60 research outputs found
Pathways of L-glutamic acid transport in cultured human fibroblasts.
The transport of L-glutamic acid has been studied in skin-derived diploid human fibroblasts. Competition analysis in the presence and absence of Na+ and mathematical discrimination by nonlinear regression indicated that L-glutamic acid enters the cell by at least three transport systems: 1) a high affinity Na+-dependent system which has been found to be identical to the previously described system for anionic amino acids (Gazzola, G. C., Dall'Asta, V., Bussolati, O., Makowske, M., and Christensen, H. N. (1981) J. Biol. Chem. 256, 6054-6059) and which is provisionally designated as System X-AG; this route was shared by L-aspartic acid; 2) a low affinity Na+-dependent system resembling the ASC System for neutral amino acids (Franchi-Gazzola, R., Gazzola, G. C., Dall'Asta, V., and Guidotti, G. G. (1982) J. Biol. Chem. 257, 9582-9587); its reactivity toward L-glutamic acid was strongly inhibited by L-serine, but not by 2-(methyl-amino)isobutyric acid; and 3) a Na+-independent system similar to System XC- described in fetal human lung fibroblasts (Bannai, S., and Kitamura, E. (1980) J. Biol. Chem. 255, 2372-2376). The XC- system served for L-glutamic acid and L-cystine, the latter amino acid behaving as a potent inhibitor of L-glutamic acid uptake. Amino acid starvation did not change the uptake of L-glutamic acid by the two Na+-dependent systems, but enhanced the activity of System XC- by increasing its Vmax. L-Glutamic acid transport was also affected by the density of the culture. An increased cell density lowered the uptake of the amino acid by Systems ASC and XC- and promoted the uptake by System X-AG. All these variations were dependent upon changes in Vmax
Desmopressin Stimulates Nitric Oxide Production in Human Lung Microvascular Endothelial Cells
Desmopressin (dDAVP) is the best characterized analogue of vasopressin, the endocrine regulator of water balance endowed with potent vasoconstrictive effects. Despite the use of dDAVP in clinical practice, ranging from the treatment of nephrogenic diabetes insipidus to bleeding disorders, much remains to be understood about the impact of the drug on endothelial phenotype. The aim of this study was, thus, to evaluate the effects of desmopressin on the viability and function of human pulmonary microvascular endothelial cells (HLMVECs). The results obtained demonstrate that the vasopressor had no cytotoxic effect on the endothelium; similarly, no sign of endothelial activation was induced by dDAVP, indicated by the lack of effect on the expression of inflammatory cytokines and adhesion molecules. Conversely, the drug significantly stimulated the production of nitric oxide (NO) and the expression of the inducible isoform of nitric oxide synthase, NOS2/iNOS. Since the intracellular level of cAMP also increased, we can hypothesize that NO release is consequent to the activation of the vasopressin receptor 2 (V2R)/guanylate cyclase (Gs)/cAMP axis. Given the multifaceted role of NOS2-deriving NO for many physio-pathological conditions, the meanings of these findings in HLMVECs appears intriguing and deserves to be further address
Organic cation transporters (OCTs/OCTNs) in human primary alveolar epithelial cells.
Abstract Alveolar epithelium, besides exerting a key role in gas exchange and surfactant production, plays important functions in host defense and inflammation. Pathological conditions associated to alveolar dysfunction include Acute Respiratory Distress Syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). The use of predictive in vitro models of human alveolar epithelium is nowadays required for the study of disease mechanisms, as well as of pharmacokinetic parameters of pulmonary drugs delivery. Here, we employed a novel 3D model of human alveoli, namely EpiAlveolar™, consisting of primary alveolar epithelial cells, pulmonary endothelial cells and fibroblasts, that reflects properly the in vivo-like conditions. In EpiAlveolar™ we performed a characterization of Organic Cation Transporters (OCTs and OCTNs) expression and activity and we found that OCTN2, OCT1 and OCT3 are expressed on the basolateral membrane; instead, ATB0,+ transporter for cationic and neutral amino acids, which shares with OCTN2 the affinity for carnitine as substrate, is readily detectable and functional at the apical side. We also show that these transporters differentially interact with anticholinergic drugs. Overall, our findings reveal close similarities of EpiAlveolar™ with the tracheal/bronchial epithelium (EpiAirway™ model) and entrust this alveolar tissue as a potential tool for the screening of biopharmaceuticals molecules
In Lysinuric Protein Intolerance system y+L activity is defective in monocytes and in GM-CSF-differentiated macrophages
<p>Abstract</p> <p>Background</p> <p>In the recessive aminoaciduria Lysinuric Protein Intolerance (LPI), mutations of <it>SLC7A7</it>/y+LAT1 impair system y<sup>+</sup>L transport activity for cationic amino acids. A severe complication of LPI is a form of Pulmonary Alveolar Proteinosis (PAP), in which alveolar spaces are filled with lipoproteinaceous material because of the impaired surfactant clearance by resident macrophages. The pathogenesis of LPI-associated PAP remains still obscure. The present study investigates for the first time the expression and function of y+LAT1 in monocytes and macrophages isolated from a patient affected by LPI-associated PAP. A comparison with mesenchymal cells from the same subject has been also performed.</p> <p>Methods</p> <p>Monocytes from peripheral blood were isolated from a 21-year-old patient with LPI. Alveolar macrophages and fibroblastic-like mesenchymal cells were obtained from a whole lung lavage (WLL) performed on the same patient. System y<sup>+</sup>L activity was determined measuring the 1-min uptake of [<sup>3</sup>H]-arginine under discriminating conditions. Gene expression was evaluated through qRT-PCR.</p> <p>Results</p> <p>We have found that: 1) system y<sup>+</sup>L activity is markedly lowered in monocytes and alveolar macrophages from the LPI patient, because of the prevailing expression of <it>SLC7A7</it>/y+LAT1 in these cells; 2) on the contrary, fibroblasts isolated from the same patient do not display the transport defect due to compensation by the <it>SLC7A6</it>/y+LAT2 isoform; 3) in both normal and LPI monocytes, GM-CSF induces the expression of <it>SLC7A7</it>, suggesting that the gene is a target of the cytokine; 4) GM-CSF-induced differentiation of LPI monocytes is comparable to that of normal cells, demonstrating that GM-CSF signalling is unaltered; 5) general and respiratory conditions of the patient, along with PAP-associated parameters, markedly improved after GM-CSF therapy through aerosolization.</p> <p>Conclusions</p> <p>Monocytes and macrophages, but not fibroblasts, derived from a LPI patient clearly display the defect in system y<sup>+</sup>L-mediated arginine transport. The different transport phenotypes are referable to the relative levels of expression of <it>SLC7A7 </it>and <it>SLC7A6</it>. Moreover, the expression of <it>SLC7A7 </it>is regulated by GM-CSF in monocytes, pointing to a role of y+LAT1 in the pathogenesis of LPI associated PAP.</p
Extending in vitro digestion models to specific human populations: Perspectives, practical tools and bio-relevant information
[EN] Background
In vitro digestion models show great promise in facilitating the rationale design of foods. This paper provides a look into the current state of the art and outlines possible future paths for developments of digestion models recreating the diverse physiological conditions of specific groups of the human population.
Scope and approach
Based on a collective effort of experts, this paper outlines considerations and parameters needed for development of new in vitro digestion models, e.g. gastric pH, enzymatic activities, gastric emptying rate and more. These and other parameters are detrimental to the adequate development of in vitro models that enable deeper insight into matters of food luminal breakdown as well as nutrient and nutraceutical bioaccessibility. Subsequently, we present an overview of some new and emerging in vitro digestion models mirroring the gastro-intestinal conditions of infants, the elderly and patients of cystic fibrosis or gastric bypass surgery.
Key findings and conclusions
This paper calls for synchronization, harmonization and validation of potential developments in in vitro digestion models that would greatly facilitate manufacturing of foods tailored or even personalized, to a certain extent, to various strata of the human population.Shani-Levi, C.; Alvito, P.; Andrés Grau, AM.; Assunção, R.; Barbera, R.; Blanquet-Diot, S.; Bourlieu, C.... (2017). Extending in vitro digestion models to specific human populations: Perspectives, practical tools and bio-relevant information. Trends in Food Science & Technology. 60:52-63. https://doi.org/10.1016/j.tifs.2016.10.017S52636
The cluster-tray method for rapid measurement of solute fluxes in adherent cultured cells
Gliadin activates arginase pathway in RAW264.7 cells and in human monocytes
Celiac disease (CD) is an autoimmune enteropathy triggered in susceptible individuals by the ingestion of gliadin-containing grains. Recent studies have demonstrated that macrophages play a key role in the pathogenesis of CD through the release of inflammatory mediators such as cytokines and nitric oxide (NO). Since arginine is the obliged substrate of iNOS (inducible nitric oxide synthase), the enzyme that produces large amount of NO, the aim of this work is to investigate arginine metabolic pathways in RAW264.7 murine macrophages after treatment with PT-gliadin (PTG) in the absence and in the presence of IFNÎł. Our results demonstrate that, besides strengthening the IFNÎł-dependent activation of iNOS, gliadin is also an inducer of arginase, the enzyme that transforms arginine into ornithine and urea. Gliadin treatment increases, indeed, the expression and the activity of arginase, leading to the production of polyamines through the subsequent induction of ornithine decarboxylase. This effect is strengthened by IFNÎł. The activation of these pathways takes advantage of the increased availability of arginine due to a decreased system y+l-mediated efflux, likely ascribable to a reduced expression of Slc7a6 transporter. A significant induction of arginase expression is also observed in human monocytes from healthy subject upon treatment with gliadin, thus demonstrating that gluten components trigger changes in arginine metabolism in monocyte/macrophage cell
Analysis of the expression of genes related to oxylipin biosynthesis in fusarium verticillioides and maize kernels during their interaction
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Journal of Plant Pathology
Volume 97, Issue 1, 2015, Pages 193-197
Analysis of the expression of genes related to oxylipin biosynthesis in fusarium verticillioides and maize kernels during their interaction (Article)
Scala, V.a ,
Beccaccioli, M.a,
Dall’Asta, C.b,
Giorni, P.c,
Fanelli, C.a
a Department of Environmental Biology, University of Rome Sapienza, Rome, Italy
b Department of Organic and Industrial Chemistry, Food Chemistry and Natural Substances Unit, University of Parma, Italy
c Istitute of Entomology and Plant Pathology, Catholic University Sacro Cuore, Piacenza, Italy
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Abstract
Fusarium verticillioides is a pathogen that can cause ear and stalk rot in maize. Under suitable environmental conditions, this fungus produces large amount of fumonisins, a potential carcinogenic to humans and animals classified as IARC2B. Recent studies have shown that pathogen and host exchange an oxylipin-based cross talk during their interaction. This study was aimed at investigating whether environmental conditions, namely water activity (aw) at 0.90 and 20°C, which are the thresholds for F. verticillioides development, affect the maize/fungal oxylipin gene expression profile. Fungal Fvlds1-3, Fvlox, Zmlox3 and the maize defense-related ZmPR1 genes significantly changed their expression after infection at 0.90 aw and 20°C. Moreover, the expression of maize genes peaked after that of fungal genes, indicating that, under these experimental conditions, plant and pathogen coordinate the oxylipin gene expression reciprocally. This kind of modulation of fungal and plant gene expression is suggestive of the “zig zag model” proposed for other plant-pathogen interaction systems
Characterization of a bioassay for transepithelial inhibition of nitric oxide production by inflammatory cells
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