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1.
The goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for the hydroxylation of a variety of steroidal compounds, using H2O2 as the only cosubstrate. Two of them are wild-type enzymes from Agrocybe aegerita and Marasmius rotula, and the third one is a recombinant enzyme from Coprinopsis cinerea. The enzymatic reactions on free and esterified sterols, steroid hydrocarbons, and ketones were monitored by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating. Interestingly, antiviral and other biological activities of 25-hydroxycholesterol have been reported recently (M. Blanc et al., Immunity 38:106–118, 2013, http://dx.doi.org/10.1016/j.immuni.2012.11.004). However, hydroxylation in the ring moiety and terminal hydroxylation at the side chain also was observed in some steroids, the former favored by the absence of oxygenated groups at C-3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active-site geometry and hydrophobicity favors the entrance of the steroid side chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side chain entrance ratio could be established that explains the various reaction yields observed.  相似文献   

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Salinity is one of the most common abiotic stresses in agriculture production. Salt tolerance of rice (Oryza sativa) is an important trait controlled by various genes. The mechanism of rice salt tolerance, currently with limited understanding, is of great interest to molecular breeding in improving grain yield. In this study, a gene regulatory network of rice salt tolerance is constructed using a systems biology approach with a number of novel computational methods. We developed an improved volcano plot method in conjunction with a new machine-learning method for gene selection based on gene expression data and applied the method to choose genes related to salt tolerance in rice. The results were then assessed by quantitative trait loci (QTL), co-expression and regulatory binding motif analysis. The selected genes were constructed into a number of network modules based on predicted protein interactions including modules of phosphorylation activity, ubiquity activity, and several proteinase activities such as peroxidase, aspartic proteinase, glucosyltransferase, and flavonol synthase. All of these discovered modules are related to the salt tolerance mechanism of signal transduction, ion pump, abscisic acid mediation, reactive oxygen species scavenging and ion sequestration. We also predicted the three-dimensional structures of some crucial proteins related to the salt tolerance QTL for understanding the roles of these proteins in the network. Our computational study sheds some new light on the mechanism of salt tolerance and provides a systems biology pipeline for studying plant traits in general.  相似文献   

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Toxoplasma gondii is a human pathogen prevalent worldwide that poses a challenging and unmet need for novel treatment of toxoplasmosis. Using a semi-automated reconstruction algorithm, we reconstructed a genome-scale metabolic model, ToxoNet1. The reconstruction process and flux-balance analysis of the model offer a systematic overview of the metabolic capabilities of this parasite. Using ToxoNet1 we have identified significant gaps in the current knowledge of Toxoplasma metabolic pathways and have clarified its minimal nutritional requirements for replication. By probing the model via metabolic tasks, we have further defined sets of alternative precursors necessary for parasite growth. Within a human host cell environment, ToxoNet1 predicts a minimal set of 53 enzyme-coding genes and 76 reactions to be essential for parasite replication. Double-gene-essentiality analysis identified 20 pairs of genes for which simultaneous deletion is deleterious. To validate several predictions of ToxoNet1 we have performed experimental analyses of cytosolic acetyl-CoA biosynthesis. ATP-citrate lyase and acetyl-CoA synthase were localised and their corresponding genes disrupted, establishing that each of these enzymes is dispensable for the growth of T. gondii, however together they make a synthetic lethal pair.  相似文献   

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The surface plasmon resonance (SPR)-induced local field effect in Al-Au-Ag trimetallic three-layered nanoshells has been studied theoretically. Because of having three kinds of metal, three plasmonic bands have been observed in the absorption spectra and the local electric field factor spectra. The local electric field enhancement and the corresponding resonance wavelength for different plasmon coupling modes and spatial positions of the Al-Au-Ag nanoshells with various geometry dimensions are investigated to find the maximum local electric field enhancement. The calculation results indicate that the giant local electric field enhancement could be stimulated by the plasmon coupling in the middle Au shell or the outer Ag shell and could be optimized by increasing the Ag shell thickness and decreasing the Au shell thickness. What is more, the local electric field enhancement also nonmonotonously depends on the dielectric constant of the environment; the local electric field intensity will be weakened when the surrounding dielectric constant is too small or too large.

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Thermostable proteins are advantageous in industrial applications, as pharmaceuticals or biosensors, and as templates for directed evolution. As protein-design methodologies improve, bioengineers are able to design proteins to perform a desired function. Although many rationally designed proteins end up being thermostable, how to intentionally design de novo, thermostable proteins is less clear. UVF is a de novo-designed protein based on the backbone structure of the Engrailed homeodomain (EnHD) and is highly thermostable (Tm > 99°C vs. 52°C for EnHD). Although most proteins generally have polar amino acids on their surfaces and hydrophobic amino acids buried in their cores, protein engineers followed this rule exactly when designing UVF. To investigate the contributions of the fully hydrophobic core versus the fully polar surface to UVF’s thermostability, we built two hybrid, chimeric proteins combining the sets of buried and surface residues from UVF and EnHD. Here, we determined a structural, dynamic, and thermodynamic explanation for UVF’s thermostability by performing 4 μs of all-atom, explicit-solvent molecular dynamics simulations at 25 and 100°C, Tanford-Kirkwood solvent accessibility Monte Carlo electrostatic calculations, and a thermodynamic analysis of 40 temperature runs by the weighted-histogram analysis method of heavy-atom, structure-based models of UVF, EnHD, and both chimeric proteins. Our models showed that UVF was highly dynamic because of its fully hydrophobic core, leading to a smaller loss of entropy upon folding. The charged residues on its surface made favorable electrostatic interactions that contributed enthalpically to its thermostability. In the chimeric proteins, both the hydrophobic core and charged surface independently imparted thermostability.  相似文献   

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Abstract

The success rate of reconstructing the Anterior Cruciate Ligament (ACL) with prosthetic ligaments is currently low both in humans and animals. The stress distribution in prosthetic ligaments that causes failure is very complex and not yet understood. Therefore, we have begun to develop a Finite Element Model of a prosthetic ACL. Here we describe the normal and contact stresses in DacronTM yarn (a multi-fibrillar structure) using input data based on experimental measurements of the load and strain of six designed yarns.

The results show that the normal and contact stresses in the fibres of the ACL yarn are directly proportional to the yam strains. Increasing the twisting length (transverse deformation) of the yarn increases the normal stress in the fibres and the yarn modulus, but decreases the contact stresses between the fibres. The structural properties of a yarn are dependent on the specific arrangement of various filament types. Increasing the distance between the longitudinal (symmetry) axes of the filaments and the axis of symmetry of the yarn decreases the stresses.  相似文献   

11.
The success rate of reconstructing the Anterior Cruciate Ligament (ACL) with prosthetic ligaments is currently low both in humans and animals. The stress distribution in prosthetic ligaments that causes failure is very complex and not yet understood. Therefore, we have begun to develop a Finite Element Model of a prosthetic ACL. Here we describe the normal and contact stresses in Dacron(TM) yarn (a multi-fibrillar structure) using input data based on experimental measurements of the load and strain of six designed yarns. The results show that the normal and contact stresses in the fibres of the ACL yarn are directly proportional to the yarn strains. Increasing the twisting length (transverse deformation) of the yarn increases the normal stress in the fibres and the yarn modulus, but decreases the contact stresses between the fibres. The structural properties of a yarn are dependent on the specific arrangement of various filament types. Increasing the distance between the longitudinal (symmetry) axes of the filaments and the axis of symmetry of the yarn decreases the stresses.  相似文献   

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Excessive salt intake is associated with hypertension and cardiovascular diseases. Salt intake exceeds the World Health Organization population nutrition goal of 5 grams per day in the European region. We assessed the health impact of salt reduction in nine European countries (Finland, France, Ireland, Italy, Netherlands, Poland, Spain, Sweden and United Kingdom). Through literature research we obtained current salt intake and systolic blood pressure levels of the nine countries. The population health modeling tool DYNAMO-HIA including country-specific disease data was used to predict the changes in prevalence of ischemic heart disease and stroke for each country estimating the effect of salt reduction through its effect on blood pressure levels. A 30% salt reduction would reduce the prevalence of stroke by 6.4% in Finland to 13.5% in Poland. Ischemic heart disease would be decreased by 4.1% in Finland to 8.9% in Poland. When salt intake is reduced to the WHO population nutrient goal, it would reduce the prevalence of stroke from 10.1% in Finland to 23.1% in Poland. Ischemic heart disease would decrease by 6.6% in Finland to 15.5% in Poland. The number of postponed deaths would be 102,100 (0.9%) in France, and 191,300 (2.3%) in Poland. A reduction of salt intake to 5 grams per day is expected to substantially reduce the burden of cardiovascular disease and mortality in several European countries.  相似文献   

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Study of protein adsorption to solid surfaces continues to be substantial because of its role in cellular responses to biomaterials, interest in molecular aspects such as conformation and orientation, new methods for making protein repellent surfaces, and new application areas such as nanoparticles and microfluidics. This brief review is based only on very recent articles of particular interest to the authors, who each have worked in this area for some time. Simulations of protein interactions with surfaces and protein repellent surfaces are the only subtopics reviewed here.  相似文献   

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Dilution of a fatty acid micellar solution at basic pH toward neutrality results in spontaneous formation of vesicles with a broad size distribution. However, when vesicles of a defined size are present before dilution, the size distribution of the newly formed vesicles is strongly biased toward that of the seed vesicles. This so-called matrix effect is believed to be a key feature of early life. Here we reproduced this effect for oleate micelles and seed vesicles of either oleate or dioleoylphosphatidylcholine. Fluorescence measurements showed that the vesicle contents do not leak out during the replication process. We hypothesized that the matrix effect results from vesicle fission induced by an imbalance of material across both leaflets of the vesicle upon initial insertion of fatty acids into the outer leaflet of the seed vesicle. This was supported by experiments that showed a significant increase in vesicle size when the equilibration of oleate over both leaflets was enhanced by either slowing down the rate of fatty acid addition or increasing the rate of fatty acid transbilayer movement. Coarse-grained molecular-dynamics simulations showed excellent agreement with the experimental results and provided further mechanistic details of the replication process.  相似文献   

17.
The relationship between salt bridges and stability/enzymatic activity is unclear. We studied this relationship by systematic alanine-scanning mutation analysis using the typical M4 family metalloprotease Pseudomonas aeruginosa elastase (PAE, also known as pseudolysin) as a model. Structural analysis revealed seven salt bridges in the PAE structure. We constructed ten mutants for six salt bridges. Among these mutants, six (Asp189Ala, Arg179Ala, Asp201Ala, Arg205Ala, Arg245Ala and Glu249Ala) were active and four (Asp168Ala, Arg198Ala, Arg253Ala, and Arg279Ala) were inactive. Five mutants were purified, and their catalytic efficiencies (k cat/K m), half-lives (t 1/2) and thermal unfolding curves were compared with those of PAE. Mutants Asp189Ala and Arg179Ala both showed decreased thermal stabilities and increased activities, suggesting that the salt bridge Asp189-Arg179 stabilizes the protein at the expense of catalytic efficiency. In contrast, mutants Asp201Ala and Arg205Ala both showed slightly increased thermal stability and slightly decreased activity, suggesting that the salt bridge Asp201-Arg205 destabilizes the protein. Mutant Glu249Ala is related to a C-terminal salt bridge network and showed both decreased thermal stability and decreased activity. Furthermore, Glu249Ala showed a thermal unfolding curve with three discernable states [the native state (N), the partially unfolded state (I) and the unfolded state (U)]. In comparison, there were only two discernable states (N and U) in the thermal unfolding curve of PAE. These results suggest that Glu249 is important for catalytic efficiency, stability and unfolding cooperativity. This study represents a systematic mutational analyses of salt bridges in the model metalloprotease PAE and provides important insights into the structure-function relationship of enzymes.  相似文献   

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通过氨基酸定点突变技术提高灵芝免疫调节蛋白LZ-8的热稳定性。通过分子动力学模拟结合温度因子预测对LZ-8氨基酸突变位点进行理性设计,在毕赤酵母X33菌株内构建并表达LZ-8突变体蛋白,采用HeLa细胞生长抑制实验和差示量热扫描分析检测并比较了LZ-8突变前后生物学活性及热力学参数。结果显示,LZ-8 N-端α螺旋为理论预测的温度敏感区域,在该区域进行F8W和R9K氨基酸双位点突变,突变后的LZ-8热稳定性提高,相变温度Tm上升0.92℃,相转变焓值ΔH提高23.14 kJ/mol,但突变后LZ-8生物学活性基本不变,LZ-8和LZ-8突变体对HeLa细胞生长抑制的IC50值分别是2.238μg/mL和2.407μg/mL。通过理性设计氨基酸突变位点,获得了稳定性提高但生物学活性不变的灵芝免疫调节蛋白LZ-8的突变体。  相似文献   

19.
盐胁迫下小麦过氧化物酶同工酶、全蛋白变化的研究   总被引:3,自引:1,他引:3  
以蛋白质含量不同的两种小麦幼苗为材料,研究了经高浓度的盐胁迫后过氧化物酶(POD)同工酶、全蛋白质的变化。结果表明,植物处于高盐环境时,会诱导或抑制某些同工酶的产生,控制蛋白质的增加和降低,以适应盐胁迫下细胞内特殊的代谢反应,从而达到抵抗盐渍侵害的目的。  相似文献   

20.
The germination of lentil seeds was gradually reduced when seeds were exposed to temperature of 30 or 40 °C, either alone or combined with 0.1, 0.2 or 0.3 M NaCl or 34.1 % (m/v) PEG 8000, during 6 –12 h imbibition. [35S]-methionine incorporation in 12 h imbibed lentil axes also decreased with increasing NaCl concentration at 20 and 40 °C, whereas at 30 °C only 0.3 M NaCl treatment partially inhibited protein synthesis. An analysis of newly synthesized proteins by 1-D SDS PAGE, showed that the expression of most polypeptides decreased following increasing stress. Among these, low molecular mass heat-shock proteins declining, higher in 40 °C treated axes than those treated at 30 °C, supports the hypothesis that at this temperature maximal level of expression of these proteins was achieved.  相似文献   

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