of hydrogen sulfide (H2S) on the cardiovascular and nervous system have recently been re-evaluated. It has been shown that lanthionine, a side product of H2S biosynthesis, previously used as a marker for H2S production, is dramatically increased in circulation in uremia, while H2S 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 H2S 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 H2S release; reduces protein content and glutathionylation of transsulfuration enzyme cystathionine--synthase; modifies the expression of miR-200c and miR-423; lowers expression of vascular endothelial growth factor VEGF; increases Ca2+ 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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Testing trans-phyletic conservation of neuronal differentiation regulatory states in deuterostomes
SoxB2 in sea urchin development: implications in neurogenesis, ciliogenesis and skeletal patterning
Abstract Background Current studies in evolutionary developmental biology are focused on the reconstruction of gene regulatory networks in target animal species. From decades, the scientific interest on genetic mechanisms orchestrating embryos development has been increasing in consequence to the fact that common features shared by evolutionarily distant phyla are being clarified. In 2011, a study across eumetazoan species showed for the first time the existence of a highly conserved non-coding element controlling the SoxB2 gene, which is involved in the early specification of the nervous system. This discovery raised several questions about SoxB2 function and regulation in deuterostomes from an evolutionary point of view. Results Due to the relevant phylogenetic position within deuterostomes, the sea urchin Strongylocentrotus purpuratus represents an advantageous animal model in the field of evolutionary developmental biology. Herein, we show a comprehensive study of SoxB2 functions in sea urchins, in particular its expression pattern in a wide range of developmental stages, and its co-localization with other neurogenic markers, as SoxB1, SoxC and Elav. Moreover, this work provides a detailed description of the phenotype of sea urchin SoxB2 knocked-down embryos, confirming its key function in neurogenesis and revealing, for the first time, its additional roles in oral and aboral ectoderm cilia and skeletal rod morphology. Conclusions We concluded that SoxB2 in sea urchins has a neurogenic function; however, this gene could have multiple roles in sea urchin embryogenesis, expanding its expression in non-neurogenic cells. We showed that SoxB2 is functionally conserved among deuterostomes and suggested that in S. purpuratus this gene acquired additional functions, being involved in ciliogenesis and skeletal patterning
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Additional file 4: Figure S3. Analysis of the cilia length in MO-injected embryos performed at 72 hpf pluteus. AcTubulin staining is shown in green and DAPI in blue. a control non injected embryo, b MO-1 injected embryo, c MO-2 injected embryo, d MO-Fluo injected embryo. Measurements of longest cilia length (l) are indicated in white. All cilia length measurements were performed using Ziess LSM Image Browser software. 10–12 cilia from at least 33 embryos in every experimental group were measured; scale bar is 20 µm
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Additional file 2: Figure S2. Statistical analysis of serotonergic neurons number and cilia length in 72 hpf S. purpuratus SoxB2 knock-down experiments. a Embryonic nervous system (Synaptotagmin B by 1e11 immunohistochemistry) of uninjected control, MO-1, MO-2 and MO-Fluo embryos. The fluorescence of 1e11 positive neurons is shown in %, normalized by control uninjected embryos (100%). The intensity of staining from 10 embryos of each group was measured using ImageJ in three independent experiments. b Number of serotonergic neurons observed in six independent experiments using uninjected control, MO-1, MO-2 and MO-Fluo embryos. Serotonergic positive neurons were measured from at least 33 embryos in each experimental group. c Cilia length in uninjected control, MO-1, MO-2 and MO-Fluo embryos measured using the Zeiss confocal laser scanning LSM 510 microscope software. 10–12 cilia from at least 33 embryos were used in three independent experiments. Statistical analysis was performed using Prism 5 GraphPad software: P value versus uninjected controls = *P < 0.05, **P < 0.01, ***P < 0.001, while P value versus MO-Fluo = +P<0.05, ++P < 0.01, +++P < 0.001
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Additional file 1: Figure S1. Control MO-Fluo in 72 hpf S. purpuratus embryos. Sea urchin embryos at 72 hpf from oral (a, b, c), lateral (a′, b′, c′) and vegetal (a″, b″, c″) views depict the tissues where fluorescence deriving from the fluorescent MO is visible (c–c″). Morphant embryos imaged with microscope (b–b″) present a phenotype similar to uninjected control embryos (a–a″)
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Additional file 5: Table S1. List of oligonucleotides used for qPCR experiments
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Additional file 3: Figure S4. Injection of MO-Fluo did not affect the development of Synaptotagmin B expressing neurons
Novel applications of lead acetate and flow cytometry methods for detection of sulfur-containing molecules
Hydrogen sulfide (H2S) is the most recently established gaseous vasodilator, enzymatically
produced from cysteine metabolism, involved in a number of pathophysiological processes. However,
its accurate detection in vivo is critical due to its volatility and tendency to form sulfane sulfur
derivatives, thus limiting the data interpretation of its biological roles. We developed new applications
of the simple and rapid method to measure H2S release in cell culture systems, based on the lead
acetate strip test. This test, previously prevalently used in microbiology, was compared with the
agar trap method, applied, in parallel, on both cell cultures and cell-free samples. Sulfane sulfur
represents the major species derived from intracellular H2S. Various fluorescent probes are available
for quantitation of H2S derivatives intracellularly. We present here an alternative to the classic
imaging method for sulfane sulfur evaluation, running on a flow cytometer, based on SSP4 probe
labeling. Flow cytometry turned out to be more direct, fully quantitative and less time-consuming
compared to microscopy and more precise with respect to the fluorescence multi-plate reader assay.
The new application methods for H2S determination appear to be fully suitable for the analysis of
H2S release and sulfane sulfur content in biological samples
Zebrafish, a Novel Model System to Study Uremic Toxins: The Case for the Sulfur Amino Acid Lanthionine
The non-proteinogenic amino acid lanthionine is a byproduct of hydrogen sulfide biosynthesis: the third endogenous vasodilator gas, after nitric oxide and carbon monoxide. While hydrogen sulfide is decreased in uremic patients on hemodialysis, lanthionine is increased and has been proposed as a new uremic toxin, since it is able to impair hydrogen sulfide production in hepatoma cells. To characterize lanthionine as a uremic toxin, we explored its effects during the early development of the zebrafish (Danio rerio), a widely used model to study the organ and tissue alterations induced by xenobiotics. Lanthionine was employed at concentrations reproducing those previously detected in uremia. Light-induced visual motor response was also studied by means of the DanioVision system. Treatment of zebrafish embryos with lanthionine determined acute phenotypical alterations, on heart organogenesis (disproportion in cardiac chambers), increased heart beating, and arrhythmia. Lanthionine also induced locomotor alterations in zebrafish embryos. Some of these effects could be counteracted by glutathione. Lanthionine exerted acute effects on transsulfuration enzymes and the expression of genes involved in inflammation and metabolic regulation, and modified microRNA expression in a way comparable with some alterations detected in uremia. Lanthionine meets the criteria for classification as a uremic toxin. Zebrafish can be successfully used to explore uremic toxin effects