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The ability of ricin, a type II ribosome-inactivating protein, to induce hepatoma cell (BEL7404) to apoptosis in vitro was examined by fluorescence microscopy, flow cytometry, and DNA fragmentation assay. As a Bcl-2 lacking model, BEL7404 bore unique advantage to study the effect of over-expressing Bcl-2 on the apoptosis induced by the inhibitor of protein synthesis. By establishing a Bcl-2 over-expressing cell line (BEL7404/ Bcl-2), we found that Bcl-2 could promote the survival of the hepatoma cell against ricin insult. The ricin-induced apoptosis of BEL7404 was accompanied by increased expression of Bak and decreased levels of Bcl-xl and Bax. Caspases and PARP cleavage activity were found to be implicated in the death process. Through the inhibitor tests, our results excluded the participation of calcium-dependent proteases or protein kinase C in the apoptotic process induced by ricin, though an elevation of intracellular calcium did occur as an immediate response to ricin treatment. Cycloheximide, another protein synthesis inhibitor, did synergistically enhance rather than inhibit the cytotoxicity of ricin to hepatoma cell BEL7404. Actually, cycloheximide alone was able to induce hepatoma cell BEL7404 to death that could also be inhibited by over-expressing Bcl-2. The elevation of apoptotic protein Bak was discussed to challenge the notion that ricin exerted its cytotoxicity through nonspecific inhibition of all the de novo protein synthesis. 相似文献
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Roland Lehmann Johann Meyer Michael Schuemann Eberhard Krause Christian Freund 《Proteomics》2009,9(23):5288-5295
The identification of modular units of cellular function is a major goal for proteomic research. Protein complexes represent important building blocks defining functionality and deciphering their composition remains a major challenge. Here, we have designed a new tandem affinity purification (TAP) tag (termed S3S‐tag) for the isolation of protein complexes. Specifically, the immune cell protein ADAP that regulates integrin adhesion was fused either C‐ or N‐terminally to the S3S‐tag. After retroviral transduction of a vector containing S3S‐tagged ADAP and internal ribosomal entry site encoded enhanced green fluorescent protein (eGFP), Jurkat T cells were sorted according to eGFP expression and further selected for expression of TAP‐tagged protein close to endogenous levels. The combination of a cleavable S‐tag and a Strep‐tag II allowed for the isolation of ADAP and associated proteins. Subsequently, stable isotope labeling with amino acids in cell culture‐based mass spectrometric analysis was performed to identify potentially specific interaction partners. Co‐purification of the known interaction partner Src kinase‐associated phosphoprotein of 55 kDa indicates the validity of our approach, while the identification of the ENA/VASP family member EVL, the guanine nucleotide exchange factor GEF‐H1 and the adaptor protein DOCK2 corroborates a link between ADAP‐mediated integrin regulation and the cytoskeleton. 相似文献
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Loss of STI1‐mediated neuronal survival and differentiation in disease‐associated mutations of prion protein 下载免费PDF全文
Michele Christine Landemberger Gabriela Pintar de Oliveira Cleiton Fagundes Machado Kenneth John Gollob Vilma Regina Martins 《Journal of neurochemistry》2018,145(5):409-416
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Follistatin‐like protein 1 contributes to dendritic cell and T‐lymphocyte activation in nasopharyngeal carcinoma patients by altering nuclear factor κb and Jun N‐terminal kinase expression 下载免费PDF全文
Hong Wang Senyong Wu Shiping Huang Shaolin Yin Guilong Zou Kuan'en Huang Zhe Zhang Anzhou Tang Wensheng Wen 《Cell biochemistry and function》2016,34(8):554-562
Follistatin‐like protein 1 (FSTL1) is a newly characterized protein that can regulate the immune response in various ways. Dendritic cells (DCs) are central to immune regulation. In this study, we explored the impact of FSTL1 on DC activity in nasopharyngeal carcinoma (NPC) patients. The surface expression of CD40, CD86, and HLA‐DR on DCs was analyzed and showed significantly elevated expression levels, indicating DC maturity. After FSTL1 was added to DCs collected from NPC patients (n = 50), controls (n = 47), and healthy donors (n = 10), interferon γ secretion and T‐cell receptor expression in cytotoxic T lymphocytes were also investigated. In the experimental groups, the expression of the critical immune protein nuclear factor (NF)‐κb was upregulated, whereas Jun N‐terminal kinase (JNK) was downregulated. Our findings demonstrate that FSTL1 plays a critical role in immune regulation, enhancing the antigen presentation ability of DCs by up‐regulating NF‐κb expression and down‐regulating JNK expression. 相似文献
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This study is aimed at elucidating the structure of a novel T‐cell adhesion inhibitor, cyclo(1,8)‐CPRGGSVC using one‐ and two‐dimensional (2D) 1H NMR and molecular dynamics (MD) simulation. The peptide is derived from the sequence of its parent peptide cIBR (cyclo(1,12)‐PenPRGGSVLVTGC), which is a fragment of intercellular adhesion molecule‐1 (ICAM‐1). Our previous results show that the cyclo(1,8)‐CPRGGSVC peptide binds to the LFA‐1 I‐domain and inhibits heterotypic T‐cell adhesion, presumably by blocking the LFA‐1/ICAM‐1 interactions. The structure of the peptide was determined using NMR and MD simulation in aqueous solution. Our results indicate that the peptide adopts type‐I β‐turn conformation at the Pro2‐Arg3‐Gly4‐Gly5 (PRGG) sequence. The β‐turn structure at the PRGG motif is well conserved in cIBR peptide and ICAM‐1 receptor, which suggests the importance of the PRGG motif for the biological activity of cyclo(1,8)‐CPRGGSVC peptide. Meanwhile, the Gly5‐Ser6‐Val7‐Cys8‐Cys1 (GSVCC) sequence forms a “turn‐like” random coil structure that does not belong to any structured motif. Therefore, cyclo(1,8)‐CPRGGSVC peptide has only one structured region at the PRGG sequence, which may play an important role in the binding of the peptide to the LFA‐1 I‐domain. The conserved β‐turn conformation of the PRGG motif in ICAM‐1, cIBR, and cyclo(1,8)‐CPRGGSVC peptides can potentially be used to design peptidomimetics. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 633–641, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com 相似文献
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Raisa Vermasvuori Jani Koskinen Katri Salonen Noora Sirén Jan Weegar John Dahlbacka Nisse Kalkkinen Niklas von Weymarn 《Biotechnology progress》2009,25(1):95-102
Three popular expression host systems Escherichia coli, Pichia pastoris and Drosophila S2 were analyzed techno‐economically using HIV‐1 Nef protein as the model product. On scale of 100 mg protein, the labor costs corresponded to 52–83% of the manufacturing costs. When analyzing the cost impact of the different phases (strain/cell line construction, bioreactor production, and primary purification), we found that with the microbial host systems the strain construction phase was most significant generating 56% (E. coli) and 72% (P. pastoris) of the manufacturing costs, whereas with the Drosophila S2 system the cell line construction and bioreactor production phases were equally significant (46 and 47% of the total costs, respectively). With different titers and production goal of 100 mg of Nef protein, the costs of P. pastoris and Drosophila S2 systems were about two and four times higher than the respective costs of the E. coli system. When equal titers and bioreactor working volumes (10 L) were assumed for all three systems, the manufacturing costs of the bioreactor production of the P. pastoris and Drosophila S2 systems were about two and 2.5 times higher than the respective costs of the E. coli system. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
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Pravin Kumar Ardeschir Vahedi‐Faridi Wolfram Saenger Andreas Ziegler Barbara Uchanska‐Ziegler 《Protein science : a publication of the Protein Society》2009,18(1):37-49
Although there is X‐ray crystallographic evidence that the interaction between major histocompatibility complex (MHC, in humans HLA) class I molecules and T cell receptors (TCR) or killer cell Ig‐like receptors (KIR) may be accompanied by considerable changes in the conformation of selected residues or even entire loops within TCR or KIR, conformational changes between receptor‐bound and ‐unbound MHC class I molecules of comparable magnitude have not been observed so far. We have previously determined the structure of the MHC class I molecule HLA‐A1 bound to a melanoma antigen‐encoding gene (MAGE)‐A1‐derived peptide in complex with a recombinant antibody fragment with TCR‐like specificity, Fab‐Hyb3. Here, we compare the X‐ray structure of HLA‐A1:MAGE‐A1 with that complexed with Fab‐Hyb3 to gain insight into structural changes of the MHC molecule that might be induced by the interaction with the antibody fragment. Apart from the expulsion of several water molecules from the interface, Fab‐Hyb3 binding results in major rearrangements (up to 5.5 Å) of heavy chain residues Arg65, Gln72, Arg145, and Lys146. Residue 65 is frequently and residues 72 and 146 are occasionally involved in TCR binding‐induced conformational changes, as revealed by a comparison with MHC class I structures in TCR‐liganded and ‐unliganded forms. On the other hand, residue 145 is subject to a reorientation following engagement of HLA‐Cw4 and KIR2DL1. Therefore, conformational changes within the HLA‐A1:MAGE‐A1:Fab‐Hyb3 complex include MHC residues that are also involved in reorientations in complexes with natural ligands, pointing to their central importance for the peptide‐dependent recognition of MHC molecules. 相似文献
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Stanley M. Jeram Tharan Srikumar Xiang‐Dong Zhang H. Anne Eisenhauer Richard Rogers Patrick G. A. Pedrioli Michael Matunis Brian Raught 《Proteomics》2010,10(2):254-265
Ubiquitin (Ub) and the ubiquitin‐like proteins (Ubls) comprise a remarkable assortment of polypeptides that are covalently conjugated to target proteins (or other biomolecules) to modulate their intracellular localization, half‐life, and/or activity. Identification of Ub/Ubl conjugation sites on a protein of interest can thus be extremely important for understanding how it is regulated. While MS has become a powerful tool for the study of many classes of PTMs, the identification of Ub/Ubl conjugation sites presents a number of unique challenges. Here, we present an improved Ub/Ubl conjugation site identification strategy, utilizing SUMmOn analysis and an additional protease (lysyl endopeptidase C), as a complement to standard approaches. As compared with standard trypsin proteolysis‐database search protocols alone, the addition of SUMmOn analysis can (i) identify Ubl conjugation sites that are not detected by standard database searching methods, (ii) better preserve Ub/Ubl conjugate identity, and (iii) increase the number of identifications of Ub/Ubl modifications in lysine‐rich protein regions. Using this methodology, we characterize for the first time a number of novel Ubl linkages and conjugation sites, including alternative yeast (K54) and mammalian small ubiquitin‐related modifier (SUMO) chain (SUMO‐2 K42, SUMO‐3 K41) assemblies, as well as previously unreported NEDD8 chain (K27, K33, and K54) topologies. 相似文献
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Proteomic analysis of host cell protein dynamics in the supernatant of Fc‐fusion protein‐producing CHO DG44 and DUKX‐B11 cell lines in batch and fed‐batch cultures 下载免费PDF全文
Jin Hyoung Park Jong Hwa Jin In Jung Ji Hyun Joo An Jong Won Kim Gyun Min Lee 《Biotechnology and bioengineering》2017,114(10):2267-2278
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Characterization of the co‐elution of host cell proteins with monoclonal antibodies during protein A purification 下载免费PDF全文
Qingchun Zhang Andrew M. Goetze Huanchun Cui Jenna Wylie Ben Tillotson Art Hewig Michael P. Hall Gregory C. Flynn 《Biotechnology progress》2016,32(3):708-717
Protein A chromatography is commonly used as the initial step for purifying monoclonal antibody biotherapeutics expressed in mammalian tissue culture cells. The purpose of this step, as well as later chromatography steps, is, in part, to remove host cell proteins (HCPs) and other related impurities. Understanding the retention mechanism for the subset of HCPs retained during this step is of great interest to monoclonal antibody (mAb) process developers because it allows formation of a guided HCP clearance strategy. However, only limited information is available about the specific HCPs that co‐purify with mAbs at this step. In this study, a comprehensive comparison of HCP subpopulations that associated with 15 different mAbs during protein A chromatography was conducted by a 2D‐LC‐HDMSE approach. We found that a majority of CHO HCPs binding to and eluting with the mAbs were common among the mAbs studied, with only a small percentage (~10% on average) of a mAb's total HCP content in the protein A (PrA) eluate specific for a particular antibody. The abundance of these HCPs in cell culture fluids and their ability to interact with mAbs were the two main factors determining their prevalence in protein A eluates. Potential binding segments for HCPs to associate with mAbs were also studied through their co‐purification with individual Fc and (Fab′)2 antibody fragments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:708–717, 2016 相似文献
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The dual effect of ultrasound‐targeted microbubble destruction in mediating recombinant adeno‐associated virus delivery in renal cell carcinoma: transfection enhancement and tumor inhibition 下载免费PDF全文
Fan Li Lifang Jin Huiping Wang Fang Wei Min Bai Qiusheng Shi Lianfang Du 《The journal of gene medicine》2014,16(1-2):28-39
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Evaluation of a novel methacrylate‐based protein a resin for the purification of immunoglobulins and Fc‐fusion proteins 下载免费PDF全文
Protein A affinity chromatography is a central part of most commercial monoclonal antibody and Fc‐fusion protein purification processes. In the last couple years an increasing number of new Protein A technologies have emerged. One of these new Protein A technologies consists of a novel, alkaline‐tolerant, Protein A ligand coupled to a macroporous polymethacrylate base matrix that has been optimized for immunoglobulin (Ig) G capture. The resin is interesting from a technology perspective because the particle size and pore distribution of the base beads are reported to have been optimized for high IgG binding and fast mass transfer, while the Protein A ligand has been engineered for enhanced alkaline tolerance. This resin was subjected to a number of technical studies including evaluating dynamic and static binding capacities, alkaline stability, Protein A leachate propensity, impurity clearance, and pressure–flow behavior. The results demonstrated similar static binding capacities as those achieved with industry standard agarose Protein A resins, but marginally lower dynamic binding capacities. Removal of impurities from the process stream, particularly host cell proteins, was molecule dependent, but in most instances matched the performance of the agarose resins. This resin was stable in 0.1 M NaOH for at least 100 h with little loss in binding capacity, with Protein A ligand leakage levels comparable to values for the agarose resins. Pressure–flow experiments in lab‐scale chromatography columns demonstrated minimal resin compression at typical manufacturing flow rates. Prediction of resin compression in manufacturing scale columns did not suggest any pressure limitations upon scale up. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1125–1136, 2014 相似文献
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Jennifer D. Stone Adam S. Chervin Hans Schreiber David M. Kranz 《Biotechnology progress》2012,28(6):1588-1597
To avoid high systemic doses, strategies involving antigen‐specific delivery of cytokine via linked antibodies or antibody fragments have been used. Targeting cancer‐associated peptides presented by major histocompatibility complex (MHC) molecules (pepMHC) increases the number of potential target antigens and takes advantage of cross‐presentation on tumor stroma and in draining lymph nodes. Here, we use a soluble, high‐affinity single‐chain T cell receptor Vα‐Vβ (scTv), to deliver cytokines to intracellular tumor‐associated antigens presented as pepMHC. As typical wild‐type T cell receptors (TCRs) exhibit low affinity (Kd = 1–100 μM or more), we used an engineered TCR, m33, that binds its antigenic peptide SIYRYYGL (SIY) bound to the murine class I major histocompatability complex protein H2‐Kb (SIY/Kb) with nanomolar affinity (Kd = 30 nM). We generated constructs consisting of m33 scTv fused to murine interleukin 2 (IL‐2), interleukin 15 (IL‐15), or IL‐15/IL‐15Rα (IL‐15 linked to IL‐15Rα sushi domain, called “superfusion”). The fusions were purified with good yields and bound specifically to SIY/Kb with high affinity. Proper cytokine folding and binding were confirmed, and the fusions were capable of stimulating proliferation of cytokine‐dependent cells, both when added directly and when presented in trans, bound to cells with the target pepMHC. The m33 superfusion was particularly potent and stable and represents a promising design for targeted antitumor immunomodulation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012 相似文献
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David J. Swan John A. Kirby Simi Ali 《Journal of cellular and molecular medicine》2010,14(9):2211-2222
The vascular and immune systems of mammals are closely intertwined: the individual components of the immune system must move between various body compartments to perform their function effectively. Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, exerts effects on the two organ systems and influences the interaction between them. In the resting state, the vascular S1P gradient contributes to control of lymphocyte recirculation through the blood, lymphoid tissue and lymphatic vasculature. The high level of S1P in blood helps maintain endothelial barrier integrity. During the inflammatory process, both the level of S1P in different immune compartments and S1P receptor expression on lymphocytes and endothelial cells are modified, resulting in functionally important changes in endothelial cell and lymphocyte behaviour. These include transient arrest of lymphocytes in secondary lymphoid tissue, crucial for generation of adaptive immunity, and subsequent promotion of lymphocyte recruitment to sites of inflammation. This review begins with an outline of the basic biochemistry of S1P. S1P receptor signalling is then discussed, followed by an exploration of the roles of S1P in the vascular and immune systems, with particular focus on the interface between them. The latter part concerns crosstalk between S1P and other signalling pathways, and concludes with a look at therapies targeting the S1P-S1P receptor axis. 相似文献
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PsHint1, associated with the G‐protein α subunit PsGPA1, is required for the chemotaxis and pathogenicity of Phytophthora sojae 下载免费PDF全文
Xin Zhang Chunhua Zhai Chenlei Hua Min Qiu Yujuan Hao Pingping Nie Wenwu Ye Yuanchao Wang 《Molecular Plant Pathology》2016,17(2):272-285
Zoospore chemotaxis to soybean isoflavones is essential in the early stages of infection by the oomycete pathogen Phytophthora sojae. Previously, we have identified a G‐protein α subunit encoded by PsGPA1 which regulates the chemotaxis and pathogenicity of P. sojae. In the present study, we used affinity purification to identify PsGPA1‐interacting proteins, including PsHint1, a histidine triad (HIT) domain‐containing protein orthologous to human HIT nucleotide‐binding protein 1 (HINT1). PsHint1 interacted with both the guanosine triphosphate (GTP)‐ and guanosine diphosphate (GDP)‐bound forms of PsGPA1. An analysis of the gene‐silenced transformants revealed that PsHint1 was involved in the chemotropic response of zoospores to the isoflavone daidzein. During interaction with a susceptible soybean cultivar, PsHint1‐silenced transformants displayed significantly reduced infectious hyphal extension and caused a strong cell death in plants. In addition, the transformants displayed defective cyst germination, forming abnormal germ tubes that were highly branched and exhibited apical swelling. These results suggest that PsHint1 not only regulates chemotaxis by interacting with PsGPA1, but also participates in a Gα‐independent pathway involved in the pathogenicity of P. sojae. 相似文献
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Michael Beisswenger Taku Yoshiya Yoshiaki Kiso Chiara Cabrele 《Journal of peptide science》2010,16(6):303-308
Synthetic peptides reproducing the helix‐loop‐helix (HLH) domains of the Id proteins fold into highly stable helix bundles upon self‐association. Recently, we have shown that the replacement of the dipeptide Val‐Ser at the loop–helix‐2 junction with the corresponding O‐acyl iso‐dipeptide leads to a completely unfolded state that only refolds after intramolecular O → N acyl migration. Herein, we report on an Id HLH analog based on the substitution of the Pro‐Ser motif at the helix‐1–loop junction with the corresponding O‐acyl iso‐dipeptide. This analog has been successfully synthesized by solid‐phase Fmoc chemistry upon suppression of DKP formation. No secondary structure could be detected for the O‐acyl iso‐peptide before its conversion into the native form by O → N acyl shift. These results show that the loop–helix junctions are determinant for the folded/unfolded state of the Id HLH domain. Further, despite the high risk of DKP formation, peptides containing O‐acyl iso‐Pro‐Ser/Thr units are synthetically accessible by Fmoc chemistry. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd. 相似文献