Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding similar to42 and 36 mg EPF from 300 ml bacterial and I L Sf9 cultures, respectively. The preparations were highly purified ( greater than or equal to99% purity on SDS-PAGE for the bacterial products and greater than or equal to97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immuno suppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity. (C) 2003 Elsevier Inc. All rights reserved.

An SNP-based PCR assay to differentiate between Listeria monocytogenes lineages derived from phylogenetic analysis of the sigB gene

The alternative sigma factor sigB gene is involved in the stress response regulation of Listeria monocytogenes, and contributes towards growth and survival in adverse conditions. This gene was examined to determine if it could be a useful indicator of lineage differentiation, similar to the established method based on ribotyping. The sigB sequence was resolved in four local L. monocytogenes strains and the phylogenetic relationship among these, and a further 21 sigB gene sequences from strains of different serotype and lineage including two Listeria innocua strains, obtained from the GenBank database were determined. The sigB nucleotide sequences of these 25 Listeria strains were then examined for single nucleotide polymorphic (SNP) sites that could differentiate between the three lineages. Based on nucleotide sequences L. monocytogenes lineage F serotype 1/2b and 4b clustered together, lineage II/serotype 1/2a and 1/2c strains clustered together, lineage III/serotypes 4a and 4c strains clustered together and L. innocua strains clustered together as an outgroup. SNPs differentiating the three lineages were identified. Individual allele-specific PCR reactions based on these polymorphisms were successful in grouping known and a further 37 local L. monocytogenes isolates into the three lineages. (C) 2003 Elsevier B.V. All fights reserved.

Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors. (C) 2003 Elsevier Science (USA). All rights reserved.

Empoderamiento de mujeres de una ONG colombiana. Un estudio de caso simple

Resumen:Este artículo es un estudio de caso sobre cómo un grupo de mujeres se empoderan, conforman una ONG y generan empoderamiento en su comunidad, argumentando que el empoderamiento es un proceso en el tiempo, el cual se da en tres niveles psicológico, organizacional y comunitario. El empoderamiento en las mujeres es interdependiente, emerge como producto de la acción de ellas sobre los problemas sociales y públicos de su comunidad, e impacta el desarrollo social en la medida en que se transforman los valores que tienen las mujeres sobre qué es una mujer y cómo es en su realidad. La ONGD de mujeres genera empoderamiento a partir de la Teología de la liberación basada en la eliminación de la opresión de género. Los hallazgos del estudio son un aporte a la comprensión del empoderamiento en organizaciones de mujeres que desarrollan políticas públicas sociales.

Simple thermodynamics of jet engines

We use the first and second laws of thermodynamics to analyze the behavior of an ideal jet engine. Simple analytical expressions for the thermal efficiency, the overall efficiency, and the reduced thrust are derived. We show that the thermal efficiency depends only on the compression ratio r and on the velocity of the aircraft. The other two performance measures depend also on the ratio of the temperature at the turbine to the inlet temperature in the engine, T-3/T-i. An analysis of these expressions shows that it is not possible to choose an optimal set of values of r and T-3/T-i that maximize both the overall efficiency and thrust. We study how irreversibilities in the compressor and the turbine decrease the overall efficiency of jet engines and show that this effect is more pronounced for smaller T-3/T-i.

A dramatic effect of double bond configuration in N-oxy-3-aza Cope rearrangements: a simple synthesis of functionalised allenes

The first examples of low temperature N-oxy-3-aza Cope rearrangements, leading to functionalised allenes are described, where the Z-configuration of the enaminic double bond in the rearranging system proves critical.

Development of a biosensor for urea assay based on amidase inhibition, using an ion-selective electrode

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; anion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensorresponse and showed that 30 mu L of cell-free extract containing 7.47 mg protein mL(-1), 2 mu L of glutaraldehyde (5%, v/v) and 10 mu L of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation ofmembranes in urea. The biosensor exhibited a linear response in the range of 4.0-10.0 mu M urea, a detection limit of 2.0 mu M for urea, a response timeof 20 s, a sensitivity of 58.245 % per mu M urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.

Determination of chitin content in fungal cell wall: an alternative flow cytometric method

The conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3Δ/chs3Δ) and genes encoding predicted Glycosyl Phosphatidyl Inositol (GPI)-anchored proteins (pga31Δ/Δ and pga62Δ/Δ), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P < 0.001) than chs3Δ/chs3Δ and pga31Δ/Δ especially in the presence of caspofungin. Ca. parapsilosis, Ca. tropicalis, and Ca. albicans showed higher cell wall chitin content. Although no relationship between chitin content and antifungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate cell wall chitin content of fungi.

Pour une approche contrastive du présent simple en français et en portugais

Les temps du passé ont toujours constitué une pierre d’achoppement aussi bien dans l’enseignement du français aux étudiants lusophones que dans celui du portugais aux francophones. Le flot continu d’articles sur ce sujet attesterait, à lui seul, la difficulté du problème. Mais aussi étonnant que cela puisse paraître, il n’existe à ce jour aucune étude systématique qui aborde sur une base théorique le présent simple dans le passage du français vers le portugais et/ou vice-versa. Or, une telle étude nous paraît une voie de recherche intéressante et fructueuse qui ouvre de nombreuses perspectives de recherche, dont les possibilités d’application, que ce soit au domaine de l’enseignement des langues étrangères ou celui de la traduction, ne sont pas négligeables. Cette réflexion est d’autant plus urgente que l’emploi du présent simple pose de sérieux problèmes aux élèves lusophones plus inexpérimentés. En effet, ces derniers ont parfois beaucoup de peine à trouver les bonnes correspondances du présent simple en portugais. Afin d’aboutir à des conclusions fiables sur les similarités et les divergences entre le français et le portugais quant aux conditions d’apparition et au mode de fonctionnement du présent simple, nous nous attacherons, dans l’étude contrastive qui suit, à examiner des échantillons de textes littéraires français traduits en portugais et des extraits de textes littéraires portugais traduits en français1. Il s’agira essentiellement de montrer, en recourant à la Théorie des Opérations Énonciatives d’Antoine Culioli (Culioli, 1990; Campos & Xavier, 1991; Campos, 1997, 1998, entre autres), par quels moyens linguistiques le portugais exprime les valeurs aspectuo-temporelles associées au présent simple en français.

Application of alkaline comet assay in human biomonitoring for genotoxicity: a study on occupational exposure to cytostatics

The use of cytostatics drugs in anticancer therapy is increasing. Health care workers can be occupationally exposed to these drugs classified as carcinogenic, mutagenic or teratogenic. Cytostatics drugs are a heterogeneous group of chemicals widely used in the treatment of cancer, nevertheless have been proved to be also mutagens, carcinogens and teratogens. Workers may be exposed to this drug, being in the hospital settings the main focus dwelled upon the pharmacy, and nursing personnel. Alkaline comet assay is one of the most promising short-term genotoxicity assays for human risk assessment, being recommended to monitor populations chronically exposed to genotoxic agents. DNA glycosylase (OGG1) represents the main mechanism of protecting the integrity of the human DNA with respect to 8-OHdG, the most well studied biomarker of oxidative damage.

Ensaio sobre um sistema de um painel de uma divisória pré-fabricada

O setor da construção civil em Portugal sofre atualmente uma crise, como consequência da crise instalada no país. Além disso, a construção tradicional devido à sua rigidez espacial contribuiu para que os edifícios não se pudessem adaptar de uma forma simples, rápida e económica. Para contrabalançar esses factos a industrialização deverá ser encarada como uma das áreas com maior potencial de crescimento no futuro. A possibilidade de conceber um sistema que se adapta às constantes mudanças das necessidades humanas torna-se um dos grandes desafios da indústria da construção civil onde a pré-fabricação terá um papel fundamental. Neste ensaio estuda-se um sistema de divisórias leves amovíveis pré-fabricadas. No primeiro capítulo deste ensaio é elaborada uma síntese de conceitos que estão relacionados com as divisórias leves pré-fabricadas (industrialização e pré-fabricação), aborda-se a avaliação do sistema previsto ao nível da legislação e os passos a dar para verificação da viabilidade do sistema. No segundo capítulo faz-se uma distinção entre os vários tipos de pré-fabricação, definemse as exigências de desempenho e funcionais do sistema, aborda-se o conceito de coordenação modular e das tolerâncias de construção referindo a sua tremenda importância na pré-fabricação. Na parte final deste capítulo abordam-se exaustivamente as exigências de desempenho das paredes interiores não resistentes. No terceiro capítulo desenvolveu-se o sistema. Descrevem-se todos os componentes do sistema e a justificação da sua utilização. Elaboram-se três pré-avaliações: mecânica, acústica e térmica. Definem-se as ligações a serem utilizadas e pormenorizam-se todos os materiais das ligações. É apresentada uma avaliação económica de custos do sistema. Definem-se os procedimentos de: fabricação, transporte, montagem e desmontagem do sistema. No último capítulo apresentam-se as principais conclusões e os possíveis desenvolvimentos futuros.

Development of a simple analytical method for the simultaneousdetermination of paracetamol, paracetamol-glucuronide andp-aminophenol in river water

Paracetamol is among the most worldwide consumed pharmaceuticals. Although its occurrence in the environment is well documented, data about the presence of its metabolites and transformation products is very scarce. The present work describes the development of an analytical method for the simultaneous determination of paracetamol, its principal metabolite (paracetamol-glucuronide) and its main transformation product (p-aminophenol) based on solid phase extraction (SPE) and high performance liquid chromatography coupled to diode array detection (HPLC-DAD). The method was applied to analysis of river waters, showing to be suitable to be used in routine analysis. Different SPE sorbents were compared and the use of two Oasis WAX cartridges in tandem proved to be the most adequate approach for sample clean up and pre-concentration. Under optimized conditions, limits of detection in the range 40–67 ng/L were obtained, as well as mean recoveries between 60 and 110% with relative standard deviations (RSD) below 6%. Finally, the developed SPE-HPLC/DAD method was successfully applied to the analysis of the selected compounds in samples from seven rivers located in the north of Portugal. Nevertheless all the compounds were detected, it was the first time that paracetamol-glucuronide was found in river water at concentrations up to 3.57 μg/L.

Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitata

The present work describes the optimization of a short-term assay, based on the inhibition of the esterase activity of the alga Pseudokirchneriella subcapitata, in a microplate format. The optimization of the staining procedure showed that the incubation of the algal cells with 20 μmolL−1 fluorescein diacetate (FDA) for 40 min allowed discrimination between metabolic active and inactive cells. The shortterm assay was tested using Cu as toxicant. For this purpose, algal cells, in the exponential or stationary phase of growth, were exposed to the heavy metal in growing conditions. After 3 or 6 h, cells were subsequently stained with FDA, using the optimized procedure. For Cu, the 3- and 6-h EC50 values, based on the inhibition of the esterase activity of algal cells in the exponential phase of growth, were 209 and 130 μg L−1, respectively. P. subcapitata cells, in the stationary phase of growth, displayed higher effective concentration values than those observed in the exponential phase. The 3- and 6-h EC50 values for Cu, for cells in the stationary phase, were 443 and 268 μgL−1, respectively. This short-term microplate assay showed to be a rapid endpoint for testing toxicity using the alga P. subcapitata. The small volume required, the simplicity of the assay (no washing steps), and the automatic reading of the fluorescence make the assay particularly well suited for the evaluation of the toxicity of a high number of environmental samples.

A simple approach to numerical modelling of propane combustion in fluidized beds

A mathematical model is proposed for the evolution of temperature, chemical composition, and energy release in bubbles, clouds, and emulsion phase during combustion of gaseous premixtures of air and propane in a bubbling fluidized bed. The analysis begins as the bubbles are formed at the orifices of the distributor, until they explode inside the bed or emerge at the free surface of the bed. The model also considers the freeboard region of the fluidized bed until the propane is thoroughly burned. It is essentially built upon the quasi-global mechanism of Hautman et al. (1981) and the mass and heat transfer equations from the two-phase model of Davidson and Harrison (1963). The focus is not on a new modeling approach, but on combining the classical models of the kinetics and other diffusional aspects to obtain a better insight into the events occurring inside a fluidized bed reactor. Experimental data are obtained to validate the model by testing the combustion of commercial propane, in a laboratory-scale fluidized bed, using four sand particle sizes: 400–500, 315–400, 250–315, and 200–250 µm. The mole fractions of CO2, CO, and O2 in the flue gases and the temperature of the fluidized bed are measured and compared with the numerical results.