• 대한의생명과학회지
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• 제23권4호
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• pp.416-420
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• 2017

Endoplasmic reticulum (ER) stress induces unfolded protein response (UPR) via inositol-requiring enzyme 1 (IRE1) activation, which sends a molecular signal for X box-binding protein 1 (XBP1) mRNA splicing in the cytosol. IRE1 endoribonuclease activity induces cleavage of XBP1 mRNA. The XBP1 mRNA is then ligated by an uncharacterized RNA ligase and translated to produce spliced XBP1 by 23 nt removed in which contains the PstI restriction enzyme site. The splicing of XBP1 mRNA can be detected by semiquantitative RT-PCR, and then splicing of XBP1 is a useful tool to measure the genetic variability in ER stress. In this study, we have estimated IRE1-dependent splicing of XBP1 mRNA under conditions of various hypothermia. The results indicated that hypothermia regulated ER stress. This study demonstrated that hypothermia is closely related to ER stress and may be useful for early diagnosis of ER-associated disease.

• 생명과학회지
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• 제24권10호
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• pp.1039-1045
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• 2014

본 연구의 목적은 마우스 레이디히 세포인 mLTC-1 세포에 사람 융모성 성선자극호르몬인 hCG를 처리하여 유도되는 소포체 스트레스가 IRE1/XBP1 경로를 통하는지 분석하는 것 이다. 이전 연구에서 hCG처리에 의해 레이디히 세포는 소포체 스트레스 매개의 세포자멸사가 유도될 뿐만 아니라 ATF6경로를 조절함으로써 UPR이 성호르몬 합성효소의 발현에 중요한 역할을 하는 것을 증명하였다. UPR 경로는 또한 IRE1/XBP1 경로를 통하여 조절되는 것이 알려져 있지만 레이디히 세포에서 hCG에 의한 소포체 스트레스에 의해 IRE1/XBP1 경로의 활성화가 유도되는지에 대해서는 거의 알려져 있지 않다. mLTC-1세포에서 hCG 처리 후 IRE1/XBP1경로의 활성을 조사하기 위하여, 인산화된 IRE1 단백질 확인하기 위한 western blot, XBP1 mRNA splicing 확인하기 위한 RT-PCR을 수행하였다. 또한 우리는 IRE의 활성을 관찰하기 위하여 소포체 스트레스-활성 표지자(ERAI) construct를 이용하고 이를 형광현미경과 flow cytometry를 이용하여 분석하였다. 결과적으로, hCG 처리에 의해 인산화된 IRE1 단백질의 발현 수준이 두드러지게 증가하였다. F-XBP1-venus/F-$XBP1{\Delta}DBD$-venus가 도입된 mLTC-1 세포에서, hCG 처리에 의해 녹색 형광을 띄는 세포들이 유도되었고 각각 핵/세포질에서 발현하는 것을 확인할 수 있었다. 게다가 hCG 처리 후에 XBP1 mRNA의 splicing 또한 상당히 증가되는 것을 확인 할 수 있었다. 이 결과들을 통하여 종합해 볼 때 레이디히 세포에서 hCG 처리에 의해 유도되는 소포체 스트레스가 IRE1/XBP1 경로의 활성을 유발하는 것을 확인 할 수 있었다.

• Asian-Australasian Journal of Animal Sciences
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• 제32권8호
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• pp.1095-1103
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• 2019

Objective: Among stress responses, the unfolded protein response (UPR) is a well-known mechanism related to endoplasmic reticulum (ER) stress. ER stress is induced by a variety of external and environmental factors such as starvation, ischemia, hypoxia, oxidative stress, and heat stress. Inositol requiring enzyme $1{\alpha}$ ($IRE1{\alpha}$)-X-box protein 1 (XBP1) is the most conserved pathway involved in the UPR and is the main component that mediates $IRE1{\alpha}$ signalling to downstream ER-associated degradation (ERAD)- or UPR-related genes. XBP1 is a transcription factor synthesised via a novel mechanism called 'frame switch splicing', and this process has not yet been studied in the horse XBP1 gene. Therefore, the aim of this study was to confirm the frame switch splicing of horse XBP1 and characterise its dynamics using Thoroughbred muscle cells exposed to heat stress. Methods: Primary horse muscle cells were used to investigate heat stress-induced frame switch splicing of horse XBP1. Frame switch splicing was confirmed by sequencing analysis. XBP1 amino acid sequences and promoter sequences of various species were aligned to confirm the sequence homology and to find conserved cis-acting elements, respectively. The expression of the potential XBP1 downstream genes were analysed by quantitative real-time polymerase chain reaction. Results: We confirmed that splicing of horse XBP1 mRNA was affected by the duration of thermal stress. Twenty-six nucleotides in the mRNA of XBP1 were deleted after heat stress. The protein sequence and the cis-regulatory elements on the promoter of horse XBP1 are highly conserved among the mammals. Induction of putative downstream genes of horse XBP1 was dependent on the duration of heat stress. We confirmed that both the mechanisms of XBP1 frame switch splicing and various binding elements found in downstream gene promoters are highly evolutionarily conserved. Conclusion: The frame switch splicing of horse XBP1 and its dynamics were highly conserved among species. These results facilitate studies of ER-stress in horse.

• 대한의생명과학회지
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• 제11권2호
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• pp.249-252
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• 2005

The mammalian unfolded protein response (UPR) protects the cell. against the stress of unfolded or misfolded proteins in the endoplasmic reticulum (ER). It has recently demonstrated that IRE1, PERK, ATF6, and X-box protein 1 (XBP-l) directly or indirectly participate in this process. Upon accumulation of unfolded/misfolded proteins in the ER lumen, release of BiP from Ire1p permits dimerization and autophosphorylation to activate its kinase and endoribonulease activities to initiate XBP-1 mRNA splicing. Spliced XBP-1 mRNA removed middle part of 23 bp and encodes a potent transcription factor, XBP-l protein that binds to the unfolded protein response element (UPRE) or endoplasmic reticulum stress element (ERSE) sequence of many UPR target genes and produces several kind of ER chaperones. In this study, we described both the result and the detailed experimental procedures of XBP-1 mRNA splicing induced by ER stress, this result might help to elucidate the roles of the UPR and early diagnosis in a number of human diseases involving endoplasmic reticulum storage disease (ERSD).

• Nutrition Research and Practice
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• 제13권2호
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• pp.95-104
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• 2019

BACKGROUND/OBJECTIVES: Inflammatory Bowel Disease (IBD) has rapidly escalated in Asia (including Korea) due to increasing westernized diet patterns subsequent to industrialization. Factors associated with endoplasmic reticulum (ER) stress are demonstrated to be one of the major causes of IBD. This study was conducted to investigate the effect of Lycium barbarum (L. barbarum) on ER stress. MATERIALS/METHODS: Mouse embryonic fibroblast (MEF) cell line and polarized Caco-2 human intestinal epithelial cells were treated with crude extract of the L. chinense fruit (LF). Paracellular permeability was measured to examine the effect of tight junction (TJ) integrity. The regulatory pathways of ER stress were evaluated in MEF knockout (KO) cell lines by qPCR for interleukin (IL) 6, IL8 and XBP1 spliced form (XBP1s). Immunoglobulin binding protein (BiP), XBP1s and CCAAT/enhancer-binding homologous protein (CHOP) expressions were measured by RT-PCR. Scanning Ion Conductance Microscopy (SICM) at high resolution was applied to observe morphological changes after treatments. RESULTS: Exposure to LF extract strengthened the TJ, both in the presence and absence of inflammation. In polarized Caco-2 pretreated with LF, induction in the expression of proinflammatory marker IL8 was not significant, whereas ER stress marker XBP1s expression was significantly increased. In wild type (wt) MEF cells, IL6, CHOP and XBP1 spliced form were dose-dependently induced when exposed to $12.5-50{\mu}g/mL$ extract. However, absence of XBP1 or $IRE1{\alpha}$ in MEF cells abolished this effect. CONCLUSION: Results of this study show that LF treatment enhances the barrier function and reduces inflammation and ER stress in an $IRE1{\alpha}$-XBP1-dependent manner. These results suggest the preventive effect of LF on healthy intestine, and the possibility of reducing the degree of inflammatory symptoms in IBD patients.

• Journal of Veterinary Science
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• 제25권2호
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• pp.21.1-21.15
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• 2024

Background: Peste des petits ruminants (PPR) is a contagious and fatal disease of sheep and goats. PPR virus (PPRV) infection induces endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR). The activation of UPR signaling pathways and their impact on apoptosis and virus replication remains controversial. Objectives: To investigate the role of PPRV-induced ER stress and the IRE1-XBP1 and IRE1-JNK pathways and their impact on apoptosis and virus replication. Methods: The cell viability and virus replication were assessed by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, immunofluorescence assay, and Western blot. The expression of ER stress biomarker GRP78, IRE1, and its downstream molecules, PPRV-N protein, and apoptosis-related proteins was detected by Western blot and quantitative reverse transcription-polymerase chain reaction, respectively. 4-Phenylbutyric acid (4-PBA) and STF-083010 were respectively used to inhibit ER stress and IRE1 signaling pathway. Results: The expression of GRP78, IRE1α, p-IRE1α, XBP1s, JNK, p-JNK, caspase-3, caspase-9, Bax and PPRV-N were significantly up-regulated in PPRV-infected cells, the expression of Bcl-2 was significantly down-regulated. Due to 4-PBA treatment, the expression of GRP78, p-IRE1α, XBP1s, p-JNK, caspase-3, caspase-9, Bax, and PPRV-N were significantly downregulated, the expression of Bcl-2 was significantly up-regulated. Moreover, in PPRV-infected cells, the expression of p-IRE1α, p-JNK, Bax, and PPRV-N was significantly decreased, and the expression of Bcl-2 was increased in the presence of STF-083010. Conclusions: PPRV infection induces ER stress and IRE1 activation, resulting in apoptosis and enhancement of virus replication through IRE1-XBP1s and IRE1-JNK pathways.

• 대한의생명과학회지
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• 제12권4호
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• pp.435-438
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• 2006

In eukaryotes, ER stress induces UPR (unfolded protein response) via IRE1 activation which sends a molecular signal for XBP1 mRNA splicing in the cytosol. During this mRNA splicing, 23 nt removed in which contains PstI site and then resulting XBP1 product is not digested with PstI restriction enzyme. In this study, using this XBP1 mRNA splicing mechanism, the effect of heat shock on thyrocytes is studied, because heat shock response in the thyrocytes needs more study to understand thyroid physiology under alternative environments. ER inducible drugs (tunicamycin, DTT, $Ca^{2+}$ ionopore A23187, BFA) induce ER stress in the thyrocytes. From 3 hours after heat shock, ER stress is induced and which is reversible when heat shock is without. While $Ca^{2+}$ ionopore A23187 is reversible from ER stress by washing out the drug, thapsigagin is irreversible. Other ER inducible drugs are not so sensitive to ER stress repairing. XBP1 mRNA splicing in a cell is very available method to detect ER stress. It needs only a small quantity of total RNA and processing also very easy.

• 한국결정성장학회지
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• 제32권5호
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• pp.199-204
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• 2022

유연한 구조의 p-XBP4 리간드를 활용하여 새로운 3차원 구조의 Gd-MOF를 합성하고 단결정 회절 분석기를 활용하여 정확한 구조를 분석하였다. 중심 금속인 Gd는 6개의 p-XBP4와 1개의 물 분자에 의해 총 7배위 되어 있으며, W(CN)₈는 배위되지 않고 전하 균형을 맞추기 위해 구조체 내에 위치하고 있다. 중심 금속인 Gd는 배위된 p-XBP4에 의해 이웃한 Gd와 연결되어 3차원의 입체구조를 형성한다. 란탄 금속의 특성을 고려하여 직류 자화율 측정을 활용하여 자성 특성에 대한 연구를 진행하였다.

• BMB Reports
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• 제39권6호
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• pp.677-685
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• 2006

Proteins that are unfolded or misfolded in the endoplasmic reticulum (ER) must be targeted for refolding or degradation to maintain the homeostasis of the ER. Derlin-1 was reportedly implicated in the retro-translocation of misfolded proteins from the ER to the cytosol for degradation. In this report, we showed that Derlin-1 was down-regulated in the endothelial cells derived from human hepatic cavernous hemangioma (CHEC) compared with other tested cells. Electron microscopy analysis showed that ER was aberrantly enlarged in CHEC cells, but not in other tested cells. When overexpressed, Derlin-1 induced the dilated ER to return normal size. This ER dynamic was associated with the activation of unfolded protein response (UPR). In CHEC cells where Derlin-1 was down-regulated, increased expression of the immunoglobulin heavy chain-binding protein (Bip) and UPR-specific splicing of X-box DNA-binding protein 1 (XBP1) mRNA were detected, as compared with that in other tested cells, indicating that UPR was activated. After Derlin-1 overexpression, the extent of UPR activation diminished, as evidenced by decreased expression of Bip, reduced amount of the spliced form of XBP1 ($XBP1_S$), and elevated expression of the unspliced form of XBP1 ($XBP1_U$). Taken together, these findings provide another example of a single protein being able to affect ER dynamic in mammalian cells, and an insight into the possible molecular mechanism(s).

• 한국발생생물학회지:발생과생식
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• 제22권1호
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• pp.73-83
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• 2018

This study investigates the endoplasmic reticulum (ER) stress and subsequent apoptosis in duced during somatic cell nuclear transfer (SCNT) process of porcine SCNT embryos. Porcine SCNT and in vitro fertilization (IVF) embryos were sampled at 3 h and 20 h after SCNT or IVF and at the blastocyst stage for mRNA extraction. The x-box binding protein 1 (Xbp1) mRNA and the expressions of ER stress-associated genes were confirmed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. Before commencing SCNT, somatic cells treated with tunicamycin (TM), an ER stress inducer, confirmed the splicing of Xbp1 mRNA and increased expressions of ER stress-associated genes. In all the embryonic stages, the SCNT embryos, when compared with the IVF embryos, showed slightly increased expression of spliced Xbp1 (Xbp1s) mRNA and significantly increased expression of ER stress-associated genes (p<0.05). In all stages, apoptotic gene expression was slightly higher in the SCNT embryos, but not significantly different from that of the IVF embryos except for the Bax/Bcl2L1 ratio in the 1-cell stage (p<0.05). The result of this study indicates that excessive ER stress can be induced by the SCNT process, which induce apoptosis of SCNT embryos.