新型聚乙烯亚胺衍生物在COS-7细胞中转染和毒性的研究

目的：研究以乙二醛为连接剂的聚乙烯亚胺（Polyethyleneimine,PEI）衍生物Polyimine-PEI对非洲绿猴肾癌细胞COS-7的转染活性和细胞毒性的影响。方法：以荧光素酶质粒为报告基因,研究高分子与DNA的复合物在COS-7细胞的转染活性,用MTT方法研究高分子对COS-7细胞的毒性。结果：COS-7细胞实验显示,Polyimine-PEI具有很低细胞毒性,其毒性显著低于PEI25kDa,同时也具有高效输送质粒的能力。结论：Polyimine-PEI是一种新型的高效,低毒在基因治疗领域有相当前景的非病毒载体。     

Membrane stretch augments the cardiac muscarinic K+ channel activity

Arachidonic acid has been shown to activate K⁺-selective, mechanosensitive ion channels in cardiac, neuronal and smooth muscle cells. Since the cardiac G protein (G _K )-gated, muscarinic K⁺ (K_ACh) channel can also be activated by arachidonic acid, we investigated whether the K_ACh channel was also sensitive to membrane stretch. In the absence of acetylcholine (ACh), K_ACh channels were not active, and negative pressure failed to activate these channels. With ACh (10 m) in the pipette, applying negative pressure (0 to –80 mm Hg) to the membrane caused a reversible, pressure-dependent increase in channel activity in cell-attached and inside-out patches (100 m GTP in bath). Membrane stretch did not alter the sensitivity of the K_ACh channel to GTP. When G _K was maximally activated with 100 m GTPS in inside-out patches, the K_ACh channel activity could be further increased by negative pressure. Trypsin (0.5 mg/ ml) applied to the membrane caused activation of the K_ACh channel in the absence of ACh and GTP; K_ACh channel activity was further increased by stretch. These results indicate that the atrial muscarinic K⁺ channels are modulated by stretch independently of receptor/G protein, probably via a direct effect on the channel protein/ lipid bilayer.     

新型聚乙烯亚胺衍生物在COS-7 细胞中转染和毒性的研究

目的:研究以乙二醛为连接剂的聚乙烯亚胺(Polyethyleneimine,PEI)衍生物Polyimine-PEI对非洲绿猴肾癌细胞COS-7的转染活性和细胞毒性的影响。方法:以荧光素酶质粒为报告基因,研究高分子与DNA的复合物在COS-7细胞的转染活性,用MTT方法研究高分子对COS-7细胞的毒性。结果:COS-7细胞实验显示,Polyimine-PEI具有很低细胞毒性,其毒性显著低于PEI25kDa,同时也具有高效输送质粒的能力。结论:Polyimine-PEI是一种新型的高效,低毒在基因治疗领域有相当前景的非病毒载体。     

Efficient cellular uptake of recombinant murine Hoxc8 homeoprotein in COS-7 cells

In order to analyze the self-delivery activity of Hoxc8, recombinant Hoxc8 protein (rHoxc8) was designed to be expressed and purified in E. coli as a glutathione S-transferase and green fluorescent protein-fused form (GST-GFP-Hoxc8). After purification using glutathione sepharose beads, the 82 kDa fusion protein was separated on the SDS-PAGE gel and confirmed by detecting the fluorescence through luminescent image analyzer. When rHoxc8 was added to culture media for 30 h, most of the COS-7 cells contained the fusion proteins, showing green fluorescence under the fluorescent microscope. When the efficiency of cellular uptake was examined after Hoechst staining, almost 100% of the cells exhibited the GFP signal, revealing that rHoxc8 can traverse the cellular membrane of COS-7 cells efficiently, suggesting that the rHoxc8 could be applied in the development of efficient and useful delivery vectors for therapeutic molecules.     

pcDNA3.1（＋）-LYVE-1真核表达质粒的构建及其在COS-7细胞中的表达

目的 构建人淋巴管内皮细胞特异标志物LYVE-1融合基因表达质粒,观察其在COS-7细胞中的表达,为进一步探讨该标志物在肿瘤淋巴转移中的作用提供工具.方法 从本院结肠癌根治术患者术所取组织中的淋巴结抽提总RNA,RT-PCR扩增LYVE-1基因片段,并将其插入pMD19-T Simple Vector进行测序,鉴定正确后构建pcDNA3.1（＋）-LYVE-1并转染COS-7细胞,RT-PCR、Western印迹检测目的 蛋白表达,间接免疫荧光检测该基因表达在COS-7细胞上.结果 成功获取了人淋巴管内皮细胞特异标志物LYVE-1全长cDNA,构建了其真核表达载体pcDNA3.1（＋）-LYVE-1,转染COS-7细胞后检测出目的 蛋白的表达,并且证明该基因表达在细胞上.结论 成功构建了pcDNA3.1（＋）-LYVE-1重组质粒,为进一步研究LYVE-1在肿瘤淋巴管转移中的功能提供了重要的实验材料.     

Subcellular localization of MAK-V/Hunk protein kinase expressed in COS-1 cells

MAK-V/Hunk is a MARK/Par-1-related protein kinase, whose function is unknown. We studied the subcellular localization of MAK-V/Hunk in COS-1 cells by immunofluorescence. It has a nucleocytoplasmic distribution and is localized to the centrosome, as indicated by co-localization with gamma-tubulin. A putative kinase-deficient mutant, with a mutation in the invariant lysine residue in the catalytic domain, was not targeted to the nucleus or centrosome. These results suggest that the nuclear and centrosomal targeting of MAK-V/Hunk is specific, and is likely to be coupled to its catalytic activity.     

The M3 muscarinic receptor i3 domain confers oxidative stress protection on calcium regulation in transfected COS-7 cells

Evidence suggests that muscarinic receptors (MAChRs) are involved in various aspects of neuronal and vascular functioning, and that there is selective oxidative stress sensitivity (OSS) among MAChR subtypes. COS-7 cells transfected with M1, M2 and M4 subtypes show greater OSS than the M1 and M3 subtypes, as seen by the decreased ability of cells to extrude or sequester calcium (Ca(2+)) following exposure to dopamine (DA) or A beta 25-35, and depolarization by oxotremorine. We sought to determine which receptor domain may be responsible for the differential vulnerability to OS between 'OS-sensitive' (M1) and 'non-sensitive' (M3) subtypes. Comparison of the amino acid sequences of each receptor has shown that the third cytoplasmic loop (i3 loop) is the domain with the most variability between the two subtypes. Therefore, mutations were made by either deleting or exchanging the i3 loop of M1 and M3 receptors. Experiments revealed that deletions of the i3 loop increased DA sensitivity (a lower percentage of cells showing recovery of [Ca(2+)](i) following depolarization) in both receptors. Chimerics of M1 in which the i3 loop of the M3 was exchanged with the i3 loop of the M1 (M1M3i3) showed that DA sensitivity was reduced (a greater percentage of cells showing increases in calcium clearance) following depolarization. The M3 chimerics containing the M1 i3 loop (M3M1i3) offered no protection against DA-induced decrements in calcium buffering. Results suggest that the longer i3 loop of the M3 may decrease OSS, possibly playing a role in targeting antioxidants to specific receptor sites that impart OSS.     

Single-channel properties and pH sensitivity of two-pore domain K+ channels of the TALK family

The two-pore K2P channel family comprises TASK, TREK, TWIK, TRESK, TALK, and THIK subfamilies, and TALK-1, TALK-2, and TASK-2 are functional members of the TALK subfamily. Here we report for the first time the single-channel properties of TALK-2 and its pHo sensitivity, and compare them to those of TALK-1 and TASK-2. In transfected COS-7 cells, the three TALK K2P channels could be identified easily by their differences in single-channel conductance and gating kinetics. The single-channel conductances of TALK-1, TALK-2, and TASK-2 in symmetrical 150 mM KCl were 21, 33, and 70 pS (-60 mV), respectively. TALK-2 was sensitive mainly to the alkaline range (pH 7-10), whereas TALK-1 and TASK-2 were sensitive to a wider pHo range (6-10). The effect of pH changes was mainly on the opening frequency. Thus, members of the TALK family expressed in native tissues may be identified based on their single-channel kinetics and pHo sensitivity.     

Overexpression of oxidized protein hydrolase protects COS-7 cells from oxidative stress-induced inhibition of cell growth and survival

Oxidized protein hydrolase (OPH) preferentially degrades oxidatively damaged proteins in vitro and is widely distributed in various cells and tissues. The role of OPH in intact cells exposed to oxidative stress was examined. For this purpose, using COS-7, a cell line derived from African green monkey kidney, COS-7-OPH cells that stably overexpressed OPH were established. When COS-7-OPH cells were exposed to oxidative stress induced by H(2)O(2) and paraquat, accumulation of protein carbonyls in the cells was apparently lower than that of parental COS-7 cells, and COS-7-OPH cells were significantly resistant to the oxidative stress compared with parental COS-7 cells. The majority of overexpressed OPH in the cells was found to be located uniformly in cytosol, and its location was not altered by H(2)O(2)-induced oxidative stress. Above results indicate that OPH in intact cells plays a preventive role against oxidative stress and suggest that OPH relieves cells from accumulation of oxidatively damaged proteins.     

家蝇抗菌肽Defensin基因在COS-7细胞中的瞬时表达

目的:研究家蝇抗菌肽Defensin cDNA在非洲绿猴肾细胞株COS-7中的表达情况,并对表达产物的抗菌作用进行初步检测。方法:以Defensin基因为模板设计特异性引物,扩增在C端含6×His标签的Defensin开放阅读框序列,将此序列与真核表达载体pcDNA3.1( )进行重组,构建重组质粒pcDNA3.1( )/Defensin-His 6。以阳离子脂质体LipofectamineTM2000为载体,对宿主细胞COS-7进行重组质粒pcDNA3.1( )/Defensin-His 6和空载体pcDNA3.1( )的转染,72h后收集细胞培养上清液,表达产物经His-Trap HP亲合层析柱分离纯化和Western blotting鉴定后,进行杀菌活性的初步检测。结果:重组质粒pcDNA3.1( )/Defensin-His 6组细胞培养上清液的纯化物,行Western blotting得到了分子量大小约为10.0kD的单一目的条带,与预期相符;杀菌活性试验中发现:该纯化物对大肠杆菌E.coliK12D31具有一定的杀菌活性。结论:家蝇抗菌肽Defensin基因在宿主细胞COS-7中得到了正确表达。     

花生四烯酸对钾离子通道的调节作用

花生四烯酸是一个重要的炎症介质,它可以对广泛存在于各种细胞膜表面的外离子通道进行直接的或间接的调节。花生四烯酸与通道的直接作用,改变了通道蛋白的构象;直接学可与通道周围的细胞膜作用,影响钾离子通道的功能。花生四烯酸还可以通过其环氧酶和脂氧酶的代谢产物、蛋白激酶Ｃ（ＰＫＣ）及「Ｃａ＾２＋」间接影响了子通道。这些环节为药理学研究提供了新的可能的靶点。     

The role of tau phosphorylation in transfected COS-1 cells

Tau cDNAs from each of the six human isoforms were transfected into COS- 1 cells and, in every case, more than one peptide was observed. The diversity of expressed isoforms was due to different levels of tau phosphorylation. Tau phosphorylation results in a decrease of the protein electrophoretic mobility. The major contribution to this mobility shift is due to the phosphorylation at the at the C-terminus of the molecule, as inferred from the expression of tau fragments. Phosphorylation takes place in some of the sites modified in neural cells and in the basis of AD patients. Copolymerization studies indicate that the level of phosphorylation, as well as the localization of the modified residues, may affect the binding of the protein to microtubules. These results indicate that phosphorylation regulates tau function inside the cell.     

Coactivation of an endogenous progesterone receptor by TIF2 in COS-7 cells

Transfection experiments, a powerful tool to study the function of steroid hormone receptors and their coregulators, are often performed in COS-7 cells, because of high transfection efficiencies and expression levels. Here we report on the presence in COS-7 cells of an endogenous steroid hormone receptor, which is highly responsive to progesterone and the synthetic steroids R1881 and ORG2058, but not to 5 alpha-DHT. A 10-fold excess of the progesterone antagonist RU486 abolishes the stimulation by progesterone, while cotransfection with the coactivator TIF2 increases its activity 6- to 7-fold. A comparison of the ligand specificity with transfected androgen or progesterone receptors indicates that the endogenous receptor is a progesterone receptor. Its presence is confirmed by steroid-binding experiments, RT-PCR and Northern blot analysis. Consequently, progesterone receptor function may be studied conveniently in COS-7 cells without cotransfection of receptor, but the endogenous receptor may interfere in studies of ligand specificity and coactivation of cotransfected receptors.     

Ion selectivity of the Kat1 K+ channel pore

Kat1 is a highly selective inward-rectifying K⁺ channel that opens for extended periods under conditions of extreme hyperpolarization. Over 200 point mutants in the pore region of the Kat1 K⁺ channel were generated and examined in the yeast Saccharomyces cerevisiae and Xenopus oocytes to assess the effect of the mutations on ion selectivity. Substitutions at the tyrosine of the signature sequence G-Y-G resulted in the most significant alterations in ion selectivity, consistent with its role in the selectivity filter. However, greater than 80% of the mutations throughout the greater pore region also conferred a defect in selectivity demonstrating that the entire pore of Kat1 contributes to the ion selectivity of this channel. Surprisingly, we identified a novel class of mutant channel that conferred enhanced selectivity of K⁺ over Na⁺. Mutants of this class frequently displayed sensitivity to the competing ion Cs⁺. This finding has led us to speculate that the Kat1 channel pore has evolved to balance not only K⁺/Na⁺ selectivity, but selectivity over Cs⁺, and possibly a wide spectrum of potential competing ions.     

TASK (TWIK-related acid-sensitive K+ channel) is expressed in glomerulosa cells of rat adrenal cortex and inhibited by angiotensin II

The present study was conducted to explore the possible contribution of a recently described leak K+ channel, TASK (TWIK-related acid-sensitive K+ channel), to the high resting K+ conductance of adrenal glomerulosa cells. Northern blot analysis showed the strongest TASK message in adrenal glomerulosa (capsular) tissue among the examined tissues including heart and brain. Single-cell PCR demonstrated TASK expression in glomerulosa cells. In patch-clamp experiments performed on isolated glomerulosa cells the inward current at -100 mV in 30 mM [K+] (reflecting mainly potassium conductance) was pH sensitive (17+/-2% reduction when the pH changed from 7.4 to 6.7). In Xenopus oocytes injected with mRNA prepared from adrenal glomerulosa tissue the expressed K+ current at -100 mV was virtually insensitive to tetraethylammonium (3 mM) and 4-aminopyridine (3 mM). Ba2+ (300 microM) and Cs+ (3 mM) induced voltage-dependent block. Lidocaine (1 mM) and extracellular acidification from pH 7.5 to 6.7 inhibited the current (by 28% and 16%, respectively). This inhibitory profile is similar (although it is not identical) to that of TASK expressed by injecting its cRNA. In oocytes injected with adrenal glomerulosa mRNA, TASK antisense oligonucleotide reduced significantly the expression of K+ current at -100 mV, while the sense oligonucleotide failed to have inhibitory effect. Application of angiotensin II (10 nM) both in isolated glomerulosa cells and in oocytes injected with adrenal glomerulosa mRNA inhibited the K+ current at -100 mV. Similarly, in oocytes coexpressing TASK and ATla angiotensin II receptor, angiotensin II inhibited the TASK current. These data together indicate that TASK contributes to the generation of high resting potassium permeability of glomerulosa cells, and this background K+ channel may be a target of hormonal regulation.     

Chitosan lactate as a nonviral gene delivery vector in COS-1 cells

The purpose of this research was to evaluate chitosan lactate (CL) of different molecular weights (MWs) as a DNA complexing agent for its efficiency in transfecting COS-1 cells (green monkey fibroblasts) and its effect on cell viability compared with polyethylenimine (PEI), a commercially available cationic polymer. CL and chitosan base dissolved in dilute acetic acid (chitosan acetate, [CA]) of different MWs (20, 45, 200, 460 kDa) and N/P ratios (2∶1, 4∶1, 8∶1, 12∶1, 24∶1) formed complexes with pSV β-galactosidase plasmid DNA. The complexes were characterized by agarose gel electrophoresis and investigated for their ability to transfect COS-1 cells compared with PEI. Additionally, the effect of CL on the viability of COS-1 cells was investigated using 3-(4,5-dimethyliazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The binding of CL/DNA and CA/DNA was dependent on chitosan MWs. The N/P ratio of CL to completely form the complex with the DNA was higher than that of CA. Both CL and CA were comparable in transfection efficiencies at an N/P ratio of 12∶1, but less efficient than PEI (P<.05). The cell viability in the presence of CL and CA at all MWs was over 90%, whereas that of PEI-treated cells was ≈50%. These results suggest the advantage of CL for in vitro gene transfection, with the ease of preparation of polymer/DNA complexes and low cytotoxicity. Published: August 4, 2006     

大鼠促甲状腺激素释放激素受体1基因的克隆及其在COS-7细胞系中的表达

目的:克隆大鼠促甲状腺激素释放激素受体1(TRH-R1)基因,构建其真核表达载体,并检测该基因在非洲绿猴肾细胞系COS-7中的表达。方法:应用RT-PCR方法,以大鼠脑源RNA为模板,扩增获得TRH-R1基因,定向克隆到pDsRed2-N1中,以LipofectAMINE 2000试剂转染pDsRed2-N1-TRH-R1表达载体至COS-7细胞系中进行瞬时表达。结果:测序结果表明,从大鼠脑源总RNA中克隆到正确的TRH-R1基因全长编码序列;显微照相观察到所构建的TRH-R1表达载体质粒在COS-7细胞系中获得有效表达。结论:大鼠TRH-R1基因的克隆、真核表达载体的构建及在COS-7细胞系中表达获得成功,为进一步研究其功能奠定了基础。     

Temperature and pressure dependence of Shaker K+ channel N- and C-type inactivation

Shaker B potassium channels undergo rapid N-type and slow C-type inactivation. While N-type inactivation is supposed to be mediated by occlusion of the pore by the N-terminal protein structure, the molecular mechanisms leading to C-type inactivation are less well understood. Considering N-type inactivation as a model for a protein conformational transition, we investigated inactivation of heterologously expressed Shaker B potassium channels and mutants thereof, showing various degrees of C-type inactivation, under high hydrostatic (oil) pressure. In addition to the derived apparent activation and reaction volumes (ΔV), experiments at various temperatures yielded estimates for enthalpic (ΔH) and entropic (TΔS) contributions. N-type inactivation was accelerated by increasing temperature and slowed by high hydrostatic pressure yielding at equilibrium ΔH = 76 kJ/mole, TΔS = 82 kJ/mole, and ΔV = 0.18 nm³ indicating that the transition to the N-type inactivated state is accompanied by an increase in volume and a decrease in order. N-terminally deleted ShΔ6–46 constructs with additional mutations at either position 449 or 463 were used to investigate C-type inactivation. In particular at high temperatures, inactivation occurred in two phases indicating more than one process. At equilibrium the following values were estimated for the major inactivation component of mutant ShΔ6–46 T449A: ΔH = –64 kJ/mole, TΔS = –60 kJ/mole, and ΔV = –0.25 nm³, indicating that the C-type inactivated state occupies a smaller volume and is more ordered than the noninactivated state. Thus, hydrostatic pressure affects N- and C-type inactivation in opposite ways. Received: 17 May 1997 / Accepted: 18 July 1997     

N-linked glycosylation determines cell surface expression of two-pore-domain K channel TRESK

Within the first external loop of mouse and human TRESK subunits one or two N-glycosylation consensus sites were identified, respectively. Using site directed mutagenesis and Western immunoblotting a single residue of both orthologues was found to be glycosylated upon heterologous expression. Two-electrode voltage-clamp recordings from Xenopus oocytes revealed that current amplitudes of N-glycosylation mutants were reduced by 80% as compared to wildtype TRESK. To investigate membrane targeting, GFP-tagged TRESK subunits were expressed in Xenopus oocytes and fluorescence intensity at the cell surface was measured by confocal microscopy. Signals of the N-glycosylation mutants were reduced by >50%, indicating that their lower current amplitudes substantially result from inadequate surface expression of the channel.     

15-HETE suppresses K+ channel activity and inhibits apoptosis in pulmonary artery smooth muscle cells

15-Hydroxyeicosatetraenoic acid (15-HETE) is an important hypoxic product from arachidonic acid (AA) in the wall of pulmonary vessels. Although its effects on pulmonary artery constriction are well known, it remains unclear whether 15-HETE acts on the apoptotic responses in pulmonary artery smooth muscle cells (PASMCs) and whether K⁺ channels participate in this process. These hypothesises were validated by cell viability assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, mitochondrial potentials assay, caspase activity assay and western blot. We found that 15-HETE enhanced cell survival, suppressed the expression and activity of caspase-3, upregulated bcl-2 and attenuated mitochondrial depolarization, prevented chromatin condensation and partly reversed K⁺ channel opener-induced apoptosis in PASMCs under serum-deprived conditions. Our data indicated that 15-HETE inhibits the apoptosis in PASMCs through, at least in part, inactivating K⁺ channels. Yumei Li and Qian Li contributed equally to this work.