Single-cell resolution imaging of membrane-anchored hepatitis C virus NS3/4A protease activity

The study of host and viral membrane-associated proteases has been hampered due to a lack of in vivo assays. We report here the development of a cell-based fluorescence assay for detecting hepatitis C virus (HCV) NS3/4A juxtamembrane protease activity. Intracellular membrane-anchored protein substrates were engineered comprising: (1) an endoplasmic reticulum targeting domain, the HCV NS5A N-terminal amphipathic alpha-helix; (2) a NS3/4A-specific cleavage site; and (3) a red fluorescent reporter group, DsRed. The results of our immunofluorescence and Western blotting studies demonstrate that our membrane-bound fluorescent probe was cleaved specifically and efficiently by NS3/4A expressed in human cells.     

Hourglass SiO2 coating increases the performance of planar patch-clamp

Obtaining high-throughput electrophysiological recordings is an ongoing challenge in ion channel biophysics and drug discovery. One particular area of development is the replacement of glass pipettes with planar devices in order to increase throughput. However, successful patch-clamp recordings depend on a surface coating which ideally should promote and stabilize giga-seal formation. Here, we present data supporting the use of a structured SiO(2) coating to improve the ability of cells to form a "seal" with a planar patch-clamp substrate. The method is based on a correlation study taking into account structure and size of the pores, surface roughness and chip capacitance. The influence of these parameters on the quality of the seal was assessed. Plasma-enhanced chemical vapour deposition (PECVD) of SiO(2) led to an hourglass structure of the pore and a tighter seal than that offered by a flat, thermal SiO(2) surface. The performance of PECVD chips was validated by recording recombinant potassium channels, BK(Ca), expressed in stable HEK-293 cell lines and in inducible CHO cell lines and low conductance IRK1, and endogenous cationic currents from CHO cells. This multiparametric investigation led to the production of improved chips for planar patch-clamp applications which allow electrophysiological recordings from a wide range of cell lines.     

The stable carbon and nitrogen isotopic composition of vegetation in tropical forests of the Amazon Basin, Brazil

Here we present the within-site, seasonal, and interannual variations of the carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope ratios of leaves, wood, bark and litter from four sites in the Amazon region, Brazil. Samples were collected in Manaus (3° 06′07′′ S; 60°01′30′′ W), Ji-Paraná (10°53′07′′ S; 61°57′06′′ W), and Santarém (2°26′35′′ S; 54°42′30′′ W) with mean annual precipitation of 2207, 2040 and 1909 mm respectively. The overall average for all leaf samples was for δ¹³C and for δ¹⁵N (n=756). The leaf δ values at these sites were often but not always statistically distinct from each other. The δ¹³C values varied from to . Pronounced differences in δ¹³C values occurred with height associated with differences in forest structure. The δ¹³C of leaf dry matter showed seasonal variations associated with the length of the dry season, despite the fact that total annual precipitation was similar among the studied sites. Leaf δ¹⁵N values ranged from to a maximum value of , and the Santarém sites showed more enriched values than Manaus and Ji-Paraná sites. No seasonal variation was detected in the δ¹⁵N of leaves, but significant differences were observed among sites and with changes in canopy height. The isotope ratio data are consistent with our current understanding of the roles of light, water availability, and recycling of soil-respired CO₂ influences on δ¹³C and consistent with our understanding that an open nitrogen cycle can lead to high δ¹⁵N values despite a significant number of legumes in the vegetation.     

Interactions of earthworms with Atrazine-degrading bacteria in an agricultural soil

In the last 10 years, accelerated mineralization of Atrazine (2-chloro-ethylamino-6-isopropylamino-s-triazine) has been evidenced in agricultural soils repeatedly treated with this herbicide. Here, we report on the interaction between earthworms, considered as soil engineers, and the Atrazine-degrading community. The impact of earthworm macrofauna on Atrazine mineralization was assessed in representative soil microsites of earthworm activities (gut contents, casts, burrow linings). Soil with or without earthworms, namely the anecic species Lumbricus terrestris and the endogenic species Aporrectodea caliginosa, was either inoculated or not inoculated with Pseudomonas sp. ADP, an Atrazine-degrading strain, and was either treated or not treated with Atrazine. The structure of the bacterial community, the Atrazine-degrading activity and the abundance of atzA, B and C sequences in soil microsites were investigated. Atrazine mineralization was found to be reduced in representative soil microsites of earthworm activities. Earthworms significantly affected the structure of soil bacterial communities. They also reduced the size of the inoculated population of Pseudomonas sp. ADP, thereby contributing to the diminution of the Atrazine-degrading genetic potential in representative soil microsites of earthworm activities. This study illustrates the regulation produced by the earthworms on functional bacterial communities involved in the fate of organic pollutants in soils.     

Comparative studies of duodenal and macrophage ferroportin proteins

Intestinal epithelial cells and reticuloendothelial macrophages are, respectively, involved in diet iron absorption and heme iron recycling from senescent erythrocytes, two critical processes of iron homeostasis. These cells appear to use the same transporter, ferroportin (Slc40a1), to export iron. The aim of this study was to compare the localization, expression, and regulation of ferroportin in both duodenal and macrophage cells. Using a high-affinity purified polyclonal antibody, we analyzed the localization and expression of ferroportin protein in the spleen, liver, and duodenum isolated from normal mice as well as from well-characterized mouse models of altered iron homeostasis. Ferroportin was found to be predominantly expressed in enterocytes of the duodenum, in splenic macrophages, and in liver Kupffer cells. Interestingly, the protein species detected in these cells migrated differently on SDS-PAGE. These differences in apparent molecular masses were partly explained by posttranslational complex N-linked glycosylations. In addition, in enterocytes, the transporter was mostly expressed at the basolateral membrane, whereas in bone marrow-derived macrophages, ferroportin was found predominantly localized in the intracellular vesicular compartment. However, some microdomains positive for ferroportin were also detected at the plasma membrane of macrophages. Despite these differences, we observed a parallel upregulation of ferroportin expression in tissue macrophages and enterocytes in response to iron-restricted erythropoiesis, suggesting that iron homeostasis is likely maintained through coordinate expression of the iron exporter in both intestinal and phagocytic cells. Our data also confirm a predominant regulation of ferroportin through systemic regulator(s) likely including hepcidin.     

A peptide inhibitor of MurA UDP-N-acetylglucosamine enolpyruvyl transferase: the first committed step in peptidoglycan biosynthesis

The MurA enzyme from Pseudomonas aeruginosa was purified to homogeneity and found to be biologically active as a UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase in a coupled enzyme assay where ATPase activity was measured by the release of inorganic phosphate. A microtiter plate assay coupled to competitive biopanning using the UDP-N-acetylglucosamine was used to screen 10⁹ C-7-C and 12-mers peptides from phage display libraries. From 60 phage-encoded peptides identified after the fourth round of biopanning, deduced amino acid sequences were aligned and two peptides were synthesized and tested for inhibition of the MurA-catalyzed reaction. The PEP 1354 peptide inhibited the ATPase activity of MurA with an IC₅₀ value of 200 μM and was found to be a competitive inhibitor of UNAG. The pre-incubation of MurA with inhibitor indicated a time-independent inhibition. This time-dependent inhibition is the first report of peptide inhibitors of MurA, which represent the scaffold for the synthesis of inhibitory peptidomimetic molecules.     

Reduced pulsatility does not explain renal hyporesponsiveness to cardiac natriuretic peptides in pulmonary hypertension

A pulsatile secretory pattern is assumed to improve hormonal efficiency. We examined the short-term time courses of circulating atrial (ANP) and brain natriuretic peptides (BNP) in patients with pulmonary hypertension (PH) and reduced renal efficiency of ANP-BNP, reflected by low natriuresis/ANP or BNP ratios. Compared to controls, we observed a persistence of ANP and BNP pulsatility in PH patients with a similar periodicity of 20min. Pulse amplitude increased proportionally to the rise in mean plasma level observed in patients (around 27%). In PH patients, the decrease in ANP-BNP renal efficiency is not attributable to a loss of the rhythmic pulsatility of these hormones.     

Neurohormones and inflammatory mediators in patients with heart failure undergoing cardiac resynchronization therapy: time courses and prediction of response

Despite interest in neurohormonal activation as a determinant of prognosis in chronic heart failure (CHF) and as a target for pharmacological treatments, data are lacking on the time-related effects of electrical cardiac resynchronization therapy (CRT) on a broad spectrum of neurohormones and cytokines. The aim of this study was to assess time-courses and extents of changes within the neurohormonal profile of CHF patients treated with CRT. We performed a prospective follow-up study in 32 patients with NYHA class III-IV CHF to investigate the effects of CRT on a broad panel of neurohormones proposed for characterization of CHF patients. Levels of atrial and brain natriuretic peptides (ANP, BNP), epinephrine, norepinephrine, aldosterone, plasma renin activity, IL-6, TNF, soluble receptors sTNFR1 and 2, and chromogranin A were assessed before implantation and after 3 months of CRT; when feasible, measurements were also performed at 1 week, 1 month and 12 months (clinical evaluation, echocardiography and ECG were also performed at each time-point). The results showed that at 3 months improvement in NYHA class and echographically assessed left ventricular (LV) reverse structural remodeling were accompanied by significant reductions versus baseline in ANP and BNP, but not in other neurohormones. Moreover a baseline ANP concentration < or = 150 pg/ml was a good predictor of response to CRT in terms of NYHA class reduction and reverse LV remodeling. In conclusion 3 months of CRT significantly reduce natriuretic peptides concentrations, while values of other neurohormones and inflammatory cytokines are relatively unvaried. A baseline ANP concentration < or = 150 pg/ml might be a clinically useful predictor of medium-term response to CRT.     

The Drosophila inhibitor of apoptosis protein DIAP2 functions in innate immunity and is essential to resist gram-negative bacterial infection

The founding member of the inhibitor of apoptosis protein (IAP) family was originally identified as a cell death inhibitor. However, recent evidence suggests that IAPs are multifunctional signaling devices that influence diverse biological processes. To investigate the in vivo function of Drosophila melanogaster IAP2, we have generated diap2 null alleles. diap2 mutant animals develop normally and are fully viable, suggesting that diap2 is dispensable for proper development. However, these animals were acutely sensitive to infection by gram-negative bacteria. In Drosophila, infection by gram-negative bacteria triggers the innate immune response by activating the immune deficiency (imd) signaling cascade, a NF-kappaB-dependent pathway that shares striking similarities with the pathway of mammalian tumor necrosis factor receptor 1 (TNFR1). diap2 mutant flies failed to activate NF-kappaB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumbed to bacterial infection. Our genetic epistasis analysis places diap2 downstream of or in parallel to imd, Dredd, Tak1, and Relish. Therefore, DIAP2 functions in the host immune response to gram-negative bacteria. In contrast, we find that the Drosophila TNFR-associated factor (Traf) family member Traf2 is dispensable in resistance to gram-negative bacterial infection. Taken together, our genetic data identify DIAP2 as an essential component of the Imd signaling cascade, protecting the organism from infiltrating microbes.