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Roles of miR-128 in Myogenic Differentiation and Insulin Signaling in Rat L6 Myoblasts (쥐L6 근원세포에서 miR-128의 근육세포 분화와 인슐린신호에서의 역할)

  • Oh, Myung-Ju;Kim, So-Hyeon;Kim, Ji-Hyun;Jhun, Byung H.
    • Journal of Life Science
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    • v.30 no.9
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    • pp.772-782
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    • 2020
  • Skeletal muscle differentiation or myogenesis is important to maintain muscle mass and metabolic homeostasis. Muscle-specific microRNAs (miRNAs) are known to play a critical role in skeletal myogenic differentiation. In this study, we examined the expression profiling of miRNAs during myogenic differentiation in rat L6 myoblasts using rat miRNA microarrays. We identified the upregulated expression of miR-128 as well as several well-known myogenic miRNAs, including miR-1, miR-133b, and miR-206. We additionally confirmed the increased expression of miR-128 observed on microarray through quantitative real-time PCR (qRT-PCR), which showed similarly upregulated expression of both primary miR-128 and mature miR-128, consistent with the microarray findings. Furthermore, transfection of miR-128 into rat L6 myoblasts induced gene expression of myogenic markers such as muscle creatine kinase (MCK), myogenin, and myosin heavy chain (MHC). Protein expression of MHC was increased as well. Inhibition of miR-128 by inhibitory peptide nucleic acids (PNAs) blocked the expression of those myogenic markers. In addition, the transfection of miR-128 into rat L6 myoblasts enhanced the phosphorylation of Erk and Akt proteins stimulated by insulin, while simultaneously reversing the inhibited phosphorylation of Erk and Akt due to insulin resistance. These findings suggest that miR-128 may play important roles in myogenic differentiation and insulin signaling.

Inhibition of miR-128 Abates Aβ-Mediated Cytotoxicity by Targeting PPAR-γ via NF-κB Inactivation in Primary Mouse Cortical Neurons and Neuro2a Cells

  • Geng, Lijiao;Zhang, Tao;Liu, Wei;Chen, Yong
    • Yonsei Medical Journal
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    • v.59 no.9
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    • pp.1096-1106
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    • 2018
  • Purpose: Alzheimer's disease (AD) is the sixth most common cause of death in the United States. MicroRNAs have been identified as vital players in neurodegenerative diseases, including AD. microRNA-128 (miR-128) has been shown to be dysregulated in AD. This study aimed to explore the roles and molecular mechanisms of miR-128 in AD progression. Materials and Methods: Expression patterns of miR-128 and peroxisome proliferator-activated receptor gamma ($PPAR-{\gamma}$) messenger RNA in clinical samples and cells were measured using RT-qPCR assay. $PPAR-{\gamma}$ protein levels were determined by Western blot assay. Cell viability was determined by MTT assay. Cell apoptotic rate was detected by flow cytometry via double-staining of Annexin V-FITC/PI. Caspase 3 and $NF-{\kappa}B$ activity was determined by a Caspase 3 Activity Assay Kit or $NF-{\kappa}B$ p65 Transcription Factor Assay Kit, respectively. Bioinformatics prediction and luciferase reporter assay were used to investigate interactions between miR-128 and $PPAR-{\gamma}$ 3'UTR. Results: MiR-128 expression was upregulated and $PPAR-{\gamma}$ expression was downregulated in plasma from AD patients and $amyloid-{\beta}$ $(A{\beta})-treated$ primary mouse cortical neurons (MCN) and Neuro2a (N2a) cells. Inhibition of miR-128 decreased $A{\beta}-mediated$ cytotoxicity through inactivation of $NF-{\kappa}B$ in MCN and N2a cells. Moreover, $PPAR-{\gamma}$ was a target of miR-128. $PPAR-{\gamma}$ upregulation attenuated $A{\beta}-mediated$ cytotoxicity by inactivating $NF-{\kappa}B$ in MCN and N2a cells. Furthermore, $PPAR-{\gamma}$ downregulation was able to abolish the effect of anti-miR-128 on cytotoxicity and $NF-{\kappa}B$ activity in MCN and N2a cells. Conclusion: MiR-128 inhibitor decreased $A{\beta}-mediated$ cytotoxicity by upregulating $PPAR-{\gamma}$ via inactivation of $NF-{\kappa}B$ in MCN and N2a cells, providing a new potential target in AD treatment.

Nucleotide Sequence and Secondary Structure of 16S rRNA from Sphingomonas chungbukensis DJ77 (Sphingomonas chungbukensis DJ77의 16S rRNA 염기서열과 이차구조)

  • Lee Kwan-Young;Kwon Hae-Ryong;Lee Won-Ho;Kim Young-Chang
    • Korean Journal of Microbiology
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    • v.41 no.2
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    • pp.125-128
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    • 2005
  • A 16S ribosomal RNA gene from S. chungbukensis DJ77 has been sequenced. This sequence had a length of 1,502 bp and was extended for 29 bp at 5' and for 37 bp at 3' from the partial sequence (1,435 bp) registered in 2000 year. Besides, 1 bp was newly added near to the 3' end. We made the secondary structure of the 16S rRNA based on E. coli model and found four specific regions. We found constant and variable regions in genus Sphingomonas as the result of multiple alignment of 16S rRNA gene sequences from Sphingomonas spp. and S. chungbukensis DJ77. We found a stem loop structure in S. chungbukensis DJ77, which was only discovered in C. jejuni to date. It showed the structural agreement despite the difference of the sequences from the both organisms. Finally, S. chungbukensis DJ77 belonged to cluster II (Sphingobium) group, after the classification using phylogenetic analysis and nucleotide signature analysis.

Morphogenetic Identification of Eel's Larva (Leptocephalus) Collected by Set net in Namhae, Korea (남해 정치망에서 채집한 엽상자어(Leptocephalus)의 형태 및 유전학적 특성)

  • Chang-Gi Hong;Kyeong-Ho Han
    • Journal of Marine Life Science
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    • v.8 no.2
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    • pp.128-135
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    • 2023
  • The present study was tried to identify whether the eel's larva was close to a conger (Conger myriaster), a pipe conger (Muraenesox cinereus) or four species of Anguilla. Experimental fishes were collected by set net in the gulf of enggang, Namhae, Korea from May to June. Their morphological characteristics were compared with adult fishes of a conger, a pipe conger and four species of Anguilla. For genetic classification, DNA was isolated and amplified by using 12S rRNA and 16S rRNA primer set. The PCR products were direct sequencing in both directions. The nucleotide sequences were analyzed using softwares. As results of morphological measurement on eel's larva, the percentages of head length and preanal length against total length were similar with a conger. Based on the nucleotide sequences, the phylogenetic tree also revealed a close relationship to a conger. Therefore, eel's larva, caught in Namhae from May to June, was identified into a conger's larva.

Analysis of miRNA expression in the trachea of Ri chicken infected with the highly pathogenic avian influenza H5N1 virus

  • Suyeon Kang;Thi Hao Vu;Jubi Heo;Chaeeun Kim;Hyun S. Lillehoj;Yeong Ho Hong
    • Journal of Veterinary Science
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    • v.24 no.5
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    • pp.73.1-73.16
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    • 2023
  • Background: Highly pathogenic avian influenza virus (HPAIV) is considered a global threat to both human health and the poultry industry. MicroRNAs (miRNA) can modulate the immune system by affecting gene expression patterns in HPAIV-infected chickens. Objectives: To gain further insights into the role of miRNAs in immune responses against H5N1 infection, as well as the development of strategies for breeding disease-resistant chickens, we characterized miRNA expression patterns in tracheal tissues from H5N1-infected Ri chickens. Methods: miRNAs expression was analyzed from two H5N1-infected Ri chicken lines using small RNA sequencing. The target genes of differentially expressed (DE) miRNAs were predicted using miRDB. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis were then conducted. Furthermore, using quantitative real-time polymerase chain reaction, we validated the expression levels of DE miRNAs (miR-22-3p, miR-146b-3p, miR27b-3p, miR-128-3p, miR-2188-5p, miR-451, miR-205a, miR-203a, miR-21-3p, and miR-200a3p) from all comparisons and their immune-related target genes. Results: A total of 53 miRNAs were significantly expressed in the infection samples of the resistant compared to the susceptible line. Network analyses between the DE miRNAs and target genes revealed that DE miRNAs may regulate the expression of target genes involved in the transforming growth factor-beta, mitogen-activated protein kinase, and Toll-like receptor signaling pathways, all of which are related to influenza A virus progression. Conclusions: Collectively, our results provided novel insights into the miRNA expression patterns of tracheal tissues from H5N1-infected Ri chickens. More importantly, our findings offer insights into the relationship between miRNA and immune-related target genes and the role of miRNA in HPAIV infections in chickens.

Molecular Identification, Ontogeny and Evolutionary Note of Echelus uropterus Leptocephali (날붕장어(Echelus uropterus) 엽상자어의 분자동정, 개체발생 및 진화학적 고찰)

  • Ji, Hwan-Sung;Lee, Soo-Jeong;Kim, Jin-Koo
    • Korean Journal of Ichthyology
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    • v.23 no.3
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    • pp.217-224
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    • 2011
  • The seven leptocephali (14.6~68.0 mm in total length) of the family Ophichthidae, from the East Sea of Korea, were identified using 12S rRNA 861-bp sequences and their morphological development were described. Our seven leptocephali were identified to Echelus uropterus based on genetic distance (d=0.000) being well consistent with that of adult E. uropterus. Our seven leptocephali of E. uropterus are characterized by morphological combinations: caudal fin present; the melanophores present in series of nine gut swellings; total myomeres 154~158; body depth in total length less than 10%; eight black horizontal bands present from anus to caudal fin base. Our morphological and molecular results suggest that E. uropterus may be an intermediate group between subfamily Ophichthinae and Myrophinae.

Isolation and Identification of Vibrio Species Contaminated in Imported Frozen Seafoods (수입냉동 어패류에 오염되어 있는 Vibrio속 세균의 분리 및 동정)

  • 윤영준;김도연;이실한;이우윤;고영환;김승곤;김정완
    • Journal of Food Hygiene and Safety
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    • v.15 no.2
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    • pp.128-136
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    • 2000
  • Twenty-four Vibrio strains were isolated from imported frozen seafoods and identified according to their physiological and biochemical properties. They included two V cholerae non-01 sp., two V. diazotrophicus sp., one V. hollisae sp., five V. natriegens sp., eight V. fluvialis sp., and four V. nereis sp.. Two of them were not identified as Vibrio species. When these strains were tested using API-2OE kit fur identification, however, only the results for two V. cholerae and five of the V. fluvialis strains matched the results obtained previously. Due to the importance of detecting V cholerae from foods, phylogenetic identification of the strains was attempted based on restriction fragment length polymorphism (RFLP) of the 16S rDNAs amplified by PCR. The results suggested that the two strains had identical RFLP patterns which were more closely related to that of V. proteolyticus than V. cholerae. The problems associated with identification of pathogens originated from seafoods demand development of accurate and rapid identification methods.

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Isolation and Characterization of Lactic Acid Bacteria from Kimchi, Korean Traditional Fermented Food to Apply into Fermented Dairy Products

  • Cho, Young-Hee;Hong, Sung-Moon;Kim, Cheol-Hyun
    • Food Science of Animal Resources
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    • v.33 no.1
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    • pp.75-82
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    • 2013
  • This study aimed to isolate lactic acid bacteria (LAB) from Kimchi and to identify suitable probiotic strain for application in fermented dairy product as a commercial starter culture. A total of 106 (LAB) strains were isolated from Kimchi collected from different regions in Korea and their phenotypic characteristics were assayed. Four isolates from MRS agar plates were selected and designated as DKL109, DKL119, DKL121 and DKL128. They were identified first by API 50 CHL kit and then 16S rRNA gene sequencing. DKL121 and DKL128 were identified as Lactobacillus paracasei and Lactobacillus casei, respectively. Other two isolates (DKL109 and DKL119) were identified as Lactobacillus plantarum. To estimate their applicability in dairy products, the characteristics including acid and bile tolerance, cold shock induced cryotolerance and enzymatic activities were determined. There was wide variation in ability of strains to acid tolerance, but no significant differences in bile tolerance, cold shock induced cryotolerance within selected strains. DKL119 and DKL121 showed the highest resistance to acid and bile and the highest ${\beta}$-galactosidase activity, respectively. When these two strains were used for yogurt preparation as a single starter culture, their viable cell counts reached to $1.0{\times}10^9CFU/mL$. Lactobacillus plantarum DKL119 showed faster acid development than commercial starter culture. Also storage trials at $10^{\circ}C$ showed that the viability of these strains was retained over 15 d. With these results, it was indicated that probiotics isolated from Kimchi can be used in yogurt manufacturing as a starter culture.

A report of 29 unrecorded bacterial species belonging to the phylum Bacteroidetes in Korea

  • Cho, Sang Hyun;Yoon, Jung-Hoon;Kim, Seung-Bum;Jahng, Kwang-Yeop;Cho, Jang-Cheon;Joh, Ki-seong;Cha, Chang-Jun;Seong, Chi-Nam;Bae, Jin-Woo;Im, Wan-Taek;Jeon, Che Ok
    • Journal of Species Research
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    • v.6 no.2
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    • pp.119-128
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    • 2017
  • Within a comprehensive, widescale investigation of indigenous prokaryotic species in Korea, 29 bacterial strains in the phylum Bacteroidetes were isolated from diverse environmental habitats that included soil, plant roots, natural caves, tidal flats, freshwater from lakes, and seawater. Based on their high 16S rRNA gene sequence similarities (>99.1%) and the formation of robust phylogenetic clades with the closest type species, each strain likely belonged to an independent and predefined bacterial species. There are no publications or official reports of the isolation of these 29 species in Korea. Our study provides strong evidence that seven species in three genera in the order Cytophagales, 15 species in 13 genera in the order Flavobacteriales and seven species in five genera in the order Sphingobacteriales, all within the phylum Bacteriodetes, are new reports of bacterial species in Korea. Gram reaction, colony and cell morphology, basic biochemical characteristics, isolation source, and strain IDs are described in the species description section.

Characterization of Extracellular $\alpha$-Galactosidase Produced by Bacillus licheniformis YB-42. ($\alpha$-Galactosidase를 생산하는 Bacillus lichennformis YB-42의 분리와 효소 특성)

  • 김현숙;이경섭;소재호;이미성;최준호;윤기홍
    • Microbiology and Biotechnology Letters
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    • v.32 no.2
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    • pp.128-134
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    • 2004
  • A bacterium producing the $\alpha$-galactosidase was isolated from Korean soybean paste. The isolate YB-42 has been identified as Bacillus licheniformis on the basis on its 16S rRNA sequence, morphology and biochemical properties. The $\alpha$-galactosidase activity was detected in both the culture supernatant and the cell extract of B. licheniformis YB-42. The partially purified extracellular $\alpha$-galactosidase was obtained from the culture supernatant by DEAE-Sepharose column and Q-Sepharose column chromatography. The enzyme showed the maximum activity for hydrolysis of para-nitrophenyl-$\alpha$-D-galactopyranoside (pNP-$\alpha$Gal) at pH 6.5 and $45^{\circ}C$. It was able to hydrolyze oligomeric substrates such as melibiose, raffmose and stachyose to liberate galactose residue, indicating that the a-galactosidase of B. licheniformis YB-42 hydrolyzed $\alpha$-1,6 linkage. The hydrolyzing activity of $\alpha$-galactosidase for both pNP-$\alpha$Gal and melibiose was dramatically decreased by galactose. Both glucose and mannose inhibited the activity for pNP-$\alpha$Gal less than galactose.