Insights into the mechanism of progressive RNA degradation by the archaeal exosome

Initially identified in yeast, the exosome has emerged as a central component of the RNA maturation and degradation machinery both in Archaea and eukaryotes. Here we describe a series of high-resolution structures of the RNase PH ring from the Pyrococcus abyssi exosome, one of them containing three 10-mer RNA strands within the exosome catalytic chamber, and report additional nucleotide interactions involving positions N5 and N7. Residues from all three Rrp41-Rrp42 heterodimers interact with a single RNA molecule, providing evidence for the functional relevance of exosome ring-like assembly in RNA processivity. Furthermore, an ADP-bound structure showed a rearrangement of nucleotide interactions at site N1, suggesting a rationale for the elimination of nucleoside diphosphate after catalysis. In combination with RNA degradation assays performed with mutants of key amino acid residues, the structural data presented here provide support for a model of exosome-mediated RNA degradation that integrates the events involving catalytic cleavage, product elimination, and RNA translocation. Finally, comparisons between the archaeal and human exosome structures provide a possible explanation for the eukaryotic exosome inability to catalyze phosphate-dependent RNA degradation.

Nanoparticle Platform to Modulate Reaction Mechanism of Phenothiazine Photosensitizers

Herein, we report on the synthesis of photosensitizing nanoparticles in which the generation of different oxidizing species, i.e., singlet oxygen ((1)O(2)) or radicals, was modulated. Sol gel and surface chemistry were used to obtain nanoparticles with specific ratios of dimer to monomer species of phenothiazine photosensitizers (PSs). Due to competition between the reactions involving electron transfer within dimer species and energy transfer from monomer triplets to oxygen, the efficiency of (1)O(2) generation could be controlled. Nanoparticles with an excess of dimer have an (1)O(2) generation efficiency (S(Delta)) of 0.01 while those without dimer have a S, value of 0.4. Furthermore, we demonstrate that the PS properties of the nanoparticles are not subjected to interference from the external medium as is commonly the case for free PSs, i.e., PS ground and triplet states are not reduced by NADH and ascorbate, respectively, and singlet excited states are less suppressed by bromide. The modulated (1)O(2) generation and the PS protection from external interferences make this nanoparticle platform a promising tool to aid in performing mechanistic studies in biological systems. Also, it offers potential application in technological areas in which photo-induced processes take place.

Mechanism of Trypanosoma cruzi death induced by Cratylia mollis seed lectin

Incubation of T. cruzi epimastigotes with the lectin Cramoll 1,4 in Ca(2+) containing medium led to agglutination and inhibition of cell proliferation. The lectin (50 A mu g/ml) induced plasma membrane permeabilization followed by Ca(2+) influx and mitochondrial Ca(2+) accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (Delta I(m)) and impaired ADP phosphorylation. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus Ca(2+) treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using T. cruzi mitochondrial fractions showed that, in contrast to digitonin, the lectin significantly decreased Delta I(m) by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll 1,4 induced epimastigotes death by necrosis.

Determination of carbaryl in tomato ""in natura"" using an amperometric biosensor based on the inhibition of acetylcholinesterase activity

This work reports the utilization of two methodologies for carbaryl determination in tomatoes. The measurements were carried out using an amperometric biosensor technique based on the inhibition of acetylcholinesterase activity due to carbaryl adsorption and a HPLC procedure. The electrochemical experiments were performed in 0.1 mol L-1 phosphate buffer solutions at pH 7.4 with an incubation time of 8 min. The analytical curve obtained in pure solutions showed excellent linearity in the 5.0 x 10(-5) to 75 x 10(-5) mol L-1 range, with the limit of detection at 0.4 x 10(-3) gL(-1). The application of such a methodology in tomato samples involved solely liquidising the samples, which were spiked with 6.0 x 10(-6) and 5.0 x 10(-5) mol L-1 carbaryl. Recovery in such samples presented values of 99.0 and 92.4%, respectively. In order to obtain a comparison, HPLC experiments were also conducted under similar conditions. However, the tomato samples have to be manipulated by an extraction procedure (MSPD), which yielded much lower recovery values (78.3 and 84.8%, respectively). On the other hand, the detection limit obtained was much lower than that for the biosensor, i.e., 3.2 x 10(-6) g L-1. Finally, the biosensor methodology was employed to analyze carbaryl directly inside the tomato, without any previous manipulation. In this case, the biosensor was immersed in the tomato pulp, which had previously been spiked with the pesticide for 8 min, removed and inserted in the electrochemical cell. A recovery of 83.4% was obtained, showing very low interference of the matrix constituents. (C) 2007 Elsevier B.V. All rights reserved.

Mechanism of Muscle Vibrations During Stimulated and Voluntary Isometric Contractions of Mammalian Skeletal Muscle

When a muscle contracts it produces vibrations. The origin of these vibrations is not known in detail. The purpose of this study was to determine the mechanism associated with muscle vibrations. Mechanisms which have been proposed in the literature were described as theories (cross-bridge cycling, vibrating string and unfused motor unit theories). Specific predictions were derived from each theory, and tested in three conceptually different studies. In the first study, the influence of recruitment strategies of motor units (MUs) on the vibromyographic (VMG) signal was studied in the in-situ cat soleus using electrical stimulation of the soleus nerve. VMG signals increased with increasing recruitment and decreased with increasing firing rates of MUs. Similar results were obtained for the human rectus femoris (RF) muscle using percutaneous electrical stimulation of the femoral nerve. The influence of MU activation on muscle vibrations was studied in RF by analyzing VMG signals at different percentages (0-100%) of the maximal voluntary contraction (MVC). In our second study, we tested the effects of changing the material properties of the in-situ cat soleus (through muscle length changes) on the VMG signal. The magnitude of the VMG signal was higher for intermediate muscle lengths compared to the longest and the shortest muscle lengths. The decreased magnitude of the VMG signal at the longest and at the shortest muscle lengths was associated with increased passive stiffness and with decreased force transients during unfused contractions, respectively. In the third study, the effect of fatigue on muscle vibrations was studied in human RF and vastus lateralis (VL) musc1es during isometric voluntary contractions at a leveI of 70% MVC. A decrease in the VMG signal magnitude was observed in RF (presumably due to derecruitment of MUs) and an increase in VL (probably related to the enhancement of physiological tremor, which may have occurred predorninantly in a mediolateral direction) with fatigue. The unfused MU theory, which is based on the idea that force transients produced by MUs during unfused tetanic contraction is the mechanism for muscle vibrations, was supported by the results obtained in the above three studies.

Constructing coincident and leading indices of economic activity for the brazilian economy

This paper has three original contributions. The first is the reconstruction effort of the series of employment and income to allow the creation of a new coincident index for the Brazilian economic activity. The second is the construction of a coincident index of the economic activity for Brazil, and from it, (re)establish a chronology of recessions in the recent past of the Brazilian economy. The coincident index follows the methodology proposed by TCB and it covers the period 1980:1 to 2007:11. The third is the construction and evaluation of many leading indicators of economic activity for Brazil which fills an important gap in the Brazilian Business Cycles literature.

Essays on the general equilibrium effects of barriers to trade on economic growth, foreign trade and the location of economic activity in Brazil

This work presents a fully operational interstate CGE model implemented for the Brazilian economy that tries to quantify both the role of barriers to trade on economic growth and foreign trade performance and how the distribution of the economic activity may change as the country opens up to foreign trade. Among the distinctive features embedded in the model, modeling of external scale economies, port efficiency and land-maritime transport costs provides an innovative way of dealing explicitly with theoretical issues related to integrated regional systems. In order to illustrate the role played by the quality of infrastructure and geography on the country‟s foreign and interregional trade performance, a set of simulations is presented where barriers to trade are significantly reduced. The relative importance of trade policy, port efficiency and land-maritime transport costs for the country trade relations and regional growth is then detailed and quantified, considering both short run as well as long run scenarios. A final set of simulations shed some light on the effects of liberal trade policies on regional inequality, where the manufacturing sector in the state of São Paulo, taken as the core of industrial activity in the country, is subjected to different levels of external economies of scale. Short-run core-periphery effects are then traced out suggesting the prevalence of agglomeration forces over diversion forces could rather exacerbate regional inequality as import barriers are removed up to a certain level. Further removals can reverse this balance in favor of diversion forces, implying de-concentration of economic activity. In the long run, factor mobility allows a better characterization of the balance between agglomeration and diversion forces among regions. Regional dispersion effects are then clearly traced-out, suggesting horizontal liberal trade policies to benefit both the poorest regions in the country as well as the state of São Paulo. This long run dispersion pattern, on one hand seems to unravel the fragility of simple theoretical results from recent New Economic Geography models, once they get confronted with more complex spatially heterogeneous (real) systems. On the other hand, it seems to capture the literature‟s main insight: the possible role of horizontal liberal trade policies as diversion forces leading to a more homogeneous pattern of interregional economic growth.

Constructing coincident and leading indices of economic activity for the brazilian economy

This paper has three original contributions. The first is the reconstruction effort of the series of employment and income to allow the creation of a new coincident index for the Brazilian economic activity. The second is the construction of a coincident index of the economic activity for Brazil, and from it, (re) establish a chronology of recessions in the recent past of the Brazilian economy. The coincident index follows the methodology proposed by The Conference Board (TCB) and it covers the period 1980:1 to 2007:11. The third is the construction and evaluation of many leading indicators of economic activity for Brazil which fills an important gap in the Brazilian Business Cycles literature.

Constructing coincident and leading indices of economic activity for the Brazilian economy

This paper has three original contributions. The fi rst is the reconstruction effort of the series of employment and income to allow the creation of a new coincident index for the Brazilian economic activity. The second is the construction of a coincident index of the economic activity for Brazil, and from it, (re) establish a chronology of recessions in the recent past of the Brazilian economy. The coincident index follows the methodology proposed by The Conference Board (TCB) and it covers the period 1980:1 to 2007:11. The third is the construction and evaluation of many leading indicators of economic activity for Brazil which fills an important gap in the Brazilian Business-Cycle literature.

Mechanism of infection and colonization of Rhipicephalus sanguineus eggs by Mertarhizium anisopliae as revealed by scanning eletron microscopy and histopathology

O presente trabalho teve como objetivo verificar a forma de penetração do fungo Metarhizium anisopliae em ovos do carrapato Rhipicephalus sanguineus, assim como as lesões infringidas no interior do ovo. A aderência e penetração do fungo foram estudadas por meio da microscopia eletrônica de varredura e a ação do fungo nos tecidos internos avaliada em secções histológicas convencionais. Para observação destes eventos, realizaram-se infecções experimentais em 11 grupos de ovos do R. sanguineus contendo 25 mg cada. Os ovos foram banhados durante 3 minutos, sob agitação manual, em suspensão com concentração de 10(8) conídios/mL. Nos grupos controle o banho foi realizado apenas no veículo da suspensão. Os ovos foram processados para análise histopatológica e microscopia eletrônica de varredura nos seguintes tempos após a infecção: 1 e 18h, e um, dois, três, quatro, cinco, seis, sete, nove e onze dias. Observou-se grande germinação de conídios em 67% dos ovos 18h após a inoculação e o fungo penetrou em 92,6% dos ovos 5 dias após a infecção. A extrusão do patógeno ocorreu em 87% dos ovos 7 dias após a infecção, chegando a 100% no 9º dia. Nas análises histopatológicas não foram observadas lesões dignas de nota, porem deve-se ressaltar que houve significativa redução (53,9%) na eclosão a partir dos ovos infectados.

The zymogen-enteropeptidase system: A practical approach to study the regulation of enzyme activity by proteolytic cleavage

The present research describes an efficient procedure to obtain high levels of trypsinogen and chymotrypsinogen by using a simple, rapid, and easily reproducible method. The extraction process and the time-course of activation of zymogens can be carried out in a single laboratory period, without sophisticated equipment. The main objective was to prepare a laboratory class that would stimulate student interest in enzyme regulation, exploring the fact that the catalytic activity of some enzymes is regulated by different mechanisms. The regulation of proteolytic enzymes requires the synthesis of an inactive zymogen and its being irreversibly switched on by specific proteolytic cleavage.

Allosteric modulation of pyrophosphatase activity of rat osseous plate alkaline phosphatase by magnesium ions

Pyrophosphatase activity of rat osseous plate alkaline phosphatase was studied at different concentrations of calcium and magnesium ions. with the aim of characterizing the modulation of enzyme activity by these metals. In the absence of metal ions, the enzyme hydrolysed pyrophosphate following Michaelian kinetics with a specific activity of 36.7 U/mg and K-0.5 = 88 mu M. In the presence of low concentrations (0.1 mM) of magnesium (or calcium) ions, the enzyme also exhibited Michaclian kinetics for the hydrolysis of pyrophosphate, but a significant increase in specific activity (123 U/mg) was observed. K-m values remained almost unchanged. Quite different behavior occurred in the presence of 2 mM magnesium (or calcium) ions. In addition to low-affinity sites (K-0.5 = 40 and 90 mu M, for magnesium and calcium, respectively), high-affinity sites were also observed with K-0.5 values 100-fold lower. The high-affinity sites observed in the presence of calcium ions represented about 10% of those observed for magnesium ions. This was correlated with the fact that only magnesium ions triggered conformational changes yielding a fully active enzyme. These results suggested that the enzyme could hydrolyse pyrophosphate, even at physiological concentrations (4 mu M), since magnesium concentrations are high enough to trigger conformational changes increasing the enzyme activity. A model, suggesting the involvement of magnesium ions in the hydrolysis of pyrophosphate by rat osseous plate alkaline phosphatase is proposed. (C) 1998 Published by Elsevier B.V. Ltd. All rights reserved.

Lipodystrophy in HIV/AIDS patients with different levels of physical activity while on antiretroviral therapy

INTRODUÇÃO: A lipodistrofia relacionada ao uso de terapia antirretroviral (TARV) pode causar estigma estético e elevar o risco de doenças cardiovasculares. A atividade física pode ser uma alternativa válida para o tratamento e prevenção da lipodistrofia. Entretanto, poucos estudos tratam dessa temática. O objetivo deste estudo foi verificar a ocorrência de lipodistrofia relacionada ao uso de TARV em portadores de HIV/AIDS, com diferentes hábitos de atividades físicas. MÉTODOS: A casuística foi formada por 42 portadores de HIV em uso de TARV, do Centro de Testagem e Aconselhamento de Presidente Prudente. Para obtenção do nível de atividade física aplicou-se o Questionário Internacional de Atividade Física (IPAQ); a lipodistrofia foi diagnosticada pelo autorrelato do paciente e a confirmação médica. O percentual de gordura de tronco foi estimado pela absortometria por raio-X de dupla energia (DEXA). Foram coletados também dados referentes a sexo, idade, tempo de uso de TARV, valores de CD4 e carga viral. RESULTADOS: Verificou-se maior ocorrência de lipodistrofia no grupo sedentário quando comparado ao ativo, além de fator protetor da prática da atividade física em relação à ocorrência da lipodistrofia. O grupo com valores mais elevados de CD4 também apresentou maior proporção de sujeitos com lipodistrofia, além de maior proporção de ativos e de indivíduos com menor faixa etária. Os acometidos pela lipodistrofia apresentaram maiores valores de percentual de gordura de tronco, bem como, os sedentários em relação aos ativos. CONCLUSÕES: O estilo de vida fisicamente ativa resultou em efeito protetor para ocorrência da lipodistrofia relacionada ao uso da TARV.

Interaction mechanism of poly (o-ethoxyaniline) and collagen blends

Blend films of poly (o-ethoxyaniline) (POEA) and collagen were fabricated by casting under optimized conditions and characterized by Raman scattering and UV-vis absorption spectroscopies. The UV-vis spectra showed that the addition of collagen in the aqueous solution of POEA promotes a dedoping of the POEA. This effect was also observed for the blend films as supported by Raman scattering and a mechanism for the chemical interaction between POEA-collagen is proposed. The influences of different percentage of collagen as well as the pH of stock solutions during the fabrication process of the blend films were also investigated. It was found that the preparation method plays an important role in the flexibility and freestanding properties of the films. Complementary, the surface morphology was studied by atomic force microscopy and the conductivity by dc measurements. (C) 2003 Elsevier Ltd. All rights reserved.