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91.
Swelling and Ca2+-activated Anion Conductances in C127 Epithelial Cells Expressing WT and ΔF508-CFTR
CFTR is a chloride channel that is required for fluid secretion and salt absorption in many exocrine epithelia. Mutations
in CFTR cause cystic fibrosis. CFTR expression influences some ion channels, but the range of channels influenced, the mechanism
of the interaction and the significance for cystic fibrosis are not known. Possible interactions between CFTR and other ion
channels were studied in C127 mouse mammary epithelial cell lines stably transfected with CFTR, ΔF508-CFTR, or vector. Cell
lines were compared quantitatively using an 125I efflux assay and qualitatively using whole-cell patch-clamp recording. As expected, 125I efflux was significantly increased by forskolin only in the CFTR line, and forskolin-stimulated whole-cell currents were
time- and voltage independent. All three lines responded to hypotonic challenge with large 125I efflux responses of equivalent magnitude, and whole-cell currents were outwardly rectified and inactivated at positive voltages.
Unexpectedly, basal 125I efflux was significantly smaller in the ΔF508-CFTR cell line than in either the CFTR or control cell lines (P < 0.0001), and the magnitude of the efflux response to ionomycin was largest in the vector cell line and smallest in the
cell line expressing ΔF508-CFTR (P < 0.01). Whole-cell responses to ionomycin had a linear instantaneous I-V relation and activated at depolarizing voltages. Forskolin responses showed simple summation with responses to ionomycin
or hypotonic challenge. Thus, we found no evidence for interactions between CFTR and the channels responsible for swelling-mediated
responses. Differences were found in basal and ionomycin-stimulated efflux, but these may arise from variations in the clonally
selected cell lines that are unrelated to CFTR expression.
Received: 15 November 1995/Revised: 16 February 1996 相似文献
92.
Role of protein kinase A and the serine-rich region of herpes simplex virus type 1 ICP4 in viral replication. 总被引:8,自引:6,他引:2 下载免费PDF全文
Efficient expression of herpes simplex virus genes requires the synthesis of functional ICP4, a nuclear phosphoprotein that contains a prominent serine-rich region between amino acids 142 and 210. Residues in this region not only are potential sites for phosphorylation but also are involved in the functions of ICP4. By comparing the growth of a virus in which this region is deleted (d8-10) with wild-type virus (KOS) in PC12 cells or PC12 cells that are deficient in cyclic AMP-dependent protein kinase (PKA), two observations were made: (i) the growth of wild-type virus was impaired by 1 to 2 orders of magnitude in the PKA-deficient cells, indicating the involvement of PKA in the growth cycle of herpes simplex virus type 1, and (ii) while the growth of d8-10 was impaired by almost 2 orders of magnitude in wild-type cells, it was not further impaired (as was that of wild-type virus) in PKA-deficient cells, implicating the region deleted in d8-10 as a possible target for cellular PKA. In trigeminal'ganglia of mice, the d8-10 mutant virus grew poorly; however, it established latency in nearly 90% of ganglia tested. Studies of the phosphorylation of wild-type and d8-10 ICP4 proteins revealed that the serine-rich region is a major determinant for phosphorylation of ICP4 in vivo and that the phosphorylation state could change as a function of the PKA activity. Consistent with this observation, the serine-rich region of ICP4 was shown to be a target for PKA in vitro. While intact ICP4 was readily phosphorylated by ICP4 in vitro, the d8-10 mutant ICP4 was not. Moreover, a synthethic peptide representing a sequence in the serine tract that is predicted to be a substrate for PKA was phosphorylated by PKA in vitro, having a Km within the physiological range. These data suggest that PKA plays a role in viral growth through phosphorylation of one or more sites on the ICP4 molecule. 相似文献
93.
Inhibition of human and simian immunodeficiency virus protease function by targeting Vpx-protease-mutant fusion protein into viral particles. 总被引:4,自引:3,他引:1 下载免费PDF全文
The human immunodeficiency virus type I (HIV-1) Vpr and HIV-2 Vpx proteins package into virions through interactions with their cognate Gag polyprotein precursor. The targeting properties of Vpr and Vpx have been exploited to incorporate foreign proteins into virions by expression as heterologous fusion molecules (X. Wu, H.-M. Liu, H. Xiao, J. Kim, P. Seshaiah, G. Natsoulis, J. D. Boeke, B. H. Hahn, and J. C. Kappes, J. Virol. 69:3389-3398, 1995). To explore the possibility of utilizing Vpx and Vpr to target dominant negative mutants of the HIV Pol proteins into virions, we fused HIV-2 Vpx with an enzymatically defective protease (PR) mutant. Using a vector system to facilitate transient coexpression with HIV provirus, Vpx-PR-mutant (VpxPR(M)) fusion protein was expressed and packaged efficiently into HIV-2 and simian immunodeficiency virus virions. Immunoblot analysis of purified virions demonstrated that the packaging of VpxPR(M) interfered with the processing of the Gag and Gag/Pol precursor proteins, similar to that of a well-characterized active-site PR inhibitor. The incomplete processing of Gag and Gag/Pol was consistent with a 25-fold reduction in virion infectivity. The coexpression of a packaging defective VpxPR(M) fusion protein with HIV-2 provirus produced virions with fully processed Gag protein, similar to wild-type virions. Importantly, virions trans complemented with a Vpx-chloramphenicol acetyltransferase fusion protein were normal with respect to the processing of Gag protein and the ability to infect and replicate in vitro. These results indicate that VpxPR(M) specifically inhibited the function of the viral protease and provide for the first time proof of principle that the incorporation of foreign proteins into virions via fusion with Vpx can inhibit HIV replication. The use of accessory proteins as vehicles to deliver deleterious proteins to virions, including dominant negative mutants of Pol proteins, may provide new opportunities for application of gene therapy-based antiretroviral strategies. The ability to package PR by expression in trans, independent of the Gag/Pol precursor, also represents a novel approach that may be exploited to study the function of the Pol proteins. 相似文献
94.
95.
中国野豌豆属的分类研究 总被引:8,自引:0,他引:8
本文报道了国产野豌豆属43种,4变种及6变型,其中包括4个新种(多叶野豌豆,三尖野豌豆,武山野豌豆,长齿野豌豆);一个新变种(三叶歪头菜)及一个新等级(千山野豌豆)。 相似文献
96.
黄皮种子发育过程中脱水敏感性与细胞膜透性的关系 总被引:3,自引:0,他引:3
黄皮(Clausena lansium (Lour.) Skeels)胚轴与完整种子的发育模式以及发育中电解质渗漏率变化有些不同. 种子生理成熟前、后的胚轴对脱水的反应也不同,前者经轻微脱水可提高萌发率和活力指数,后者不耐任何程度的脱水.活力指数的急剧下降伴随着电解质渗漏率的迅速上升.实验表明,黄皮种子在发育过程中没有形成耐脱水性. 细胞膜透性变化可反映脱水对种子的伤害程度 相似文献
97.
可育的抗除草剂溴苯腈转基因小麦 总被引:21,自引:0,他引:21
报道了采用微粒轰击(Microprojectile bom bardm ent) 幼胚将除草剂抗性基因导入小麦(Triticumaestivum L.)的转化研究。实验共使用了13 个小麦品种, 从开花后14~18 d 的籽粒中剥取幼胚, 植物表达质粒含有CaMV 35S启动子控制的除草剂溴苯腈抗性基因bxn 以及筛选标记基因NTPⅡ。采用高压放电基因枪,用质粒DNA 包被的钨粒轰击预培养3 d 的幼胚。在含有卡那霉素类似物geneticin G418sulphate 的MS培养基上, 经过多步骤筛选和分化, 从800 多个幼胚中获得了16 株转化苗。除草剂抗性鉴定和Southern 杂交分析证明, 其中4 株为转基因植物,具有溴苯腈抗性, 并且自交可育。转化工作从分离幼胚到转化苗鉴定完毕, 最短时间为6 个月, 因此, 该方法是一项快速有效的基因导入技术 相似文献
98.
水稻幼芽细胞生物膜上的赤霉素结合蛋白的结合特性 总被引:1,自引:0,他引:1
在水稻 (Oryza sativa)幼芽中存在膜结合的赤霉素结合蛋白 ,其与 GA3 结合的平衡解离常数(Kd)为 6.5× 1 0 -8mol/ L,总浓度为 0 .3 pmol· mg-1 蛋白质。结合蛋白与 GA3 结合活力在 0℃时比 2 5℃时高 1 4 0 %。它与 GA3 结合的最适 p H为 5。 GA3 与此结合蛋白的结合量随反应时间延长而增加 ,1 h达最大值 ,以后又逐渐下降。 IAA、ABA可与 GA3 竞争赤霉素结合蛋白。 相似文献
99.
100.
Xianzhen Li Yunzhan Huang Degui Xu Dongping Xiao Fengxie Jin Peiji Gao 《Biotechnology letters》1996,18(2):205-210
Summary The cellobiose oxidizing enzyme of the newly isolated cellulolytic bacterium Cytophaga sp. LX-7 was produced extracellularly when grown on cellulose or other saccharides, which was previously noted only in fungi. The enzyme could use not only cellobiose, maltose, glucose and other saccharides but also cellulose as substrates, and use dichlorophenol indophenol and oxygen as electron acceptors. 相似文献