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Phorbol-induced surface expression of NR2A subunit homologues in hek293 cells

Acta Pharmacologica Sinica 2006年第12期27卷 1580-1585页

作者： Chan-ying ZHENG~2 Xiu-juan YANG~3 Zhan-yan FU~4 Jian-hong LUO~(2,5)2 Department of Neurobiology,Zhejiang University School of Medicine,Hangzhou 310031,China 3 Institute of Neuroscience,Shanghai Institutes for Biological Sciences,the Chinese Academy of Sciences,Shanghai 200031,China 4 Department of Physiology and Biophysics,Georgetown University School of Medicine Washington 20057,USA DepartmentofNeurobiology ZhejiangUniversitySchoolofMedicineHangzhou310031China InstituteofNeuroscience ShanghaiInstitutesforBiologicalSciencestheChineseAcademyofSciencesShanghai200031China

Aim:N-methyl-D-aspartate receptors(NMDAR)are heteromeric complexes pri-marily assembled from NR1 and NR2 *** normal conditions,NR2 sub-units assemble into homodimers in the endoplasmic reticulum(ER).Thesehomodimers remain in the ER until they coassemble with NR1 dimers and aretrafficked to the cell ***,it still remains unclear whether functionalhomomeric NMDAR exist in physiological or pathological ***:We transfected GFP-NR2A alone into hek293 cells,treated the cells with PKCactivator 12-myristate-13 acetate(PMA),and then detected surface NR2A sub-units with a live cell immunostaining *** also used a series of NR2Amutants with a partial deletion of its C-terminus to identify the regions that areinvolved in the PMA-mediated surface expression of NR2A ***:NR2A subunits were expressed on the cell membrane after incubation with PMA(200 nmol/L,30 min),although no functional NMDA channels were detected afterPMA-induced membrane *** with an ER marker alsorevealed that NR2A subunits were exported from the ER after PMA ***,the deletion of amino acids between 1149-1347 or 1354-1464 ofNR2A inhibited PMA-induced surface expression ofNR2A ***:First,our data suggests that PMA treatment can induce the surface expression ofhomomeric NR2A ***,this process is probably mediated bythe NR2A C-terminal region between positions 1149 and 1464.

关键词： NMDA receptor NR2A subunit PMA surface expression hek293 cells

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Inhibitory Effects of Neferine on Na_v1.5 Channels Expressed in hek293 cells

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2016年第4期36卷 487-493页

作者：王琛王换肖军花王嘉陵向继洲汤强 Department of Pharmacology School of Basic Medicine Tongji Medical College Huazhong University of Science and Technology Department of Pharmacology The Third Affiliated Hospital of Zhengzhou University

Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of neferine on Nav1.5 channels that are stably expressed in hek 293 cells. We found that neferine potently and reversibly inhibited Nav1.5 currents in a concentration dependent manner with a half-maximal inhibition（IC50） being 26.15 μmol/L. The inhibitory effects of neferine on Nav1.5 currents were weaker than those of quinidine at the same concentration. The steady-state inactivation curve was significantly shifted towards hyperpolarizing direction in the presence of 30 μmol/L neferine, while the voltage-dependent activation was unaltered. Neferine prolonged the time to peak of activation, increased the inactivation time constants of Nav1.5 currents and markedly slowed the recovery from inactivation. The inhibitory effect of neferine could be potentiated in a frequency-dependent manner. These results suggested that neferine can block Nav1.5 channels under the open state and inactivating state and it is an open channel blocker of Nav1.5 channels.

关键词： neferine Nay1.5 channel whole-cell patch-clamp hek293 cells

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Exposure to extremely low-frequency electromagnetic fields inhibits T-type calcium channels in hek293 cells

Exposure to extremely low-frequency electromagnetic fields i...

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中国神经科学学会第十届全国学术会议

作者： Yu-Jie CUI Chang-Long HU Institutes of Brain Science School of Life Sciences and State Key Laboratory of Medical NeurobiologyFudan University

The modulation of a variety of ion channel activities by extremely low-frequency electromagnetic fields（ELF-EMF） has been reported previously,few reports have researched the effects of ELF-EMF on the activity of voltage-activated T-type calcium *** this report,we investigated the effects of ELF-EMF on all three subtypes of T-type calcium channels transfected in hek293 *** results demonstrate that the exposure of hek293 cells to ELF-EMF for 60 min significantly decreased the currents of T-type calcium channels（31%）,time-dependent patterns have been observed *** phenomenon is very similar to the effect of intracellular application of arachidonic acid（AA） on currents of T-type calcium channels in hek293 *** increases in intracellular AA（15.4%） and extracellular Cysteinyl leukotrienes（31%） were observed following ELF-EMF *** blottings indicated that the total expression levels of T-type calcium channel protein were not affected after exposure to ELF-EMF.5-lipoxygenase inhibitors were able to ehminate this ELF-EMF-induced decrease in T-type calcium channel *** addition,perfusion of CysLTs significantly inhibited the currents of T-type calcium channels.

关键词： extremely low-frequency electromagnetic fields T-type calcium channel currents Cysteinyl leukotrienes hek293 cells

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Construction of pEYFP-hApelin-R and pRluc-hNTSRI-pcDNA3.1 eukaryotic expression vectors and their expression in hek293 cells

Construction of pEYFP-hApelin-R and pRluc-hNTSRI-pcDNA3.1 eu...

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中国神经科学学会第九届全国学术会议暨第五次会员代表大会

作者： Xin CAI~1,Jing CHEN~(2,3) Bo BAI~(2,*) 1 Taishan Medical University,Tai''an 271000,China 2 Jingning Medical University,Jining 272000,China 3 University of Warwick,England 999020,United Kingdom

＜正＞Objective To construct expression vectors that enhanced yellow fluorescent protein（EYFP）fused with Apelin receptor（Apelin-R）and Renilla reniformis（Rluc）fused with Neurotensin type 1 receptor（NTSR1）,to investigate the heterdimerization of human Apelin-R and NTSRl and its role in signal *** The human NTSRl gene was amplified by PCR using the plasmid pcDNA3.1-hNTSRl as *** PCR product was digested,ligased with the plasmid pRluc and then be transformed into the competent cell Top *** construct was identified by DNA *** recombinant plasmid was transiently transfected into human embryonic kidney 293（hek293）cells,and the expression of pRluc-hNTSRl-pcDNA3.1 was detected by confocal microscopy and Western ***-hApelin-R was also constructed by the same *** Two fragments of 1257 bp and 1143 bp were amplified by PCR,and the DNA sequences were identical with the gene in GenBank（NM002531, NM005161）,*** blot showed 91 kDa and 69 kDa *** microscopy showed that NTSRl and Apelin-R were expressed on the plasma *** The pRluc-hNTSRl-pcDNA3.1 and pEYFP-hApelin -R eukaryotic expression vectors were successfully constructed,and the expression vector could be used to investigate the heterdimerization of human Apelin-R and NTSRl and its role in signal transduction,which will provide new target for drug development.

关键词： Apelin receptor EYFP neurotensin type 1 receptor renilla reniformis eukaryotic expression vector hek293 cells

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Construction of recombinant plasmid pEGFP-C2-L539fs/47 and its expression in hek293 cells

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Journal of Medical Colleges of PLA(China) 2012年第3期27卷 125-133页

作者： Lue Ying Zhang Aifeng Han Wenqi Li Guoliang Zhang Junbo Gao Jie Pan Junqiang Zhang Yong Sun Chaofeng Department of Cardiovascular Diseases First Affiliated HospitalMedical SchoolXi'an Jiaotong UniversityXi'an 710061China

Objective:To reconstruct pEGFP-C2-L539fs/47,a HERG nonsense mutant in eukaryotic expression plasmid,and observe the fusion protein expressed in hek293 cells(human embryo kidney cells).Methods:After double digestion of pcDNA3-L539fs/47 and pEGFP-C2-HERG with sbf I and Eco91 I,the small product fragment,from pcDNA3-L539fs/47,was subcloned into the big fragment of pEGFP-C2-HERG under T4 ***-C2-L539fs/47 was identified by agarose gel electrophoresis and ***3-L539fs/47 and pEGFP-C2-L539fs/47 were transiently transfected into hek293 cells by Lipofect,*** expression of fusion protein in hek293 cells was detected through immunofluorescence,laser confocal imaging scanning in vivo,Western blot and ***:Mutation region cDNA fragment(about 1 kb) and target vector fragment(about 7.2 kb) were ligated after purification and gel *** gel electrophoresis and sequencing successfully demonstrated eukaryotic expression plasmid pEGFP-C2-L539fs/47,constructed approximately 8.2 kb,sequencing consistent with template *** transfection efficiency of recombinant plasmid by fluorescence microscopy was more than60%.Western blot analysis detected pcDNA3-L539fs/47 expression of the protein size 60 KD,the expression of pEGFP-C2 fusion protein size of approximately 90 *** L539fs/47 gene expression in hek293 cells was significant by PCR *** laser imaging showed that pEGFP-C2-L539fs/47 protein was successfully expressed in cytoplasm and cytomembrane of hek293 ***:pEGFP-C2-L539fs/47 containing the HERG gene mutant was successfully constructed by double digestion method and expressed fusion protein in hek293 cells,which laid a foundation for the further study on L539fs/47.

关键词： HERG gene Nonsense mutations Eukaryotic expression vector pEGFP hek293 cells

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Construction of an Expression Plasmid pEGFP-N1-boTLR2 for Full-length Bovine TLR2 and Its Expression in hek293 cells

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Agricultural Science & Technology 2012年第6期13卷 1194-1197页

作者：王玉明王静萱酒泉职业技术学院甘肃酒泉735000

[Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in hek293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and ligated into the pMD18-T simple vector and then subcloned into the pEGFP-N1 vector. A recombinant eukaryotic expression plasmid containing the full-length CDS region of bovine TLR2 was constructed and transiently transfected into hek293 cells. The transfection efficiency and the location of recombinant protein were examined by FCM and confocal microscopy. Then the bovine TLR2 mRNA expression in hek293/boTLR2 was detected by qRT-PCR. Finally, we analyzed the biological activity through the response that lipoteichoic acid stimulates hek293/boTLR2 cells. [Result] The full-length TLR2 gene was successfully cloned and ligated into eukaryotic expression vector. The recombinant expression vector expressed bovine TLR2 in hek293 cells. hek293/boTLR2 cells produced higher levels of IL-8 secretion than nontransfected hek293 cells when stimulated with LTA from Staphylococcus aureus. [Conclusion] The established cell model can provide a fast, flexible and convenient means for screening TLR agonists and antagonists, and may also be useful for investigating the interaction between TLR agonists and TLRs.

关键词： Toll-like receptor Bovine hek293 cells Lipoteichoic acid Interleukin-8

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Cytotoxicity assessment of a gold nanoparticle-chitosan nanocomposite as an efficient support for cell immobilization:comparison with chitosan hydrogel and chitosan-gelatin

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BIOCELL 2014年第1期38卷 11-16页

作者： Mohammad Reza RAMEZANI Hossein NADERI-MANESH Hossain-Ali RAFIEEPOUR Department of Biophysics Faculty of Biological SciencesTarbiat Modares UniversityTehranIran Department of Biotechnology Faculty of ChemistryUniversity of KashanKashanIran

Cell-based biosensors have become a research hotspot in the biosensors and bioelectronics fields. The main feature of cell-based biosensors is immobilization of living cells on the surface of transducers. Different types of polymers which are used as scaffolds for cell growth should be biocompatible and should have reac- tive functional groups for further attachment of biomolecules. In this work, cell attachment and proliferation on chitosan hydrogel, chitosan-gelatin and gold nanoparticle-chitosan nanocomposite membranes was stud- ied. Characterization of the membranes was performed using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Cytotoxicity assessment on hek293 cells was carried out for all mem- branes using the MTT assay. Cell morphology and viability were assessed to evaluate cell attachment and pro- liferation. Regarding cell studies, the findings revealed that none of the membranes induced cytotoxic effects. However, the data showed that gold nanoparticle nanocomposite membranes improved hek293 attachment and adhesion more than other membranes, indicating that it provides an effective surface for immobilizing cells for sensing applications.

关键词： gold nanoparticle-chitosan nanocomposite hek293 cells cytotoxic effects scanning electron microscopy

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Construction of recombinant human nerve growth factor beta adenovirus and evaluation of its function An in vitro and in vivo study

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Neural Regeneration Research 2010年第16期5卷 1261-1269页

作者： En-Feng Gao Si-Ho Choi Mi-Ae Sung Bo-Han Li Samir Jabaiti Sang Bae Yoo Sung-June Kim Soung-Min Kim Jeong Won Jahng Jong-Ho Lee Department of Stomatology China-Japan Friendship Hospital Beijing 100029 China Center for Stem Cell and Regenerative Medicine Keck School of Medicine University of Sourthern Californina USA Department of Oral and Maxillofacial Surgery & Dental Research Institute Seoul National University Seoul Korea Department of Plastic and Reconstructive Surgery Jordan University Hospital Amman Jordan Department of Electrical Engineering School of Electrical and Computer Science Seoul National University Seoul Korea

Exogenous delivery of nerve growth factor （NGF） promotes neural regeneration. However, the short half-life limits delivery efficacy. Therefore, a long-term, efficient, local delivery tool or scheme is needed. The purpose of this study was to construct a functioning, recombinant, adenoviral vector carrying human NGF-β （hNGF-β） DNA, and to measure expression of the constructed vector in vitro and in vivo. rhNGF-β adenoviral vector containing full-length hNGF-β cDNA was generated by homologous recombination in Escherichia CoIL The rhNGF-β adenovirus was packaged and amplified in human embryonic kidney hek293 cells. Transformation efficiency, expression and function of rhNGF-β adenovirus for primary Schwann cells, Schwann cell lines, human embryonic kidney hek 293 cells, CRH myoblasts, and NIH3T3 fibroblasts were evaluated. Subsequently, expression of rhNGF-β adenovirus at the peripheral nerve of rat was also assessed. Recombinant adenoviral vector carrying hNGF-β was successfully constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. Green fluorescent protein expression was observed in 90% of rhNGF-β adenovirus-infected cells （primary Schwann cells, Schwann cell line, human embryonic kidney hek 293 cells, CRH myoblasts, and NIH3T3 fibroblasts） compared with non-infected cells. Total mRNA isolated from rhNGF-β adenovirus-infected cells exhibited strong expression. Maximum NGF release was induced by primary cultured Schwann cells at 4 days after infection, which steadily continued for 14 days. PC-12 cells exposed to media conditioned with rhNGF-β adenovirus-infected Schwann cells exhibited increased neurite extension. In vivo experiment revealed that the injected rhNGF-β adenovirus was transfected into the cells at the injected site and promoted expression of NGF, p75NTR and brain derived neurotrophic factor at the sciatic nerve and dorsal root ganglia.

关键词： nerve growth factor Schwann cell peripheral nerve regeneration adenoviral vector hek293 cells myoblasts fibroblasts

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The Function of SeMNPV IAP3 in Mammalian cells

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Virologica Sinica 2008年第3期23卷 183-188页

作者： Li-na ZHU Chun-lei MEI Zhong-xin ZHANG State Key Laboratory of Virology Wuhan Institute of Virology Chinese Academy of Sciences Wuhan 430071China Graduate school Chinese Academy of Sciences Beijing 100039 China

The baculoviral inhibitors of apoptosis play a significant role in infectivity and viral host-range, which make them potential candidates for the engineering and improvement of baculovirus insecticidal. The iap3 gene of Spodoptera exigua nucleopolyhedrovirus （SeMNPV）, amplified by PCR, was 939 bp encoding IAP3. The PCR product was cloned into EcoR I/Barn H I of the plasmid pEGFP-C1. GFP was fused to the N-terminaus of IAP3 to study distribution in hek293. It was observed that the plasmid expressing IAP3 significantly inhibited apoptosis induced by cisplatin in hek293 cells. We conclude that the IAP3 of SeMNPV is functional in mammalian cells.

关键词： Apoptosis SeMNPV IAP3 hek293 cells Caspase inhibitor Cisplatin

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