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The plasma membrane transporter ASCT2 is a well-known Na+-dependent obligatory antiporter of neutral amino acids. The crucial role of the residue C467 in the recognition and binding of the ASCT2 substrate glutamine, has been highlighted by structure/function relationship studies. The reconstitution in proteoliposomes of the human ASCT2 produced in P. pastoris is here employed to unveil another role of the C467 residue in the transport reaction. Indeed, the site-directed mutant C467A displayed a novel property of the transporter, i.e., the ability of mediating a low but measurable unidirectional transport of [3H]-glutamine. This reaction conforms to the main features of the ASCT2-mediated transport, namely the Na+-dependence, the pH dependence, the stimulation by cholesterol included in the proteoliposome membrane, and the specific inhibition by other common substrates of the reconstituted human ASCT2. Interestingly, the WT protein cannot catalyze the unidirectional transport of [3H]-glutamine, demonstrating an unspecific phenomenon. This difference is in favor of a structural conformational change between a WT and C467A mutant that triggers the appearance of the unidirectional flux; this feature has been investigated by comparing the available 3D structures in two different conformations, and two homology models built on the basis of hEAAT1 and GLTPh.

Alanine, serine, cysteine transporter 2 (ASCT2) is a membrane amino acid transporter with relevance to human physiology and pathology, such as cancer. Notwithstanding, the study on the ASCT2 transport cycle still has unknown aspects, such as the role of Na+ in this process. We investigate this issue using recombinant hASCT2 reconstituted in proteoliposomes. Changes in the composition of purification buffers show the crucial role of Na+ in ASCT2 functionality. The transport activity is abolished when Na+ is absent or substituted by Li+ or K+ in purification buffers. By employing a Na+ fluorometric probe, we measured an inwardly directed flux of Na+ and, by combining fluorometric and radiometric assays, determined a 2Na+ : 1Gln stoichiometry. Kinetics of Na+ transport suggest that pH-sensitive residues are involved in Na+ binding/transport. Our results clarify the role of Na+ on human ASCT2 transporter activity.

The effect of cholesterol was investigated on the OCTN1 transport activity measured as [14C]-tetraethylamonium or [3H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli. Removal of cholesterol from the native transporter by M&beta, CD before reconstitution led to impairment of transport activity. A similar activity impairment was observed after treatment of proteoliposomes harboring the recombinant (cholesterol-free) protein by M&beta, CD, suggesting that the lipid mixture used for reconstitution contained some cholesterol. An enzymatic assay revealed the presence of 10 &micro, g cholesterol/mg total lipids corresponding to 1% cholesterol in the phospholipid mixture used for the proteoliposome preparation. On the other way around, the activity of the recombinant OCTN1 was stimulated by adding the cholesterol analogue, CHS to the proteoliposome preparation. Optimal transport activity was detected in the presence of 83 &micro, g CHS/ mg total lipids for both [14C]-tetraethylamonium or [3H]-acetylcholine uptake. Kinetic analysis of transport demonstrated that the stimulation of transport activity by CHS consisted in an increase of the Vmax of transport with no changes of the Km. Altogether, the data suggests a direct interaction of cholesterol with the protein. A further support to this interpretation was given by a docking analysis indicating the interaction of cholesterol with some protein sites corresponding to CARC-CRAC motifs. The observed direct interaction of cholesterol with OCTN1 points to a possible direct influence of cholesterol on tumor cells or on acetylcholine transport in neuronal and non-neuronal cells via OCTN1.

Objective The study aimed to evaluate whether high-flow oxygen therapy (HFOT) during training was more effective than oxygen in improving exercise capacity in hypoxemic chronic obstructive pulmonary disease (COPD). Methods A total of 171 patients with COPD and chronic hypoxemia were consecutively recruited in 8 rehabilitation hospitals in a randomized controlled trial. Cycle-ergometer exercise training was used in 20 supervised sessions at iso inspiratory oxygen fraction in both groups. Pre- and post-training endurance time (Tlim), 6-minute walking distance (6MWD), respiratory and limb muscle strength, arterial blood gases, Barthel Index, Barthel Dyspnea Index, COPD Assessment Test, Maugeri Respiratory Failure questionnaire, and patient satisfaction were evaluated. Results Due to 15.4% and 24.1% dropout rates, 71 and 66 patients were analyzed in HFOT and Venturi mask (V-mask) groups, respectively. Exercise capacity significantly improved after training in both groups with similar patient satisfaction. Between-group difference in post-training improvement in 6MWD (mean: 17.14 m; 95% CI = 0.87 to 33.43 m) but not in Tlim (mean: 141.85 seconds; 95% CI = −18.72 to 302.42 seconds) was significantly higher in HFOT. The minimal clinically important difference of Tlim was reached by 47% of patients in the V-mask group and 56% of patients in the HFOT group, whereas the minimal clinically important difference of 6MWD was reached by 51% of patients in the V-mask group and 69% of patients in the HFOT group, respectively. Conclusion In patients with hypoxemic COPD, exercise training is effective in improving exercise capacity. Impact Statement The addition of HFOT during exercise training is not more effective than oxygen through V-mask in improving endurance time, the primary outcome, whereas it is more effective in improving walking distance.

Unintentional poisonings are a global health problem frequently resulting in hospital admissions. Propafenone is a class 1C antiarrhythmic drug used in the second-line management of supraventricular and ventricular arrhythmias and, when unintentionally ingested, can lead to severe and life-threatening poisoning. We describe a case of a 3-year-old male patient unintentionally ingesting 300 mg (20 mg/kg) of propafenone and presenting with ventricular tachycardia with QT prolongation. Two boli of intravenous hypertonic sodium bicarbonate (total amount of 3 mEq/kg), followed by 3-hours continuous infusion of 1 mEq kg-1 h-1 sodium bicarbonate, were able to restore the clinical conditions of the patient. With this case report, we aim to highlight the existing challenge in the therapeutic management of propafenone intoxication that finds intravenous hypertonic bicarbonate to be a useful tool also in pediatric population.

The human plasma membrane transporter ASCT2 is responsible for mediating Na- dependent antiport of neutral amino acids. New insights into structure/function relationships were unveiled by a combined approach of recombinant over-expression, site-directed mutagenesis, transport assays in proteoliposomes and bioinformatics. WT and Cys mutants of hASCT2 were produced in P. pastoris and purified for functional assay. The reactivity towards SH reducing and oxidizing agents of WT protein was investigated and opposite effects were revealed; transport activity increased upon treatment with the Cys reducing agent DTE, i.e., when Cys residues were in thiol (reduced) state. Methyl-Hg, which binds to SH groups, was able to inhibit WT and seven out of eight Cys to Ala mutants. On the contrary, C467A loses the sensitivity to both DTE activation and Methyl-Hg inhibition. The C467A mutant showed a Km for Gln one order of magnitude higher than that of WT. Moreover, the C467 residue is localized in the substrate binding region of the protein, as suggested by bioinformatics on the basis of the EAAT1 structure comparison. Taken together, the experimental data allowed identifying C467 residue as crucial for substrate binding and for transport activity modulation of hASCT2.

Intestinal and systemic illnesses have been linked to increased gut permeability. Bile acids, whose luminal profile can be altered in human disease, modulate intestinal paracellular permeability. We investigated the mechanism by which selected bile acids increase gut permeability using a validated in vitro model. Human intestinal Caco-2 cells were grown in monolayers and challenged with a panel of bile acids. Transepithelial electrical resistance and luminal-to-basolateral fluxes of 10-kDa Cascade blue-conjugated dextran were used to monitor paracellular permeability. Immunoprecipitation and immunoblot analyses were employed to investigate the intracellular pathway. Redistribution of tight junction proteins was studied by confocal laser microscopy. Micromolar concentrations of cholic acid, deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) but not ursodeoxycholic acid decreased transepithelial electrical resistance and increased dextran flux in a reversible fashion. Coincubation of 50 μM CDCA or DCA with EGF, anti-EGF monoclonal antibody, or specific src inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP-2) abolished the effect. A concentration of 50 μM of either CDCA or DCA also induced EGF receptor phosphorylation, occludin dephosphorylation, and occludin redistribution at the tight junction level in the same time frame and in a reversible fashion. We conclude that selected bile acids modulate intestinal permeability via EGF receptor autophosphorylation, occludin dephosphorylation, and rearrangement at the tight junction level. The effect is mediated by the src family kinases and is abolished by EGF treatment. These data also support the role of bile acids in the genesis of necrotizing enterocolitis and the protective effect of EGF treatment.

Unconjugated bilirubin promotes intestinal secretion without affecting nutrient digestion or absorption. In the current study, the effects of unconjugated bilirubin (UCB) on the barrier function of the intestinal epithelium were investigated. The apical side of human intestinal cell line Caco-2 monolayers was challenged with purified UCB. Transepithelial electrical resistance and paracellular fluxes of 10 kD Cascade blue conjugate dextran were measured. Cell monolayer viability was studied using LDH release and trypan blue exclusion tests. Redistribution of enterocyte tight junction occludin was studied by confocal microscopy. Bilirubin induced a dose-dependent decrease of transepithelial electrical resistance (TEER). This effect was maximal at 6 h and tended to be reversed at 48 h. Oxidated bilirubin was ineffective. Bilirubin significantly increased fluorescent dextran paracellular passage. Cell viability was not affected by UCB over the 5-200 nmol/L concentration range. Finally, bilirubin triggered a reversible redistribution of tight junctional occludin. UCB increases the permeability of intestinal epithelium. This effect is reversible, dependent on the redox status of the molecule and the rearrangement of the tight junction. These data attribute to bilirubin a novel role of functional modulator of intestinal paracellular permeability in vitro.

Specific immunotherapy (SIT) efficacy and safety by subcutaneous (SCIT) and sublingual (SLIT) route is supported by literature data. Pre-coseasonal treatment is currently the more accepted option for pollen immunotherapy in terms of costs and patient's compliance. This retrospective study evaluated the patient's preference concerning subcutaneous or sublingual route in pre-coseasonal treatment.We evaluated 145 patients (79 males, 66 females, age ranging from 14 to 69 years), suffering from moderate-severe rhino-conjunctivitis or mild bronchial asthma and with homogeneous characteristic according to allergic disease severity. We proposed either SLIT, with extracts by different producers, or SCIT with Pollinex 4 (Allergy Therapeutics, Worthing, UK), a product designed for ultra-short administration in 4 injections, highlighting for each kind of SIT the major practical advantages or burdens.Of 145 patients, 72 chose Pollinex 4 SCIT and 73 chose SLIT. SCIT-treated patients received a total of 90 vaccines (18 patients had double course of SCIT). SLIT-treated patients received a total of 87 vaccines (14 patients had double course of SLIT). In the SCIT group, there were 49 males and 23 females; in the SLIT group, there were 30 males and 43 females. Mean age was 36.5 years in SCIT group and 28.5 years in SLIT group. Males preferred SCIT (49 of 72 patients) and females preferred SLIT (43 of 73 patients). No severe reaction was observed either in SCIT or SLIT group.Patients are active subjects in decisional process. Trying to apply in real life the indications coming from guidelines about patient's preference is an important matter. In our patients SCIT with ultra short schedule and SLIT are similarly preferred.

A 3-year prospective post marketing survey on the safety of the recently developed ultrashort pre-seasonal subcutaneous immunotherapy (uSCIT-MPL4) with pollen allergoids adjuvanted with monophosphoryl lipid A was performed. A total of 510 patients received uSCIT-MPL4, 61% for grass, 35.7% for birch, 13.2% for parietaria and 3% for other pollens (ragweed, mugwort, and olive). A total of 3308 injections were given and the mean duration of uSCIT-MPL-4 was 2.3 years. Overall, only 7 slight systemic reactions (SR) were observed in 510 patients (1.37%) and 2.11/1000 injections suggesting that this treatment is even safer than traditional depot injection SIT.

There is now an increasing body of evidence to support the practice of allergen-specific sublingual-swallow immunotherapy (SLIT) in the treatment of IgE-mediated respiratory allergies. Recent studies on traditional injection therapy have pointed out that this form of treatment is not only capable to decrease actual allergic symptoms, but may also have long-term clinical and preventive effects and may influence atopy natural history. In the year 2000, our group published a retrospective, multicenter study showing the efficacy and safety of SLIT in a survey of 302 patients. We now carried out a second study on the same patients, with the aim of investigating long-term and preventive effects of SLIT. Beside the well-known safety and efficacy of this treatment (80.8 % of patients reported clinical benefits), SLIT proved also to elicit long term clinical effects: over a mean follow-up of 11.6 months after the end of treatment, 80.8 % of patients still mantained the previously achieved benefits. During the follow-up period, only 1 % of non-asthma patients reported an onset of respiratory symptoms, and only 9.6 % of patients undergoing new skin tests showed new sensitizations. All the clinical benefits were strongly linked to the length of treatment: patients with long-lasting benefits were treated for a mean length of 29.1 months, while patients showing a return to pre-SLIT condition were treated for a mean 13.3 months. SLIT can obtain long-term and preventive effects so far attributed to injection immunotherapy.

Twenty-seven patients with hay fever had a carbachol inhalation challenge both out of season and during the pollen season. Eight patients with allergic asthma were used as a control group. Only three patients (11.1%) demonstrated a value of a provocative dose causing a 20% fall in FEV 1 in the asthmatic range out of pollen season, but during pollen exposure, the number of positive responses significantly increased to 13 (48.1%). We observed differences regarding mean age, age of onset of symptoms, sex, and family history between patients with positive responses and patients who failed to react to inhalation challenge. It appears reasonable that an aspecific bronchial provocation test, performed during the pollen season, can detect with greater sensitivity patients with hay fever at risk of developing asthma in the future, and it also appears reasonable that these patients should be treated differently from subjects with "pure" allergic rhinitis. We expect the ongoing follow-up to clarify the prognostic value to be attributed to these findings.