Structural and functional analysis of ypt2, an essential ras-related gene in the fission yeast Schizosaccharomyces pombe encoding a Sec4 protein homologue.

Using the cloned Saccharomyces cerevisiae YPT1 gene as hybridization probe, a gene, designated ypt2, was isolated from the fission yeast Schizosaccharomyces pombe and found to encode a 200 amino acid long protein most closely related to the ypt branch of the ras superfamily. Disruption of the ypt2 gene is lethal. The bacterially produced ypt2 gene product is shown to bind GTP. A region of the ypt2 protein corresponding to but different from the 'effector region' of ras proteins is also different from that of ypt1 proteins of different species but identical to the 'effector loop' of the S.cerevisiae SEC4 gene product, a protein known to be required for vesicular protein transport. The S.pombe ypt2 gene under control of the S.cerevisiae GAL10 promoter is able to suppress the temperature-sensitive phenotype of a S. cerevisiae sec4 mutant, indicating a functional similarity of these GTP-binding proteins from the two very distantly related yeasts

Vitamin K-dependent carboxylase: Synthesis of an inhibitor of the glutamyl binding site

AbstractLiver microsomes contain a vitamin K and O2-dependent carboxylase that converts peptide-bound glutamyl residues to γ-carboxyglutamyl residues. The peptide Boc-O-phospho—Ser-O-phospho—Ser—Leu-OMe has now been synthesized. This peptide inhibits the carboxylation of endogenous protein precursors by a detergent-solubilized preparation of the carboxylase and is an apparent competitive inhibitor of the carboxylation of Phe—Leu—Glu—Glu—Leu

Lipid-coated zinc oxide nanocrystals as innovative ROS-generators for photodynamic therapy

Photodynamic Therapy (PDT) is a medical treatment that combines the administration of a nontoxic drug, called photosensitizer (PS), with light irradiation of the targeted region. It has been proposed as a new cancer therapy, promising better selectivity and fewer side-effects compared to traditional chemo- and radio-therapies. PSs indeed can accumulate specifically within the region of interest so that when the light is directly focused only in that region the therapeutic effect is highly localized. Traditional PSs, like chlorins and porphyrins, suffer from several drawbacks such as aggregation in biological media and poor biocompatibility. Thus, the development of innovative photosensitizers able to overcome these issues is crucial to the therapeutic action of PDT. Among the others, nanostructured Zinc Oxide (ZnO) has been recently proposed as new therapeutic agent and PS thanks to its semiconducting properties, biocompatible features, and ease of functionalization [1]. Nevertheless, further efforts are needed in order to improve its colloidal stability in biological media and to unravel the effective therapeutic mechanism. Here, we propose the synthesis and characterization of lipid-coated ZnO nanoparticles as new photosensitizer for cancer PDT [2]. First, by Dynamic Light Scattering (DLS) experiments, we show that the lipid-coating increases the colloidal stability of the ZnO NPs in Phosphate buffered saline (PBS). Then, using Electron Paramagnetic Resonance (EPR) coupled with the spin-trapping technique, we demonstrate and characterize the ability of bare and lipid-coated ZnO NPs to generate Reactive Oxygen Species (ROS) in water only when remotely actuated via light irradiation. Interestingly, our results aware that the surface chemistry of the NPs greatly influence the type of photo-generated ROS. Finally, we show that our NPs are effectively internalized inside human epithelial carcinoma cells (HeLa) via a lysosomal pathway and that they are able to generate ROS inside cancer cells. [1] B. Dumontel, M. Canta, H. Engelke, A. Chiodoni, L. Racca, A. Ancona, T. Limongi, G. Canavese and V. Cauda, ‎J. Mater. Chem. B. under revision. [2] A. Ancona, H. Engelke, N. Garino, B. Dumontel, W.Fazzini and V. Cauda, to be submitted. The support from ERC Starting Grant – Project N. 678151 “Trojananohorse” is gratefully acknowledged

Intra-oral compartment pressures: a biofunctional model and experimental measurements under different conditions of posture

Oral posture is considered to have a major influence on the development and reoccurrence of malocclusion. A biofunctional model was tested with the null hypotheses that (1) there are no significant differences between pressures during different oral functions and (2) between pressure measurements in different oral compartments in order to substantiate various postural conditions at rest by intra-oral pressure dynamics. Atmospheric pressure monitoring was simultaneously carried out with a digital manometer in the vestibular inter-occlusal space (IOS) and at the palatal vault (sub-palatal space, SPS). Twenty subjects with normal occlusion were evaluated during the open-mouth condition (OC), gently closed lips (semi-open compartment condition, SC), with closed compartments after the generation of a negative pressure (CCN) and swallowing (SW). Pressure curve characteristics were compared between the different measurement phases (OC, SC, CCN, SW) as well as between the two compartments (IOS, SPS) using analysis of variance and Wilcoxon matched-pairs tests adopting a significance level of α = 0.05. Both null hypotheses were rejected. Average pressures (IOS, SPS) in the experimental phases were 0.0, −0.08 (OC); −0.16, −1.0 (SC); −48.79, −81.86 (CCN); and −29.25, −62.51 (SW) mbar. CCN plateau and peak characteristics significantly differed between the two compartments SPS and IOS. These results indicate the formation of two different intra-oral functional anatomical compartments which provide a deeper understanding of orofacial biofunctions and explain previous observations of negative intra-oral pressures at rest

Enhanced Biostability and Cellular Uptake of Zinc Oxide Nanocrystals Shielded with Phospholipid Bilayer

The widespread use of ZnO nanomaterials for biomedical applications, including therapeutic drug delivery or stimuli-responsive activation, as well as imaging, imposes a careful control over the colloidal stability and long-term behaviour of ZnO in biological media. Moreover, the effect of ZnO nanostructures on living cells, in particular cancer cells, is still under debate. This paper discusses the role of surface chemistry and charge of zinc oxide nanocrystals, of around 15 nm in size, which influence their behaviour in biological fluids and effect on cancer cells. In particular, we address this problem by modifying the surface of pristine ZnO nanocrystals (NCs), rich of hydroxyl groups, with positively charged amino-propyl chains or, more innovatively, by self-assembling a double-lipidic membrane, shielding the ZnO NCs. Our findings show that the prolonged immersion in simulated human plasma and in the cell culture medium leads to highly colloidally dispersed ZnO NCs only when coated by the lipidic bilayer. In contrast, the pristine and amine-functionalized NCs form huge aggregates after already one hour of immersion. Partial dissolution of these two samples into potentially cytotoxic Zn2+ cations takes place, together with the precipitation of phosphate and carbonate salts on the NCs’ surface. When exposed to living HeLa cancer cells, higher amounts of lipid-shielded ZnO NCs are internalized with respect to the other samples, thus showing a reduced cytotoxicity, based on the same amount of internalized NCs. These results pave the way for the development of novel theranostic platforms based on ZnO NCs. The new formulation of ZnO shielded with a lipid-bilayer will prevent strong aggregation and premature degradation into toxic by-products, and promote a highly efficient cell uptake for further therapeutic or diagnostic functions

Neural correlates and determinants of approach-avoidance conflict in the prelimbic prefrontal cortex

The recollection of environmental cues associated with threat or reward allows animals to select the most appropriate behavioral responses. Neurons in the prelimbic cortex (PL) respond to both threat-and reward-associated cues. However, it remains unknown whether PL regulates threat-avoidance vs. reward-approaching responses when an animals’ decision depends on previously associated memories. Using a conflict model in which male Long-Evans rats retrieve memories of shock and food-paired cues, we observed two distinct phenotypes during conflict: I) rats that continued to press a lever for food (Pressers); and ii) rats that exhibited a complete suppression in food seeking (Non-pressers). Single-unit recordings revealed that increased risk-taking behavior in Pressers is associated with persistent food-cue responses in PL, and reduced spontaneous activity in PL glutamatergic (PLGLUT) neurons during conflict. Activating PLGLUT neurons in Pressers attenuated foodseeking responses in a neutral context, whereas inhibiting PLGLUT neurons in Non pressers reduced defensive responses and increased food approaching during conflict. Our results establish a causal role for PLGLUT neurons in mediating individual variability in memory-based risky decision making by regulating threat-avoidance vs. reward-approach behaviors.Fil: Fernandez Leon, Jose Alberto. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigaciones en Física e Ingeniería del Centro de la Provincia de Buenos Aires. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigaciones en Física e Ingeniería del Centro de la Provincia de Buenos Aires. - Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigaciones en Física e Ingeniería del Centro de la Provincia de Buenos Aires; Argentina. University of Texas Health Science Center at Houston; Estados UnidosFil: Engelke, Douglas S.. University of Texas Health Science Center at Houston; Estados UnidosFil: Aquino Miranda, Guillermo. University of Texas Health Science Center at Houston; Estados UnidosFil: Goodson, Alexandria. University of Texas Health Science Center at Houston; Estados Unidos. Rice University; Estados UnidosFil: Rasheed, María N.. University of Texas Health Science Center at Houston; Estados UnidosFil: Do Monte, Fabricio H.. University of Texas Health Science Center at Houston; Estados Unidos. Rice University; Estados Unido

The interface between the stellar wind and interstellar medium around R Cassiopeiae revealed by far-infrared imaging

The circumstellar dust shells of intermediate initial-mass (about 1 to 8 solar masses) evolved stars are generated by copious mass loss during the asymptotic giant branch phase. The density structure of their circumstellar shell is the direct evidence of mass loss processes, from which we can investigate the nature of mass loss. We used the AKARI Infrared Astronomy Satellite and the Spitzer Space Telescope to obtain the surface brightness maps of an evolved star R Cas at far-infrared wavelengths, since the temperature of dust decreases as the distance from the star increases and one needs to probe dust at lower temperatures, i.e., at longer wavelengths. The observed shell structure and the star's known proper motion suggest that the structure represents the interface regions between the dusty wind and the interstellar medium. The deconvolved structures are fitted with the analytic bow shock structure to determine the inclination angle of the bow shock cone. Our data show that (1) the bow shock cone of 1 - 5 x 10^-5 solar masses (dust mass) is inclined at 68 degrees with respect to the plane of the sky, and (2) the dust temperature in the bow shock cone is raised to more than 20 K by collisional shock interaction in addition to the ambient interstellar radiation field. By comparison between the apex vector of the bow shock and space motion vector of the star we infer that there is a flow of interstellar medium local to R Cas whose flow velocity is at least 55.6 km/s, consistent with an environment conducive to dust heating by shock interactions.Comment: 7 pages, 2 figures, accepted for publication in Astronomy and Astrophysic

DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk.

The aminopeptidase DPP9 removes dipeptides from N-termini of substrates having a proline or alanine in second position. Although linked to several pathways including cell survival and metabolism, the molecular mechanisms underlying these outcomes are poorly understood. We identified a novel interaction of DPP9 with Filamin A, which recruits DPP9 to Syk, a central kinase in B-cell signalling. Syk signalling can be terminated by degradation, requiring the ubiquitin E3 ligase Cbl. We show that DPP9 cleaves Syk to produce a neo N-terminus with serine in position 1. Pulse-chases combined with mutagenesis studies reveal that Ser1 strongly influences Syk stability. Furthermore, DPP9 silencing reduces Cbl interaction with Syk, suggesting that DPP9 processing is a prerequisite for Syk ubiquitination. Consistently, DPP9 inhibition stabilizes Syk, thereby modulating Syk signalling. Taken together, we demonstrate DPP9 as a negative regulator of Syk and conclude that DPP9 is a novel integral aminopeptidase of the N-end rule pathway