• 생명과학회지
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• 제25권5호
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• pp.594-600
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• 2015

단백질-단백질 결합은 다양한 세포내 반응 조절에서 중요한 역할을 한다. Postsynaptic density-95/disks large/ zonula occludens-1 (PDZ) 도메인은 널리 알려진 단백질-단백질 결합 매개 도메인 중 하나이다. PDZ 도메인은 결합 단백질의 카르복실(C)-말단의 특정 motif와 결합한다. Multi-PDZ domain protein 1 (MUPP1)은 13개 PDZ 도메인을 가지는 단백질로서 다양한 구조단백질 및 신호단백질에 대한 scaffold로 작용한다고 알려져 있지만 MUPP1의 세포 내 기능은 아직 명확히 밝혀지지 않았다. 본 연구에서 MUPP1의 PDZ 도메인과 결합하는 단백질을 규명하기 위하여 효모 two-hybrid 방법을 이용하였고 muskelin이 MUPP1과 결합하는 것을 확인하였다. Muskelin은 GAB_AA 수용체(GABA_AR)의 α1 subunit와 결합하며 수용체의 endocytosis와 분해에 관여하는 것으로 알려져 있다. Muskelin은 MUPP1의 3번째 PDZ 도메인과 결합하지만, 다른 PDZ 도메인과는 결합하지 않았다. 또한 MUPP1과의 결합에 muskelin의 C-말단부위가 필수적임을 효모 two-hybrid 방법으로 확인하였다. HEK-293T 세포에 MUPP1과 muskelin을 동시에 발현하여 면역 침강한 결과 두 단백질은 같이 면역 침강하였다. 반면에 C-말단 결손 muskelin은 MUPP1과 같이 면역 침강하지 않았다. 또한 muskelin과 MUPP1은 세포내의 같은 위치에서 발현하였다. 이러한 결과들은, muskelin과의 결합을 통해, MUPP1 혹은 MUPP1과 결합하는 단백질이 GABA_AR의 세포내이동과 회전(turnover)을 조절할 가능성을 시사한다.

• 생명과학회지
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• 제26권3호
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• pp.282-288
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• 2016

단백질-단백질 결합은 수용체 단백질, 효소, 세포 골격 단백질의 세포내 위치 결정 및 기능 조절에 중요한 역할을 한다. Postsynaptic density-95/disks large/zonula occludens-1 (PDZ) 도메인을 가진 단백질들은 시냅스 가소성, 신경세포 성장과 분화뿐만 아니라 많은 질병의 병태생리에 중요하게 관여하는 scaffold 단백질로 작용한다. Multi-PDZ domain protein 1 (MUPP1)은 13개 PDZ 도메인을 가지는 단백질로서 세포막 수용체 군집화, 신호전달 복합체 구성, 세포 골격 조정에 대한 매개 역할을 하는 것으로 알려지고 있지만 MUPP1의 세포 내 기능은 아직 명확히 밝혀지지 않았다. 본 연구에서 MUPP1의 아미노 말단 PDZ 도메인과 결합하는 새로운 단백질을 규명하기 위하여 효모 two-hybrid 방법을 이용하였고 Wdpcp (전에 Fritz로 알려짐)이 MUPP1과 결합하는 것을 확인하였다. Wdpcp는 planar cell polarity (PCP) effector로서 세포 이동과 섬모형성에 관여하는 것으로 알려져 있다. Wdpcp는 MUPP1의 첫 번째 PDZ 도메인과 결합하지만, 다른 PDZ 도메인과는 결합하지 않았다. 또한 MUPP1와 Wdpcp의 결합에서 Wdpcp의 C-말단부위가 결합에 필수적임을 효모 two-hybrid 방법으로 확인하였다. 이러한 단백질간 결합은 glutathione S-transferase (GST) pull-down assay, 공동면역침강, HEK-293T 세포에서의 발현위치를 통하여 추가적으로 확인하였다. 이러한 결과들은, MUPP1과 Wdpcp 결합은 세포내 액틴 다이내믹스(dynamics)와 세포이동 조절에 역할을 할 가능성을 시사한다.

• Asian-Australasian Journal of Animal Sciences
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• 제29권5호
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• pp.731-738
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• 2016

Glucagon-like peptide-2 (GLP-2) is important for intestinal barrier function and regulation of tight junction (TJ) proteins, but the intracellular mechanisms of action remain undefined. The purpose of this research was to determine the protective effect of GLP-2 mediated TJ and transepithelial electrical resistance (TER) in lipopolysaccharide (LPS) stressed IPEC-J2 cells and to test the hypothesis that GLP-2 regulate TJ and TER through the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in IPEC-J2 cells. Wortmannin and LY294002 are specific inhibitors of PI3K. The results showed that $100{\mu}g/mL$ LPS stress decreased TER and TJ proteins occludin, claudin-1 and zonula occludens protein 1 (ZO-1) mRNA, proteins expressions (p<0.01) respectively. GLP-2 (100 nmol/L) promote TER and TJ proteins occludin, claudin-1, and zo-1 mRNA, proteins expressions in LPS stressed and normal IPEC-J2 cells (p<0.01) respectively. In normal cells, both wortmannin and LY294002, PI3K inhibitors, prevented the mRNA and protein expressions of Akt and mTOR increase induced by GLP-2 (p<0.01) following with the significant decreasing of occludin, claudin-1, ZO-1 mRNA and proteins expressions and TER (p<0.01). In conclusion, these results indicated that GLP-2 can promote TJ's expression and TER in LPS stressed and normal IPEC-J2 cells and GLP-2 could regulate TJ and TER through the PI3K/Akt/mTOR pathway.

• Animal Bioscience
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• 제34권7호
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• pp.1157-1168
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• 2021

Objective: The aim of this study was to investigate the effects of different amounts of wheat bran (WB) inclusion and postbiotics form by Saccharomyces cerevisiae and phytase co-fermented wheat bran (FWB) on the growth performance and health status of broilers. Methods: Study randomly allocated a total of 300 male broilers to a control and 4 treatment groups (5% WB, 5% FWB, 10% WB, and 10% FWB inclusion, respectively) with each pen having 20 broilers and 3 pens per treatment. Results: The WB does not contain enzymes, but there are 152.8, 549.2, 289.5, and 147.1 U/g dry matter xylanase, protease, cellulase and β-glucanase in FWB, respectively. Furthermore, FWB can decrease nitric oxide release of lipopolysaccharide stimulated chicken peripheral blood mononuclear cells by about two times. Results show that 10% FWB inclusion had significantly the highest weight gain (WG) at 1 to 21 d; 5% FWB had the lowest feed conversion rate at 22 to 35 d; 10% WB and 10% FWB inclusion have the highest villus height and Lactobacillus spp. number in caecum; and both 5% and 10% FWB can increase ash content in femurs. Compared to control group, all treatments increase mucin 2, and tight junction (TJ), such as occludin, claudin-1, zonula occludens-1, and mRNA expression in ileum by at least 5 folds. In chicken peripheral blood mononuclear cells, nicotinamide adenine dinucleotide phosphate-oxidase-1 mRNA expression decreases from 2 to 5 times, and glutamate-cysteine ligase catalytic subunit mRNA expression also increases in all treatment groups compared to control group. The mRNA expression of pro-inflammatory cytokines, including interleukin-6 (IL-6), nuclear factor-κB, and IL-1β, decreases in 5% and 10% FWB groups compared to control group. Conclusion: To summarize, both WB and FWB inclusion in broilers diets increase TJ mRNA expression and anti-oxidation and anti-inflammation, but up to 10% FWB groups have better WG in different stages of broiler development.

• Asian-Australasian Journal of Animal Sciences
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• 제27권5호
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• pp.733-742
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• 2014

The glucagon-like peptide 2 (GLP-2) that is expressed in intestine epithelial cells of mammals, is important for intestinal barrier function and regulation of tight junction (TJ) proteins. However, there is little known about the intracellular mechanisms of GLP-2 in the regulation of TJ proteins in piglets' intestinal epithelial cells. The purpose of this study is to test the hypothesis that GLP-2 regulates the expressions of TJ proteins in the mitogen-activated protein kinase (MAPK) signaling pathway in piglets' intestinal epithelial cells. The jejunal tissues were cultured in a Dulbecco's modified Eagle's medium/high glucose medium containing supplemental 0 to 100 nmol/L GLP-2. At 72 h after the treatment with the appropriate concentrations of GLP-2, the mRNA and protein expressions of zonula occludens-1 (ZO-1), occludin and claudin-1 were increased (p<0.05). U0126, an MAPK kinase inhibitor, prevented the mRNA and protein expressions of ZO-1, occludin, claudin-1 increase induced by GLP-2 (p<0.05). In conclusion, these results indicated that GLP-2 could improve the expression of TJ proteins in weaned pigs' jejunal epithelium, and the underlying mechanism may due to the MAPK signaling pathway.

• Asian-Australasian Journal of Animal Sciences
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• 제33권11호
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• pp.1797-1808
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• 2020

Objective: This study assessed the effects of probiotics on cecal microbiota, gene expression of intestinal tight junction proteins, and immune response in the cecal tonsil of broiler chickens challenged with Salmonella enterica subsp. enterica. Methods: One-day-old broiler chickens (n = 240) were randomly allocated to four treatments: negative control (Cont), multi-strain probiotic-treated group (Pro), Salmonella-infected group (Sal), and multi-strain probiotic-treated and Salmonella-infected group (ProSal). All chickens except those in the Cont and Pro groups were gavaged with 1×10⁸ cfu/mL of S. enterica subsp. enterica 4 days after hatching. Results: Our results indicated that body weight, weight gain, and feed conversion ratio of birds were significantly reduced (p<0.05) by Salmonella challenge. Chickens challenged with Salmonella decreased cecal microbial diversity. Chickens in the Sal group exhibited abundant Proteobacteria than those in the Cont, Pro, and ProSal groups. Salmonella infection downregulated gene expression of Occludin, zonula occludens-1 (ZO1), and Mucin 2 in the jejunum and Occludin and Claudin in the ileum. Moreover, the Sal group increased gene expression of interferon-γ (IFN-γ), interleukin-6 (IL-6), IL-1β, and lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF) and reduced levels of transforming growth factor-β4 and IL-10 compared with the other groups (p<0.05). However, chickens receiving probiotic diets increased Lactobacillaceae abundance and reduced Enterobacteriaceae abundance in the ceca. Moreover, supplementation with probiotics increased the mRNA expression of Occludin, ZO1, and Mucin 2 in the ileum (p<0.05). In addition, probiotic supplementation downregulated the mRNA levels of IFN-γ (p<0.05) and LITAF (p = 0.075) and upregulated IL-10 (p = 0.084) expression in the cecal tonsil. Conclusion: The administration of multi-strain probiotics modulated intestinal microbiota, gene expression of tight junction proteins, and immunomodulatory activity in broiler chickens.

• The Korean Journal of Physiology and Pharmacology
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• 제28권3호
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• pp.239-252
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• 2024

Dexmedetomidine displays multiple mechanisms of neuroprotection in ameliorating ischemic brain injury. In this study, we explored the beneficial effects of dexmedetomidine on blood-brain barrier (BBB) integrity and neuroinflammation in cerebral ischemia/reperfusion injury. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 1.5 h and reperfusion for 24 h to establish a rat model of cerebral ischemia/reperfusion injury. Dexmedetomidine (9 ㎍/kg) was administered to rats 30 min after MCAO through intravenous injection, and SB203580 (a p38 MAPK inhibitor, 200 ㎍/kg) was injected intraperitoneally 30 min before MCAO. Brain damages were evaluated by 2,3,5-triphenyltetrazolium chloride staining, hematoxylin-eosin staining, Nissl staining, and brain water content assessment. BBB permeability was examined by Evans blue staining. Expression levels of claudin-5, zonula occludens-1, occludin, and matrix metalloproteinase-9 (MMP-9) as well as M1/M2 phenotypes-associated markers were assessed using immunofluorescence, RT-qPCR, Western blotting, and gelatin zymography. Enzyme-linked immunosorbent assay was used to examine inflammatory cytokine levels. We found that dexmedetomidine or SB203580 attenuated infarct volume, brain edema, BBB permeability, and neuroinflammation, and promoted M2 microglial polarization after cerebral ischemia/reperfusion injury. Increased MMP-9 activity by ischemia/reperfusion injury was inhibited by dexmedetomidine or SB203580. Dexmedetomidine inhibited the activation of the ERK, JNK, and p38 MAPK pathways. Moreover, activation of JNK or p38 MAPK reversed the protective effects of dexmedetomidine against ischemic brain injury. Overall, dexmedetomidine ameliorated brain injury by alleviating BBB permeability and promoting M2 polarization in experimental cerebral ischemia/reperfusion injury model by inhibiting the activation of JNK and p38 MAPK pathways.

• Journal of Periodontal and Implant Science
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• 제48권5호
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• pp.284-294
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• 2018

Purpose: Epithelial barrier dysfunction is involved in the pathophysiology of periodontitis and oral lichen planus. Estrogens have been shown to enhance the physical barrier function of intestinal and esophageal epithelia, and we aimed to investigate the effect of estradiol (E2) on the regulation of physical barrier and tight junction (TJ) proteins in human oral epithelial cell monolayers. Methods: HOK-16B cell monolayers cultured on transwells were treated with E2, an estrogen receptor (ER) antagonist (ICI 182,780), tumor necrosis factor alpha ($TNF{\alpha}$), or dexamethasone (Dexa), and the transepithelial electrical resistance (TER) was then measured. Cell proliferation was measured by the cell counting kit (CCK)-8 assay. The levels of TJ proteins and nuclear translocation of nuclear factor $(NF)-{\kappa}B$ were examined by confocal microscopy. Results: E2 treatment increased the TER and the levels of junctional adhesion molecule (JAM)-A and zonula occludens (ZO)-1 in a dose-dependent manner, without affecting cell proliferation during barrier formation. Treatment of the tight-junctioned cell monolayers with $TNF{\alpha}$ induced decreases in the TER and the levels of ZO-1 and nuclear translocation of $NF-{\kappa}B$. These $TNF{\alpha}-induced$ changes were inhibited by E2, and this effect was completely reversed by co-treatment with ICI 182,780. Furthermore, E2 and Dexa presented an additive effect on the epithelial barrier function. Conclusions: E2 reinforces the physical barrier of oral epithelial cells through the nuclear ER-dependent upregulation of TJ proteins. The protective effect of E2 on the $TNF{\alpha}-induced$ impairment of the epithelial barrier and its additive effect with Dexa suggest its potential use to treat oral inflammatory diseases involving epithelial barrier dysfunction.

• Journal of Cancer Prevention
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• 제21권2호
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• pp.95-103
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• 2016

Background: Excess energy supply induces chronic low-grade inflammation in association with oxidative stress in various tissues including intestinal epithelium. The objective of this study was to investigate the effect of high-fat diet (HFD) on intestinal cell membrane integrity and intestinal tumorigenesis in $Apc^{Min/+}$ mice. Methods: Mice were fed with either normal diet (ND) or HFD for 12 weeks. The number of intestinal tumors were counted and biomarkers of endotoxemia, oxidative stress, and inflammation were determined. Changes in intestinal integrity was measured by fluorescein isothiocyanate (FITC)-dextran penetration and membrane gap junction protein expression. Results: HFD group had significantly higher number of tumors compared to ND group (P < 0.05). Blood total antioxidant capacity was lower in HFD group, while colonic 8-hydroxy-2'-deoxyguanosine level, a marker of oxidative damage, was higher in HFD group compared to that of ND group (P < 0.05). The penetration of FITC-dextran was substantially increased in HFD group (P < 0.05) while the expressions of membrane gap junction proteins including zonula occludens-1, claudin-1, and occludin were lower in HFD group (P < 0.05) compared to those in ND group. Serum concentration of lipopolysaccharide (LPS) receptor (CD14) and colonic toll-like receptor 4 (a LPS receptor) mRNA expression were significantly higher in HFD group than in ND group (P < 0.05), suggesting that significant endotoxemia may occur in HFD group due to the increased membrane permeability. Serum interleukin-6 concentration and myeloperoxidase activity were also higher in HFD group compared to those of ND group (P < 0.05). Conclusions: HFD increases oxidative stress disrupting intestinal gap junction proteins, thereby accelerating membrane permeability endotoxemia, inflammation, and intestinal tumorigenesis.

• 한국식품과학회지
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• 제53권2호
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• pp.165-173
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• 2021

본 연구에서는 UPM에 의해 유발되는 NCI-H441 세포 내 ROS 생성과 그에 따른 tight junction 단백질(N-cadherin, fibronectin, occluding, ZO-1, 및 claudin-4 등)의 발현 억제를 확인하였고 ECL의 전처리로 인하여 해당 ROS 생성 및 tight junction 단백질 발현 억제 현상이 완화되는 보호효과를 확인하였다. 그 결과, 폐 세포에서 UPM에 의해 유발된 산화 스트레스 및 tight junction 단백질의 발현 감소가 ECL을 선 처리함으로써 억제되어 결과적으로 산화 스트레스와 같은 미세먼지에 의한 폐 손상으로부터 보호 효과를 나타내고 tight junction 수준의 유지를 확인하였다. 이러한 결과를 통해 ECL 추출물은 폐와 기관지의 보호효과를 나타내며 폐 관련 질병의 예방효과가 있는 기능성 천연 물질로써 활용이 가능할 것으로 기대된다.