Nitroxide spin labels and EPR spectroscopy: A powerful association for protein dynamics studies.

Site-Directed Spin Labelling (SDSL) technique is based on the attachment of a paramagnetic label onto a specific position of a protein (or other bio-molecules) and the subsequent study by Electron Paramagnetic Resonance (EPR) spectroscopy. In particular, continuous-wave EPR (cw-EPR) spectra can detect the local conformational dynamics for proteins under various conditions. Moreover, pulse-EPR experiments on doubly spin-labelled proteins allow measuring distances between spin centres in the 1.5-8 nm range, providing information about structures and functions. This review focuses on SDSL-EPR spectroscopy as a structural biology tool to investigate proteins using nitroxide labels. The versatility of this spectroscopic approach for protein structural characterization has been demonstrated through the choice of recent studies. The main aim is to provide a general overview of the technique, particularly for non-experts, to spread the applicability of this technique in various fields of structural biology.

A combined NMR and EPR investigation on the effect of the disordered RGG regions in the structure and the activity of the RRM domain of FUS.

Structural disorder represents a key feature in the mechanism of action of RNA-binding proteins (RBPs). Recent insights revealed that intrinsically disordered regions (IDRs) linking globular domains modulate their capability to interact with various sequences of RNA, but also regulate aggregation processes, stress-granules formation, and binding to other proteins. The FET protein family, which includes FUS (Fused in Sarcoma), EWG (Ewing Sarcoma) and TAF15 (TATA binding association factor 15) proteins, is a group of RBPs containing three different long IDRs characterized by the presence of RGG motifs. In this study, we present the characterization of a fragment of FUS comprising two RGG regions flanking the RNA Recognition Motif (RRM) alone and in the presence of a stem-loop RNA. From a combination of EPR and NMR spectroscopies, we established that the two RGG regions transiently interact with the RRM itself. These interactions may play a role in the recognition of stem-loop RNA, without a disorder-to-order transition but retaining high dynamics.

Is the sugar intestinal permeability test a reliable investigation for coeliac disease screening?

BACKGROUND: The lactulose/mannitol (L/M) intestinal permeability test is a simple, non-invasive screening test for coeliac disease. The reliability of the L/M test has so far only been tested in selected groups of patients with coeliac disease. AIM: To evaluate the reliability of the L/M test in a group of patients with coeliac disease who had been diagnosed during mass serological screening of the general population. PATIENTS AND METHODS: Twenty nine patients with coeliac disease detected by screening and 54 age matched coeliac disease free controls aged 11-15 years underwent an L/M test with 5 g lactulose and 2 g mannitol in isotonic aqueous solution. Urinary sugars were measured by high performance liquid chromatography. RESULTS: The median % urinary recovery of lactulose (lactulose UR) was significantly higher in patients with coeliac disease than in controls (0.63 v 0.18, p < 0.001). The mean mannitol % UR was lower in patients with coeliac disease than in controls (17.6 v 18.5) but the difference was not significant. The median urinary L%/M% ratio was significantly higher in patients with coeliac disease than in controls (0.038 v 0.014, p < 0.001). However, 16 of the 29 patients with coeliac disease showed an L%/M% ratio within normal limits (< 0.044). CONCLUSIONS: The L/M intestinal permeability test is not a valuable tool for screening of coeliac disease in the general population. The pattern of the urinary probe recovery suggests that many patients with coeliac disease could remain symptomless because the extent of their intestinal mucosal damage is small ("short" coeliac disease).

[Protein-losing gastroenteropathy (Ménétrier's disease) in childhood: a report of 3 cases]. / Gastroenteropatia protido-disperdente (malattia di Ménétrier) nell'età pediatrica: descrizione di tre casi.

So far, Ménétrier's disease has been thought to be an uncommon disorder in children. It is characterized by hypertrophic gastritis, protein-losing enteropathy, hypoproteinemia and edema. During childhood, the main features of this condition include an abrupt onset and a spontaneous recovery. In this paper we describe three children, aging between 3 months and 3 years, who presented with protracted vomiting, generalized edema, colitis (one case) and elevated serum aminotransferases (one case). The diagnosis of Ménétrier's disease was made by finding the typical endoscopic and histological picture of the gastric mucosa (two cases) or by the radiological findings (one case). The fecal alpha-1-antitrypsin excretion, which is a marker of the protein-losing enteropathy, was high in all patients. Two cases showed evidences for a primary CMV infection as the possible cause of Ménétrier's disease, due to the presence of cytomegalic inclusions in the gastric mucosa and the IgM class anti-CMV antibodies positivity. All 3 cases, who received only a support treatment (plasma and albumin intravenous infusions), completely recovered in a 2-3 weeks time. In conclusion, it is confirmed that in children a protein-losing gastroenteropathy may be caused by a primary infection with CMV.

Intestinal permeability changes during the first month: effect of natural versus artificial feeding.

The aim of the study was to investigate the effect of age and feeding pattern on intestinal permeability during the first month of life. The subjects were 72 full-term, healthy neonates who were (a) exclusively breast-fed (BF group, n = 36) or (b) artificially fed (CM group, n = 36) with either an adapted formula (AF group, n = 17) or a partly hydrolyzed (hypoantigenic) formula (HA group, n = 19). A lactulose/mannitol (lac/man) intestinal permeability test was performed at 1 and 7 days (steady-state method, n = 72), then at 30 days of life (single oral load, n = 47). Urinary lactulose and mannitol were measured by HPLC. The mean lac/man urinary ratio dropped from 1.27 +/- 0.73 (day 1) to 0.34 +/- 0.36 at day 7 and to 0.22 +/- 0.21 at day 30. At 7 days BF infants showed a significantly lower lac/man urinary ratio (0.22 +/- 0.25) than the CM group (0.47 +/- 0.41). The human neonate shows a developmental pattern of sugar intestinal permeability that resembles gut closure observed in other mammals. Intestinal permeability decreases faster in breast-fed babies than in those fed with adapted or HA formulas.

Modifications of Intestinal Permeability Test Induced by Biliopancreatic Diversion: Preliminary Results.

BACKGROUND: Biliopancreatic Diversion (BPD), excluding the jejunum and part of the ileum from the transit of the food, reduces the absorptive intestinal area available to 250 cm of the distal ileum. The Intestinal Permeability Test (IPT) with Lactulose/Mannitol is performed to assess the intestinal mucosa function. The aim of this study was to investigate the effect of intestinal anatomical modifications induced by BPD on IPT in a group of severely obese patients. METHODS: Group A: 18 severely obese subjects who underwent BPD; the IPT was performed before (T0) and 8.2 +/- 0.9 days after BPD (T1). Group B: nine subjects of Group A; IPT was repeated 96.1 +/- 12.7 days (T2) and 180.4 +/- 19.7 days (T3) after BPD, respectively. IPT was expressed as the %Lactulose/%Mannitol ratio in the urine collected during 5 h after oral administration (normal %L/%M < 0.04). RESULTS: Data from Group A (paired Student's t-test) exhibited significantly higher values in T, with respect to T0 for %L/%M (p < 0.05) and for %L (p < 0.05), and significantly lower values in T, with respect to T0 (p < 0.001) for %M. In the Group B analysis of Variance from T0-T1-T2-T3 resulted statistically significant (p < 0.05) for % L/ % M and % M. CONCLUSIONS: The reduction of intestinal absorption surface induced by BPD causes a significant increase of the Lactulose/Mannitol IPT values, showing an intestinal mucosa function impairment. The IPT values improve progressively at 3 and 6 months after this surgical procedure.