• Animal cells and systems
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• v.1 no.1
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• pp.143-150
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• 1997

Transcription factor CP2 was identified initially to bind the promoter region of the murine a-globin gene and its activity was shown to increase 2 to 3 fold during the induced differentiation of murine erythroleukemia (MEL) cells. To get further insight into the role of CP2 during development and differentiation, steady-state levels of CP2 message were monitored by using reverse transcriptase (RT)-PCR and in situ hybridization assays in the cultured MEL cells and differentiating embryonic stem (ES) cells in vitro, and in fetal and adult mouse tissues. The amount of CP2 messages increased 3 to 5 fold during induced differentiation of MEL cells, suggesting that the increment of CP2 activity during induced differentiation of MEL cells is originated from the increase of transcription initiation. On the other hand, CP2 expression is not restricted to the erythroid lineage cells; CP2 expressed ubiquitously from the undifferentiated ES cells to adult tissue cells. CP2 transcript was observed even in the undifferentiated ES cells and the level of expression increased from day 8 of the differentiating embryoid bodies. RT-PCR assay in the total RNAs prepared from several tissues of the adult mouse also showed ubiquitous expression profile, although the levels of expression were variable among tissues. When non-radioactive in situ hybridization assay was performed to the paraffin-sectioned whole body mouse embryos at days 11.5, 13.5, and 16.5 after fertilization, variable amounts of positive signals were also detected in different tissues.

• Korean Journal of Pharmacognosy
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• v.53 no.2
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• pp.102-110
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• 2022

Alzheimer's disease (AD) is the most common form of dementia, and the accumulation of β-amyloid (Aβ) in the brain triggers AD, followed by hyperphosphorylation of tau protein, neurofibrillary tangles, and synapses loss, neuronal cell death, and cognitive decline occur in a chain. In APPswe neuronal cell line, 50 ㎍/ml of Campbell early (Vitis labruscana B.) leaves 50% ethanol extract (VLL) treatment inhibited the secretion of Aβ1-42 by about 63% and the secretion of Aβ1-40 by about 50%. VLL did not target the enzymatic activity of the amyloidogenic pathway and decreased the protein expression of APP. As a result of RT-qPCR (Reverse transcription-quantitative real-time PCR) of the APPswe cell line treated with VLL, it is thought that the protein expression of APP was reduced by inhibiting the transcription process of the APP gene. In addition, VLL inhibited acetylcholinesterase (AChE) enzyme activity in vitro by 27.6% and 54.7%, respectively, at 50 and 100 ㎍/ml concentrations. We found that VLL inhibited the production of Aβ, a dementia-inducing substance, by suppressing the transcription of the APP gene, and that VLL inhibited AChE activity. We suggest that VLL has the potential as a natural drug material that modulates the alleviation of dementia symptoms.

• Development and Reproduction
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• v.4 no.2
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• pp.231-236
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• 2000

Recent studies have clearly shown that the expression of genes for gonadotropin-releasing hormone (GnRH) and its receptor in the rat reproductive organs including ovary, testis, placenta uterus and mammary gland. Moreover, luteinizing hormone (LH) classically known to be a main target product of GnRH in anterior pituitary has been found in rat gonads. These findings suggested the presence of local circuit composed of GnRH and LH in the rat gonads. The present study was undertaken to elucidate whether the genes for LH and its receptor are expressed in rat mammary gland. Expression of LH and its receptor genes in the rat mammary gland was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) and specific LH radioimmunoassay (RIA). The LH${\beta}$ transcripts in the mammary gland from cycling rats contained the pituitary type of LH${\beta}$ exons 1~3 encoding the entire LH${\beta}$ polypeptide but lacked the rat testis-specific LH${\beta}$ exon(s). Presence of ${\alpha}$ -subunit transcripts in the rat mammary gland were determined by RT-PCR. The cDNA fragments encoding exons 2~7 of rat LH receptor transcripts were amplified in both rat ovary and mammary gland samples. We could detect the GnRH expression in mammary gland from cycling virgin rats, and this result disagreed with previous report that mammary GnRH expression is occured in lactating rats only. Considerable amounts of immunoreactive LH molecules with good RIA parallelism in standard curve were detected in crude extracts from the rat mammary gland, indicating that the immunoreactive LH materials in the gland might be identical to authentic pituitary LH. To our knowledge, the present study demonstrated for the first time the expression of LH subunits and LH receptor in the rat mammary gland. Our findings suggested that the mammary gland might be the novel source and target of LH and the mammary LH could be act as a local regulator with auto-and/or paracrine manner under the regulation of local GnRH.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.10
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• pp.1550-1557
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• 2018

Objective: Circular RNAs (circRNAs) are a newfound class of non-coding RNA in animals and plants. Recent studies have revealed that circRNAs play important roles in cell proliferation, differentiation, autophagy and apoptosis during development. However, there are few reports about muscle development-related circRNAs in livestock. Methods: RNA sequencing analysis was employed to identify and annotate circRNAs from longissimus dorsi of sheep. Reverse transcription followed by real-time quantitative (q) polymerase chain reaction (PCR) analysis verified the presence of these circRNAs. Targetscan7.0 and miRanda were used to analyse the interaction of circRNA-microRNA (miRNA). To investigate the function of circRNAs, an experiment was conducted to perform enrichment analysis hosting genes of circRNAs using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. Results: About 75.5 million sequences were obtained from RNA libraries of sheep skeletal muscle. These sequences were mapped to 729 genes in the sheep reference genome. We identified 886 circRNAs, including numerous circular intronic RNAs and exonic circRNAs. Reverse transcription PCR (RT-PCR) and DNA sequencing analysis confirmed the presence of several circRNAs. Real-Time RT-PCR analysis exhibited resistance of sheep circRNAs to RNase R digestion. We found that many circRNAs interacted with muscle-specific miRNAs involved in growth and development of muscle, especially circ776. The GO and KEGG enrichment analysis showed that hosting genes of circRNAs was involved in muscle cell development and signaling pathway. Conclusion: The study provides comprehensive expression profiles of circRNAs in sheep skeletal muscle. Our study offers a large number of circRNAs to facilitate a better understanding of their roles in muscle growth. Meanwhile, we suggested that circ776 could be analyzed in future study.

• The Journal of Korean Society of Virology
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• v.29 no.4
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• pp.247-259
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• 1999

Small round structured viruses (SRSV) are the major ethological agents which can cause outbreaks of non-bacterial gastroenteritis or food poisoning both in children and adults. The classification of family Caliciviridae to which SRSV belong, is based on the genome encoding three open reading frames. The rotavirus is another major pathogen which causes diarrhea in young children. We examined stool specimens obtained from diarrheal patients in Wonju from which bacterial pathogens were not found. To detect causative viruses from stool specimens of patients, reverse transcription (RT)-polymerase chain reaction (PCR) or nested PCR using rotavirus or SRSV specific primers was performed. In this study, RT-nested PCR procedure which can amplify a 330 bp fragment derived from RNA dependent RNA polymerase (RDRP) region within ORF1 was applied for the detection of SRSV. For the detection of rotaviruses, a 877 bp fragment from the VP4 region of rotavirus genome was amplified. As a result, rotavirus was not detected while SRSV sequences were detected from one out of five specimens. The nucleotide and amino acid sequences of the Wonju isolate were compared with other 6 Korean isolates which have been isolated and sequenced in our laboratory. Sequence analysis revealed that the Wonju isolate was rather distinct from other Korean isolates: the Wonju isolate was closer to genogroup I of SRSV while other 6 Korean isolates belonged to genogroup II.

• Journal of Animal Science and Technology
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• v.50 no.2
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• pp.177-184
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• 2008

Acid-labile subunit(ALS) is a 85-kDa glycosylated plasma protein which forms a 150-kDa ternary complex with 7.5-kDa insulin-like growth factor(IGF) and 40~45-kDa IGF-binding protein-3. In a previous study, the present authors prepared a porcine ALS(pALS) expression construct by inserting a pALS coding sequence into a plasmid vector following synthesis of the sequence by reverse transcription-polymerase chain reaction(RT-PCR). The expression construct, however, was subsequently found to have a mis-sense mutation at two bases of the pALS coding sequence which is presumed to have occurred through a PCR error. In the present study, the correct coding sequence was synthesized by the site-directed mutagenesis and inserted into the pET-28a(+) plasmid expression vector containing the His-tag sequence flanking the last codon of the insert DNA. After induction of the expression construct in E. coli BL21(DE3) cells, the resulting presumptive recombinant peptide was purified by the Ni-affinity chromatography. Upon SDS- PAGE, the affinity-purified peptide was resolved as a single band at a 66-kDa position which is consistent with the expected molecular mass of the presumptive recombinant pALS. Collectively, results indicate that a recombinant pALS peptide was successfully expressed and purified in the present study.

• Journal of Life Science
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• v.31 no.12
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• pp.1100-1109
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• 2021

The halophyte Artemisia scoparia is an edible medicinal plant, with insecticidal, anti-inflammatory, anticholesterol, antipyretic, and antibacterial effects. The aim of this study was to assess the inhibitory effect of crude extract and solvent-partitioned fractions obtained from A. scoparia on MMP-2 and MMP-9 activity in phorbol-12-myristate-13-acetate (PMA)-stimulated human fibrosarcoma HT-1080 cells using four different activity tests: gelatin zymography, MMP enzyme-linked immunosorbent assay (ELISA), wound healing assay, reverse transcription-polymerase chain reaction (RT-PCR), and Western blot assay. A. scoparia samples were extracted twice with methylene chloride (MC) and twice with methanol (MeOH). After the MC and MeOH crude extracts were combined, the combined crude extracts showed a significant inhibitory effect against MMP-2 and MMP-9 enzymes. They were then fractionated into n-hexane, 85% (v/v) aqueous methanol (85% (v/v) aq.MeOH), n-butanol, and water according to solvent polarity. Among the four solvent-partitioned fractions, n-hexane and 85% (v/v) aq. MeOH fractions significantly inhibited MMP-2 and MMP-9 activity and cell mobility. In addition, the n-hexane and 85% (v/v) aq.MeOH fractions effectively inhibited MMP-2 and -9 activity in the gelatin zymography and MMP ELISA assay. In the wound healing assay, RT-PCR, and Western blot assay, all solvent-partitioned fractions, except the H₂O fraction, significantly suppressed cell migration, as well as the expression levels of MMP-2 and -9 mRNA and proteins.

• Journal of Ginseng Research
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• v.45 no.6
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• pp.717-725
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• 2021

Background: Korean Red Ginseng (KRG) is a traditional herb that has several beneficial properties including anti-aging, anti-inflammatory, and autophagy regulatory effects. However, the mechanisms of these effects are not well understood. In this report, the underlying mechanisms of anti-inflammatory and autophagy-promoting effects were investigated in aged mice treated with KRG-water extract (WE) over a long period. Methods: The mechanisms of anti-inflammatory and autophagy-promoting activities of KRG-WE were evaluated in kidney, lung, liver, stomach, and colon of aged mice using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), and western blot analysis. Results: KRG-WE significantly suppressed the mRNA expression levels of inflammation-related genes such as interleukin (IL)-1β, IL-8, tumor necrosis factor (TNF)- α, monocyte chemoattractant protein-1 (MCP-1), and IL-6 in kidney, lung, liver, stomach, and colon of the aged mice. Furthermore, KRG-WE downregulated the expression of transcription factors and their protein levels associated with inflammation in lung and kidney of aged mice. KRG-WE also increased the expression of autophagy-related genes and their protein levels in colon, liver, and stomach. Conclusion: The results suggest that KRG can suppress inflammatory responses and recover autophagy activity in aged mice.

• Microbiology and Biotechnology Letters
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• v.49 no.3
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• pp.305-315
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• 2021

The cotton leafworm, Spodoptera littoralis, is a major pest in Egypt and many countries worldwide, and causes heavy economic losses. As a result, management measures to control the spread of the worm are required. S. littoralis nucleopolyhedrovirus (SpliNPV) is one of the most promising bioagents for the efficient control of insect pests. In this study, a chitinase gene (chitA) of a 1.8 kb DNA fragment was cloned and fully characterized from SpliNPV-EG1, an Egyptian isolate. A sequence of 601 amino acids was deduced when the gene was completely sequenced with a predicted molecular mass of 67 kDa for the preprotein. Transcriptional analyses using reverse transcription polymerase chain reaction (RT-PCR) revealed that chitA transcripts were detected first at 12 h post infection (hpi) and remained detectable until 168 hpi, suggesting their transcriptional regulation from a putative late promoter motif. In addition, quantitative analysis using quantitative RT-PCR showed a steady increase of 7.86-fold at 12 hpi in chitA transcription levels, which increased up to 71.4-fold at 120 hpi. An approximately 50 kDa protein fragment with chitinolytic activity was purified from ChitA-induced bacterial culture and detected by western blotting with an anti-recombinant SpliNPV chitinase antibody. Moreover, purification of the expressed ChitA recombinant protein showed in vitro growth inhibition of two different fungi species, Fusarium solani and F. oxysporum, confirming that the enzyme assembly and activity was correct. The results supported the potential role and application of the SpliNPV-ChitA protein as a synergistic agent in agricultural fungal and pest control programs.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.16
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• pp.7031-7038
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• 2015

Background: Arginine may play important roles in tumor progression by providing ornithine for polyamine biosynthesis, required for cell growth. The aim of this work was to determine the expression of arginine metabolic pathway enzymes in head and neck squamous cell carcinoma (HNSCC) in northeast India. Materials and Methods: The expressions of arginase isoforms (ARG1 and ARG2), ornithine aminotransferase (OAT) and ornithine decarboxylase (ODC) were examined in fifty paired HNSCC and adjacent non-tumor tissues by immunohistochemistry. Immunocytochemistry, semiquantitative reverse transcription sq-PCR and quantitative real-time qPCR were used to assess protein and mRNA expressions in peripheral blood of fifty HNSCC patients and hundred controls. Results: ARG1 and ODC protein and mRNA were strongly expressed in peripheral blood from HNSCC patients. No ARG2 expression was observed. In vivo, expression of ARG1, ARG2 and ODC was significantly higher in tumor than in non-tumor tissues. Most tumors expressed low levels of OAT, with no difference in tissues or blood, compared to controls. The absolute extent of maximal ARG1 upregulation with qPCR showed 6.23 fold increase in HNSCC. Conclusions: These findings strongly suggest that in HNSCCs, the ARG1 pathway is stimulated leading to the formation of polyamines as indicated by higher ODC expression, which promote tumor growth.