• Korean Circulation Journal
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• v.53 no.6
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• pp.387-403
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• 2023

Background and Objectives: Myocardial ischemia and reperfusion injury (MIRI) has high morbidity and mortality worldwide. We aimed to explore the role of long noncoding RNA lysyl oxidase like 1 antisense RNA 1 (LOXL1-AS1) in cardiomyocyte pyroptosis. Methods: Hypoxia/reoxygenation (H/R) injury was constructed in human cardiomyocyte (HCM). The level of LOXL1-AS1, miR-761, phosphatase and tensin homolog (PTEN) and pyroptosis-related proteins was monitored by quantitative real-time polymerase chain reaction or western blot. Flow cytometry examined the pyroptosis level. Lactate dehydrogenase (LDH), creatine kinase-MB and cardiac troponin I levels were detected by test kits. Enzyme-linked immunosorbent assay measured the release of inflammatory cytokines. Dual-luciferase assay validated the binding relationship among LOXL1-AS1, miR-761, and PTEN. Finally, ischemia/reperfusion (I/R) animal model was constructed. Hematoxylin and eosin staining assessed morphological changes of myocardial tissue. NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and casepase-1 expression was determined by immunohistochemistry. Results: After H/R treatment, LOXL1-AS1 and PTEN were highly expressed but miR-761 level was suppressed. LOXL1-AS1 inhibition or miR-761 overexpression increased cell viability, blocked the release of LDH and inflammatory cytokines (interleukin [IL]-1β, IL-18), inhibited pyroptosis level, and downregulated pyroptosis-related proteins (ASC, cleaved caspase-1, gasdermin D-N, NLRP3, IL-1β, and IL-18) levels in HCMs. LOXL1-AS1 sponged miR-761 to up-regulate PTEN. Knockdown of miR-761 reversed the effect of LOXL1-AS1 down regulation on H/R induced HCM pyroptosis. LOXL1-AS1 aggravated the MIRI by regulating miR-761/PTEN axis in vivo. Conclusions: LOXL1-AS1 targeted miR-761 to regulate PTEN expression, then enhance cardiomyocyte pyroptosis, providing a new alternative target for the treatment of MIRI.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.8
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• pp.1103-1109
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• 2018

Objective: This study aimed to screen and identify the target genes of miR-375 in pig Sertoli (ST) cells and to elucidate the effect of miR-375 on the proliferation of ST cells. Methods: In this study, bioinformatics software was used to predict and verify miR-375 target genes. Quantitative polymerase chain reaction (PCR) was used to detect the relationship between miR-375 and its target genes in ST cells. Enzyme-linked immunosorbent assay (ELISA) of rearranged L-myc fusion (RLF) and hypoxia-induced gene domain protein 1A (HIGD1A) was performed on porcine ST cells, which were transfected with a miR-375 mimics and inhibitor to verify the results. Dual luciferase reporter gene assays were performed to assess the interactions among miR-375, RLF, and HIGD1A. The effect of miR-375 on the proliferation of ST cells was analyzed by CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS). Results: Five possible target genes of miR-375, including RLF, HIGD1A, colorectal cancer associated 2, POU class 3 homeobox 1, and WW domain binding protein 1 like, were found. The results of quantitative PCR suggested that mRNA expression of RLF and HIGD1A had a negative correlation with miR-375, indicating that RLF and HIGD1A are likely the target genes of miR-375. The ELISA results revealed that RLF and HIGD1A were negatively correlated with the miR-375 protein level. The luminescence results for the miR-375 group cotransfected with wild-type RLF and HIGD1A vector were significantly lower than those of the miR-375 group co-transfected with the blank vector or mutant RLF and HIGD1A vectors. The present findings suggest that RLF and HIGD1A are target genes of miR-375 and that miR-375 inhibits ST cell proliferation according to MTS analysis. Conclusion: It was speculated that miR-375 affects cell proliferation through its target genes, which play an important role in the development of testicular tissue.

• Journal of the Korean Society of Food Science and Nutrition
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• v.35 no.8
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• pp.990-996
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• 2006

This research was conducted to evaluate the changes in allergenicity of shrimp by physical treatments using competitive indirect enzyme-linked immunosorbent assay (Ci-ELISA). Shrimp was subjected to physical treatments such as high hydrostatic pressure (HHP), sonication, autoclave and microwave. Heat-stable protein (HSP) purified from raw shrimp was used as a major allergen. The binding ability of monoclonal IgG and shrimp-allergic patients' IgE to HSP treated with HHP decreased, increasing the pressure up to 400 MPa. Especially, it became less than 50% at 400 MPa. The binding ability of mAb to HSP treated with sonication (10, 20, 30 and 60 min) decreased with the treated time. Especially, it became less than 60% with the treatment for 60 min. The allergenicity change of HSP treated with autoclave and microwave little decreased. The binding ability of mAb to HSP during the treatment for 20 min became more than 70%. The results suggest that allergenicity of HSP in raw shrimp be more easily lost by HHP or sonication treatment than by autoclave or microwave treatment.

• International Journal of Oral Biology
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• v.33 no.3
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• pp.77-82
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• 2008

Innate immune response is initiated by the recognition of unique microbial molecular patterns through pattern recognition receptors (PRRs). The purpose of this study is to dissect the expression of various PRRs in gingival epithelial cells of differentiated versus undifferentiated states. Differentiation of immortalized human gingival epithelial HOK-16B cells was induced by culture in the presence of high $Ca^{2+}$ at increased cell density. The expression levels of various PRRs in HOK-16B cells were examined by realtime reverse transcription polymerase chain reaction (RTPCR) and flow cytometry. In addition, the expression of human beta defensins (HBDs) was examined by real time RT-PCR and the amounts of secreted cytokines were measured by enzyme linked immunosorbent assay. In undifferentiated HOK-16B cells, NACHT-LRR-PYDcontaining protein (NALP) 2 was expressed most abundantly, and toll like receptor (TLR) 2, TLR4, nucleotide-binding oligomerization domain (NOD) 1, and NOD2 were expressed in substantial levels. However, TLR3, TLR7, TLR8, TLR9, ICE protease-activating factor (IPAF), and NALP6 were hardly expressed. In differentiated cells, the levels of NOD2, NALP2, and TLR4 were different from those in undifferentiated cells at RNA but not at protein levels. Interestingly, differentiated cells expressed the increased levels of HBD-1 and -3 but secreted reduced amount of IL-8. In conclusion, the repertoire of PRRs expressed by gingival epithelial cells is limited, and undifferentiated and differentiated cells express similar levels of PRRs.

• Journal of Applied Biological Chemistry
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• v.58 no.3
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• pp.227-232
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• 2015

A microarray study has been employed to understand changes of gene expression in E. coli KD43162 resistant to ampicillin, ampicillin-sulbactam, piperacillin, piperacillin-tazobactam, cefazolin, cefepime, aztreonam, imipenem, meropenem, gentamicin, tobramycin, ciprofloxacin, levofloxacin, moxifloxacin, fosfomycin, and trimethoprim-sulfamethoxazole except for amikacin using disk diffusion assay. Using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and MALDI-TOF MS analyses, 36 kDa of outer membrane proteins (OMPs) was found to be deleted in the multidrug resistant E. coli KD 43162. Microarray analysis was used to determine up- and down-regulated genes in relation to multidrug resistant E. coli KD43162. Among the up-regulated genes, these genes were corresponded to express the proteins as penicillin-binding proteins (PBPs), tartronate semialdehyde reductase, ethanolamine utilization protein, shikimate kinase I, allantoinase, predicted SAM-dependent methyltransferase, L-glutamine: D-fructose-6-phosphate aminotransferase (GFAT), phospho-glucosamine mutase, predicted N-acetylmannosamine kinase, and predicted N-acetylmannosamine-6-P epimerase. Up-regulation of PBPs, one of primary target sites of antibiotics, might be responsible for the multidrug resistance in E. coli with increasing amount of target sites. Up-regulation of GFAT enzyme may be related to the up-regulation of PBPs because GFAT produces N-acetylglucosamine, a precursor of peptidoglycans. One of GFAT inhibitors, azaserine, showed a potent inhibition on the growth of E. coli KD43162. In conclusion, up-regulation of PBPs and GFATs with the loss of 36 kDa OMP refers the multidrug resistance in E. coli KD 43162.

• Journal of Veterinary Science
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• v.22 no.4
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• pp.45.1-45.13
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• 2021

Background: Peste des petits ruminants (PPR) is an infectious disease caused by the peste des petits ruminants virus (PPRV) that mainly produces respiratory symptoms in affected animals, resulting in great losses in the world's agriculture industry every year. Single-domain variable heavy chain (VHH) antibody fragments, also referred to as nanobodies, have high expression yields and other advantages including ease of purification and high solubility. Objectives: The purpose of this study is to obtain a single-domain antibody with good reactivity and high specificity against PPRV. Methods: A VHH cDNA library was established by immunizing camels with PPRV vaccine, and the capacity and diversity of the library were examined. Four PPRV VHHs were selected, and the biological activity and antigen-binding capacity of the four VHHs were identified by western blot, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) analyses. ELISA was used to identify whether the four VHHs were specific for PPRV, and VHH neutralization tests were carried out. ELISA and western blot analyses were used to identify which PPRV protein was targeted by VHH2. Results: The PPRV cDNA library was constructed successfully. The library capacity was greater than 2.0 × 10⁶ cfu/mL, and the inserted fragment size was approximately 400 bp to 2000 bp. The average length of the cDNA library fragment was about 1000 bp, and the recombination rate was approximately 100%. Four single-domain antibody sequences were selected, and proteins expressed in the supernatant were obtained. The four VHHs were shown to have biological activity, close affinity to PPRV, and no cross-reaction with common sheep diseases. All four VHHs had neutralization activity, and VHH2 was specific to the PPRV M protein. Conclusions: The results of this preliminary research of PPRV VHHs showed that four screened VHH antibodies could be useful in future applications. This study provided new materials for inclusion in PPRV research.

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.244-255
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• 2019

Xylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (PbXI) from the psychrophilic soil microorganism, Paenibacillus sp. R4. Surprisingly, activity assay results showed that PbXI is not a cold-active enzyme, but displays optimal activity at $60^{\circ}C$. We solved the crystal structure of PbXI at $1.94-{\AA}$ resolution to investigate the origin of its thermostability. The PbXI structure shows a $({\beta}/{\alpha})_8$-barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the ${\beta}4-{\alpha}6$ loop region, was newly identified and is thought to be important for the stability of PbXI. Compared with previously determined thermostable and mesophilic XI structures, the ${\beta}1-{\alpha}2$ loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible ${\beta}1-{\alpha}2$ loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate low-temperature purpose-specific XI enzymes.

• Journal of Animal Science and Technology
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• v.63 no.2
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• pp.426-439
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• 2021

It is already well known that castration improves marbling quality but exact timing of castration is still highly debated in beef cattle production industry. After castration, blood hormonal changes occur in steer and objective of this study was to investigate the effects of growth hormone (GH) levels on adipocyte differentiation in stromal vascular cells (SVCs) and transdifferentiation into adipocytes in C2C12 myoblasts. Total GH concentrations were measured via enzyme-linked immunosorbent assay (ELISA) in 24 male calves and 4 female calves. Cell proliferation, cellular triglyceride (TG) accumulation, and the cell's lipolytic capability were measured in C2C12 myoblasts and SVCs. Myogenic, adipogenic, and brown adipocyte-specific gene expression was measured via real-time polymerase chain reaction (PCR) using SYBR green. Serum GH levels were the highest in late-castrated calves. Treatment with 5 ng/mL GH resulted in greater TG accumulation as well as increased CCAAT-enhancer-binding protein (C/EBP)α and peroxisome proliferator-activated receptor (PPAR)γ expression compared to that after treatment with 15 ng/mL GH. Treatment with 5 ng/mL GH also resulted in lower myogenin (myo)G and myoD expression compared to that after treatment with 15 ng/mL GH. The expression of bone morphogenetic protein (BMP) 7 after treatment with 5 ng/mL GH was higher than that after treatment with 15 ng/mL GH. But carcass characteristics data showed no significant difference between early and late castrated steers. Therefore, our results indicate that castration timing does not seem to be inevitable determinate of carcass qualities, particularly carcass weight and marbling score in Hanwoo beef cattle.

• Nutrition Research and Practice
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• v.17 no.1
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• pp.164-173
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• 2023

BACKGROUND/OBJECTIVES: Hyperglycemia is a major cause of diabetes and diabetesrelated diseases. Sodium butyrate (NaB) is a short-chain fatty acid derivative that produces dietary fiber by anaerobic bacterial fermentation in the large intestine and occurs in foods, such as Parmesan cheese and butter. Butyrate has been shown to prevent obesity, improve insulin sensitivity, and ameliorate dyslipidemia in diet-induced obese mice. Therefore, this study examined the effects and mechanism of NaB on the secretion of inflammatory cytokines induced by high glucose (HG) in THP-1 cells. MATERIALS/METHODS: THP-1 cells were used as an in vitro model for HG-induced inflammation. The cells were cultured under normal glycemic or hyperglycemic conditions with or without NaB (0-25 μM). Western blotting and quantitative polymerase chain reaction were used to evaluate the protein and mRNA levels of nuclear factor-κB (NF-κB), interleukin-6, tumor necrosis factor-α, acetylated p65, acetyl CREB-binding protein/p300 (CBP/p300), and p300 using THP-1 cells. Histone acetyltransferase (HAT), histone deacetylase (HDAC), and pro-inflammatory cytokine secretion activity were analyzed using an enzyme-linked immunosorbent assay. RESULTS: HG significantly upregulated histone acetylation, acetylation levels of p300, NF-κB activation, and inflammatory cytokine release in THP-1 cells. Conversely, the NaB treatment reduced cytokine release and NF-κB activation in HG-treated cells. It also significantly reduced p65 acetylation, CBP/p300 HAT activity, and CBP/p300 gene expression. In addition, NaB decreased the interaction of p300 in acetylated NF-κB and TNF-α. CONCLUSIONS: These results suggest that NaB suppresses HG-induced inflammatory cytokine production through HAT/HDAC regulation in monocytes. NaB has the potential for preventing and treating diabetes and its related complications.

• Journal of Digital Convergence
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• v.17 no.8
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• pp.265-270
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• 2019

Growing evidence suggests that mediating apoptotic cell death of ER stress plays an important role in pathological development of neurodegenerative diseases including Alzheimer's disease. The ethanol extract of Rodiola sacra (ERS) investigates whether ER stress protects neuroinvasive neuro-2A cells from homocysteine (Hcy) cell death and ER stress. In neuronal cells, Hcy markedly decreased the viability of the cells and induced the death of Annexin V-positive cells as confirmed by MTT assay. The Hcy cell viability and apoptotic loss pretreated with ERS were attenuated, and Hcy showed stress in the expression of C / EBP homologous protein, 78-kDa glucose regulatory protein and the junction of X-box binding protein-1 (xbp1) mRNA. ESR decreased Hcy-induced mRNA binding, GRP78 and CHOP cells induced Hcy-induced ER stress and apoptosis, and Western blotting revealed expression of heme oxygenase-1 and HO-1 enzyme activity Inhibition is indicative of therapeutic value for neurodegenerative diseases such as decreased cell death by hemin.