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New insights into the regulation and roles of phosphatidylinositol 3,4-bisphosphate 对 3,4-二磷酸磷脂酰肌醇的调节和作用的新认识
Pub Date : 2024-09-14 DOI: 10.1093/jb/mvae063
Junya Hasegawa
Phosphoinositides (PIPs) are phospholipids and components of the cellular membrane. In mammals, seven phosphorylated derivatives of PIPs have been identified. Among them, phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] is produced by lipid phosphatases (e.g., SHIP2) or by lipid kinases PI3KC2α and PI3KC2β. Although PI(3,4)P2 is undetectable in normal mouse or human tissues and common cell lines, it appears in a mouse prostate cancer model and in cells exposed to oxidative stress, indicating that PI(3,4)P2 is involved in the pathogenesis of some diseases. Here, I summarize recent findings on the cellular roles and pathophysiological significance of PI(3,4)P2.
磷脂(PIPs)是磷脂,也是细胞膜的组成部分。在哺乳动物中,已经发现了七种磷酸化的 PIPs 衍生物。其中,磷脂酰肌醇 3,4-二磷酸[PI(3,4)P2]是由脂质磷酸酶(如 SHIP2)或脂质激酶 PI3KC2α 和 PI3KC2β 产生的。虽然 PI(3,4)P2 在正常小鼠或人体组织和普通细胞系中检测不到,但它却出现在小鼠前列腺癌模型和暴露于氧化应激的细胞中,这表明 PI(3,4)P2 与某些疾病的发病机制有关。在此,我总结了有关 PI(3,4)P2 的细胞作用和病理生理学意义的最新发现。
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引用次数: 0
The NRF2 inducer CDDO-2P-Im provokes a reduction in amyloid β levels in Alzheimer’s disease model mice NRF2 诱导剂 CDDO-2P-Im 可降低阿尔茨海默病模型小鼠的淀粉样蛋白 β 水平
Pub Date : 2024-09-11 DOI: 10.1093/jb/mvae060
Akira Uruno, Shiori Kadoguchi-Igarashi, Ritsumi Saito, Shohei Koiso, Daisuke Saigusa, Ching-Tung Chu, Takafumi Suzuki, Takashi Saito, Takaomi C Saido, Antonio Cuadrado, Masayuki Yamamoto
Alzheimer’s disease (AD) is the most common etiology of dementia. The transcription factor NF-E2-related factor 2 (NRF2) induces the expression of genes encoding phase II detoxification and antioxidant genes. NRF2 is regulated by Kelch-like ECH-associated protein 1 (KEAP1), and the KEAP1-NRF2 system is the key regulatory system involved in cytoprotection. To examine whether pharmacological induction of NRF2 expression alleviates AD phenotypes in vivo, we employed two AD mouse models, i.e., App NL-G-F/NL-G-F (AppNLGF) and APPV717I::TAUP301L (APP/TAU) mice. As the synthetic oleanane triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11-dien-28-oyl)] (CDDO)-4(-pyridin-2-yl)-imidazole (CDDO-2P-Im) exhibits strong NRF2-inducing activity, we treated AD model mice with CDDO-2P-Im. We found that Aβ42 levels were markedly greater in the brains of AppNLGF mice than in those of APP/TAU mice. CDDO-2P-Im treatment significantly decreased Aβ42 levels, but not Aβ40 levels, in APP/TAU mice. Consequently, CDDO-2P-Im also decreased the ratio of Aβ42/Aβ40, a vital marker of amyloid plaque formation. LC–MS/MS analyses revealed that CDDO-2P-Im was delivered to the brains of the APP/TAU mice. CDDO-2P-Im induced the expression of detoxification and antioxidant gene targets of NRF2 and elevated reduced glutathione (GSH) levels in the mouse brain. These results support the notion that CDDO-2P-Im ameliorates AD-related pathologic changes.
阿尔茨海默病(AD)是痴呆症最常见的病因。转录因子 NF-E2 相关因子 2(NRF2)诱导编码第二阶段解毒和抗氧化基因的表达。NRF2 受 Kelch-like ECH-associated protein 1(KEAP1)调控,而 KEAP1-NRF2 系统是参与细胞保护的关键调控系统。为了研究药理诱导 NRF2 的表达是否能缓解体内的 AD 表型,我们采用了两种 AD 小鼠模型,即 App NL-G-F/NL-G-F (AppNLGF)和 APPV717I::TAUP301L (APP/TAU)小鼠。由于合成的齐墩果烷三萜类化合物 1-[2-氰基-3,12-二氧代油橄榄-1,9(11-二烯-28-酰基)](CDDO)-4(-吡啶-2-基)-咪唑(CDDO-2P-Im)具有很强的 NRF2 诱导活性,因此我们用 CDDO-2P-Im 处理 AD 模型小鼠。我们发现,AppNLGF小鼠大脑中的Aβ42水平明显高于APP/TAU小鼠。CDDO-2P-Im 能显著降低 APP/TAU 小鼠脑中 Aβ42 的水平,但不能降低 Aβ40 的水平。因此,CDDO-2P-Im 还降低了 Aβ42/Aβ40 的比率,而 Aβ42/Aβ40 是淀粉样斑块形成的重要标志。LC-MS/MS分析显示,CDDO-2P-Im被输送到了APP/TAU小鼠的大脑中。CDDO-2P-Im 诱导了 NRF2 的解毒和抗氧化基因靶点的表达,并提高了小鼠大脑中还原型谷胱甘肽(GSH)的水平。这些结果支持了 CDDO-2P-Im 可改善注意力缺失症相关病理变化的观点。
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引用次数: 0
Cancer-associated SF3B1 Mutations Inhibit mRNA Nuclear Export by Disrupting SF3B1–THOC5 Interactions 癌症相关 SF3B1 突变通过破坏 SF3B1-THOC5 相互作用抑制 mRNA 核输出
Pub Date : 2024-09-11 DOI: 10.1093/jb/mvae061
Gang Liu, Bo Zhao, Yueru Shi, Youzhong Wan
Mutations in SF3B1 are common in many types of cancer, which promotes cancer progression through aberrant RNA splicing. Recently, mRNA nuclear export has been reported to be defective in cells with SF3B1 K700E mutation. However, the mechanism remains unclear. Our study reveals that the K700E mutation in SF3B1 attenuates its interaction with THOC5, an essential component of mRNA nuclear export complex THO. Furthermore, SF3B1 mutation caused reduced binding of THOC5 with some mRNA and inhibited the nuclear export of these mRNA. Interestingly, THOC5 overexpression restores the nuclear export of these mRNA in cells with SF3B1 K700E mutation. Importantly, other types of cancer-associated SF3B1 mutations also inhibited mRNA nuclear export similarly, suggesting that it is common for cancer-associated SF3B1 mutation to inhibit mRNA nuclear export. Our research highlights the critical role of the THOC5–SF3B1 interaction in the regulation of mRNA nuclear export and provides valuable insights into the impact of SF3B1 mutations on mRNA nuclear export.
SF3B1 基因突变在许多类型的癌症中都很常见,它通过异常的 RNA 剪接促进癌症进展。最近有报道称,在SF3B1 K700E突变的细胞中,mRNA核输出存在缺陷。然而,其机制仍不清楚。我们的研究发现,SF3B1的K700E突变削弱了它与mRNA核输出复合物THO的重要组成部分THOC5的相互作用。此外,SF3B1突变导致THOC5与一些mRNA的结合减少,并抑制了这些mRNA的核输出。有趣的是,在SF3B1 K700E突变的细胞中,过表达THOC5可恢复这些mRNA的核输出。重要的是,其他类型的癌症相关SF3B1突变也同样抑制mRNA的核输出,这表明癌症相关SF3B1突变抑制mRNA的核输出是常见的。我们的研究强调了THOC5-SF3B1相互作用在调控mRNA核输出中的关键作用,并就SF3B1突变对mRNA核输出的影响提供了有价值的见解。
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引用次数: 0
Mtc6/Ehg2 is a novel endoplasmic reticulum-resident glycoprotein essential for high-pressure tolerance Mtc6/Ehg2是一种新型内质网驻留糖蛋白,对耐受高压至关重要
Pub Date : 2024-04-15 DOI: 10.1093/jb/mvae035
Satoshi Uemura, Takahiro Mochizuki, Yusuke Kato, Tetsuo Mioka, Riseko Watanabe, Mai Fuchita, Mao Yamada, Yoichi Noda, Takashi Moriguchi, Fumiyoshi Abe
Hydrostatic pressure is a common mechanical stressor that modulates metabolism and reduces cell viability. Eukaryotic cells have genetic programs to cope with hydrostatic pressure stress and maintain intracellular homeostasis. However, the mechanism underlying hydrostatic pressure tolerance remains largely unknown. We have recently demonstrated that Maintenance of telomere capping protein 6 (Mtc6) plays a protective role in the survival of the budding yeast Saccharomyces cerevisiae under hydrostatic pressure stress by supporting the integrity of nutrient permeases. The current study demonstrate that Mtc6 acts as an endoplasmic reticulum (ER) membrane protein. Mtc6 comprises two transmembrane domains, a C-terminal cytoplasmic domain, and a luminal region with 12 Asn (N)-linked glycans attached to it. Serial mutational analyses showed that the cytoplasmic C-terminal amino acid residues GVPS are essential for Mtc6 activity. Multiple N-linked glycans in the luminal region are involved in the structural conformation of Mtc6. Moreover, deletion of MTC6 led to increased degradation of the leucine permease Bap2 under hydrostatic pressure, suggesting that Mtc6 facilitates proper folding of nutrient permeases in the ER under the stress condition. We propose a novel model of molecular function in which the glycosylated luminal domain and cytoplasmic GVPS sequences of Mtc6 cooperatively support the nutrient permease activity.
静水压是一种常见的机械应激源,可调节新陈代谢并降低细胞活力。真核细胞具有应对静水压压力和维持细胞内平衡的基因程序。然而,静水压耐受性的内在机制在很大程度上仍然未知。我们最近证明,端粒盖蛋白6(Mtc6)通过支持营养渗透酶的完整性,在静水压胁迫下对出芽酵母的存活起到保护作用。目前的研究证明,Mtc6 是一种内质网(ER)膜蛋白。Mtc6 包括两个跨膜结构域、一个 C 端胞质结构域和一个附有 12 个 Asn(N)连接糖的腔区。连续突变分析表明,胞质 C 端氨基酸残基 GVPS 对 Mtc6 的活性至关重要。腔内区域的多个 N-连接聚糖参与了 Mtc6 的结构构象。此外,缺失MTC6会导致亮氨酸渗透酶Bap2在静水压力下的降解增加,这表明Mtc6有助于营养物质渗透酶在压力条件下在ER中正确折叠。我们提出了一个新的分子功能模型,其中Mtc6的糖基化管腔结构域和胞质GVPS序列共同支持营养物质渗透酶的活性。
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引用次数: 0
Evaluation of the cyclic single-chain Fv antibody derived from nivolumab by biophysical analyses and in vitro cell-based bioassay 通过生物物理分析和体外细胞生物测定评估由 nivolumab 衍生的环状单链 Fv 抗体
Pub Date : 2024-04-09 DOI: 10.1093/jb/mvae034
Sena Kamesawa, Mizuki Ogawa, Yoshiki Funakoshi, Masaya Kato, Shosei Kai, Mana Namikawa, Kyo Okazaki, Takashi Sato, Yoshihiro Kobashigawa, Hiroshi Morioka
Single-chain Fv (scFv) is a recombinant small antibody in which a polypeptide linker connects the variable regions of the light chain (VL) and the heavy chain (VH). The practical use of scFv, however, has been prevented by its tendency to aggregate due to interchain VL–VH interactions. We recently developed a cyclic scFv whose N-terminus and C-terminus were connected by protein ligation techniques. Biophysical comparisons between cyclic and linear scFv have been conducted, but cell biological evaluations remain unexplored. Here we studied the properties of cyclic and linear scFv derived from nivolumab. Biophysical studies revealed that the thermal stability was not changed but that the antigen-binding activity was approximately 3-fold higher as a result of circularization. A cell-based PD-1/PD-L1 interaction inhibitory assay revealed that the biological activity of scFv was markedly higher in the circularized form. In addition, biophysical analysis of scFv proteins incubated in the presence of serum revealed that circularization suppressed the decrease in antigen-binding activity. It could be assumed that circularization of scFv improved stability in the presence of serum, which in turn would suggest the applicability of cyclic scFv as a biopharmaceutical format.
单链 Fv(scFv)是一种重组小抗体,其轻链(VL)和重链(VH)的可变区之间由多肽连接。然而,由于 VL-VH 链间的相互作用,scFv 容易聚集,这阻碍了它的实际应用。我们最近开发了一种环状 scFv,其 N 端和 C 端通过蛋白质连接技术连接起来。我们已对环状 scFv 和线性 scFv 进行了生物物理比较,但细胞生物学评估仍未进行。在此,我们研究了从 nivolumab 提取的环状和线性 scFv 的特性。生物物理研究表明,热稳定性没有改变,但抗原结合活性因环化而提高了约 3 倍。基于细胞的 PD-1/PD-L1 相互作用抑制试验显示,环化后的 scFv 生物活性明显更高。此外,在有血清存在的情况下培养 scFv 蛋白的生物物理分析表明,环化抑制了抗原结合活性的降低。可以认为,scFv 的环化提高了其在血清存在下的稳定性,这反过来又表明了环状 scFv 作为生物制药形式的适用性。
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引用次数: 0
Reflection 反射
Pub Date : 2022-09-01 DOI: 10.1093/jb/mvac038
Akira Kikuchi
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引用次数: 0
OUP accepted manuscript OUP接受稿件
Pub Date : 2021-01-01 DOI: 10.1093/jb/mvab138
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引用次数: 0
Crystal structure of metagenomic &bgr;-xylosidase/ &agr;-L-arabinofuranosidase activated by calcium 钙活化的宏基因组&bgr;-木糖苷酶/ &agr;- l -阿拉伯糖醛酸苷酶的晶体结构
Pub Date : 2017-09-01 DOI: 10.1093/jb/mvx012
T. Matsuzawa, S. Kaneko, N. Kishine, Z. Fujimoto, K. Yaoi
The crystal structure of metagenomic β-xylosidase/α-l-arabinofuranosidase CoXyl43, activated by calcium ions, was determined in its apo and complexed forms with xylotriose or l-arabinose in the presence and absence of calcium. The presence of calcium ions dramatically increases the kcat of CoXyl43 for p-nitrophenyl β-d-xylopyranoside and reduces the Michaelis constant for p-nitrophenyl α-l-arabinofuranoside. CoXyl43 consists of a single catalytic domain comprised of a five-bladed β-propeller. In the presence of calcium, a single calcium ion was observed at the centre of this catalytic domain, behind the catalytic pocket. In the absence of calcium, the calcium ion was replaced with one sodium ion and one water molecule, and the positions of these cations were shifted by 1.3 Å. The histidine-319 side chain, which coordinates to the 2-hydroxyl oxygen atom of the bound xylose molecule in the catalytic pocket, also coordinates to the calcium ion, but not to the sodium ion. The calcium-dependent increase in activity appears to be caused by the structural change in the catalytic pocket induced by the tightly bound calcium ion and coordinating water molecules, and by the protonation state of glutamic acid-268, the catalytic acid of the enzyme. Our findings further elucidate the complex relationship between metal ions and glycosidases.
在钙离子激活下,测定了宏基因组β-木糖苷酶/α-l-阿拉伯糖糠糖苷酶CoXyl43的载子和与木糖或l-阿拉伯糖在有钙和无钙情况下的络合形式。钙离子的存在显著提高了CoXyl43对对硝基苯β-d-木吡喃苷的kcat,降低了对硝基苯α-l-阿拉伯糖脲苷的Michaelis常数。CoXyl43由一个由五叶β-螺旋桨组成的单一催化结构域组成。在钙存在的情况下,在催化结构域的中心,在催化口袋后面,观察到一个单一的钙离子。在没有钙的情况下,钙离子被一个钠离子和一个水分子取代,这些阳离子的位置移动了1.3 Å。组氨酸-319侧链与催化袋中结合木糖分子的2-羟基氧原子配位,也与钙离子配位,但不与钠离子配位。钙依赖性的活性增加似乎是由紧密结合的钙离子和配位的水分子引起的催化口袋结构变化以及酶的催化酸谷氨酸-268的质子化状态引起的。我们的发现进一步阐明了金属离子与糖苷酶之间的复杂关系。
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引用次数: 13
Giardia duodenalis Rad52 protein: biochemical characterization and response upon DNA damage 十二指肠贾第虫Rad52蛋白的生化特性及其对DNA损伤的响应
Pub Date : 2017-08-01 DOI: 10.1093/jb/mvx009
Rosa María Martínez-Miguel, Antonio Sandoval-Cabrera, M. L. Bazán-Tejeda, A. L. Torres-Huerta, Diego A. Martínez-Reyes, R. Bermúdez-Cruz
Giardia duodenalis is a flagellated binucleated protozoan that colonizes the small intestine in mammals, causing giardiasis, acute or chronic diarrhea. DNA double strand break either endogenously or exogenously generated is a major insult to DNA and its repair by homologous recombination (HR) is crucial for genomic stability. During HR, Rad52 plays key roles in the loading of the Rad51 recombinase, and the annealing of the second double-strand break end to the displaced strand of the D-loop structure. Among the functions found in vitro in yeast and human Rad52 protein are: ssDNA or dsDNA binding activity, ability to anneal bare or RPA coated-ssDNA, as well as multimeric ring formation. In this work, we searched for conserved domains in a putative Rad52 protein from G. duodenalis (GdRad52). Its coding sequence was cloned, expressed and purified to study its biochemical properties. rGdRad52 binds to dsDNA and ssDNA, with greater affinity for the latter. Likewise, rGdRad52 promotes annealing of DNA uncoated and coated with GdRPA1. rGdRad52 interacts with GdDMC1B and with GdRPA1 protein as shown in far western blotting assay. Additionally, rGdRad52 formed multimeric rings as observed by electronic microscopy. Finally, GdRad52 is over expressed in response upon DNA damage inflicted on trophozoites.
十二指肠贾第虫是一种有鞭毛的双核原生动物,在哺乳动物的小肠中定植,引起贾第虫病,急性或慢性腹泻。无论是内源性还是外源性DNA双链断裂都是对DNA的主要损伤,同源重组对DNA的修复对基因组的稳定性至关重要。在HR过程中,Rad52在Rad51重组酶的加载和第二双链断裂端退火到D-loop结构的移位链中起着关键作用。体外在酵母和人Rad52蛋白中发现的功能包括:ssDNA或dsDNA结合活性,退火裸或RPA涂层的ssDNA的能力,以及多聚环的形成。在这项工作中,我们在十二指肠鸡中寻找一个推定的Rad52蛋白(GdRad52)的保守结构域。对其编码序列进行克隆、表达和纯化,研究其生化特性。rGdRad52结合dsDNA和ssDNA,对后者的亲和力更强。同样,rgdrpa52促进未包被和包被GdRPA1的DNA退火。rGdRad52与GdDMC1B和GdRPA1蛋白相互作用。此外,rGdRad52在电子显微镜下形成多聚环。最后,GdRad52在对滋养体DNA损伤的反应中过表达。
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引用次数: 4
GPNMB promotes proliferation of developing eosinophils GPNMB促进发育中的嗜酸性粒细胞增殖
Pub Date : 2017-08-01 DOI: 10.1093/jb/mvx002
S. Hwang, J. Kang, B. K. Kim, Tae Gi Uhm, Hye Jeong Kim, Hyune-Hwan Lee, B. Binas, I. Chung
Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane protein that is expressed in a wide variety of cell types, including haematopoietic lineages. We previously demonstrated that GPNMB is one of the most highly expressed genes at an early and intermediate stage of eosinophil development. We herein examined GPNMB expression and its possible functional effect using cord blood (CB) CD34+ haematopoietic stem cells differentiating toward eosinophils during a 24-day culture period. Western blot and confocal microscopy analyses showed that GPNMB reached its highest levels at day 12 with most GPNMB-positive cells also expressing major basic protein 1 (MBP1), an eosinophil granule protein. GPNMB declined thereafter, but was still present at an appreciable level at day 24, the time when CB eosinophils most abundantly expressed MBP1 and were thus considered fully differentiated. When the developing CB cells were cultured in the presence of a blocking anti-GPNMB antibody, cell proliferation was significantly reduced. In agreement, ectopic expression of GPNMB in heterologous cells resulted in a significant increase in cell proliferation, while small interfering RNA of GPNMB inhibited the GPNMB-mediated proliferation. Thus, GPNMB is expressed in a temporal manner during eosinophil development and delivers a proliferative signal upon activation.
糖蛋白非转移性黑色素瘤蛋白B (GPNMB)是一种I型跨膜蛋白,在多种细胞类型中表达,包括造血谱系。我们之前已经证明GPNMB是嗜酸性粒细胞发育早期和中期最高度表达的基因之一。在此,我们利用脐带血(CB) CD34+造血干细胞在24天的培养期间向嗜酸性粒细胞分化,研究了GPNMB的表达及其可能的功能影响。Western blot和共聚焦显微镜分析显示,GPNMB在第12天达到最高水平,大多数GPNMB阳性细胞也表达主要碱性蛋白1 (MBP1),一种嗜酸性粒细胞颗粒蛋白。GPNMB随后下降,但在第24天仍以可观的水平存在,这是CB嗜酸性粒细胞最丰富表达MBP1的时候,因此被认为是完全分化的。当发育中的CB细胞在阻断抗gpnmb抗体存在下培养时,细胞增殖明显降低。与此一致的是,GPNMB在异种细胞中的异位表达导致细胞增殖显著增加,而GPNMB的小干扰RNA抑制GPNMB介导的增殖。因此,GPNMB在嗜酸性粒细胞发育过程中以时间方式表达,并在激活时传递增殖信号。
{"title":"GPNMB promotes proliferation of developing eosinophils","authors":"S. Hwang, J. Kang, B. K. Kim, Tae Gi Uhm, Hye Jeong Kim, Hyune-Hwan Lee, B. Binas, I. Chung","doi":"10.1093/jb/mvx002","DOIUrl":"https://doi.org/10.1093/jb/mvx002","url":null,"abstract":"Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane protein that is expressed in a wide variety of cell types, including haematopoietic lineages. We previously demonstrated that GPNMB is one of the most highly expressed genes at an early and intermediate stage of eosinophil development. We herein examined GPNMB expression and its possible functional effect using cord blood (CB) CD34+ haematopoietic stem cells differentiating toward eosinophils during a 24-day culture period. Western blot and confocal microscopy analyses showed that GPNMB reached its highest levels at day 12 with most GPNMB-positive cells also expressing major basic protein 1 (MBP1), an eosinophil granule protein. GPNMB declined thereafter, but was still present at an appreciable level at day 24, the time when CB eosinophils most abundantly expressed MBP1 and were thus considered fully differentiated. When the developing CB cells were cultured in the presence of a blocking anti-GPNMB antibody, cell proliferation was significantly reduced. In agreement, ectopic expression of GPNMB in heterologous cells resulted in a significant increase in cell proliferation, while small interfering RNA of GPNMB inhibited the GPNMB-mediated proliferation. Thus, GPNMB is expressed in a temporal manner during eosinophil development and delivers a proliferative signal upon activation.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"17 1","pages":"85–91"},"PeriodicalIF":0.0,"publicationDate":"2017-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75881387","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
期刊
The Journal of Biochemistry
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