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Vasilenko E. A. Gorshkova E. N. Astrakhantseva I. V. Nedospasov S. A. Mokhonov V. V. 《Russian Journal of Bioorganic Chemistry》2020,46(3):299-305
Russian Journal of Bioorganic Chemistry - The aim of this work was to evaluate novel three-domain antibodies consisting of two domains specific for human tumor necrosis factor (hTNF), and of the... 相似文献
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Inhibition of interferon signaling by the New York 99 strain and Kunjin subtype of West Nile virus involves blockage of STAT1 and STAT2 activation by nonstructural proteins 下载免费PDF全文
The interferon (IFN) response is the first line of defense against viral infections, and the majority of viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile viruses, the New York 99 strain (NY99) and Kunjin virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-alpha/beta) and gamma IFN (IFN-gamma) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-alpha. IFN-alpha signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins responsible for this inhibition. 相似文献
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E.?A.?Vasilenko V.?V.?Mokhonov E.?N.?Gorshkova I.?V.?AstrakhantsevaEmail author 《Molecular Biology》2018,52(3):323-334
Bispecific antibodies capable of simultaneously binding two targets have been studied for many years with a view to their implementation in clinical practice. Unique biological and pharmacological properties, as well as the diversity of their formats, make it possible to consider bispecific antibodies as promising agents for use in various procedures: from visualization of intracellular processes to targeted anticancer therapy. Bispecific antibodies help to determine more precisely the therapeutic target, thereby increasing the efficiency of therapy and reducing the probability of side effects. The present review describes the main formats of bispecific antibodies, methods for their generation, and possibilities for practical application. 相似文献
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Role of the membrane fusion protein in the assembly of resistance-nodulation-cell division multidrug efflux pump in Pseudomonas aeruginosa 总被引:1,自引:0,他引:1
Mokhonov VV Mokhonova EI Akama H Nakae T 《Biochemical and biophysical research communications》2004,322(2):483-489
The tripartite xenobiotic-antibiotic transporter of Pseudomonas aeruginosa consists of the inner membrane transporter (e.g., MexB, MexY), the periplasmic membrane-fusion-protein (e.g., MexA, MexX), and the outer membrane channel protein (e.g., OprM). These subunits were assumed to assemble into a transporter unit during export of the substrates. However, subunit interaction and their specificity in native form remained to be elucidated. To address these important questions, we analyzed the role of the individual subunits for the assembly of MexAB-OprM by pull-down assay tagging only one of the subunits. We found stable MexA-MexB-OprM complex without chemical cross-linking that withstand all purification procedures. Results of bi-partite interactions analysis showed tight association between MexA and OprM in the absence of MexB, whereas the expression systems lacking MexA failed to co-purify MexB or OprM. None of the heterologous subunit combinations such as MexA+MexY(his)+OprM and MexX+MexB(his)+OprM showed interaction. These results implied that the membrane fusion protein is central to the tripartite xenobiotic transporter assembly. 相似文献
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Pyankov OV Agranovski IE Pyankova O Mokhonova E Mokhonov V Safatov AS Khromykh AA 《Environmental microbiology》2007,9(4):992-1000
We have recently developed a new personal sampler and demonstrated its feasibility for detection of viable airborne microorganisms including bacteria, fungi and viruses. To accelerate the time-consuming analytical procedure involving 2-5 days of biological testing, we employed a real-time PCR protocol in conjunction with the personal sampler for collection of airborne viruses. The advantage of this approach is that if the presence of a particular pathogen in the air is detected by the PCR, the remaining collecting liquid can be further analysed by more time-consuming biological methods to estimate the number of airborne infectious/live microorganisms. As sampling of bioaerosols in natural environments is likely to be associated with substantial contamination by a range of microorganisms commonly existing in an ambient air, an investigation of the specificity of detection by targeted PCR analysis is required. Here we present the results of the study on the detection of Influenza virus in the ambient air contaminated with high concentrations of bacteria and fungi using real-time PCR protocol. The combined sampling PCR detection method was found to be fully feasible for the rapid ( approximately 2.5 h) and highly specific (no cross-reactivity) identification of the labile airborne virus in the air containing elevated concentrations of other microorganisms. 相似文献
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Mokhonov V Mokhonova E Yoshihara E Masui R Sakai M Akama H Nakae T 《Protein expression and purification》2005,40(1):91-100
Structural and functional characterization of the multidrug transporter, MexB, of Pseudomonas aeruginosa is significantly restricted due to a low yield of approximately 0.1 mg/L of culture from natural sources. To facilitate structural studies of this medically important transporter protein, we developed a large-scale system for expression of the genetically engineered recombinant, MexB, in the Escherichia coli cell. Using the system, the eventual yield of MexB attained was about 10mg/L of culture. The optimized purification protocol in the presence of dodecyl beta-D-maltoside allowed isolation of highly homogeneous MexB. The oligomeric state of the protein in detergent solution has been characterized to verify that the native state of the purified protein has been preserved. The molecular mass of the protein-detergent complex was found to be 380-450kDa. The MexB-dodecyl beta-d-maltoside mass ratio was determined to be 1.8 +/- 0.05. Taking into account the monomeric MexB molecular mass deduced from its amino acid sequence (112.8 kDa), we concluded that the purified MexB exists as the homotrimer in the surfactant solution. Circular dichroism analysis of MexB showed dominance of the alpha-helix structures. High yield, homogeneity, and stability of MexB position it as a good candidate for structural and functional characterization. 相似文献
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Mokhonova EI Mokhonov VV Akama H Nakae T 《Biochemical and biophysical research communications》2005,327(3):650-655
We developed an Escherichia coli expression system for overproduction of a highly toxic membrane protein that is impossible to overexpress by traditionally used approaches. The method is based on combination of the genetic modifications of a bicistronic expression plasmid, stabilization of a synthesized protein, and selection of a compatible expression host. This enabled us to enhance the expression level of a toxic membrane protein 30-50 times compared with expression in the native state and to obtain 3-5mg of a highly purified functionally active protein per liter of culture. We describe the method for the amplified expression of membrane proteins, using the Pseudomonas aeruginosa multidrug resistance protein, MexY, as an example. The amplified MexY was correctly folded in the cytoplasmic membrane of the E. coli without forming inclusion bodies. This method can be applicable to the large-scale expression of the other problematic membrane proteins that are otherwise extremely difficult to overproduce. 相似文献
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