Synthesis and characterization of delta-atracotoxin-ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

delta-Atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. H-1 NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.

Transdermal penetration of vasoconstrictors - Present understanding and assessment of the human epidermal flux and retention of free bases and ion-pairs

Purpose. As reductions in dermal clearance increase the residence time of solutes in the skin and underlying tissues we compared the topical penetration of potentially useful vasoconstrictors (VCs) through human epidermis as both free bases and ion-pairs with salicylic acid (SA). Methods. We determined the in vitro epidermal flux of ephedrine, naphazoline, oxymetazoline, phenylephrine, and xylometazoline applied as saturated solutions in propylene glycol: water (1: 1) and of ephedrine, naphazoline and tetrahydrozoline as 10% solutions of 1: 1 molar ratio ion-pairs with SA in liquid paraffin. Results. As free bases, ephedrine had the highest maximal flux, Jmax = 77.4 +/- 11.7 mug/cm(2)/h, being 4-fold higher than tetrahydrozoline and xylometazoline, 6-fold higher than phenylephrine, 10-fold higher than naphazoline and 100-fold higher than oxymetazoline. Stepwise regression of solute physicochemical properties identified melting point as the most significant predictor of flux. As ion-pairs with SA, ephedrine and naphazoline had similar fluxes (11.5 +/- 2.3 and 12.0 +/- 1.6 mug/cm(2)/h respectively), whereas tetrahydrozoline was approximately 3-fold slower. Corresponding fluxes of SA from the ion-pairs were 18.6 +/- 0.6, 7.8 +/- 0.8 and 1.1 +/- 0.1 respectively. Transdermal transport of VC's is discussed. Conclusions. Epidermal retention of VCs and SA did not correspond to their molar ratio on application and confirmed that following partitioning into the stratum corneum, ion-pairs separate and further penetration is governed by individual solute characteristics.

Resposta da pressão arterial ao esforço em adolescentes: influência do sobrepeso e obesidade

Artigo original Ergoespirometria

Endogenous angiotensin II modulates nNOS expression in renovascular hypertension

Nitric oxide (NO) influences renal blood flow mainly as a result of neuronal nitric oxide synthase (nNOS). Nevertheless, it is unclear how nNOS expression is modulated by endogenous angiotensin II, an inhibitor of NO function. We tested the hypothesis that the angiotensin II AT1 receptor and oxidative stress mediated by NADPH oxidase contribute to the modulation of renal nNOS expression in two-kidney, one-clip (2K1C) hypertensive rats. Experiments were performed on male Wistar rats (150 to 170 g body weight) divided into 2K1C (N = 19) and sham-operated (N = 19) groups. nNOS expression in kidneys of 2K1C hypertensive rats (N = 9) was compared by Western blotting to that of 2K1C rats treated with low doses of the AT1 antagonist losartan (10 mg·kg-1·day-1; N = 5) or the superoxide scavenger tempol (0.2 mmol·kg-1·day-1; N = 5), which still remain hypertensive. After 28 days, nNOS expression was significantly increased by 1.7-fold in the clipped kidneys of 2K1C rats and by 3-fold in the non-clipped kidneys of 2K1C rats compared with sham rats, but was normalized by losartan. With tempol treatment, nNOS expression increased 2-fold in the clipped kidneys and 1.4-fold in the non-clipped kidneys compared with sham rats. The changes in nNOS expression were not followed by changes in the enzyme activity, as measured indirectly by the cGMP method. In conclusion, AT1 receptors and oxidative stress seem to be primary stimuli for increased nNOS expression, but this up-regulation does not result in higher enzyme activity.

Pesquisa de anticorpos anti-PGL-I em pacientes infectados pelo HIV em área endêmica de hanseníase

Esse estudo investiga a infecção subclínica de Mycobacterium leprae em pacientes infectados e não infectados pelo HIV, através da dosagem de anticorpos anti-PGL-I, e avalia se existe uma possível correlação dos resultados sorológicos encontrados com o estado de imunossupressão dos pacientes infectados pelo HIV. Foi realizado um estudo transversal analítico em 350 pacientes infectados pelo HIV e em 350 pacientes não infectados pelo HIV para detecção de anticorpos IgM anti-PGL-I em região endêmica para hanseníase. Avaliou-se uma possível correlação do estado de imunossupressão dos pacientes infectados pelo HIV (contagem de linfócitos CD4+, carga viral e uso ou não de terapia antirretroviral) com a soropositividade para PGLI. Dentre os pacientes infectados pelo HIV, 6% (21/350) apresentaram sorologia positiva para PGL-I e dos indivíduos não infectados pelo HIV, 29,1% (102/350) tinham PGL-I positivo. O grupo controle apresentou cerca de cinco vezes mais indivíduos com anticorpos anti-PGL-I do que o grupo infectado pelo HIV. Não houve diferença estatisticamente significativa na correlação do estado de imunossupressão do paciente com o resultado da sorologia anti-PGL-I. Houve uma menor produção de anticorpos anti-PGL-I em indivíduos infectados pelo HIV, o que pode indicar uma baixa taxa de infecção subclínica por M. leprae, ou uma baixa produtividade específica desses anticorpos, ou ambas as hipóteses. A desregulação de linfócitos B em indivíduos infectados pelo HIV pode ser a causa da baixa produção de anticorpos anti-PGL-I. Não houve correlação do estado de imunossupressão do paciente com o resultado da sorologia anti-PGL-I.

Os gêneros de discurso, cenografia e ethos em discursos da obra O templo e a forca de Luiz Guilherme Santos Neves

A pesquisa busca mostrar como o discurso religioso do gênero sermão, na voz do enunciador Frei Gregório José Maria de Bene, pode influir na estratégia de adesão e convencimento dos escravos da região de Serra, província do Espírito Santo, para a construção da igreja do Queimado. O sermão citado é o recorte fundamental do discurso literário O Templo e a Forca (1999), recriado por Luiz Guilherme Santos Neves, analisado a partir de cena englobante legitimadora. O trabalho insere-se nos estudos da Analise do Discurso (AD) de linha francesa, pelo referencial teórico de Dominique Maingueneau, que nos orientará quanto às cenografias do padre, sua imagem de enunciador, as condições de produção do sermão, ressaltando elementos que interagem no embate, visando à sociedade da época e às questões culturais dos envolvidos na eclosão da revolta. O principal objetivo é examinar as cenas de enunciação e como se constrói o ethos religioso em cada cena e suas variadas funções. É elementar dizer que o religioso se reconstrói a cada momento a partir do comprometimento com as situações de comunicação. Para maior entendi-mento, dizemos que essas diversas reconstruções apresentam nos discursos diferentes encenações desse tão fomentado religioso com suas estratégias de adesão, apresentando-se ora com a imagem daquele que fala em nome de Deus, preza a docilidade da vida do campo à sombra das andirobeiras gigantes onde se pode sentir o silêncio que convida à contemplação e à prece; ora aquele comprometido com seus propósitos interesseiros e pessoais. Fala para não ser entendido, e o que vale é erguer a casa de São José sem poupar a carne e o sangue das mortes que virão. Importa ressaltar que sempre há possibilidade de olhar ingênuo sobre texto religioso, que, conforme Main-gueneau (2010), só é legível relacionado a vasto intertexto que contribuirá para estruturar o discurso. Para enriquecer ainda mais este estudo, afora o gênero sermão usamos recortes que estruturam o discurso de Neves e sinalizam as variadas cenografias e a forma como se constituem enquanto gêneros: Diálogo interior – momentos solitários do frade – Visão do frade e Monólogo; Diálogo Compartilhado – segmenta diálogos entre o enunciador e seus vários co-enunciadores, que estruturam os acontecimentos do discurso; e o gênero exortação, do padre aos escravos. Os recortes são momentos de grandes embates recriados pelo autor, em discurso leve fazendo seu leitor transitar prazerosamente junto ao frade entre as suas variadas imagens nos discursos. Levando o leitor a acreditar tratar-se de cenas relacionadas ao gênero cômico. E talvez o fosse, se não terminasse em revolta.

Evaluation of respiratory parameters in minimally processed lettuce grown under organic or conventional system

The increased preference for minimally processed vegetables has been attributed to the health benefits associated with fresh produce and the demand for ready-to-eat salads. In this paper, lettuce (Lactuca sativa L.) was evaluated for the effects of different cropping systems on the respiratory properties. Lettuce was packaged in low density polyethylene bags and stored in a refrigerator at 4 ºC. The concentration of carbon dioxide and oxygen inside the package was monitored during the storage at zero, three, six, eight, ten and twelve days by gas chromatography. Dry matter variation was measured gravimetrically up to day fourteen of storage. Values of respiratory rate for conventional lettuce increased from day 1 to 3 and remained low, while respiratory rate of the organic lettuce increased three-fold up to day 8, stabilizing at a high level. Variation in dry matter during storage also resulted from differences between the two cultivation systems. The highest content of dry matter was achieved by organic lettuce.

Improving fECG signal acquisition in the evaluation of fetal well-being

The ability to monitor fetal heart rate is vital during late pregnancy and labor in order to evaluate fetal well-being. Current monitoring practice is essentially based on external cardiotocography and, less frequently, during labor, invasive fetal scalp electrocardiography. Many current and envisaged applications could benefi t from simpler devices using a 3-lead ECG confi guration. We are designing a maternity support belt with an embedded wireless 3-lead ECG sensor, and have investigated the infl uence of the ground electrode position on signal quality. Data from over 100 pregnant women was collected with the ground electrode placed in 3 locations in order to determine optimum electrode placement and belt form factor.

The effects of place of residence on tourist motivations: a revised case of the Urban Quadrangle of Minho

This study seeks to capture the underlying reasons for the travel decisions of residents of the Urban Quadrangle of Minho (composed of the municipalities of Barcelos, Braga, Guimarães, and Vila Nova de Famalicão). The aim of the research is three-fold. Firstly, the study identifies the push and pull motivational factors of residents of the Urban Quadrangle of Minho. Secondly, the study examines whether there are differences between the tourist motivations of residents of the four different municipalities of the Urban Quadrangle. Finally, the study investigates if there are any differences in the motivations of those who choose national and international destinations. The methodology comprises quantitative research based on questionnaires administered in 2012 to residents of the Urban Quadrangle of Minho. A principal component factor analysis is employed to identify six push and seven pull factors. The comparison of the mean scores of these factors across municipalities and across residents that choose national and international destinations reveals that the most valued and least valued factors are common to all four municipalities and both groups of residents (that choose national and international destinations).

The effects of place of residence on tourist motivations: the case of Urban Quadrangle of Minho

The aim of this preliminary study is three-fold. Firstly, the study identifies the push and pull motivational factors of residents of the Urban Quadrangle of Minho. Secondly, the study examines whether there are differences between the tourist motivations of residents of the four different municipalities. Finally, the study investigates if there are any differences in the motivations of those who choose national and international destinations.

Leaf area and epicuticular wax content of Sida spp

Morpho-physiological characteristics and chemical composition are directly related to superior competitive ability of crops. This study intended to make a comparative analysis of dry matter production, leaf area and amount of epicuticular wax of three species of Sida spp: S. urens L., S. rhombifolia L. and S. spinosa L. Plants were collected at three growth stages: V1: stage described as up to 10 fully expanded leaves; V2: between 11 leaves and flowering; and R: after flowering. At stages V2 and R, the highest number of leaves was recorded for S. rhombifolia, followed by S. spinosa at V2 and S. urens at R. These results were relatively proportional to leaf area for all species. S. spinosa at the vegetative stage produced the highest values of specific leaf area (SLA), with no significant differences between species at the stage R. The amount of wax per unit of leaf area between species at the same developmental stage was significantly different only at the reproductive stage, where S. spinosa produced 23.18 and 6.23 fold more wax than S. urens and S. rhombifolia respectively. Between the growth stages of each species, there was decrease in the amount of wax with plant age and increase in leaf area (AFE), number of leaves and dry matter. The leaves of the Sida species exhibit different characteristics and this information can be used to optimize the use of herbicides in the control of these weeds.

"Seis sigma": uma nova cultura empresarial

Existe uma miríade de ferramentas para a gestão da qualidade. A grande maioria apareceu no meio industrial, e algumas ultrapassaram barreiras, chegando a todas as áreas de negócio. O processo Seis Sigma é uma ferramenta que se destacou pela sua versatilidade e eficácia, e alcançou uma grande notoriedade na indústria fabril. O discente teve a oportunidade de contactar com o processo Seis Sigma, que evidenciou a sua validade, como ferramenta poderosa em qualquer área de negócio. Baseado nesse trabalho, pretende-se demonstrar a mais-valia que esta ferramenta apresenta, e que, com empenho e dedicação, todos podem usar. Os objectivos deste trabalho são: • Identificar o valor superior numa empresa do ramo automóvel na implementação de um processo Seis Sigma numa área de BackOffice; • Verificar se após o fim do projecto Seis Sigma, as medidas implementadas continuam a produzir efeitos. Metodologia usada para a execução deste trabalho • O discente, com a função de Green Belt, foi o responsável pela implementação de um projecto Seis Sigma. No seguimento do que já havia sido efectuado no projecto Seis Sigma, este trabalho usou a ferramenta DMAIC (ver p.5) como metodologia para a sua execução. Conclusões Ficou demonstrado com este projecto, em que o discente teve um profundo envolvimento, que a metodologia Seis Sigma ao ser aplicada num qualquer processo repetitivo de trabalho vai: • Obrigar a repensar todos os procedimentos instalados procurando todas as oportunidades demelhoria; • Criar novas ferramentas para dar solução às oportunidades encontradas; • Verificar a validade e mais-valia nas novas ferramentas e procedimentos; • Em função da constante verificação, melhorar as ferramentas e procedimentos instalados, ou criar novos procedimentos ou ferramentas; • De uma forma permanente, continuar a analisar o processo, monitorizando a qualidade e a procura de novas oportunidades de melhoria. O facto de o processo Seis Sigma obrigar a uma introspecção ao trabalho realizado, e, tornar todo o processo de melhoria sistemático, faz com que seja sempre uma mais-valia a sua aplicação, num ambiente fabril, ou de BackOffice. Ainda que o processo Seis Sigma seja terminado a dada altura, as melhorias que trouxe e as ferramentas que foram desenvolvidas para a sua execução ficam, deixando um legado de melhoria de processos, que, por si só, justificam a sua implementação.

The folding of knotted proteins: insights from lattice simulations

We carry out systematic Monte Carlo simulations of Go lattice proteins to investigate and compare the folding processes of two model proteins whose native structures differ from each other due to the presence of a trefoil knot located near the terminus of one of the protein chains. We show that the folding time of the knotted fold is larger than that of the unknotted protein and that this difference in folding time is particularly striking in the temperature region below the optimal folding temperature. Both proteins display similar folding transition temperatures, which is indicative of similar thermal stabilities. By using the folding probability reaction coordinate as an estimator of folding progression we have found out that the formation of the knot is mainly a late folding event in our shallow knot system.

Arrow Plot: a new graphical tool for selecting up and down regulated genes and genes differentially expressed on samples subgroups

Background: A common task in analyzing microarray data is to determine which genes are differentially expressed across two (or more) kind of tissue samples or samples submitted under experimental conditions. Several statistical methods have been proposed to accomplish this goal, generally based on measures of distance between classes. It is well known that biological samples are heterogeneous because of factors such as molecular subtypes or genetic background that are often unknown to the experimenter. For instance, in experiments which involve molecular classification of tumors it is important to identify significant subtypes of cancer. Bimodal or multimodal distributions often reflect the presence of subsamples mixtures. Consequently, there can be genes differentially expressed on sample subgroups which are missed if usual statistical approaches are used. In this paper we propose a new graphical tool which not only identifies genes with up and down regulations, but also genes with differential expression in different subclasses, that are usually missed if current statistical methods are used. This tool is based on two measures of distance between samples, namely the overlapping coefficient (OVL) between two densities and the area under the receiver operating characteristic (ROC) curve. The methodology proposed here was implemented in the open-source R software. Results: This method was applied to a publicly available dataset, as well as to a simulated dataset. We compared our results with the ones obtained using some of the standard methods for detecting differentially expressed genes, namely Welch t-statistic, fold change (FC), rank products (RP), average difference (AD), weighted average difference (WAD), moderated t-statistic (modT), intensity-based moderated t-statistic (ibmT), significance analysis of microarrays (samT) and area under the ROC curve (AUC). On both datasets all differentially expressed genes with bimodal or multimodal distributions were not selected by all standard selection procedures. We also compared our results with (i) area between ROC curve and rising area (ABCR) and (ii) the test for not proper ROC curves (TNRC). We found our methodology more comprehensive, because it detects both bimodal and multimodal distributions and different variances can be considered on both samples. Another advantage of our method is that we can analyze graphically the behavior of different kinds of differentially expressed genes. Conclusion: Our results indicate that the arrow plot represents a new flexible and useful tool for the analysis of gene expression profiles from microarrays.

Production of hydroxamic acids by immobilized Pseudomonas aeruginosa cells Kinetic analysis in reverse micelles

Intact cells from Pseudomonas aeruginosa strain L10 containing amidase were used as biocatalysts both free and immobilized in a reverse micellar system. The apparent kinetic constants for the transamidation reaction in hydroxamic acids synthesis, were determined using substrates such as aliphatic, amino acid and aromatic amides and esters, in both media. In reverse micelles, K-m values decreased 2-7 fold relatively to the free biocatalyst using as substrates acetamide, acrylamide, propionamide and glycinamide ethyl ester. We have concluded that overall the affinity of the biocatalyst to each substrate increases when reactions are performed in the reversed micellar system as opposed to the buffer system. The immobilized biocatalyst in general, exhibits higher stability and faster rates of reactions at lower substrates concentration relatively to the free form, which is advantageous. Additionally, the immobilization revealed to be suitable for obtaining the highest yields of hydroxamic acids derivatives, in some cases higher than 80%. (C) 2013 Elsevier B.V. All rights reserved.