Proper names and movement to the determiner position: A neurolinguistic study

3nonenoneSEMENZA C.; LONGOBARDI G; COCOLO R; GRANÀ ASemenza, Carlo; Longobardi, Giuseppe; Cocolo, R; Granà, A

Determination of bilirubin by thermal lens spectrometry and studies of its transport into hepatic cells

The liver is responsible for clearance of bilirubin, the end product of heme catabolism, from the bloodstream. The main aim of our investigation was to determine the role of the carrier protein bilitranslocase in bilirubin uptake into the liver. Our experiments consisted of exposing cell cultures to bilirubin solutions under different conditions and measuring the uptake of bilirubin into the cells. However, since bilirubin is only slightly soluble in aqueous solution (< 70 nM at pH 7.4), we had to use bilirubin concentrations that are far below the limit of detection of the commonly used techniques (e.g. LOD for HPLC with UV-Vis detection $\cong 10$ $\mu$M). TLS showed up to be a suitable technique for investigation of bilirubin uptake with an LOD of 2 nM. Under basal conditions, bilirubin uptake did not occur. However, increase of cytosolic NADH due to catabolism of specific substrates (e.g. lactate or ethanol) seemed to trigger bilirubin uptake. Furthermore, bilirubin uptake was completely inhibited by addition of specific anti-bilitranslocase antibodies. We can thus infer that, under these conditions, bilitranslocase is the main bilirubin transporter

BILIRUBIN ANALYSIS BY THERMAL LENS SPECTROMETRY: A TOOL TO INVESTIGATE THE MOLECULAR MECHANISM OF ITS MEMBRANE TRANSPORT

Bilirubin (Br), the product of heme catabolism, is formed in all cells and shed into the blood, where it is transported by albumin to the liver. At this level, it is taken up, glucurono-conjugated and excreted into the bile by an ATP-dependent efflux pump. Uptake of Br from the blood into the liver has been proposed to be mediated by OATP1B1 (Cui Y et al 2001). Later, this Br transport function has been questioned (Wang Y et al 2003). Bilitranslocase (BTL) is a plasma membrane organic anion carrier that binds Br with high affinity (Kd=2nM) (Battiston L et al 1998). The aim of this work was to test directly the Br transport capacity of BTL in liver cells. A cell transport assay was set up, based on the measurement of the time-dependent disappearance of Br from a medium bathing a monolayer of cultured human liver cells (HepG2). The involvement of BTL was investigated by testing the effect of an anti-sequence antibody on the kinetics of Br disappearance. The medium containing Br was a simple phosphate buffered saline solution (pH 7.4). Under these conditions, its solubility is <70nM, i.e. high enough to saturate BTL, but far too low to be detected by radioactive counting or conventional UV-Vis spectrometry. The samples were therefore assayed by thermal lens spectrometry (Franko M 2001), a technique that enabled to measure Br in the range 2-50nM, avoiding the confounding presence of albumin. Br uptake was found to be a quite fast phenomenon, that was abolished not only by the anti- sequence anti-BTL antibody, but also by nicotinic acid, that binds to BTL with high affinity (Kd=11 nM) at the same level as Br. The serine reagent PMSF, that binds to the Br- and nicotinic acid binding site of BTL (Passamonti S et al 1997), abolished Br uptake into cells as well. Taurocholate and digoxin, two OATP reference substrates, did not affect Br uptake in our assay. Thus, BTL is a Br carrier, as also accepted by the Transport Classification Database (http://tcdb.ucsd.edu/)

Environmental risk from digested manure in relation to their pathogen content and gaseous emissions

The primary objective of this study was to measure levels of ammonia and GHG emitted by pig and cattle manure samples before and after the anaerobic digestion process. Concurrently, to study the effect of anaerobic digestion on microorganisms\u2019 survival in manure, emission measurements were performed together with microbiological essays to determine the microbial load (E. Coli O157 for bovine samples, Coliforms, Salmonella Species, Sulphite Reducing Anaerobes, Enterococci and Lactobacilli) in manure samples before and after treatment at different sampling times. Samples were taken before and after manure treatment: in the reception pit and after the digestion process. In the laboratory, 0.2 l of each sample was placed in a tank to measure gaseous emissions through the dynamic chamber method, using an infrared photo-acoustic detector IPD (Br\ufcel & Kjaer, Multi-gas Monitor Type 1302), while collecting data every minute. These preliminary results highlight the efficacy of digestion treatment on bacteria abatement. Pathogens in cattle slurry were investigated, except for sulphite-reducing anaerobic bacteria (Clostridia) that, in some cases, were enhanced by anaerobic digestion (up to +41 %) and did not survive anaerobic digestion. Ammonia emission increased (up to +130 %) in cattle digested manure in comparison to fresh slurry. Carbon dioxide declined (-16 %) and, as expected, methane emission was considerably reduced (-82 %) by the anaerobic treatment. The current study is still in process, the expected results are to find a relation between ammonia, GHG emissions and microbial load before and after anaerobic digestion