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Nunghathai Sawasdee Mutita Junking Piengpaga Ngaojanlar Nattakan Sukomon Thawornchai Limjindaporn Varaporn Akkarapatumwong Pa-thai Yenchitsomanus 《Biochemical and biophysical research communications》2010,401(1):85-91
Kidney anion exchanger 1 (kAE1) mediates chloride (Cl−) and bicarbonate (HCO3−) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl−/HCO3− exchange at the basolateral membrane and failure of proton (H+) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXØ motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells. 相似文献
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Liu N Zhang QY Vakharia D Dunbar D Kaminsky LS 《Archives of biochemistry and biophysics》2001,389(1):130-134
While fresh human hepatocyte cultures are widely used to model hepatic cytochrome P450 (CYP) regulation and activity, their CYP1A subfamily composition induced by, e.g., polycyclic aromatic hydrocarbons is ambiguous. CYP1A1, CYP1A2, or both have been reported to be expressed, and their varied roles in chemical carcinogenesis makes resolution of which CYPs are expressed essential. We have used an immunoblot system with Bis-Tris-HCl-buffered polyacrylamide gel, which clearly resolves human CYP1A1 and CYP1A2, and polyclonal goat anti-human CYP1A1/CYP1A2 and rabbit anti-human CYP1A2 antibodies to probe the expressed CYP1A1 and CYP1A2 composition of seven individual human hepatocyte cultures induced with 5 microM benzo[k]fluoranthene (BKF) for 24 h. In six of the cultures only CYP1A1 was detected, and in the seventh both CYPs were detected. In most vehicle-treated hepatocyte cultures, neither CYP1A1 nor CYP1A2 was detected. In three additional hepatocyte cultures treated individually with BKF and 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD), the resultant induced CYP1A1/1A2 profiles were essentially not influenced by the nature of the inducing agents. To develop an activity-based assay to differentiate between CYP1A1 and CYP1A2 expression in human hepatocytes, our previously published R warfarin assay (Drug Metab. Disp. (1995) 23, 1339-1345) was applied to TCDD (10 nM)-treated hepatocyte culture. The low concentration of TCDD did not produce inhibition of the warfarin metabolism-such inhibition could confound the results. Based on the ratios of 6- to 8-hydroxywarfarin formed in two cultures, the ratios of CYP1A1/CYP1A2 expressed in these cultures were determined and they agreed with the ratios determined by immunoblot analysis. Thus each individual human hepatocyte culture must be characterized for induced CYP1A1 and CYP1A2 expression in studies of CYP1A activity. The warfarin assay provides a means of characterizing the cultures. 相似文献
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核不均一核糖核蛋白(heterogeneous nuclear ribonucleoprotein,hnRNP)是一类多功能RNA结合蛋白家族,能与RNA聚合酶Ⅱ合成的新生转录本结合,并以复合体形式参与转录本稳定与成熟调控过程. hnRNP A1是hnRNPs家族重要成员,不仅广泛参与癌症与神经系统疾病相关基因的可变剪接调控,还在病毒侵染、细胞衰老及应激恢复中发挥重要作用.此外,hnRNP A1作为典型的RNA结合蛋白,在转录与可变剪接调控过程中,可通过动态三维结构识别特定序列.本文总结了hnRNP A1的最新研究进展,以期为进一步探究hnRNP A1在疾病发生中的功能研究提供参考. 相似文献
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Ragnar Österberg Per Elias Jörgen Kjems Rogert Bauer 《Journal of biomolecular structure & dynamics》2013,31(6):1111-1120
Abstract The complex formation between elongation factor Tu (EF-Tu), GTP, and valyl-tRNAVal 1A has been investigated in a hepes buffer of “pH” 7.4 and 0.2 M ionic strength using the small-angle neutron scattering method at concentrations of D2O where EF-Tu (42% D2O) and tRNA (71% D2O) are successively matched by the solvents. The results indicate that EF-Tu undergoes a conformational change and contracts as a result of the complex formation, since the radius of gyration decreases by 15% from 2.82 to 2.39 nm. tRNAVal 1A, on the other hand, seems to mainly retain its conformation within the complex, since the radii of gyration for the free (after correction for interparticular scattering) and complexed form are essentially the same. 2.38 and 2.47 nm, respectively. 相似文献
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We have cloned and sequenced a 2845 bp cDNA representing the 3'-end of either a new picorna-like virus species or genotype of Solenopsis invicta virus-1 (SINV-1). Analysis of the nucleotide sequence revealed 1 large open reading frame. The amino acid sequence of the translated open reading frame was most identical to structural proteins of SINV-1 (97%), followed by the Kashmir bee virus (KBV, 30%), and acute bee paralysis virus (ABPV, 29%). A PCR-based survey for SINV-1 and the new species or genotype (tentatively named S. invicta virus-1A, SINV-1A) using RNA extracts of S. invicta collected around Gainesville, Florida, revealed a mean colony infestation rate of 25% by SINV-1 and 55% by SINV-1A. Both SINV-1 and SINV-1A were found to co-infect 17.5% of the nests surveyed. Although the data preclude definitive species or genotype assignment, there is no doubt that SINV-1A is distinct from SINV-1, identifiable, and infects S. invicta. We provide a simple RT-PCR technique capable of discerning SINV-1 and SINV-1A infection of S. invicta. 相似文献
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We have established conditions in which soluble Al is toxic to the yeast Saccharomyces cerevisiae. The major modifications to a standard synthetic medium were lowering the pH and the concentration of Mg ions. Alterations to the PO4, Ca, or K concentration had little effect on toxicity. Organic acids known to chelate Al reduced its toxicity, suggesting that Al3+ is the toxic Al species. The unique ability of Mg ions to ameliorate Al toxicity led us to investigate the hypothesis that Al inhibits Mg uptake by yeast. Yeast cells accumulate Mg, Co, Zn, Ni, and Mn ions via the same transport system (G.F. Fuhrmann, A. Rothstein [1968] Biochim Biophys Acta 163: 325-330). Al3+ inhibited the accumulation of 57Co2+ by yeast cells more effectively than Ga, La, or Mg. In addition, a mutant yeast strain with a defect in divalent cation uptake proved to be more sensitive to Al than a wild-type strain. Taken together, these results suggest that Al may cause Mg deficiency in yeast by blocking Mg transport. We discuss the relevance of yeast as a model for the study of Al toxicity in plant systems. 相似文献
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一成骨不全家系的COL1A1基因突变检测 总被引:7,自引:0,他引:7
成骨不全(Osteogenesisimperfecta,OI)是一种由于Ⅰ型胶原形成障碍,导致骨脆性增强为主要症状的 常染色体显性遗传性疾病。临床上主要表现为骨质脆弱、蓝巩膜、耳聋和中等程度的关节畸形等症状。成骨不全 基因分别定位于17q21.31 q22和7q22.1,其致病基因分别为COL1A1和COL1A2。对一常染色体显性遗传的 成骨不全家系进行连锁分析,在COL1A1遗传位点发现紧密连锁(LOD=9.31;θ=.00)。突变检测发现在 COL1A1基因第26内含子5′端剪接位点处存在一由GT转换为AT的致病突变,该突变引起的异常剪接是导致成 骨不全的致病原因之一。 相似文献
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研究微重力对COL1A1(Ⅰ型胶原α1链基因)启动子活性的影响,探讨微重力对成骨细胞相关基因表达影响的作用机制.将长为3.6 kb COL1A1启动子双酶切,获得不同长度的启动子片段,并与报告基因EGFP(增强型绿色荧光蛋白)连接,转染ROS17/2.8细胞,用G418筛选,得到稳定转染COL1A1-EGFP基因的ROS17/2.8细胞株.利用回转器模拟微重力效应,体外培养条件下,观察各细胞株报告基因的表达情况.结果显示细胞在模拟微重力下培养24,48 h后,报告基因EGFP和Ⅰ型胶原的表达升高,表明COL1A1启动子活性增强.说明短期模拟微重力条件下,成骨细胞能通过增强COL1A1启动子活性,代偿性提高Ⅰ型胶原的表达. 相似文献
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拟南芥CK1A基因功能初步研究 总被引:3,自引:0,他引:3
利用RT-PCR方法从拟南芥中分离了1个CK基因家族成员CK1A, 该基因的ORF全长2 112 bp, 编码一条703个氨基酸残基的多肽。构建了CK1A基因的植物表达载体35S: GFP: CK1A, 采用基因枪法进行的洋葱表皮细胞GFP瞬时表达实验表明, 荧光信号主要分布在细胞核上, 显示CK1A基因的产物可能在细胞核上发挥作用。半定量RT-PCR分析表明: CK1A基因在花中表达量最大, 其次是茎和根, 在叶和叶柄中表达量较弱。蓝光使CK1A基因的表达升高, 12 h时表达量明显增加, 24 h时表达量下降。酵母双杂交结果显示CK1A蛋白在蓝光下能与CRY2蛋白发生相互作用, 暗示CK1A基因可能参与拟南芥的蓝光信号传导途径。 相似文献
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Jan de Vries Filipa L. Sousa Bettina B?lter Jürgen Soll Sven B. Gould 《The Plant cell》2015,27(7):1827-1833
A pivotal step in the transformation of an endosymbiotic cyanobacterium to a plastid some 1.5 billion years ago was the evolution of a protein import apparatus, the TOC/TIC machinery, in the common ancestor of Archaeplastida. Recently, a putative new TIC member was identified in Arabidopsis thaliana: TIC214. This finding is remarkable for a number of reasons: (1) TIC214 is encoded by ycf1, so it would be the first plastid-encoded protein of this apparatus; (2) ycf1 is unique to the green lineage (Chloroplastida) but entirely lacking in glaucophytes (Glaucophyta) and the red lineage (Rhodophyta) of the Archaeplastida; (3) ycf1 has been shown to be one of the few indispensable plastid genes (aside from the ribosomal machinery), yet it is missing in the grasses; and (4) 30 years of previous TOC/TIC research missed it. These observations prompted us to survey the evolution of ycf1. We found that ycf1 is not only lacking in grasses and some parasitic plants, but also for instance in cranberry (Ericaceae). The encoded YCF proteins are highly variable, both in sequence length and in the predicted number of N-terminal transmembrane domains. The evolution of the TOC/TIC machinery in the green lineage experienced specific modifications, but our analysis does not support YCF1 to be a general green TIC. It remains to be explained how the apparent complete loss of YCF1 can be tolerated by some embryophytes and whether what is observed for YCF1 function in a member of the Brassicaceae is also true for, e.g., algal and noncanonical YCF1 homologs.Some of the best evidence we have for the monophyly of plastids is how they import proteins (McFadden and van Dooren, 2004; Zimorski et al., 2014). Establishing a machinery that translocates proteins across the two membranes separating the organelle’s stroma from the cytosol was crucial for the transition of the endosymbiotic diazotrophic-like cyanobacterium to a plastid in the heterotrophic host (Cavalier-Smith, 2000; McFadden and van Dooren, 2004; Dagan et al., 2013). It was also a prerequisite for the successful transfer of genetic material from the endosymbiont to the host nucleus through endosymbiotic gene transfer (EGT; Martin et al., 1993). Most of the EGT that stripped the plastid genome of its coding capacity occurred in the common ancestor from which the three archaeplastidal lineages—Glaucophyta, Rhodophyta (red lineage), and Chloroplastida (green lineage)—evolved (Martin et al., 1998; Zimorski et al., 2014).Two main protein complexes mediate plastid protein import: the translocon on the outer envelope of chloroplasts (TOC) and the translocon on the inner envelope of chloroplasts (TIC). In accordance with the majority of EGT events, the main TOC/TIC components evolved in the common ancestor of Archaeplastida (Timmis et al., 2004; Kalanon and McFadden, 2008). Most proteins of the TIC complex are of cyanobacterial origin. However, all previously identified TOC and TIC components are encoded on nuclear DNA (Gould et al., 2008; Kalanon and McFadden, 2008; Shi and Theg, 2013), and, thus far, only TIC40 is unique to the Chloroplastida.Recently, Kikuchi et al. (2013) found the plastid-encoded YCF1 to be part of a 1-MD membrane protein complex that included TIC20. This complex contained neither TIC40 nor TIC110 components that were previously identified (Kessler and Blobel, 1996; Chou et al., 2003), but three novel ones, including YCF1 (Kikuchi et al., 2013). YCF1 was the first plastid-encoded protein identified whose presence was shown to be essential for the survival of Chlamydomonas reinhardtii and tobacco (Nicotiana tabacum), but whose function remained elusive (Boudreau et al., 1997; Drescher et al., 2000). YCF1—or in accordance with its newly proposed function, TIC214—would represent the first plastid-encoded protein of the TIC complex. It would also add to the set of TIC components (including TIC40 and a full-length TIC62) that is unique to the green lineage. In the course of evolution, organelles have lost or transferred 1000s of genes to the host nucleus, but they hardly ever gained any. That alone is remarkable and the apparent lack of ycf1 in Glaucophytes and Rhodophytes (whose import apparatus evolved from the same ancestral TOC/TIC as that of Chloroplastida) prompted us to inspect the distribution and sequence diversity of ycf1 in more detail. 相似文献
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膜联蛋白A1(Annexin A1,ANXA1,lipocortin I)是膜联蛋白超家族中的一员,它参与细胞信号转导、分化及凋亡等多种重要的生命过程.近年来的研究表明其表达水平在不同肿瘤组织中有差异,在同一肿瘤的不同类型中有显著变化,可能与肿瘤的侵袭转移相关,如乳腺癌、胃肠癌、肝癌、头颈部肿瘤、前列腺癌等.本文结合ANXA1的基本结构及生物学特性对以上研究的进展作一综述. 相似文献