Acetylcholinesterase from the charru mussel Mytella charruana: kinetic characterization, physicochemical properties and potential as in vitro biomarker in environmental monitoring of mollusk extraction areas.

Acetylcholinesterase (AChE; EC 3.1.1.7) from aquatic organisms have been used to evaluate the exposure of specimens to pesticides and heavy metals at sublethal levels in environmental samples. AChE of Mytella charruana was extracted to characterize its physicochemical and kinetic properties as well as the effect of organophosphate (dichlorvos, diazinon, chlorpyrifos, methyl-parathion and temephos), carbamates (carbaryl, carbofuran and aldicarb), benzoylureas (diflubenzuron and novaluron), pyrethroid (cypermethrin) and juvenile hormone analog - JHA (pyriproxyfen) and the effect of metal ions: Hg2+, Cd2+, Pb2+, As3+, Cu2+ and Zn2+, in order to evaluate the potential of the enzyme as biomarker. The optimum pH of M. charruana AChE was 8.5 and the maximum activity peak occurred at 48 °C, being highly thermostable maintaining 97.8% of its activity after incubation at 60 °C. The Michaelis-Menten constants (km) for the substrates acetylthiocholine and propionylthiocholine were 2.8 ± 1.26 and 4.94 ± 6.9 mmol·L-1, respectively. The Vmax values for the same substrates were 22.6 ± 0.90 and 10.2 ± 4.94 mU·mg-1, respectively. Specific inhibition results suggest an AChE presenting active site with dimensions between those of AChE and butyrylcholinesterase (BChE). The IC20 values related to the effect of the pesticides on the enzyme showed higher inhibitory power of temephos (0.17 µmol·L-1), followed by aldicarb (0.19 µmol·L-1) and diflubenzuron (0.23 µmol·L-1). Metal ions inhibited M. charruana enzyme in the following order: Hg2+ > Pb2+ > Cd2+ > As3+ > Cu2+ > Zn2+. These data suggest that the enzyme showed potential as in vitro biomarker of the exposure to temephos, mercury, zinc and copper.

Evaluation of acute oral toxicity, embryotoxicity and cytotoxicity of the polar fraction of Parkinsonia aculeata aerial parts extract.

Ethnopharmacobotanical information reports that Parkinsonia aculeata infusion is used to control diabetes-related complications and dyslipidemia. However, few studies are reported on the safe use of this species. The aim of this study is to evaluate the acute toxicity, embryotoxicity and cytotoxicity of a polar fraction obtained from hydroethanolic extract of P. aculeata (PfrHEPA). For the acute toxicity test, we considered the Up and Down method which the guidelines are described by the Organization for Economic Cooperation and Development (OECD N°425). The animals were treated with PfrHEPA (2000 mg/kg) or with distilled water (10 ml/kg) by gavage and observed from Day 1 to14. For embryotoxicity assay, zebrafish embryos were exposed to PfrHEPA (100 mg/L) and toxicity parameters were observed during four consecutive days. The cytotoxicity of PfrHEPA (5, 10, 25, 50, 75 and 100 µg/ml, respectively) was performed on normal cell lines (mesenchymal stem cells, African green monkey renal cells and mouse pre-adipocytes 3 T3-L1 using the MTT salt reduction assay. In the acute toxicity test, no mortality was observed in mice treated with PfrHEPA (2000 mg/kg), as well as behavioral changes, histopathological abnormalities and hematological and biochemical variables. In the embryotoxicity test, no abnormal changes related to the toxicological parameters were observed in the period of 96 h. Regarding the cytotoxicity assay, PfrHEPA showed no cytotoxic effect on the normal cell lines tested, with an IC50 value > 100 µg/ml. These results suggest the safe use of P. aculeata, however, more trials are needed for PfrHEPA to be presented as new safe therapeutic proposal for the control of metabolic disorders.

Characterization of brain acetylcholinesterase of bentonic fish Hoplosternum littorale: Perspectives of application in pesticides and metal ions biomonitoring.

Acetylcholinesterase (AChE; EC 3.1.1.7) is a serine hydrolase, whose main function is to modulate neurotransmission at cholinergic synapses. It is, therefore, the primary target of some pesticides and heavy metals. Its inhibition in aquatic organisms has been used as an indicator of the presence of these pollutants in water bodies. The present study aimed to characterize physicochemical and kinetic parameters of brain AChE in the benthic fish Hoplosternum littorale and to analyze the in vitro effects of pesticides (dichlorvos, diazinon, chlorpyrifos, parathion-methyl, temephos, carbaryl, carbofuran, aldicarb, diflubenzuron, novaluron and pyriproxyfen) and metal ions (As3+, Cd2+, Cu2+, Fe2+, Mn2+, Mg2+, K+, Pb2+, Hg2+, Zn2+) investigating the potential of this enzyme as environmental biomarker based on current regulations. Specific substrates and inhibitors have indicated AChE to be the predominant cholinesterase (ChE) in the brain of H. littorale. Peak activity was observed at pH 8.0 and 30 °C. The enzymatic activity is otherwise moderately thermostable (≈ 50% activity at 45 °C). The enzyme can reduce the activation energy of acetylthiocholine hydrolysis reaction to 8.34 kcal mol-1 while reaching a rate enhancement of 106. Among the pesticides under study, dichlorvos presented an IC50 value below the maximum concentrations allowed by legislation. This study presents the first report on the inhibition of brain AChE activity from Siluriformes by the pesticides novaluron and pyriproxyfen. Mercury ion also exerted a strong inhibitory effect on its enzymatic activity. The H. littorale enzyme thus has the potential to function as an in vitro biomarker for the presence of the pesticide dichlorvos as well as mercury in areas of mining and industrial discharge.

Acetylcholinesterase of mangrove oyster Crassostrea rhizophorae: A highly thermostable enzyme with promising features for estuarine biomonitoring.

Enzyme biomarkers from several aquatic organisms have been used for assessing the exposure to contaminants at sublethal levels. Amongst them, the cholinesterases are commonly extracted from several organisms to evaluate/measure organophosphate and carbamate neurotoxic effects. Acetylcholinesterase (AChE; EC 3.1.1.7) is an enzyme of the group of serine esterases that acts on the hydrolysis of the neurotransmitter acetylcholine allowing the intermittence of the nerve impulses responsible for the neuronal communication. This enzyme is the main target for the action of some pesticides and the inhibition of its activity in bivalve mollusks may be used as biomarker due to their filter-feeding habit. In this context, the present study aimed to characterize physicochemical and kinetic parameters of the AChE extracted from gills and viscera of the oyster Crassostrea rhizophorae and investigate the in vitro effect of pesticides (dichlorvos, diazinon, chlorpyrifos, methyl-parathion, temephos, carbaryl, carbofuran, aldicarb, diflubenzuron and novaluron) in search for assessing its potential as biomarker. Specific substrates and inhibitors evidenced the predominance of AChE in both tissues. The optimum pH found for gills and viscera AChE were 8.0 and 8.5, respectively. The maximum peak of activity occurred at 70 °C for gill AChE and 75 °C for viscera AChE. The enzymes of both tissues presented remarkable thermostability. The Michaelis-Menten constant for both enzymes were 1.32 ±â€¯0.20 mM for gills and 0.43 ±â€¯0.12 mM for viscera. The Vmax values for gills and viscera were 53.57 ±â€¯1.72 and 27.71 ±â€¯1.15 mU/mg, respectively. The enzymes were able to reduce the activation energy to 9.75 kcal mol-1 (gills) and 11.87 kcal mol-1 (viscera) obtaining rate enhancements of 3.57 × 105 and 1.01 × 104, respectively, in relation to non-catalyzed reactions. Among the pesticides under study, the carbamates carbaryl and carbofuran exerted the strongest inhibitory effects on the enzyme activity achieving important degrees of inhibition at concentrations below national and international current regulations. The first observation of the effects of benzoylurea pesticides (diflubenzuron and novaluron) on AChE from mollusks is reported here. The gills AChE of C. rhizophorae showed potential to be specific biomarker for the carbamate carbaryl while the viscera AChE showed it for carbofuran. According to their features, these enzymes may be proposed as promising tools for estuarine monitoring as well as biocomponent of biosensor devices.

Potencial visual evocado por padrão reverso com tabuleiro de xadrez em gatos domésticos adultos / Pattern reversal visual evoked potential using checkerboard stimulus in adult domestic cats

O potencial visual evocado por padrão reverso (PVE-PR) é um método objetivo, não invasivo e estabelecido para as investigações funcionais. Este trabalho teve como objetivo a obtenção de dados do PVE-PR com tabuleiro de xadrez em gatos domésticos adultos, além de verificar a influência de diferentes ângulos visuais sobre os picos de tempo implícito e amplitude das ondas N75 e P100. Foram utilizados 13 gatos adultos (2-4 anos de idade), de ambos os sexos. Cada animal foi posicionado 100cm de distância de um monitor de vídeo situado a 17,2º. O estímulo gerado para a formação dos PVEs foi uma onda "quadrada" com a frequência de 6 Hz, apresentados em cinco frequências espaciais (16', 32', 64', 130' e 260'de arco). Os valores médios dos tempos implícitos (ms) e amplitudes (µV) de P100 não foram diferentes estatisticamente e nem estavam correlacionados (r=0,067). Com a mudança de tamanho do ângulo de estímulo apresentado, os picos de tempo implícito e amplitude do P100 apresentaram uma discreta variação. Aos 32' de arco, a amplitude de P100 atingiu seu pico máximo, com 14,94±1,13µV (média±erro padrão da média). N75 não mostrou nenhuma variação de amplitude. Com 16' de arco, o tempo implícito de N75 diminuiu, não havendo correlação com os valores de amplitude (r=0,26). O ângulo visual que desencadeou um maior pico de amplitude e tempo implícito constante para P100, foi o de 32' de arco. Em gatos adultos, um PVE-PR pode ser obtido sem o uso de midriáticos e sedativos, com estímulos subtendidos entre ângulos grandes como 64' e pequenos como 16'.

The pattern reversal visual evoked potential (PR-VEP) is an objective method, stablished for noninvasive functional investigations. This research aimed to obtain data from the PR-VEP with checkerboard in adult domestic cats and the influence of different visual angles on the peak latency and implicit time of waves N75 and P100. It was used 13 adult cats (2-4 years old), of both sexes. Each animal was positioned 100 cm away from a video monitor located at 17.2º. The stimulus generated for the formation of wave VEPs was "square" with the frequency of 6Hz, presented in five spatial frequencies (16 ', 32', 64 ', 130' and 260 'of arc). Mean values of implicit times (ms) and amplitudes (µvolts) of P100 were not statistically different and were not correlated (r=0.067). With the change in size of the angle of stimulus presented, the peak implicit time and amplitude of P100 showed a slight variation. At 32 'of arc amplitude of P100 reached its peak with 14,94±1,13µV (mean±SEM). N75 showed no variation in amplitude. With 16'of arc the implicit time N75 decreased and no correlation with amplitude values was observed (r=0,26). The visual angle that triggered higher peak amplitude and implicit time constant for P100, was 32'of arc. In adult cats, PR-VEP can be obtained without mydriatic and sedatives, with stimuli subtending angles between large as 64' and small as 16 '.

The effects of introducing the Amazonian squirrel monkey on the behavior of the northeast marmoset / Efeitos da introdução do macaco-de-cheiro amazônico sobre o comportamento do sagui-do-nordeste

The squirrel monkey Saimiri sciureus, an endemic primate of the Amazonian Basin, has been introduced in many localities of the Atlantic Forest biome. Introduced exotic species gain a competitive advantage in their new environment because of a lack of natural predators, competitors or diseases. This advantage can result in a reduction in the resources for the endemic species. The aims of this work were to evaluate the level of adaptation, and monitor the forms of interaction and the impact caused by S. sciureus on the behavior of the native species Callithrix jacchus in the Saltinho Biological Reserve, Tamandaré, Pernambuco, Brazil. The behavior was assessed by use of scan sampling method, every 5 min. A total of 382 h (191 h or 2,292 scans per group) of time sampling was performed. Sixteen interactions were recorded. The interaction index was low and represented 2.4% of the total observation time. Interactions were significantly higher during the dry season (58.5%) relative to the rainy season (41.5%). Callithrix jacchus was intolerant to the presence of S. sciureus and the last one was more tolerant during the interactions. In the presence of S. sciureus, C. jacchus eating, foraging, resting, socializing, and self-grooming behaviors were reduced while alertness was increased. Territorial behavior showed no significant difference. In the presence of S. sciureus, on average, the group of marmosets assembled at a higher layer of the forest stratum. The results might indicate a negative effect of S. sciureus on the native species, C. jacchus.

O mico-de-cheiro, Saimiri sciureus, uma espécie endêmica da Bacia Amazônica, tem sido introduzido com sucesso em muitas localidades do bioma Mata Atlântica. Espécies exóticas introduzidas adquirem uma vantagem competitiva em seu novo ambiente por causa da ausência de seus predadores naturais, resultando em uma redução dos recursos alimentares para as espécies endêmicas. Nossos objetivos foram avaliar o nível de adaptação e monitorar as formas de interação e do impacto causado pelo S. sciureus sobre o comportamento da espécie nativa, Callithrix jacchus, na Reserva Biológica de Saltinho, Tamandaré, Pernambuco, Brasil. O comportamento foi estudado através do método de amotragem scan sampling a cada 5 minutos. Um esforço amostral de 382 h (191 h ou 2.292 scans por grupo) foi obtido. Dezesseis interações foram registradas. O índice de interação foi baixo e representou 2,4% to total de tempo de observação. As interações foram significantemente maiores na estação seca (58,5%) do que na estação chuvosa (41,5%). Em termos de tipos de interação, C. jacchus foi intolerante à presença de S. sciureus, que por sua vez apresentou alto nível de tolerância. Na presença de S. sciureus, o C. jacchus reduziu a alimentação, forrageamento, descanso, comportamentos sociais e autocatação e aumentou o comportamento de alerta. O comportamento territorial não apresentou diferença significante. Na presença do S. sciureus, no geral, o grupo de saguis permaneceu em um estrato mais alto na floresta. Os resultados parecem indicar um possível efeito negativo do S. sciureus sobre a espécie nativa, C. jacchus.

Análise do funcionamento do Shaker à diversos níveis de fluxo / Analysis of the functioning of the Shaker to diverse levels of flow

Introdução: A fisioterapia respiratória tem importante papel em patologias respiratórias hipersecretivas, auxiliando na mobilização e remoção das secreções brônquicas. Dentre os adjuntos terapêuticos, os dispositivos oscilatórios de alta freqüência são largamente utilizados na higiene brônquica. Assim, este estudo teve por objetivo avaliar os valores de pressão expiratória positiva (PEP) e freqüência obtidos pelo aparelho Shaker® quando submetido a fluxos variáveis (de 2 a 30 L/min). Metodologia: Para a coleta dos dados foi elaborado um circuito composto por dois fluxômetros de ar comprimido, o Shaker®e um microcomputador, com um software previamente calibrado para registrar os dados de PEP e frequência. Resultados: Os resultados indicaram valores de pressão mínima constantes, 2,5 cmH2O, e valores máximos que variaram de 12,5-47,5 cmH2O. A menor freqüência registrada foi de 16,41 Hz e a máxima de 27,48 Hz. Conclusão: Considerando os resultados obtidos, fluxos expiratórios elevados produzem altos níveis pressóricos com baixas freqüências, podendo resultar em menor efeito desobstrutivo e barotrauma. Logo, o paciente deve ser orientado quanto à utilização de fluxos baixos na terapia com o Shaker®, proporcionando uma técnica de higiene brônquica, eficaz e segura.

Topographic analysis of the ganglion cell layer in the retina of the four-eyed fish Anableps anableps.

Fish of the genus Anableps (Anablepidae, Cyprinodontiformes) have eyes that are adapted for simultaneous aerial and aquatic vision. In this study we investigate some of the corresponding retinal specializations of the adult Anableps anableps eye using retinal transverse sections and wholemounts. The linear dimensions of the retina were found to be asymmetric with a greater representation of the dorsal compared to the ventral visual field. The total number of neurons in the ganglion cell layer of the ventral hemiretina was on average 3.6 times greater than the values obtained in the dorsal hemiretina. Isodensity contour maps revealed a prominent horizontal visual streak in the ventral hemiretina with an average peak cell density of 18,286 cells/mm(2). A second less-well-developed horizontal visual streak was also observed in the dorsal hemiretina. A sub-population of large cells with soma areas between 74 and 188 microm(2) was identified and found to be distributed evenly across both hemiretinas. Together, these results show that the sampling gain of the ventral retina is significantly greater than the dorsal segment, that retinal specializations important for mediating acute vision are present in the parts of the visual field immediately above and below the surface of the water, and that visual functions related with the large ganglion cells require more even sampling across the visual field. The relevance of these retinal specializations to the feeding and other behavioral strategies adopted by Anableps is discussed.