• Asian-Australasian Journal of Animal Sciences
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• v.30 no.6
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• pp.781-787
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• 2017

Objective: The early growth response (Egr) family consists of four members (Egr1, Egr2, Egr3, and Egr4) that are zinc finger transcription factors. Among them, Egr3 is involved in transcriptional regulation of target genes during muscle spindle formation and neurite outgrowth. We previously showed that the immunoreactive Egr3 is localized on oocyte spindle and accumulate near the microtubule organizing center during meiosis I in mice. Egr3 was also shown to be localized on spermatocytes. We herein investigated if Egr3 is expressed in mouse gonads and if Egr3 blockade results in any defect in oocyte maturation. Methods: Expression of Egr3 in mouse gonads was examined by reverse transcription-polymerase chain reaction. Full-length Egr3 and truncated Egr3 (${\Delta}Egr3$) complementary RNAs (cRNAs) with Xpress tag at N-terminus and DsRed2 at C-terminus, and small interfering RNA (siRNA) targeting Egr3 were microinjected into mouse oocytes at germinal vesicle stage. Localization of microinjected Egr3 was examined by confocal live imaging and immunofluorescence staining. Results: Egr3 mRNA was detected in mouse ovaries and testes from 1 to 4 week-old mice. An uncharacterized longer transcript containing 5'untranslated region was also detected in 3 and 4 week-old gonads. Microinjected Xpress-Egr3-DsRed2 or Xpress-${\Delta}Egr3$-DsRed2 localized to nuclei and chromosomes during meiotic progression. Microinjection of these cRNAs or Egr3 siRNA in oocytes did not affect meiotic maturation. Immunofluorescence staining of Egr3 in Xpress-${\Delta}Egr3$-DsRed2-injected oocytes showed a positive signal only on meiotic spindle, suggesting that this antibody does not detect endogenous or exogenous Egr3 in mouse oocytes. Conclusion: The results show that Egr3 localizes to chromosomes during meiotic progression and that certain antibodies may not faithfully represent localization of target proteins in oocytes. Egr3 seems to be dispensable during oocyte maturation in mice.

• Journal of Periodontal and Implant Science
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• v.32 no.2
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• pp.389-402
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• 2002

BMP can induce ectopic bone formation when implanted into sites such as rat muscle and can greatly enhance healing of bony defects when applied exogenously. In addition, BMP stimulated osteoblastic differentiation in vitro in various types of cells. The aim of this study was to investigate the effect of recombinant human bone morphogenetic protein(rhBMP-2) on the proliferation and osteoblastic differentiation of human periodontal ligament cells and gingival fibroblasts. The cell number and alkaline phosphatase activity were measured in 3 experimental groups of human periodontal ligament cells and gingival fibroblasts (control group, rhBMP-2 50ng/ml group, and rhBMP-2 100ng/ml group) at 1 and 2 weeks after culture. At the same time, total RNA of cultured cells were extracted and reverse trascription polymerase chain reaction(RT-PCR) was performed to determine the expression of mRNA of bone matrix protein. RhBMP-2 had no effect on the cell proliferation of human periodontal ligament cells and gingival fibroblasts. Alkaline phosphatase activity was elevated significantly by rhBMP-2 in both cells. And periodontal ligament cells showed significantly higher alkaline phosphatase activity than gingival fibroblasts. ${\beta}$-actin, type I collagen, alkaline phosphatase, BMP-2 mRNA were expressed in all of the samples. Osteopontin, osteocalcin mRNA were expressed in all periodontal ligament cell groups, and rhBMP-2 50ng/ml group, rhBMP-2 100ng/ml group of 2 week culture period of gingival fibroblasts. Bone sialoprotein mRNA was only expressed in rhBMP-2 50ng/ml group and rhBMP-2 100ng/ml group of 2-week culture period. These results suggest that rhBMP-2 stimulates osteoblastic differentiation in human periodontal ligament cells and gingival fibroblasts in vitro.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.3
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• pp.424-435
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• 2020

Objective: The study was conducted to investigate variations in the immunophysiological responses to exercise-induced stress in Jeju and Thoroughbred horses. Methods: Blood samples were collected from the jugular veins of adult Jeju (n = 5) and Thoroughbred (n = 5) horses before and after 30 min of exercise. The hematological, biochemical, and immunological profiles of the blood samples were analyzed. Blood smears were stained and observed under a microscope. The concentration of cell-free (cf) DNA in the plasma was determined using real time polymerase chain reaction (PCR). Peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells were separated using Polymorphprep, and the expression of various stress-related and chemokine receptor genes was measured using reverse transcriptase (RT) and real-time PCR. Results: After exercise, Jeju and Thoroughbred horses displayed stress responses with significantly increased rectal temperatures, cortisol levels, and muscle catabolism-associated metabolites. Red blood cell indices were significantly higher in Thoroughbred horses than in Jeju horses after exercise. In addition, exercise-induced stress triggered the formation of neutrophil extracellular traps (NETs) and reduced platelet counts in Jeju horses but not in Thoroughbred horses. Heat shock protein 72 and heat shock protein family A (Hsp70) member 6 expression is rapidly modulated in response to exercise-induced stress in the PBMCs of Jeju horses. The expression of CXC chemokine receptor 4 in PBMCs was higher in Thoroughbred horses than in Jeju horses after exercise. Conclusion: In summary, the different immunophysiological responses of Jeju and Thoroughbred horses explain the differences in the physiological and anatomical properties of the two breeds. The physiology of Thoroughbred horses makes them suitable for racing as they are less sensitive to exercise-induced stress compared to that of Jeju horses. This study provides a basis for investigating the link between exercise-induced stresses and the physiological alteration of horses. Hence, our findings show that some of assessed parameters could be used to determine the endurance performance of horses.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.3
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• pp.398-407
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• 2020

Objective: The Yip1 domain family (YIPF) proteins were proposed to function in endoplasmic reticulum (ER) to Golgi transport and maintenance of the morphology of the Golgi, which were homologues of yeast Yip1p and Yif1p. YIPF3, the member 3 of YIPF family was a homolog of Yif1p. The aim of present study was to investigate the expression and regulation mechanism of porcine YIPF3. Methods: Quantitative realtime polymerase chain reaction (qPCR) was used to analyze porcine YIPF3 mRNA expression pattern in different tissues and pig kidney epithelial (PK15) cells stimulated by polyinosine-polycytidylic acid (poly [I:C]). Site-directed mutations combined with dual luciferase reporter assays and electrophoretic mobility shift assay (EMSA) were employed to reveal transcription regulation mechanism of porcine YIPF3. Results: Results showed that the mRNA of porcine YIPF3 (pYIPF3) was widely expressed with the highest levels in lymph and lung followed by spleen and liver, while weak in heart and skeletal muscle. Subcellular localization results indicated that it expressed in Golgi apparatus and plasma membranes. Upon stimulation with poly (I:C), the level of this gene was dramatically up-regulated in a time- and concentration-dependent manner. pYIPF3 core promoter region harbored three cis-acting elements which were bound by ETS proto-oncogene 2 (ETS2), zinc finger and BTB domain containing 4 (ZBTB4), and zinc finger and BTB domain containing 14 (ZBTB14), respectively. In which, ETS2 and ZBTB4 both promoted pYIPF3 transcription activity while ZBTB14 inhibited it, and these three transcription factors all played important regulation roles in tumorigenesis and apoptosis. Conclusion: The pYIPF3 mRNA expression was regulated by ETS2, ZBTB4, and ZBTB14, and its higher expression in immune organs might contribute to enhancing ER to Golgi transport of proteins, thus adapting to the immune response.

• Korean Journal of Ichthyology
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• v.24 no.2
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• pp.118-124
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• 2012

The present study was conducted to investigate different gene expression profile between treated poMSTNpro and non-treated in rainbow trout and to identify those genes that are specifically or predominantly expressed in treated poMSTNpro by employing annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR). We isolated total RNAs in muscle tissues from the treated poMSTNpro fish by immersion bath technique with fish myostatin prodomain (Paralichthys olivaceus, poMSTNpro) for one month and the other was non-treated poMSTNpro, and synthesized cDNA using annealing control primers (ACP, Seegene, Korea). Using 20 different ACPs for PCR, were cloned sequenced, and analyzed identities of 2 differentially expressed genes (DEGs). According to BLAST analysis, sequences of 2 clones significantly matched database entries and confirmed by semi-quantitative RT-PCR. The functional roles of one up-regulated gene, cytochrome P450 mono-oxygenases 2K1v2 (CYP2K1v2), and one down-regulated gene was Profilin-1 were identified. We identified distinctive gene expression profiles in improved rainbow trout growth by treatment with a fish myostatin prodomain using ACP-based GeneFishing.

• Neonatal Medicine
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• v.17 no.2
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• pp.254-261
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• 2010

Little is known about neonatal mitochondrial disease, though mitochondrial metabolic disorders may often present in the neonatal period because of the high energy requirement of neonate. In newborn period, common presentations are not specific and the disease course may be rapid and fatal. In this study, we report three cases of neonatal mitochondrial disease. The first case was strongly suspected because of sudden seizure and mental change with severe lactic acidosis, and multiorgan failure. Plasma lactate/pyruvate (L/P) ratio was increased to 55.6 with marked lactic aciduria and increased plasma alanin up to 2,237 nmol/mL. In the second patient, a peritoneal dialysis was performed for acute adrenal and renal failure, but metabolic acidosis persisted. Plasma L/P ratio was increased to 23.9, and MRC I (mitochondrial respiratory chain defect) was diagnosed through the enzymatic analysis of the muscles. The third case showed repetitive episode of lactic acidosis during the first two months of life, hypotonia, failure to thrive and feeding difficulties. We found markedly increased cerebrospinal fluid L/P ratio up to 57 though plasma L/P ratio(19.4) was borderline with increased plasma lactate. The lactate peak was prominent in brain magnetic resonance spectroscopy (MRS). MRC II was confirmed through muscle biopsy. Plasma lactate level and lactate peak of brain MRS were normalized after conservative treatment.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.10
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• pp.1581-1590
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• 2018

Objective: The aim of this study was to clone alternative splicing isoforms of pig myoneurin (MYNN), predict the structure and function of coding protein, and study temporal and spatial expression characteristics of each transcript. Methods: Alternative splice isoforms of MYNN were identified using RNA sequencing (RNA-seq) and cloning techniques. Quantitative real-time polymerase chain reaction (qPCR) was employed to detect expression patterns in 11 tissues of Large White (LW) and Mashen (MS) pigs, and to study developmental expression patterns in cerebellum (CE), stomach (ST), and longissimus dorsi (LD). Results: The results showed that MYNN had two alternatively spliced isoforms, MYNN-1 (GenBank accession number: KY470829) and MYNN-2 (GenBank accession number: KY670835). MYNN-1 coding sequence (CDS) is composed of 1,830 bp encoding 609 AA, whereas MYNN-2 CDS is composed of 1,746 bp encoding 581 AA. MYNN-2 was 84 bp less than MYNN-1 and lacked the sixth exon. MYNN-2 was found to have one $C_2H_2$ type zinc finger protein domain less than MYNN-1. Two variants were ubiquitously expressed in all pig tissues, and there were significant differences in expression of different tissues (p<0.05; p<0.01). The expression of MYNN-1 was significantly higher than that of MYNN-2 in almost tissues (p<0.05; p<0.01), which testified that MYNN-1 is the main variant. The expression of two isoforms decreased gradually with increase of age in ST and CE of MS pig, whereas increased gradually in LW pig. In LD, the expression of two isoforms increased first and then decreased with increase of age in MS pig, and decreased gradually in LW pig. Conclusion: Two transcripts of pig MYNN were successfully cloned and MYNN-1 was main variant. MYNN was highly expressed in ST, CE, and LD, and their expression was regular. We speculated that MYNN plays important roles in digestion/absorption and skeletal muscle growth, whereas the specific mechanisms require further elucidation.

• Animal Bioscience
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• v.35 no.1
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• pp.115-125
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• 2022

Objective: Intramuscular fat (IMF) is a critical economic indicator of pork quality. Studies on IMF among different pig breeds have been performed via high-throughput sequencing, but comparisons within the same pig breed remain unreported. Methods: This study was performed to explore the gene profile and identify candidate long noncoding RNA (lncRNAs) and mRNAs associated with IMF deposition among Laiwu pigs with different IMF contents. Based on the longissimus dorsi muscle IMF content, eight pigs from the same breed and management were selected and divided into two groups: a high IMF (>12%, H) and low IMF group (<5%, L). Whole-transcriptome sequencing was performed to explore the differentially expressed (DE) genes between these two groups. Results: The IMF content varied greatly among Laiwu pig individuals (2.17% to 13.93%). Seventeen DE lncRNAs (11 upregulated and 6 downregulated) and 180 mRNAs (112 upregulated and 68 downregulated) were found. Gene Ontology analysis indicated that the following biological processes played an important role in IMF deposition: fatty acid and lipid biosynthetic processes; the extracellular signal-regulated kinase cascade; and white fat cell differentiation. In addition, the peroxisome proliferator-activated receptor, phosphatidylinositol-3-kinase-protein kinase B, and mammalian target of rapamycin pathways were enriched in the pathway analysis. Intersection analysis of the target genes of DE lncRNAs and mRNAs revealed seven candidate genes associated with IMF accumulation. Five DE lncRNAs and 20 DE mRNAs based on the pig quantitative trait locus database were identified and shown to be related to fat deposition. The expression of five DE lncRNAs and mRNAs was verified by quantitative real time polymerase chain reaction (qRT-PCR). The results of qRT-PCR and RNA-sequencing were consistent. Conclusion: These results demonstrated that the different IMF contents among pig individuals may be due to the DE lncRNAs and mRNAs associated with lipid droplets and fat deposition.

• Animal Bioscience
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• v.36 no.2
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• pp.200-208
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• 2023

Objective: Muscle acetylcholine receptors have five alpha subunits (α, β, δ, ε, or γ), and cholinergic receptor nicotinic gamma subunit (CHRNG) is the γ subunit. It may also play an essential role in biological processes, including cell differentiation, growth, and survival, while the role of CHRNG has not been studied in the literature. Therefore, the purpose of this study is to clarify the effect of CHRNG on the proliferation and differentiation of bovine preadipocytes. Methods: We constructed a CHRNG overexpression adenovirus vector and successfully overexpressed it on bovine preadipocytes. The effects of CHRNG on bovine preadipocyte proliferation were detected by Edu assay, cell counting Kit-8 (CCK-8), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), Western blot and other techniques. We also performed oil red O, RT-qPCR, Western blot to explore its effect on the differentiation of preadipocytes. Results: The results of Edu proliferation experiments showed that the number of EDU-positive cells in the overexpression group was significantly less. CCK-8 experiments found that the optical density values of the cells in the overexpression group were lower than those of the control group, the mRNA levels of proliferating cell nuclear antigen (PCNA), cyclin A2 (CCNA2), cyclin B1 (CCNB1), cyclin D2 (CCND2) decreased significantly after CHRNG gene overexpression, the mRNA levels of cyclin dependent kinase inhibitor 1A (CDKN1A) increased significantly, and the protein levels of PCNA, CCNB1, CCND2 decreased significantly. Overexpression of CHRNG inhibited the differentiation of bovine preadipocytes. The results of oil red O and triglyceride determination showed that the size and speed of lipid droplets accumulation in the overexpression group were significantly lower. The mRNA and protein levels of peroxisome proliferator activated receptor gamma (PPAR class="checkNonKBPoint">γ), CCAAT enhancer binding protein alpha (CEBPα), fatty acid binding protein 4 (FABP4), fatty acid synthase (FASN) decreased significantly. Conclusion: Overexpression of CHRNG in bovine preadipocytes inhibits the proliferation and differentiation of bovine preadipocytes.

• Journal of Korean Medicine for Obesity Research
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• v.22 no.2
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• pp.93-101
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• 2022

Objectives: This study aimed to observe the anti-diabetic effect and underlying mechanisms of Galgunhwanggumhwangryun-tang (GHH; Gegen-Qinlian-decoction) in the C2C12 myotubes. Methods: GHH (1.0 mg/ml) or metformin (0.75 mM) or insulin (100 nM) were treated in C2C12 myotubes after 4 days differentiation. The glucose uptake was assessed by 2-[N-(7-160 nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose uptake by C2C12 cells. The expression of adenosine monophosphate-activated protein kinase (AMPK) and phosphorylation AMPK (pAMPK) were measured by western blot. We also evaluated gene expression of glucose transporter type 4 (Slc2a4, formerly known as GLUT4), glucokinase (Gk), carnitine palmitoyltransferase IA (Cpt1a), nuclear respiratory factors 1 (Nrf1), mitochondrial transcription factor A (Tfam), and peroxisome proliferator-activated receptor γ coactivator 1α (Ppargc1a) by quantitative real-time polymerase chain reaction. Results: GHH promoted glucose uptake in C2C12 myotubes. The expression of AMPK protein, which plays an essential role in glucose metabolism, was increased by treatment with GHH. GHH treatment tended to increase gene expression of Slc2a4, Gk, and Nrf1 but was not statistically significant. However, GHH significantly improved Tfam and Ppargc1a gene expression in C2C12 myotubes. Conclusions: In summary, GHH treatment promoted glucose uptake in C2C12 myotubes. We suggest that these effects are associated with increased gene expression involved in mitochondrial biosynthesis and oxidative phosphorylation, such as Tfam and Ppargc1a, and increased expression of AMPK protein.