Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from Sphingobium japonicum UT26 and DmbA from Mycobacterium bovis 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies. 相似文献
The aim of this study was to investigate the effects of quercetin (QUE) on the testicular architecture as well as markers of oxidative, inflammatory, and apoptotic profile of male gonads in Zucker diabetic fatty (ZDF) rats suffering from Type 2 diabetes mellitus in the absence or presence of obesity. QUE was administered orally at a dose of 20 mg/kg/day for 6 weeks. Morphometric analysis revealed that QUE treatment led to an improvement in testicular appearance, particularly in the case of Obese ZDF rats. Furthermore, a significant stabilization of the antioxidant capacity (p < 0.05), superoxide dismutase and catalase activity (p < 0.01), with a concomitant decrease in lipid peroxidation (p < 0.05) were observed in Obese ZDF animals exposed to QUE. Our data also indicate a significant decline in the levels of interleukin (IL)-1 (p < 0.05), IL-6 (p < 0.01) and tumor necrosis factor alpha (p < 0.001) following QUE supplementation to Obese ZDF rats in comparison with their respective control. Finally, a significant down-regulation of the pro-apoptotic BAX protein (p < 0.0001) was observed in Obese ZDF rats administered with QUE, while a significant Bcl-2 protein overexpression (p < 0.0001) was recorded in Lean ZDF animals when compared to their untreated control. As such, our results suggest that QUE is a potentially beneficial agent to reduce testicular damage in ZDF rats with Type 2 diabetes mellitus by decreasing oxidative stress, chronic inflammation, and excessive cell loss through apoptosis. 相似文献
A strong difference in the physico-chemical properties of the plastic lubricants studied was found in this study through pressure drop, thermal analysis, vibration damping, texture hardness and rheological measurements. Oxidation aging of the lubricant sample containing rapeseed oil additive was proposed. Its higher thermal sensitivity was simultaneously confirmed by frequency dependent complex shear modulus of elasticity measurements as well as by rheological testing. Rapeseed oil modified lubricant showed a higher decrease in both storage and moduli losses due to a temperature increase from 16 to 26 ° C compared to the rapeseed oil free sample. Simultaneously, the flow curves were shifted to the higher shear stresses (for plastic lubricant without rapeseed oil additive) typical for rheopectic fluids. For the rapeseed oil modified lubricant, the flow curves were shifted to the lower shear stresses, indicating its thixotropic fluid behaviour. The synthetic lubricant without rapeseed oil additive exhibited higher dissipative rheological behaviour as reflected by decreasing first resonance frequency peak position compared to the rapeseed oil modified lubricant as obtained from vibration damping measurements. It was found that the synthetic lubricant exhibited better vibration damping properties and mechanical energy dissipation into heat due to its higher viscous friction than the rapeseed oil modified lubricant under experimental conditions.
The ability to form an efficient interface between material and neural cells is a crucial aspect for construction of neuroelectrodes. Diamond offers material characteristics that could, to a large extent, improve the performance of neuroelectrodes. The greatest advantage of diamond is a large variety of material and surface properties such as electrical conductivity, surface morphology, and surface chemistry. Such a variety of material characteristics can lead to various cellular responses. Here, the authors compare survival, adhesion, and neurite formation of primary neurons on diamond thin films of various morphologies and treatments with several types of polymers commonly used to enhance cell adhesion. The authors find that the variation of surface roughness of nanocrystalline diamond film when coated with polymer does not have a major influence on neuron survival or adhesion. The adhesion of neurons can be influenced by the selected type of polymer coating. High molecular weight of polyethylenimine results in lower viability, adhesion, and neurite formation. The addition of laminin to treated films do not lead to significant improvements in neuron adhesion and neurite development. Their findings emphasize the importance of the correct polymer treatment over morphological properties of diamond thin films as a material for forming interfaces with primary neurons. 相似文献
The properties of materials at the nanoscale open up new methodologies for engineering prospective materials usable in high-end applications. The preparation of composite materials with a high content of an active component on their surface is one of the current challenges of materials engineering. This concept significantly increases the efficiency of heterogeneous processes moderated by the active component, typically in biological applications, catalysis, or drug delivery. Here we introduce a general approach, based on laser-induced optomechanical processing of silver colloids, for the preparation of polymer surfaces highly enriched with silver nanoparticles (AgNPs). As a result, the AgNPs are firmly immobilized in a thin surface layer without the use of any other chemical mediators. We have shown that our approach is applicable to a broad spectrum of polymer foils, regardless of whether they absorb laser light or not. However, if the laser radiation is absorbed, it is possible to transform smooth surface morphology of the polymer into a roughened one with a higher specific surface area. Analyses of the release of silver from the polymer surface together with antibacterial tests suggested that these materials could be suitable candidates in the fight against nosocomial infections and could inhibit the formation of biofilms with a long-term effect. 相似文献
Computation of trajectories for ligand binding and unbinding via protein tunnels and channels is important for predicting possible protein–ligand interactions. These highly complex processes can be simulated by several software tools, which provide biochemists with valuable information for drug design or protein engineering applications. This paper focuses on aiding this exploration process by introducing the DockVis visual analysis tool. DockVis operates with the multivariate output data from one of the latest available tools for the prediction of ligand transport, CaverDock. DockVis provides the users with several linked views, combining the 2D abstracted depictions of ligands and their surroundings and properties with the 3D view. In this way, we enable the users to perceive the spatial configurations of ligand passing through the protein tunnel. The users are initially visually directed to the most relevant parts of ligand trajectories, which can be then explored in higher detail by the follow-up analyses. DockVis was designed in tight collaboration with protein engineers developing the CaverDock tool. However, the concept of DockVis can be extended to any other tool predicting ligand pathways by the molecular docking. DockVis will be made available to the wide user community as part of the Caver Analyst 3.0 software package ( www.caver.cz ). 相似文献