RESUMO
The neural crest (NC) is a transitory embryonic structure of vertebrates that gives rise to an astonishing variety of derivatives, encompassing both neural and mesenchymal cell types. Neural crest cells (NCCs) are an excellent model to study how environmental factors modulate features such as cell multipotentiality and differentiation. Tests with multifunctional substrates that allow NCCs to express their full potential, while promoting cell subcloning, are needed to advance knowledge about NCC self-renewal and to foster future biotechnological approaches. Here we show that a self-assembled peptide named PuraMatrixTM is an excellent substrate that allows the differentiation of NCCs based on the identification of seven different cell types. Depending on the PuraMatrixTM concentration employed, different frequencies and quantities of a given cell type were obtained. It is noteworthy that an enormous quantity and diversity of mesenchymal phenotypes, such as chondrocytes, could be observed. The quantity of adipocytes and osteocytes also increased with the use of mesenchymal differentiation factors (MDF), but PuraMatrixTM alone can support the appearance of these mesenchymal cell types. PuraMatrixTM will promote advances in studies related to multipotentiality, self-renewal and control of NCC differentiation, since it is an extremely simple and versatile material which can be employed for both in vivo and in vitro experiments.
Assuntos
Diferenciação Celular/fisiologia , Autorrenovação Celular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Crista Neural/fisiologia , Peptídeos/metabolismo , Adipócitos/citologia , Adipócitos/fisiologia , Animais , Células Cultivadas , Condrócitos/citologia , Condrócitos/fisiologia , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/fisiologia , Crista Neural/citologia , Osteócitos/citologia , Osteócitos/fisiologia , Codorniz/embriologia , Codorniz/metabolismo , Vertebrados/embriologia , Vertebrados/metabolismoRESUMO
Donepezil is a second generation acetylcholinesterase (AChE) inhibitor for treatment of Alzheimer's disease (AD). AChE is important for neurotransmission at neuromuscular junctions and cholinergic brain synapses by hydrolyzing acetylcholine into acetate and choline. In vitro data support that donepezil is a reversible, mixed competitive and noncompetitive inhibitor of AChE. The experimental fact then suggests a more complex binding mechanism beyond the molecular view in X-ray models resolved at cryogenic temperatures that show a unique binding mode of donepezil in the active site of the enzyme. Aiming at clarifying the mechanism behind that mixed competitive and noncompetitive nature of the inhibitor, we have applied molecular dynamics (MD) simulations and docking and free-energy calculations to investigate microscopic details and energetics of donepezil association for conditions of substrate-free and -bound states of the enzyme. Liquid-phase MD simulation at room temperature shows AChE transits between "open" and "closed" conformations to control accessibility to the active site and ligand binding. As shown by docking and free-energy calculations, association of donepezil involves its reversible axial displacement and reorientation in the active site of the enzyme, assisted by water molecules. Donepezil binds equally well the main-door anionic binding site PAS, the acyl pocket, and the catalytic site CAS by respectively adopting outward-inward-inward orientations regardless of substrate occupancy-the overall stability of that reaction process depends however on co-occupancy of the enzyme being preferential for its substrate-free state. All together, our findings support a physiologically relevant mechanism of AChE inhibition by donepezil involving multistable interactions modes at the molecular origin of the inhibitor's activity.
Assuntos
Acetilcolinesterase , Doença de Alzheimer , Acetilcolinesterase/metabolismo , Sítios de Ligação , Inibidores da Colinesterase/farmacologia , Donepezila , Humanos , TemperaturaRESUMO
This paper deals with molecular modeling of new therapeutic agents for treating the Alzheimer's disease. The therapeutic line adopted for this study is the cholinergic hypothesis. To modulate positively the cholinergic function through the inhibition of the acetylcholinesterase, a set of candidates was designed from a natural compound extracted from the cashew nutshell liquid, anacardic acid. In silico screening of this chemical library revealed a ligand that is more promising once it is correlated with an active drug through specific topological and electronic descriptors. The protein-ligand docking showed stable binding modes and the binding free energy computed for the active site of the receptor suggests that our ligand presents a potential biological response. Graphical Abstract Representation of the three dimensional structure of the AChE, showing the important binding sites of the Gorge and the conformation of the ligand.
Assuntos
Acetilcolinesterase/química , Doença de Alzheimer/tratamento farmacológico , Inibidores da Colinesterase/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ácidos Anacárdicos/química , Anacardium/química , Desenho de Fármacos , HumanosRESUMO
Estolides are vegetable-oil-based lubricants obtained from oleic acid or any source of hydroxy fatty acids. In this work, the estolides synthesis from oleic acid and methyl ricinoleate (biodiesel from castor oil), using immobilized commercial lipases (Novozym 435, Lipozyme RM-IM, and Lipozyme TL-IM) in a solvent-free medium was investigated. Acid value was used to monitor the reaction progress by determining the consumption of acid present in the medium. Novozym 435 showed the best performance. Water removal improved the conversion. Novozym 435 was more active at atmospheric pressure. Novozym 435 was reused four times with conversion reaching 15% after the fourth reaction at 80°C. Estolides produced under the reaction conditions used in this work presented good properties, such as, low temperature properties as pour point (-24°C), viscosity (23.9 cSt at 40°C and 5.2 cSt at 100°C), and viscosity index (153).
RESUMO
The objective of this work was to study the synthesis of ethyl esters via esterification of soybean oil deodorizer distillate with ethanol, using solid acid catalysts and commercial immobilized lipases, in a solvent-free system. Three commercially immobilized lipases were used, namely, Lipozyme RM-IM, Lipozyme TL-IM, and Novozym 435, all from Novozymes. We aimed for optimum reaction parameters: temperature, enzyme concentration, initial amount of ethanol, and its feeding technique to the reactor (stepwise ethanolysis). Reaction was faster with Novozym 435. The highest conversion (83.5%) was obtained after 90 min using 3 wt.% of Novozym 435 and two-stage stepwise addition of ethanol at 50 degrees C. Four catalysts were also tested: zeolite CBV-780, SAPO-34, niobia, and niobic acid. The highest conversion (30%) was obtained at 100 degrees C, with 3 wt.% of CBV-780 after 2.5 h. The effects of zeolite CBV 780 concentration were studied, resulting in a conversion of 49% using 9 wt.% of catalyst.
Assuntos
Biotecnologia/métodos , Enzimas Imobilizadas/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Lipase/metabolismo , Catálise , Esterificação , Etanol/metabolismo , Proteínas Fúngicas , Cinética , Temperatura , Zeolitas/metabolismoRESUMO
The aim of this study was to produce monolaurin utilizing a commercial immobilized lipase (Lipozyme IM-20; Novo Nordisk, Bagsvaerd, Denmark) through the direct esterification of lauric acid and glycerol in a solvent-free system. The influence of fatty acid/glycerol molar ratio, temperature, and Lipozyme (IM-20) concentration on the molar fraction of monolaurin were determined using an experimental design. The best conditions employed were 55 degrees C, lauric acid/glycerol molar ratio of 1.0, and 3.0% (w/w) enzyme concentration. The final product, obtained after 6 h of reaction, was 45.5% monolaurin, 26.8% dilaurin, 3.1% trilaurin, and 24.6% lauric acid. The reusability of the enzyme was also studied.
Assuntos
Biotecnologia/métodos , Glicerídeos/biossíntese , Glicerídeos/química , Lauratos/química , Cromatografia Gasosa , Enzimas/química , Enzimas Imobilizadas , Ésteres , Glicerol/química , Ácidos Láuricos/química , Lipase/química , Modelos Teóricos , Monoglicerídeos , Temperatura , Fatores de TempoRESUMO
The reference source method associated with HPGe gamma-ray spectrometric techniques has been applied to half-life determinations of radionuclides used in nuclear medicine. Simultaneous measurements were undertaken for radiopharmaceutical nuclides such as 67Ga, 99Tcm, 123I, 131I and another radionuclide as reference. Besides optimizing the analysis time, this procedure was shown to be independent of the instrumental technique, environmental and radiochemical impurity interferences for most radionuclides. However, some restrictions were observed in samples that contained impurities with the same emission energy or an energy that was very close to the energy of the radionuclide to be measured. The measurement conditions improved the quoted accuracy of the half-life by reducing the type B component uncertainty.
Assuntos
Algoritmos , Radioisótopos de Gálio/análise , Radioisótopos do Iodo/análise , Compostos Radiofarmacêuticos/análise , Espectrometria gama/métodos , Tecnécio/análise , Simulação por Computador , Radioisótopos de Gálio/química , Meia-Vida , Radioisótopos do Iodo/química , Medicina Nuclear/métodos , Radioisótopos/análise , Radioisótopos/química , Compostos Radiofarmacêuticos/química , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tecnécio/químicaRESUMO
Since 1998, the National Laboratory for Ionizing Radiation Metrology has lead an intercomparison program for activity measurements of radiopharmaceuticals administered to patients in the Nuclear Medicine Services (NMS) with the purpose of promoting the quality control. In this work, intercomparison results with the radionuclides 67Ga, 123I, 99mTc and 201Tl from the beginning of the program to the present will be presented establishing as a consequence, the radionuclide calibrators' traceability used by the participants. The analysis of the results showed that 68% were within the limits of +/-10% required by the Brazilian Regulatory Authority that demands this accuracy band for the radiopharmaceutical measurements in the NMS. The results also demonstrate an improvement in the measurement quality that this program has provided since its beginning.