排序方式: 共有62条查询结果,搜索用时 15 毫秒
41.
Megan Garvey Johannes Klinger Holger Klose Rainer Fischer Ulrich Commandeur 《Journal of visualized experiments : JoVE》2014,(88)
Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems. 相似文献
42.
Kool J van Liempd SM Harmsen S Beckman J van Elswijk D Commandeur JN Irth H Vermeulen NP 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,858(1-2):49-58
Here we describe novel on-line human CYP1A2 and CYP2D6 Enzyme Affinity Detection (EAD) systems coupled to gradient HPLC. The use of the systems lies in the detection of individual inhibitory ligands in mixtures (e.g. metabolic mixtures or herbal extracts) towards two relevant drug metabolizing human CYPs. The systems can rapidly detect individual compounds in mixtures with affinities to CYP1A2 or 2D6. The HPLC-EAD systems were first evaluated and validated in flow injection analysis mode. IC50 values of known ligands for both CYPs, tested both in flow injection and in HPLC mode, were well comparable with those measured in microplate reader formats. Both EAD systems were also connected to gradient HPLC and used to screen known compound mixtures for the presence of CYP1A2 and 2D6 inhibitors. Finally, the on-line CYP2D6 EAD system was used to screen for the inhibitory activities of stereoisomers of a mixture of five methylenedioxy-alkylamphetamines (XTC analogs) on a chiral analytical column. 相似文献
43.
Louis S. Ates Roy Ummels Susanna Commandeur Robert van der Weerd Marion Sparrius Eveline Weerdenburg Marina Alber Rainer Kalscheuer Sander R. Piersma Abdallah M. Abdallah Moataz Abd El Ghany Alyaa M. Abdel-Haleem Arnab Pain Connie R. Jiménez Wilbert Bitter Edith N.G. Houben 《PLoS genetics》2015,11(5)
Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria. 相似文献
44.
Florian Ryffel Eric JN Helfrich Patrick Kiefer Lindsay Peyriga Jean-Charles Portais J?rn Piel Julia A Vorholt 《The ISME journal》2016,10(3):632-643
The phyllosphere, which is defined as the parts of terrestrial plants above the ground, is a large habitat for different microorganisms that show a high extent of adaption to their environment. A number of hypotheses were generated by culture-independent functional genomics studies to explain the competitiveness of specialized bacteria in the phyllosphere. In contrast, in situ data at the metabolome level as a function of bacterial colonization are lacking. Here, we aimed to obtain new insights into the metabolic interplay between host and epiphytes upon colonization of Arabidopsis thaliana leaves in a controlled laboratory setting using environmental metabolomics approaches. Quantitative nuclear magnetic resonance (NMR) and imaging high-resolution mass spectrometry (IMS) methods were used to identify Arabidopsis leaf surface compounds and their possible involvement in the epiphytic lifestyle by relative changes in compound pools. The dominant carbohydrates on the leaf surfaces were sucrose, fructose and glucose. These sugars were significantly and specifically altered after epiphytic leaf colonization by the organoheterotroph Sphingomonas melonis or the phytopathogen Pseudomonas syringae pv. tomato, but only to a minor extent by the methylotroph Methylobacterium extorquens. In addition to carbohydrates, IMS revealed surprising alterations in arginine metabolism and phytoalexin biosynthesis that were dependent on the presence of bacteria, which might reflect the consequences of bacterial activity and the recognition of not only pathogens but also commensals by the plant. These results highlight the power of environmental metabolomics to aid in elucidating the molecular basis underlying plant–epiphyte interactions in situ. 相似文献
45.
Nicolas Boutard Aleksandra Sabiniarz Klaudia Czerwińska Małgorzata Jarosz Anna Cierpich Ewa Kolasińska Katarzyna Wiklik Karolina Gluza Claude Commandeur Anna Buda Agata Stasiowska Aneta Bobowska Mariusz Galek Charles-Henry Fabritius Marta Bugaj Edyta Palacz Andrzej Mazan Adrian Zarębski Piotr Kowalczyk 《Bioorganic & medicinal chemistry letters》2019,29(4):607-613
Maternal embryonic leucine zipper kinase (MELK) is involved in several key cellular processes and displays increased levels of expression in numerous cancer classes (colon, breast, brain, ovary, prostate and lung). Although no selective MELK inhibitors have yet been approved, increasing evidence suggest that inhibition of MELK would constitute a promising approach for cancer therapy. A weak high-throughput screening hit (17, IC50?≈?5?μM) with lead-like properties was optimized for MELK inhibition. The early identification of a plausible binding mode by molecular modeling offered guidance in the choice of modifications towards compound 52 which displayed a 98?nM IC50. A good selectivity profile was achieved for a representative member of the series (29) in a 486 protein kinase panel. Future elaboration of 52 has the potential to deliver compounds for further development with chemotherapeutic aims. 相似文献
46.
47.
Background
During the development of the central nervous system (CNS), patterning processes along the dorsoventral (DV) axis of the neural tube generate different neuronal subtypes. As development progresses these neurons are arranged into functional units with varying cytoarchitecture, such as laminae or nuclei for efficient relaying of information. Early in development ventral and dorsal regions are similar in size and structure. Different proliferation rates and cell migration patterns are likely to result in the formation of laminae or nuclei, eventually. However, the underlying molecular mechanisms that establish these different structural arrangements are not well understood. 相似文献48.
Alois Bonifacio André R. Groenhof Peter H. J. Keizers Chris de Graaf Jan N. M. Commandeur Nico P. E. Vermeulen Andreas W. Ehlers Koop Lammertsma Cees Gooijer Gert van der Zwan 《Journal of biological inorganic chemistry》2007,12(5):645-654
Cytochrome P450 2D6 (CYP2D6) is one of the most important cytochromes P450 in humans. Resonance Raman data from the T309V
mutant of CYP2D6 show that the substitution of the conserved I-helix threonine situated in the enzyme’s active site perturbs
the heme spin equilibrium in favor of the six-coordinated low-spin species. A mechanistic hypothesis is introduced to explain
the experimental observations, and its compatibility with the available structural and spectroscopic data is tested using
quantum-mechanical density functional theory calculations on active-site models for both the CYP2D6 wild type and the T309V
mutant.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Alois Bonifacio, André R. Groenhof and Peter H. J. Keizers contributed equally to this work. 相似文献
49.
Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi 总被引:2,自引:0,他引:2
Rasmus JN Frandsen Jens A Andersson Matilde B Kristensen Henriette Giese 《BMC molecular biology》2008,9(1):70
Background
The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. 相似文献50.
In this study, the first fluorescent assay for bacterial cytochrome P450 BM3 (BM3) and mutants is described. BM3 mutants are potentially very versatile biocatalysts for the production of fine chemicals. A fluorescent assay would be very useful for the identification of nonnatural ligands in high-throughput inhibition assays. Because of the ease and sensitivity of alkoxyresorufin O-dealkylation assays, four different alkoxyresorufins were evaluated as substrates. Wild-type BM3 showed extremely low activity toward all four alkoxyresorufins tested. Five different BM3 mutants were constructed, carrying different combinations of mutations R47L, F87V, and L188Q, which were previously shown to increase activity toward nonnatural substrates. For all mutants, a high benzyloxyresorufin O-dealkylation (BROD) activity was found. The triple mutant of BM3, R47L/F87V/L188Q, showed the highest activity, increasing 900-fold compared to wild-type BM3. The BROD assay could also be applied in whole Escherichia coli cells; permeabilization by lipopolysaccharide deficiency strongly increased activity. To demonstrate the applicability of the BROD assay to screening for novel ligands of BM3 R47L/F87V/L188Q, a library of 45 drug-like compounds was tested for inhibition. Of these compounds, 8 showed strong inhibition of the BROD activity, demonstrating for the first time that drug-like molecules also can bind with high affinity to BM3 mutants. 相似文献