The “www” of Xenopus laevis Oocytes: The Why, When, What of Xenopus laevis Oocytes in Membrane Transporters Research

After 50 years, the heterologous expression of proteins in Xenopus laevis oocytes is still essential in many research fields. New approaches and revised protocols, but also classical methods, such as the two-electrode voltage clamp, are applied in studying membrane transporters. New and old methods for investigating the activity and the expression of Solute Carriers (SLC) are reviewed, and the kinds of experiment that are still useful to perform with this kind of cell are reported. Xenopus laevis oocytes at the full-grown stage have a highly efficient biosynthetic apparatus that correctly targets functional proteins at the defined compartment. This small protein factory can produce, fold, and localize almost any kind of wild-type or recombinant protein; some tricks are required to obtain high expression and to verify the functionality. The methodologies examined here are mainly related to research in the field of membrane transporters. This work is certainly not exhaustive; it has been carried out to be helpful to researchers who want to quickly find suggestions and detailed indications when investigating the functionality and expression of the different members of the solute carrier families

Characterization of the transport of lysine-containing dipeptides by PepT1orthologs expressed in Xenopus laevis oocytes.

During digestion, dietary proteins cleaved in di and tri-peptides are translocated from the intestinal lumen into the enterocytes via PepT1 (SLC15A1) using an inwardly directed proton electrochemical gradient. The kinetic properties in various PepT1 orthologs (Dicentrarchus labrax, Oryctolagus cuniculus, Danio rerio) have been explored to determine the transport efficiency of different combinations of lysine, methionine, and glycine. Species-specific differences were observed. Lys-Met resulted the best substrate at all tested potentials in sea bass and rabbit PepT1, whereas in the zebrafish transporter all tested dipeptides (except Gly-Lys) elicited similar currents independently on the charge position or amino acid composition. For the sea bass and rabbit PepT1, kinetic parameters, K0.5 and Imax and their ratio, show the importance of the position of the charged lysine in the peptide. The PepT1 transporter of these species has very low affinity for Lys-Lys and Gly-Lys; this reduces the transport efficiency which is instead higher for Lys-Met and Lys-Gly. PepT1 from zebrafish showed relatively high affinity and excellent transport efficiency for Met-Lys and Lys-Met. These data led us to speculate about the structural determinants involved in substrate interaction according to the model proposed for this transporter

Functional characterization of iron transporter from Dictyostelium discoideum as a model of cellular iron homeostasis

Iron plays a central role in a large number of essential cellular functions but it is also potentially toxic being able to generate reactive oxygen species. The social amoeba Dictyostelium discoideum possesses several iron genes with the exception of transferrin, ferritin and TFR (Bozzaro et al., 2013; Peracino et al., 2013) and represents a model for the study of cellular iron homeostasis showing subcellular localization of iron transporters resembling that of macrophages. In particular, D. discoideum expresses the ortholog of Nramp1 transporter in phago-lysosomes and that of Nramp2 in the contractile vacuole. To better understand the function of dd Nramp1, the protein was expressed in Xenopus laevis oocytes by cRNA injection and functionally tested by radiochemical and electrophysiological techniques. To increase the surface localization of dd Nramp1, its N and C termini were replaced with the corresponding regions of the murine DMT1, which shows a high level of expression in the membrane of X. laevis oocytes (Gunshin et al., 1997). Dd Nramp1 is an electrogenic proton-dependent divalent metal ion transporter with a cation selectivity comparable to that of the murine DMT1 (Illing et al., 2012). It transports ferrous but not ferric iron and it is partially inhibited by Na+

Threonine 67 is a key component in the coupling of the NSS amino acid transporter KAAT1

The crystallizations of the prokaryotic LeuT and of the eukaryotic DAT and SERT transporters represent important steps forward in the comprehension of the molecular physiology of Neurotransmitter: Sodium Symporters, although the molecular determinants of the coupling mechanism and of ion selectivity still remain to be fully elucidated. The insect NSS homologue KAAT1 exhibits unusual physiological features, such as the ability to use K+ as the driver ion, weak chloride dependence, and the ability of the driver ion to influence the substrate selectivity; these characteristics can help to define the molecular determinants of NSS function. Two non-conserved residues are present in the putative sodium binding sites of KAAT1: Ala 66, corresponding to Gly 20 in the Na2 site of LeuT, and Ser 68, corresponding to Ala 22 in the Nal site. Thr 67 appears also to be significant since it is not conserved among NSS members, is present as threonine only in KAAT1 and in the paralogue CAATCH1 and, according to LeuT structure, is close to the amino acid binding site. Mutants of these residues were functionally characterized in Xenopus oocytes. The T67Y mutant exhibited uptake activity comparable to that of the wild type, but fully chloride-independent and with enhanced stereoselectivity. Interestingly, although dependent on the presence of sodium, the mutant showed reduced transport-associated currents, indicating uncoupling of the driver ion and amino acid fluxes. Thr 67 therefore appears to be a key component in the coupling mechanism, participating in a network that influences the cotransport of Na+ and the amino acid

Safety in wine production. A pilot study on the quality evaluation of prosecco wine in the framework of ue regulation

In Italy, wine production is considered a sector of excellence, where the wines’ appreciable sensory features are favored by environmental factors, including weather and climate conditions, which benefit territories with a specific vocation. The whole chain involves many economic and agri-food sector operators, and requires an in-depth assessment of specific risks for identifying critical points, keeping the entire production process under control, and ensuring product traceability. This article describes the results of a pilot study conducted in the Prosecco DOCG (Designations of Controlled and Guaranteed Origin) area, concerning the detection of residues of plant protection products in fifty wine bottles. Although considerably below the maximum residue levels, all the samples tested were positive, ranging from two to five active substances detected in each sample. In addition to the provisions of the European Community legislation, this paper critically evaluates some best practices models that are already used by the Wine Federations of Italy, with the aim of identifying advantages of and areas for improvement in production methods, applicable to raw materials reception, rasping, storage, and bottling phases, in order to guarantee product safety and quality

Sex- and age-related differences in the management and outcomes of chronic heart failure: an analysis of patients from the ESC HFA EORP Heart Failure Long-Term Registry

Aims: This study aimed to assess age- and sex-related differences in management and 1-year risk for all-cause mortality and hospitalization in chronic heart failure (HF) patients. Methods and results: Of 16 354 patients included in the European Society of Cardiology Heart Failure Long-Term Registry, 9428 chronic HF patients were analysed [median age: 66 years; 28.5% women; mean left ventricular ejection fraction (LVEF) 37%]. Rates of use of guideline-directed medical therapy (GDMT) were high (angiotensin-converting enzyme inhibitors/angiotensin receptor blockers, beta-blockers and mineralocorticoid receptor antagonists: 85.7%, 88.7% and 58.8%, respectively). Crude GDMT utilization rates were lower in women than in men (all differences: P\ua0 64 0.001), and GDMT use became lower with ageing in both sexes, at baseline and at 1-year follow-up. Sex was not an independent predictor of GDMT prescription; however, age >75 years was a significant predictor of GDMT underutilization. Rates of all-cause mortality were lower in women than in men (7.1% vs. 8.7%; P\ua0=\ua00.015), as were rates of all-cause hospitalization (21.9% vs. 27.3%; P\ua075 years. Conclusions: There was a decline in GDMT use with advanced age in both sexes. Sex was not an independent predictor of GDMT or adverse outcomes. However, age >75 years independently predicted lower GDMT use and higher all-cause mortality in patients with LVEF 6445%