Live-cell topology assessment of URG7, MRP6₁₀₂ and SP-C using glycosylatable green fluorescent protein in mammalian cells.

Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membranetopology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylationin the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas nofluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By usingmammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6102,SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in Cinform.MRP6102and SP-C(Leu/Val) are inserted into the membrane in Coutform. A minor population of untarget-ed SP-C is removed by proteasome dependent quality control system

Reversible adsorption on a random site surface

We examine the reversible adsorption of hard spheres on a random site surface in which the adsorption sites are uniformly and randomly distributed on a plane. Each site can be occupied by one solute provided that the nearest occupied site is at least one diameter away. We use a numerical method to obtain the adsorption isotherm, i.e. the number of adsorbed particles as a function of the bulk activity. The maximum coverage is obtained in the limit of infinite activity and is known exactly in the limits of low and high site density. An approximate theory for the adsorption isotherms, valid at low site density, is developed by using a cluster expansion of the grand canonical partition function. This requires as input the number of clusters of adsorption site of a given size. The theory is accurate for the entire range of activity as long as the site density is less than about 0.3 sites per particle area. We also discuss a connection between this model and the vertex cover problem.Comment: 16 pages, 10 figure

Characterization of a Functional NTPDase in the Endoplasmic Reticulum of Rat Submandibular Salivary Gland

Nucleotidase activity and Ca-uptake were characterized in endoplasmic reticulum (ER) enriched rat submandibular gland (SMG) microsomal preparations. (i) Ca-uptake had characteristics of an ER Ca-ATPase. (ii) Nucleotidase activity was equally stimulated by calcium, magnesium and manganese, but with different Km values. (iii) Specific inhibitors of P-type Ca-ATPases were ineffective on nucleotidase activity, demonstrating that this activity was not related to calcium uptake and did not correspond to classical Ca2+ pumps. (iv) ATP and UTP were more efficient substrates, whereas ADP and UDP were hydrolyzed at significantly slower rate. (v) Nucleotidase activity was sensitive to mild detergent solubilization and insensitive to ionophore addition. (vi) Nucleotidase activity was strongly inhibited by suramin, a nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitor. (vii) Nucleotidase activity exponentially diminished as function of time. All these observations are consistent with a NTPDase identity. The presence of a NTPDase was demonstrated by immunohistochemistry in rat SMG. Immunoreactivity was stronger in ductal cells than in mucous and serous acini. Although this enzyme was observed in the plasma membrane, colocalization with the ER marker calnexin revealed a specific subcellular localization in this organelle of all three types of cell. The putative function of this NTPDase activity in salivary glands is discussed.Fil: Ostuni, M. A.. Inserm; Francia. Université Paris; Francia. Universidad de Buenos Aires. Facultad de Odontología; ArgentinaFil: Egido, P.. Universidad de Buenos Aires. Facultad de Odontología; ArgentinaFil: Peranzi, G.. Inserm; Francia. Université Paris; FranciaFil: Alonso, Guillermo Luis. Universidad de Buenos Aires. Facultad de Odontología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lacapere, J. J.. Inserm; Francia. Université Paris; FranciaFil: Gonzalez, D. A.. Inserm; Francia. Université Paris; Franci

Extracellular ATP hydrolysis in Caco-2 human intestinal cell line

Extracellular nucleotides and nucleosides activate signaling pathways that play major roles in the physiology and pathophysiology of the gastrointestinal tract. Ectonucleotidases hydrolyze extracellular nucleotides and thus regulate ligand exposure to purinergic receptors. In this study, we investigated the expression, localization and activities of ectonucleotidases using Caco-2 cells, a model of human intestinal epithelial cells. In addition, by studying ATP release and the rates of extracellular ATP (eATP) hydrolysis, we analyzed the contribution of these processes to the regulation of eATP in these cells. Results show that Caco-2 cells regulate the metabolism of eATP and by-products by ecto-nucleoside triphosphate diphosphohydrolase-1 and -2, a neutral ecto-phosphatase and ecto-5′-nucleotidase. All these ectoenzymes were kinetically characterized using intact cells, and their presence confirmed by denatured and native gels, western blot and cytoimmunofluorescence techniques. In addition, regulation of eATP was studied by monitoring the dynamic balance between intracellular ATP release and ectoATPase activity. Following mechanical and hypotonic stimuli, Caco-2 cells triggered a strong but transient release of intracellular ATP, with almost no energy cost, leading to a steep increase of eATP concentration, which was later reduced by ectoATPase activity. A data-driven algorithm allowed quantifying and predicting the rates of ATP release and ATP consumption contributing to the dynamic accumulation of ATP at the cell surface.Fil: Schachter, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Alvarez, Cora Lilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Bazzi, Zaher. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Faillace, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Corradi, Gerardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Hattab, C.. Universite de Paris. Institut National de la Transfusion Sanguine.; FranciaFil: Rinaldi, Debora Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez-Lebrero, Rodolfo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Pucci Molineris, Melisa Eliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Sévigny, J.. Laval University; CanadáFil: Ostuni, M. A.. Universite de Paris; Francia. Universite Paris D. Diderot - Paris 7. French National Institute Of Blood Transfusion.; FranciaFil: Schwarzbaum, Pablo Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

A Linked Data Recommender System Using a Neighborhood-Based Graph Kernel

Abstract. The ultimate mission of a Recommender System (RS) is to help users discover items they might be interested in. In order to be really useful for the end-user, Content-based (CB) RSs need both to harvest as much information as possible about such items and to effectively han-dle it. The boom of Linked Open Data (LOD) datasets with their huge amount of semantically interrelated data is thus a great opportunity for boosting CB-RSs. In this paper we present a CB-RS that leverages LOD and profits from a neighborhood-based graph kernel. The proposed ker-nel is able to compute semantic item similarities by matching their local neighborhood graphs. Experimental evaluation on the MovieLens dataset shows that the proposed approach outperforms in terms of accuracy and novelty other competitive approaches.

Facile one-spot synthesis of highly branched polycaprolactone

Reported is the first solvent-free (bulk) synthesis of degradable/bioresorbable, highly branched polymers via tin octanoate Sn(Oct2) catalysed controlled ring opening co-polymerisation (ROP) of mono and di-functional lactone monomers that proceed to near quantitative conversion. The successful isolation of solvent soluble, highly branched structures was shown to be dependent on both the concentration of the di-functional monomer and the overall reaction time. Comparison with analogous systems utilising controlled radical polymerisation (CRP) to form the highly/hyper branched polymers suggested significant experimental differences between the two chain growth methods. The maximum proportion of di-functional monomer without gelation ensuing was found to be 0.6 equivalents w.r.t. mono-functional monomer (c.f. 1 with CRP) and the onset of significant levels of branching occurred at approximately 90% conversion (c.f. ~70% with CRP). These differences and significant disparity in reaction times were attributed to (a) the coordination and insertion (C+I) propagation mechanism adopted by the Sn catalyst and (b) the presence of additional trans-esterification reactions at high conversion. Evidence is presented to support the conclusion that there are two mechanisms contributing to the overall branching process in the ROP system at high conversion. First, the C+I mechanism promotes growth of linear polymer until approximately 90% conversion, after which both the C+I and trans-esterification processes contribute to the interchain branching process. The branched nature of the molecular structures was supported by confirmation plots generated from static light scattering. This data demonstrated that the polymers synthesised exhibit varying degrees of branching, consistent with the di-functional monomer (4,4’-bioxepanyl-7,7’-dione - BOD) concentration in the feed. The degree of branching was calculated using 3 different methods and the results were shown to be independent of method. Finally, DSC analysis of the polymers demonstrated correlation between the degree of branching achieved and the observed Tm for the material where increased branching leads to a drop in the recorded Tm

ATPe Dynamics in Protozoan Parasites: Adapt or Perish

In most animals, transient increases of extracellular ATP (ATPe) are used for physiological signaling or as a danger signal in pathological conditions. ATPe dynamics are controlled by ATP release from viable cells and cell lysis, ATPe degradation and interconversion by ecto-nucleotidases, and interaction of ATPe and byproducts with cell surface purinergic receptors and purine salvage mechanisms. Infection by protozoan parasites may alter at least one of the mechanisms controlling ATPe concentration. Protozoan parasites display their own set of proteins directly altering ATPe dynamics, or control the activity of host proteins. Parasite dependent activation of ATPe conduits of the host may promote infection and systemic responses that are beneficial or detrimental to the parasite. For instance, activation of organic solute permeability at the host membrane can support the elevated metabolism of the parasite. On the other hand ecto-nucleotidases of protozoan parasites, by promoting ATPe degradation and purine/pyrimidine salvage, may be involved in parasite growth, infectivity, and virulence. In this review, we will describe the complex dynamics of ATPe regulation in the context of protozoan parasite–host interactions. Particular focus will be given to features of parasite membrane proteins strongly controlling ATPe dynamics. This includes evolutionary, genetic and cellular mechanisms, as well as structural-functional relationships.Instituto de Investigaciones Bioquímicas de La Plat

Multidimensional analysis of the frequencies and rates of cytokine secretion from single cells by quantitative microengraving

The large diversity of cells that comprise the human immune system requires methods that can resolve the individual contributions of specific subsets to an immunological response. Microengraving is process that uses a dense, elastomeric array of microwells to generate microarrays of proteins secreted from large numbers of individual live cells ([similar]10⁴–10⁵ cells/assay). In this paper, we describe an approach based on this technology to quantify the rates of secretion from single immune cells. Numerical simulations of the microengraving process indicated an operating regime between 30 min–4 h that permits quantitative analysis of the rates of secretion. Through experimental validation, we demonstrate that microengraving can provide quantitative measurements of both the frequencies and the distribution in rates of secretion for up to four cytokines simultaneously released from individual viable primary immune cells. The experimental limits of detection ranged from 0.5 to 4 molecules/s for IL-6, IL-17, IFNγ, IL-2, and TNFα. These multidimensional measures resolve the number and intensities of responses by cells exposed to stimuli with greater sensitivity than single-parameter assays for cytokine release. We show that cells from different donors exhibit distinct responses based on both the frequency and magnitude of cytokine secretion when stimulated under different activating conditions. Primary T cells with specific profiles of secretion can also be recovered after microengraving for subsequent expansion in vitro. These examples demonstrate the utility of quantitative, multidimensional profiles of single cells for analyzing the diversity and dynamics of immune responses in vitro and for identifying rare cells from clinical samples.National Institute of Allergy and Infectious Diseases (U.S.) (Award no. 5U19AI050864-07)National Institute of Allergy and Infectious Diseases (U.S.) (Award no. F32AI651003)National Institute of Allergy and Infectious Diseases (U.S.) (Award no. U19AI070352)National Institute of Allergy and Infectious Diseases (U.S.) (Award no. U19AI046130)National Institute of Allergy and Infectious Diseases (U.S.) (Award no. P01AI045757)National Institute of Neurological Disorders and Stroke (U.S.) (Jacob Javits Merit Award (NS2427))Massachusetts Institute of Technology (Texaco- Mangelsdorf Career Development Professor

Preparation and Characterization of Covalently Binding of Rat Anti-human IgG Monolayer on Thiol-Modified Gold Surface

The 16-mercaptohexadecanoic acid (MHA) film and rat anti-human IgG protein monolayer were fabricated on gold substrates using self-assembled monolayer (SAM) method. The surface properties of the bare gold substrate, the MHA film and the protein monolayer were characterized by contact angle measurements, atomic force microscopy (AFM), grazing incidence X-ray diffraction (GIXRD) method and X-ray photoelectron spectroscopy, respectively. The contact angles of the MHA film and the protein monolayer were 18° and 12°, respectively, all being hydrophilic. AFM images show dissimilar topographic nanostructures between different surfaces, and the thickness of the MHA film and the protein monolayer was estimated to be 1.51 and 5.53 nm, respectively. The GIXRD 2θ degrees of the MHA film and the protein monolayer ranged from 0° to 15°, significantly smaller than that of the bare gold surface, but the MHA film and the protein monolayer displayed very different profiles and distributions of their diffraction peaks. Moreover, the spectra of binding energy measured from these different surfaces could be well fitted with either Au4f, S2p or N1s, respectively. Taken together, these results indicate that MHA film and protein monolayer were successfully formed with homogeneous surfaces, and thus demonstrate that the SAM method is a reliable technique for fabricating protein monolayer