1,721,088 research outputs found
Unveiling of the AS-NMD-mediated regulation of Peptide/Histidine Transporter 1 (SLC15A4/PHT1) gene products under physiological or inflammatory challenges
No full textIn vertebrates, the SLC15A4 gene codes for the peptide/histidine transporter 1 (PHT1) protein, which transport activity of di-/tripeptides and histidine across plasma and subcellular membranes has been generally described [1]. Recently, several studies have pointed out that the human SLC15A4 gene codes for a late endosome/lysosome carrier taking part in TLR7-, TLR9-, and NOD1-mediated pathways, thus putatively involved in inflammatory, immune and autoimmune diseases. Accordingly, SLC15A4/PHT1 altered activity has been associated to conditions such as type 2 diabetes, inflammatory bowel diseases (IBD) and systemic lupus erythematosus [2]. In mammals, the Slc15a4 genes are abundantly expressed by immune and nervous cells, in which they undergo alternative splicing (AS) events which physiological meaning is still unknown. Here, we report of a unique SLC15A4/PHT1 AS-mediated regulation which we found conserved from zebrafish to human. We show that this regulation is physiologically facilitated by the Nonsense-Mediated mRNA Decay (NMD) surveillance pathway, in human nervous-derived and immune-derived cells too; in these latter, both the canonical and AS variants are sensitive to inflammatory molecular triggers. Furthermore, in an IBD murine model, we show that GI inflammation seems to differentially affect the expression levels of the alternative SLC15A4/PHT1 gene products in different GI tracts. Overall, by unveiling the AS-NMD-mediated gene regulation, our findings might introduce a key analytical element for stepping forward the comprehension of the mechanism of intervention of SLC15A4/PHT1 in inflammatory and immune processes. [1] Verri T, Barca A, et al. (2017) Di- and tripeptide transport in vertebrates: the contribution of teleost fish models. J Comp Physiol B. 187(3):395-462. [2] Griffith AD, Zaidi AK, et al. (2018) A requirement for slc15a4 in imiquimod-induced systemic inflammation and psoriasiform inflammation in mice. Sci Rep. 8(1):14451
Allograft inflammatory factor-1 in metazoans: Focus on invertebrates
No full textAllograft inflammatory factor-1 (AIF-1) is a calcium-binding scaffold/adaptor protein often associated with inflammatory diseases. Originally cloned from active macrophages in humans and rats, this gene has also been identified in other vertebrates and in several invertebrate species. Among metazoans, AIF-1 protein sequences remain relatively highly conserved. Generally, the highest expression levels of AIF-1 are observed in immunocytes, suggesting that it plays a key role in immunity. In mammals, the expression of AIF-1 has been reported in different cell types such as activated macrophages, microglial cells, and dendritic cells. Its main immunomodulatory role during the inflammatory response has been highlighted. Among invertebrates, AIF-1 is involved in innate immunity, being in many cases upregulated in response to biotic and physical challenges. AIF-1 transcripts result ubiquitously expressed in all examined tissues from invertebrates, suggesting its participation in a variety of biological processes, but its role remains largely unknown. This review aims to present current knowledge on the role and modulation of AIF-1 and to highlight its function along the evolutionary scale
Comparison of the electrophysiological properties of the two paralogues of Salmo salar oligopeptide transporter PepT1: new insights from PepT1a vs. PepT1b
No full textDi and tripeptide (di/tripeptide) absorption in the intestine occurs via PepT1, an electrogenic transporter that uses an inwardly-directed proton electrochemical gradient to drive the transport of more than 8,000 different molecules inside the cells. Studies in mammals indicate that PepT1 also exhibits peptide sensing function(s). In teleost fish, two genes, namely PepT1b and more recently PepT1a, have been identified. The two paralogues – the result of (at least one) round of whole-genome duplication during the early evolution of the ray-finned fish lineage – encode proteins that share 64-67% similarity at the amino acid level. While PepT1b has been widely studied in several teleost fish species, including S. salar, PepT1a function is fully unknown to date. The aim of this work was to characterize and compare S. salar PepT1a and Pept1b after heterologous expression in Xenopus laevis oocytes, and study their role(s) in peptide transport and/or sensing. Injection of cRNAs into X. laevis oocytes allowed high functional “in membrane” expression of both proteins. Function was verified by Two Electrode Voltage Clamp (TEVC), as currents elicited by perfusion of di/tripeptides were regularly recorded for both proteins. Notably, ours is the first experimental evidence that PepT1a is an electrogenic transporter of di/tripeptides. Measuring the transport currents at two different pH (6.5 and 7.6) and in the presence of increasing concentrations of glycine-L-glutamine (GQ) (from 0.01 to 30 mM) allowed to investigate kinetic values, as the maximal current (Imax) and the substrate apparent affinity (K0.5), for each transporter and pH. Results suggest that the two transporters interact differently with the substrate and that the external pH influence the substrate affinity and consequently the transport efficiency, which is definitely lower for PepT1a with respect to PepT1b. Moreover, preliminary results suggest that L-lysine-containing substrates elicit transport-associated currents of different amplitude in the two isoforms. Large currents are recorded from PepT1b in the presence of L-lysine-glycine (KG), and currents similar to GQ are recorded for glycine-L-lysine (GK); conversely, small responses for both substrates are obtained in PepT1a. Overall, these data support the idea of a different role of the two isoforms, opening the possibility to investigate on PepT1a as a “transceptor” involved in nutrient sensing.
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Survey of genes involved in mitochondrial biogenesis in early development of zebrafish as candidates for mitochondrial pathologies
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Flow cytometric analysis of monocytes polarization and reprogramming from inflammatory to immunosuppressive phase during sepsis
No full textSepsis outcome is determined by a balance between inflammation and immune suppression. We aimed to evaluate monocytes polarization and reprogramming during these processes. We analyzed 93 patients with procalcitonin level >0.5 ng/mL (hPCT) and suspected/confirmed sepsis, and 84 controls by analysis of CD14, CD16 and HLA-DR expression on blood monocytes using fluorescent labeled monoclonal antibodies and BD FACS CANTO II. Complete blood cell count, procalcitonin and other biochemical markers were evaluated. Intermediate monocytes CD14++CD16+ increased in hPCT patients (including both positive and negative culture) compared to controls (13.6% ± 0.8 vs 6.2% ± 0.3, p<0.001), while classical monocytes CD14++CD16- were significantly reduced (72.5% ± 1.6 vs 82.6% ± 0.7, p<0.001). Among hPCT patients having positive microbial culture, the percentage of intermediate monocytes was significantly higher in septic compared with non-septic/localized-infection patients (17.4% vs 11.5%; p<0.05) whilst the percentage of classical monocytes was lower (68.0% vs 74.5%). Three-four days following the diagnosis of sepsis, HLA-DR expression on monocyte (mHLA-DR) was lower (94.3%) compared to controls (99.4%) (p<0.05). Septic patients with the worst clinical conditions showed higher incidence of secondary infections, longtime hospitalization and lower HLA-DR+monocytes compared to septic patients with better clinical outcome (88.4% vs 98.6%, p=0.05). The dynamic nature of sepsis correlates with monocytes functional polarization and reprogramming from a pro-inflammatory CD14++CD16+ phenotype in non-septic hPCT patients to a decrease of HLA-DR surface expression in hPCT patients with confirmed sepsis, making HLA-DR reduction a marker of immune-paralysis and sepsis outcome. Analysis of monocytes plasticity opens to new mechanisms responsible for pro/anti-inflammatory responses during sepsis, and new im - muno therapies
The colon epithelium as a target for the intracellular antioxidant activity of hydroxytyrosol: a study on rat colon explants
Full text linkOxidative stress is involved in the genesis and progress of many disorders of the gastrointestinal (GI) tract. In particular, the colon epithelium is one of the GI tract segments more exposed to pro-oxidant conditions. We aimed to study the intracellular antioxidant activity of hydroxytyrosol (HT), one of the most potent natural antioxidant phenolic compounds typically present in olive oil, directly on the colon epithelium, under basal physiological and pro-oxidant conditions. Our approach was based on the application of in situ confocal microscopy on rat colon explants loaded with the fluorescent probe 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluoresceindiacetate, which is sensitive to intracellular oxidative stress. In the intact mucosa, HT exerted a dose-dependent decrease of the basal intracellular reactive oxygen species (ROS) production of superficial colonocytes. Also, it induced a direct dose-dependent antioxidant action on the colon mucosa exposed to a pro-oxidant condition such as the H2O2 challenge. The effect of 100 μM HT was comparable to that of 10 μM Trolox, which is widely used as a standard in in vitro assays for the determination of antioxidant activity. The intracellular antioxidant activity of HT on the intact mucosa was also tested against tert-butyl peroxide, another pro-oxidant. The results show that HT can directly contribute to the redox balance of colonic epithelium by reducing ROS in both basal and pro-oxidant conditions, and support the potential of HT as a functional food ingredient with applications in protecting the intestinal mucosa against oxidative stress
Analysis of the accumulation and transit along the zebrafish gut lumen of quantum rod/glycyl-L-sarcosine conjugates
No full textThe zebrafish (Danio rerio) model is increasingly gaining relevance in research as a model for vertebrate gastrointestinal (GI) physiology, mainly for the ease with which it is possible to analyse basic functions at the gut level. Also, it is commonly used to test the fate of drugs, novel pharmacological compounds and/or xenobiotics interacting with the epithelial barrier along the GI tract. In vertebrates, the gastrointestinal homeostasis and functionality depend on proper physicochemical, mechanical and enzymatic processes. Alterations of such processes directly elicit pathophysiological onsets. For studying and monitoring GI processes e.g. ingestion, transit, absorption, the zebrafish ‘toolbox’ can be easily implemented by up-to-date nanotechnology tools i.e. colloidal semiconductor nanocrystals aiming at elucidating the gut dynamics. Here, to detect and monitor digestive and absorptive processes along the functional gut of the zebrafish we exploit PEG-functionalized Quantum Rods (QRs) with high photostability and biocompatibility. More in detail, QRs were conjugated with glycyl-L-sarcosine (QRs-GlySar) dipeptide molecules, as potential substrates of transport/sensing systems functionally expressed on the luminal plasma membranes of the enterocytes constituting the intestinal epithelial barrier. QRs conjugated with GlySar were administered to 5 dpf (days post fertilization) zebrafish larvae for 48-72 hours, to investigate their differential accumulation, transit, and interaction with the gut lumen and the epithelial barrier. Control experiments were carried out with QRs not conjugated. By tracing the signal fluorescence QRs-GlySar, we easily detected a time- and dose-dependent trend of accumulation and peristaltic progression of the QRs bioconjugates along the gut lumen; at the same time, no QRs were detected in gills or other tissues/organs. Interestingly, the accumulation, transit, and local interactions appeared to have differential trends based on the presence or absence of the conjugated dipeptide. Moreover, when larvae were treated with QRs-GlySar, fluorescent signals were detected in the circulatory blood flow, indicating the occurrence of physiological absorptive processes which might drive QRs across transepithelial pathways, remarkably. Overall, these preliminary results give hints on the effectiveness of the combination of QRs (as imaging probes) with specific dipeptides or dipeptide-like molecules (as physiologically active substrates) both in investigating functional processes of the zebrafish GI tracts, as well as in disclosing mechanisms potentially triggered by the interactions of dipeptides at epithelial level
Allograft inflammatory factor-1 (AIF-1) in the common sea urchin Paracentrotus lividus: molecular and expression analysis
No full textAllograft Inflammatory Factor1 (AIF1), alias ionized calcium-binding adapter molecule 1 (IBA1), is a highly conserved Ca2+-binding cytokine that has been identified as a key regulator of the immune response in vertebrates. AIF1 is highly expressed in activated macrophages during inflammatory responses, thus representing an accurate indicator of macrophage activation in the body and a pathogenic factor in several inflammatory diseases. Proteins of the AIF1 superfamily are also present in invertebrates, from sponges to echinoderms.
Here, we describe the Paracentrotus lividus Aif-1, which encodes a predicted protein of 151 amino acids with high similarity to vertebrate AIF1. In the common sea urchin, molecular and immunocytochemical analyses showed the constitutive expression of Aif-1 in the coelomocytes. Aif-1 localizes in the perinuclear area of amoebocytes and inside the granules of red cells, but it is not present in vibratile cells and colorless spherula cells. Moreover, significant increase of P. lividus Aif-1 expression, at both mRNA and protein level, are observed in coelomocytes after Gram+ bacterial challenge.
BLAST searches across Echinoderm databases resulted in identification of orthologous proteins from 24 species (8 sea urchins, 1 brittle star, 12 starfishes and 3 sea cucumbers). Among these, P. lividus Aif-1 shared a high identity with several species, e.g., 85.4% with the sea urchin Strongylocentrotus purpuratus, 60.9% with the brittle star Ophiocoma echinata, 59.6% with the starfish Achantaster planci, and 52.3% with the sea cucumber Apostichopus japonicus.
Our study on P. lividus Aif-1 will contribute to elucidate AIF1 function along the evolutionary scale and to consolidate the key evolutionary position of echinoderms throughout metazoans with respect to the common immune paths
Electroneutral Na+/H+ exchange in brush-border membrane vesicles from Penaeus japonicus hepatopancreas
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