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A novel L-fucose-binding lectin from Fenneropenaeus indicus induced cytotoxicity in breast cancer cells 一种新型的L-焦点结合凝集素来自于indicus Fenneropenaeus诱导乳腺癌细胞毒性
Pub Date : 2017-01-01 DOI: 10.1093/jb/mvw057
Biji Chatterjee, K. Ghosh, N. Yadav, Santosh R. Kanade
Lectins are omnipresent in almost all life forms, being the proteins which specifically bind to carbohydrate moieties on the cell surface; they have been explored for their anti-tumour activities. In this study, we purified a fucose specific-lectin (IFL) from Fenneropenaeus indicus haemolymph using fucose-affinity column and characterized for its haemagglutination activity, carbohydrate specificity, dependency on cations and cytotoxicity against cancer cells. The lectin showed non-specificity against human erythrocytes. It was a Ca2+-dependent lectin which remained stable over wide pH and temperature ranges. The lectin showed effective dose dependent cytotoxicity against different human cancer cell lines and induced apoptosis in MCF-7 cells as evidenced by DNA ladder assay and PARP cleavage in a dose dependent manner. Moreover, an increased p21 level corresponding to cyclin D downregulation in response to IFL treatment was observed which might work as probable factors to inhibit cell growth and induce apoptosis of MCF-7 cells. Therefore, we report a novel lectin from the prawn haemolymph with high specificity for L-fucose and antiproliferative towards human cancer cells. However, further establishment of the modus operandi of this lectin is required to enable its biotechnological applications.
凝集素在几乎所有的生命形式中都无处不在,是一种与细胞表面的碳水化合物部分特异性结合的蛋白质;人们已经探索了它们的抗肿瘤活性。在这项研究中,我们利用聚焦亲和柱从indicus Fenneropenaeus血淋巴中纯化了一种聚焦特异性凝集素(IFL),并对其血液凝集活性、碳水化合物特异性、阳离子依赖性和对癌细胞的细胞毒性进行了表征。凝集素对人红细胞无特异性。它是一种Ca2+依赖性凝集素,在较宽的pH和温度范围内保持稳定。DNA阶梯实验和PARP切割实验表明,凝集素对不同的人癌细胞具有剂量依赖性的细胞毒性,并能诱导MCF-7细胞凋亡。此外,在IFL处理下,p21水平升高与cyclin D下调相对应,这可能是抑制MCF-7细胞生长和诱导细胞凋亡的可能因素。因此,我们报道了从对虾血淋巴中提取的一种新的凝集素,这种凝集素具有高特异性的L-聚焦和对人类癌细胞的抗增殖作用。然而,需要进一步建立这种凝集素的操作方法,使其能够在生物技术上应用。
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引用次数: 8
Wnt-expressing cells in the intestines: guides for tissue remodeling 肠道中表达wnt的细胞:组织重塑的指南
Pub Date : 2017-01-01 DOI: 10.1093/jb/mvw070
H. Miyoshi
The crypt is a minimal functional unit in the intestinal epithelium. This unique structure is maintained by surrounding mesenchymal cells that focally interact with associated epithelial cells. Canonical and non-canonical Wnt ligands enable specific microenvironments localized to each end of the crypt major axis. While canonical Wnt-expressing cells are localized near the crypt bottom where intestinal stem cells reside, non-canonical Wnt-expressing cells are positioned beneath the luminal surface of epithelial cells. During wound healing, propagation and appropriate relocation of each cell population are thought to ensure subsequent crypt regeneration. In this review, I integrate information from recent studies on Wnt-expressing cells and intestinal fibroblast lineages and discuss their roles in homeostasis and wound healing. More information on the lineages of Wnt-expressing cells will help clarify the mechanisms of epithelial tissue formation.
隐窝是肠上皮中最小的功能单位。这种独特的结构是由周围的间充质细胞维持的,它们局部地与相关的上皮细胞相互作用。规范和非规范Wnt配体使特定的微环境定位于隐窝长轴的每一端。典型表达wnt的细胞位于肠干细胞所在的隐窝底部附近,而非典型表达wnt的细胞位于上皮细胞的管腔表面以下。在伤口愈合过程中,每个细胞群的繁殖和适当的迁移被认为是确保后续隐窝再生的关键。在这篇综述中,我整合了最近关于wnt表达细胞和肠成纤维细胞谱系的研究信息,并讨论了它们在体内平衡和伤口愈合中的作用。更多关于wnt表达细胞谱系的信息将有助于阐明上皮组织形成的机制。
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引用次数: 17
Context-dependent regulation of the &bgr;-catenin transcriptional complex supports diverse functions of Wnt/&bgr;-catenin signaling &bgr;-catenin转录复合体的环境依赖性调控支持Wnt/&bgr;-catenin信号传导的多种功能
Pub Date : 2017-01-01 DOI: 10.1093/jb/mvw072
Takamasa Masuda, T. Ishitani
Wnt/&bgr;-catenin signaling is activated repeatedly during an animal’s lifespan, and it controls gene expression through its essential nuclear effector, &bgr;-catenin, to regulate embryogenesis, organogenesis, and adult homeostasis. Although the &bgr;-catenin transcriptional complex has the ability to induce the expression of many genes to exert its diverse roles, it chooses and transactivates a specific gene set from among its numerous target genes depending on the context. For example, the &bgr;-catenin transcriptional complex stimulates the expression of cell cycle-related genes and consequent cell proliferation in neural progenitor cells, while it promotes the expression of neural differentiation-related genes in differentiating neurons. Recent studies using animal and cell culture models have gradually improved our understanding of the molecular basis underlying such context-dependent actions of the &bgr;-catenin transcriptional complex. Here, we describe eight mechanisms that support &bgr;-catenin-mediated context-dependent gene regulation, and their spatio-temporal regulation during vertebrate development. In addition, we discuss their contribution to the diverse functions of Wnt/&bgr;-catenin signaling.
Wnt/ -catenin信号在动物的一生中被反复激活,它通过其基本的核效应物-catenin控制基因表达,调节胚胎发生、器官发生和成体体内平衡。虽然&bgr;-catenin转录复合体有能力诱导许多基因的表达来发挥其不同的作用,但它会根据环境从众多靶基因中选择并激活特定的一组基因。例如,&bgr;-catenin转录复合体在神经祖细胞中刺激细胞周期相关基因的表达和细胞增殖,同时在分化神经元中促进神经分化相关基因的表达。最近使用动物和细胞培养模型的研究逐渐提高了我们对&bgr;-catenin转录复合物的上下文依赖作用的分子基础的理解。在这里,我们描述了支持&bgr;-catenin介导的环境依赖基因调控的八种机制,以及它们在脊椎动物发育过程中的时空调控。此外,我们还讨论了它们对Wnt/&bgr;-catenin信号传导的各种功能的贡献。
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引用次数: 28
The Y54(L)W mutation of anti-leukotriene C4 single-chain antibody increases affinity to leukotriene E4 抗白三烯C4单链抗体Y54(L)W突变增加了对白三烯E4的亲和力
Pub Date : 2017-01-01 DOI: 10.1093/jb/mvw055
Y. Kawakami, Mai Kinoshita, Y. Mori, Shuji Okochi, Shiori Hirano, Ichika Shimoda, Keita Kanzaki, T. Suzuki-Yamamoto, M. Kimoto, M. Sugahara, T. Hori, H. Saino, M. Miyano, Shozo Yamamoto, Yoshitaka Takahashi
The X-ray crystal structure of an anti-leukotriene (LT) C4 monoclonal antibody (mAbLTC) in complex with LTC4 was determined, however, crystallographic studies alone are not enough to fully understand the structures of the antigen-binding site. To elucidate the individual contribution of Tyr-54 and Asn-58 in the light chain of mAbLTC, both of which formed a hydrogen bond with glutamic acid of LTC4, we examined whether substitution of the residues affects the antigen binding affinity and specificity using an anti-LTC4 single chain variable fragment (scFvLTC). Among the Tyr-54(L) mutants, Y54(L)W showed a dramatic increase in the affinity to LTE4 which was comparable to that to LTD4. Essentially the same results were obtained using the Y54(L)W mutant expressed in Escherichia coli and Pichia pastoris. The structural modeling suggested the formation of a novel hydrogen bond between the substituted tryptophan in the antibody and the cysteine residue in LTE4. The affinity of Y54(L)R, Y54(L)E and Y54(L)L to LTC4 was markedly reduced, whereas other tested Tyr-54(L) mutants as well as Asn-58(L) mutants did not show significant change in LT binding. The results may provide an insight into the molecular basis of specific LT recognition by the antibody.
测定了一种与LTC4复合物的抗白三烯(LT) C4单克隆抗体(mAbLTC)的x射线晶体结构,但单靠晶体学研究不足以完全了解抗原结合位点的结构。为了阐明tyrr -54和Asn-58在与LTC4的谷氨酸形成氢键的mAbLTC轻链中的单独贡献,我们使用抗LTC4单链可变片段(scFvLTC)检测了残基的取代是否影响抗原结合亲和力和特异性。在Tyr-54(L)突变体中,Y54(L)W对LTE4的亲和力显著增加,与LTD4的亲和力相当。在大肠杆菌和毕赤酵母中表达的Y54(L)W突变体获得了基本相同的结果。结构模型表明在抗体中的取代色氨酸和LTE4中的半胱氨酸残基之间形成了一个新的氢键。Y54(L)R、Y54(L)E和Y54(L)L对LTC4的亲和力明显降低,而其他被试的Tyr-54(L)突变体和Asn-58(L)突变体对LT的结合没有明显变化。结果可能提供了一个深入了解特异性LT识别抗体的分子基础。
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引用次数: 1
EPR studies of wild type and mutant Dre2 identify essential [2Fe–-2S] and [4Fe–-4S] clusters and their cysteine ligands 野生型和突变型Dre2的EPR研究鉴定了必需的[2Fe—2S]和[4Fe—4S]簇及其半胱氨酸配体
Pub Date : 2017-01-01 DOI: 10.1093/jb/mvw054
Yan Zhang, Chunyu Yang, A. Dancis, Eiko Nakamaru-Ogiso
Yeast Dre2 (anamorsin or CIAPIN1) is an essential component for cytosolic Fe/S cluster biosynthesis. The C-terminal domain contains eight evolutionarily conserved cysteine residues, and we previously demonstrated that the yeast Dre2 overexpressed in Escherichia coli contains one binuclear ([2Fe–2S]) cluster and one tetranuclear ([4Fe–4S]) cluster. In this study, we replaced each conserved cysteine with alanine and analyzed the effects by Electron Paramagnetic Resonance. Although the C311A mutant lacked both signals, our data clearly suggest that the [2Fe–2S] cluster is ligated to Cys252, Cys263, Cys266 and Cys268, whereas the [4Fe–4S] cluster is ligated to Cys311, Cys314, Cys322 and Cys325. By simulation analysis of the C263A and C322A data, we obtained the g-values for the [4Fe–4S] cluster (gx,y,z = 1.830, 1.947 and 2.018) and for the [2Fe–2S] cluster (gx,y,z =1.919, 1.962 and 2.001). We also observed spin–spin interaction between the two clusters, suggesting their close proximity. Chemically reconstituted Dre2 showed air sensitivity of the [4Fe–4S] cluster converting to a [2Fe–2S] cluster. Furthermore, using a yeast shuffle strain, we demonstrated for the first time that each of the Cys Fe–S cluster ligands with the exception of C252 is essential, indicating that both Dre2 clusters are needed for cell viability.
酵母Dre2 (anamorsin或CIAPIN1)是胞质铁/硫簇生物合成的重要成分。c端结构域包含8个进化上保守的半胱氨酸残基,我们之前已经证明在大肠杆菌中过表达的酵母Dre2包含一个双核([2Fe-2S])簇和一个四核([4Fe-4S])簇。在本研究中,我们用丙氨酸代替了每一个保守的半胱氨酸,并通过电子顺磁共振分析了效果。虽然C311A突变体缺乏这两个信号,但我们的数据清楚地表明,[2Fe-2S]簇与Cys252、Cys263、Cys266和Cys268相连,而[4Fe-4S]簇与Cys311、Cys314、Cys322和Cys325相连。通过对C263A和C322A数据的模拟分析,我们得到了[4Fe-4S]簇的g值(gx,y,z = 1.830, 1.947和2.018)和[2Fe-2S]簇的g值(gx,y,z =1.919, 1.962和2.001)。我们还观察到两个团簇之间的自旋-自旋相互作用,表明它们的距离很近。化学重组的Dre2显示出[4Fe-4S]团簇转化为[2Fe-2S]团簇的空气敏感性。此外,利用酵母shuffle菌株,我们首次证明了除了C252之外,每个Cys Fe-S簇配体都是必需的,这表明两个Dre2簇都是细胞生存所必需的。
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引用次数: 18
Structural basis of cucumisin protease activity regulation by its propeptide 黄瓜蛋白酶前肽调控其活性的结构基础
Pub Date : 2017-01-01 DOI: 10.1093/jb/mvw053
Ami Sotokawauchi, M. Kato‐Murayama, K. Murayama, T. Hosaka, I. Maeda, M. Onjo, N. Ohsawa, D. Kato, K. Arima, M. Shirouzu
Cucumisin [EC 3.4.21.25], a subtilisin-like serine endopeptidase, was isolated from melon fruit, Cucumis melo L. Mature cucumisin (67 kDa, 621 residues) is produced by removal of the propeptide (10 kDa, 88 residues) from the cucumisin precursor by subsequence processing. It is reported that cucumisin is inhibited by its own propeptide. The crystal structure of mature cucumisin is reported to be composed of three domains: the subtilisin-like catalytic domain, the protease-associated domain and the C-terminal fibronectin-III-like domain. In this study, the crystal structure of the mature cucumisin•propeptide complex was determined by the molecular replacement method and refined at 1.95 Å resolution. In this complex, the propeptide had a domain of the &agr;–&bgr; sandwich motif with four-stranded antiparallel &bgr;-sheets, two helices and a strand of the C-terminal region. The &bgr;-sheets of the propeptide bind to two parallel surface helices of cucumisin through hydrophobic interaction and 27 hydrogen bonds. The C-terminus of the propeptide binds to the cleft of the active site as peptide substrates. The inhibitory assay suggested that the C-terminal seven residues of the propeptide do not inhibit the cucumisin activity. The crystal structure of the cucumisin•propeptide complex revealed the regulation mechanism of cucumisin activity.
黄瓜素[EC 3.4.21.25]是从甜瓜果实中分离得到的一种类似枯草杆菌素的丝氨酸内肽酶。通过后续加工,将黄瓜素前体的前肽(10 kDa, 88个残基)去除,得到成熟的黄瓜素(67 kDa, 621个残基)。据报道,黄瓜素被其自身的前肽所抑制。成熟黄瓜素的晶体结构由三个结构域组成:枯草菌素样催化结构域、蛋白酶相关结构域和c端纤维连接蛋白- iii样结构域。本研究采用分子置换法测定成熟黄瓜素•前肽配合物的晶体结构,并以1.95 Å分辨率进行细化。在这个复合物中,前肽具有&agr; -&bgr;三明治图案,四股反平行片,两个螺旋和一股c端区域。前肽的&bgr;-片通过疏水相互作用和27个氢键与黄瓜素的两个平行表面螺旋结合。前肽的c端作为肽底物与活性位点的间隙结合。抑制实验表明,前肽c端7残基对黄瓜素活性无抑制作用。黄瓜素•前肽复合物的晶体结构揭示了黄瓜素活性的调控机制。
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引用次数: 12
Differences in the secretion and transport of Wnt proteins Wnt蛋白分泌和转运的差异
Pub Date : 2017-01-01 DOI: 10.1093/jb/mvw071
S. Takada, S. Fujimori, Takuma Shinozuka, R. Takada, Yusuke Mii
During the last three decades, our understanding about Wnt signaling has progressed greatly, especially with regards to the molecular mechanism of intracellular transmission of this signaling, as well as its physiological roles. In parallel, the molecular nature of Wnt proteins has gradually but surely been clarified. Wnt proteins are post-translationaly modified with fatty acid and glycosaminoglycans, resulting in constraint of the 3D structure and behavior of the proteins. Specific binding proteins or extracellular vesicles, which appear to shield the lipid moiety from the aquatic environment, enable Wnt proteins to be transported in the extracellular space. Equally, Wnt-interacting proteins in the extracellular space, including heparan sulfate proteoglycan, are also involved in its spreading. Recent studies also show that intercellular transmission of Wnt proteins occurs by cell migration and extension of cell protrusions. Here, we will show the molecular and cellular bases of the trafficking of Wnt proteins and discuss questions that remain to be answered.
在过去的三十年中,我们对Wnt信号传导的理解有了很大的进展,特别是关于该信号在细胞内传递的分子机制及其生理作用。与此同时,Wnt蛋白的分子性质也逐渐明确了。Wnt蛋白在翻译后被脂肪酸和糖胺聚糖修饰,从而限制了蛋白质的三维结构和行为。特异性结合蛋白或细胞外囊泡似乎可以保护脂质部分不受水生环境的影响,使Wnt蛋白能够在细胞外空间运输。同样,细胞外空间的wnt相互作用蛋白,包括硫酸肝素蛋白聚糖,也参与了其扩散。最近的研究也表明,Wnt蛋白的细胞间传递是通过细胞迁移和细胞突起的延伸发生的。在这里,我们将展示Wnt蛋白运输的分子和细胞基础,并讨论仍有待回答的问题。
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引用次数: 36
Molecular characterization of tomato &agr;1,3/4-fucosidase, a member of glycosyl hydrolase family 29 involved in the degradation of plant complex type N-glycans 番茄&agr 1,3/4-聚焦酶的分子特征,该酶是糖基水解酶家族29的成员,参与植物复合体型n -聚糖的降解
Pub Date : 2016-12-30 DOI: 10.1093/jb/mvw089
Mohammad Ziaur Rahman, Megumi Maeda, Satsuki Itano, Md. Anowar Hossain, T. Ishimizu, Y. Kimura
In this study, we identified a gene in tomato that encodes an acidic α-fucosidase (LOC101254568 or Solyc03g006980, α-Fuc'ase S1-1), which may be involved in the turnover of plant complex-type N-glycans. Recombinant α-Fuc'ase S1-1 (rFuc'ase S1-1) was expressed using a baculovirus-insect cell expression system. rFuc'ase Sl-1 is 55 kDa in size and has an optimum pH around 4.5. It substantially hydrolyzed the non-reducing terminal α1,3-fucose residue on LNFP III and α1,4-fucose residues of Lea epitopes on plant complex-type N-glycans, but not the α1,2-fucose residue on LNFP I or the α1,3-fucose residue on pyridylaminated Fucα1-3GlcNAc. Furthermore, we found that this tomato α-Fuc'ase S1-1 was inactive toward the core penta-oligosaccharide unit [Manβ1-4(Xylβ1-2)GlcNAcβ1-4(Fucα1-3)GlcNAc-PA] of plant complex-type N-glycans. Molecular 3D modelling of α-Fuc'ase Sl-1 and structure/sequence interpretation based on comparison with a homologous α-fucosidase from Bifidobacterium longum subsp. infantis (Blon_2336) indicated that residues Asp193 and Glu237 might be important for substrate binding.
在这项研究中,我们在番茄中发现了一个编码酸性α-聚焦酶(LOC101254568或Solyc03g006980, α-聚焦酶S1-1)的基因,该基因可能参与植物复合体型n -聚糖的转换。利用杆状病毒-昆虫细胞表达系统表达重组α-Fuc酶S1-1 (rFuc酶S1-1)。rFuc酶Sl-1的大小为55 kDa,其最佳pH值约为4.5。它能有效水解LNFP III上的非还原末端α1,3-聚焦残基和植物n-聚糖上Lea表位的α1,4-聚焦残基,但不能水解LNFP I上的α1,2-聚焦残基或吡啶层合Fucα1-3GlcNAc上的α1,3-聚焦残基。此外,我们发现该番茄α-Fuc'酶S1-1对植物复合体n -聚糖的核心五寡糖单元[Manβ1-4(Xylβ1-2)GlcNAcβ1-4(Fucα1-3)GlcNAc-PA]无活性。长双歧杆菌α-聚焦酶Sl-1的分子三维建模及结构/序列分析infantis (Blon_2336)表明,残基Asp193和Glu237可能对底物结合很重要。
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引用次数: 5
Biochemical characterization of the flagellar stator-associated inner membrane protein FliL from Vibrio alginolyticus 溶藻弧菌鞭毛定子相关内膜蛋白FliL的生化特性研究
Pub Date : 2016-12-24 DOI: 10.1093/jb/mvw076
Ananthanarayanan Kumar, M. Isumi, M. Sakuma, Shiwei Zhu, Yuuki Nishino, Yasuhiro Onoue, S. Kojima, Y. Miyanoiri, K. Imada, M. Homma
The flagellar motor is embedded in the cell envelope and rotates upon interaction between the stator and the rotor. The rotation is powered by ion flow through the stator. A single transmembrane protein named FliL is associated with torque generation in the flagellar motor. We established an Escherichia coli over-expression system for FliL of Vibrio alginolyticus, a marine bacterium that has a sodium-driven polar flagellum. We successfully expressed, purified, and crystallized the ca. 17 kDa full-length FliL protein and generated a construct that expresses only the ca. 14 kDa periplasmic region of FliL (ΔTM FliL). Biochemical characterization and NMR analysis revealed that ΔTM FliL weakly interacted with itself to form an oligomer. We speculate that the observed dynamic interaction may be involved in the role of FliL in flagellar motor function.
鞭毛马达嵌入细胞包膜中,在定子和转子之间的相互作用下旋转。旋转是由通过定子的离子流驱动的。一种名为FliL的跨膜蛋白与鞭毛马达中的扭矩产生有关。我们建立了溶藻弧菌(一种具有钠驱动极性鞭毛的海洋细菌)FliL的大肠埃希氏过表达系统。我们成功地表达、纯化和结晶了约17 kDa的全长FliL蛋白,并生成了一个仅表达约14 kDa的FliL质周区域的构建体(ΔTM FliL)。生化表征和核磁共振分析表明ΔTM FliL与自身弱相互作用形成低聚物。我们推测观察到的动态相互作用可能与FliL在鞭毛运动功能中的作用有关。
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引用次数: 10
The emerging complexity of ubiquitin architecture 泛素结构的新兴复杂性
Pub Date : 2016-12-23 DOI: 10.1093/jb/mvw088
F. Ohtake, Hikaru Tsuchiya
Ubiquitylation is an essential post-translational modification (PTM) of proteins with diverse cellular functions. Polyubiquitin chains with different topologies have different cellular roles, and are referred to as a 'ubiquitin code'. Recent studies have begun to reveal that more complex ubiquitin architectures function as important signals in several biological pathways. These include PTMs of ubiquitin itself, such as acetylated ubiquitin and phospho-ubiquitin. Moreover, important roles for heterogeneous polyubiquitin chains, such as mixed or branched chains, have been reported, which significantly increase the diversity of the ubiquitin code. In this review, we describe mass spectrometry-based methods to characterize the ubiquitin signal. We also describe recent advances in our understanding of complex ubiquitin architectures, including our own findings concerning ubiquitin acetylation and branching within polyubiquitin chains.
泛素化是具有多种细胞功能的蛋白质的重要翻译后修饰(PTM)。具有不同拓扑结构的多泛素链具有不同的细胞作用,被称为“泛素代码”。最近的研究已经开始揭示更复杂的泛素结构在几种生物学途径中起着重要的信号作用。这些包括泛素本身的PTMs,如乙酰化泛素和磷酸化泛素。此外,异质多泛素链(如混合链或支链)的重要作用已被报道,它们显著增加了泛素代码的多样性。在这篇综述中,我们描述了基于质谱的方法来表征泛素信号。我们还描述了我们对复杂泛素结构的理解的最新进展,包括我们自己关于泛素乙酰化和多泛素链分支的发现。
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引用次数: 77
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The Journal of Biochemistry
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