• The Korean Journal of Mycology
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• v.26 no.4 s.87
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• pp.450-457
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• 1998

The cryparin of Cryphonectria parasitica belongs to a cell wall associated fungal hydrophobin. The cryparin, though it is encoded by a single copy gene, is known for the high expression during the liquid culture of C. parasitica, and it turns out that 22% of total mRNA was transcribed for cryparin at 48hr after the liquid culture. In addition, it is also known as one of down-regulated fungal proteins by the presence of double stranded RNA virus, Cryphonectria hypovirus 1. In previous studies (Kim et al., 1999), we have constructed a cryparin-null mutant by replacing the cryparin gene with hygromycin B resistance gene due to site directed homologous recombination. In order for the promoter analysis of cryparin which seems to be very strong as well as mycoviral specific, it is preferable to have a strain with only a target promoter replaced and a discernable target site for incoming vectors. However, the cryparin-null mutant revealed the presence of an additional copy of transforming vector except the one which replaced the cryparin gene. In addition, the cryparin-null mutant did not contain any markers for targeted integration of incoming vectors. This prompts us to design an experiment to obtain a strain for promoter analysis of cryparin gene. A different mating type strain EP6(Mata, $met^-$) was mated with the cryparin-null mutant ${\triangle}$Crp194-7(MatA, Crp${\triangle}$::hph) to make the progenies with only a single replacement vector and $met^-$ characteristic remained. Nutritional assay as well as Southern blot analysis revealed that the progeny, ${\triangle}$Crp194-a6, was the methionine auxotroph with a single replacing vector in genome. Northern blot analysis and PAGE showed that there was no cryparin produced in this bred strain either.

• Korean Journal of Clinical Laboratory Science
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• v.51 no.3
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• pp.309-315
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• 2019

Cervical cancer is the fourth most common malignant neoplasm in women worldwide. Most cases of cervical cancer are caused by an infection by the human papillomavirus. Molecular diagnostic methods have emerged to detect the HPV for sensitivity, specificity, and objectivity. In particular, real-time PCR has been introduced to acquire a more sensitive target DNA or RNA. RNA extraction and complementary DNA synthesis are proceeded before performing real-time PCR targeting RNA. To identify an adequate and sensitive cDNA synthesis kit, this study evaluated the two commonly used kits for cDNA synthesis. The results show that the $R^2$ and efficiency (%) of the two cDNA synthesis kits were similar in the cervical cancer cell lines. On the other hand, the Takara kit compared to Invitrogen kit showed P<0.001 in the $10^2$ and $10^3$ SiHa cell count. The Takara kit compared to the Invitrogen kit showed P<0.001 in the $10^1$ and $10^2$ HeLa cell count. Furthermore, 8, 4, 2, 1, and 0.5 ml of forty exfoliated cell samples were used to compare the cDNA synthesis kits. The Takara kit compared to the Invitrogen kit showed P<0.01 in 8, 4, and 1 ml and P<0.05 in 0.5 mL. The study was performed to identify the most appropriate cDNA synthesis kit and suggests that a cDNA synthesis kit could affect the real-time PCR results.

• Journal of Microbiology and Biotechnology
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• v.31 no.2
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• pp.197-206
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• 2021

microRNA-361-3p (miR-361-3p) is involved in the carcinogenesis of oral cancer and pancreatic catheter adenocarcinoma, and has anti-carcinogenic effects on non-small cell lung cancer (NSCLC). However, its effect on multiple myeloma (MM) is less reported. Here, we found that upregulating the expression of miR-361-3p inhibited MM cell viability and promoted MM apoptosis. We measured expressions of tumor necrosis factor receptor-associated factor 6 (TRAF6) and miR-361-3p in MM cells and detected the viability, colony formation rate, and apoptosis of MM cells. In addition, we measured expressions of apoptosis-related genes Bcl-2, Bax, and Cleaved caspase-3 (C caspase-3). The binding site between miR-361-3p and TRAF6 was predicted by TargetScan. Our results showed that miR-361-3p was low expressed in the plasma of MM patients and cell lines, while its overexpression inhibited viability and colony formation of MM cells and increased the cell apoptosis. Furthermore, TRAF6, which was predicted to be a target gene of miR-361-3p, was high-expressed in the plasma of patients and cell lines with MM. Rescue experiments demonstrated that the effect of TRAF6 on MM cells was opposite to that of miR-361-3p. Upregulation of miR-361-3p induced apoptosis and inhibited the proliferation of MM cells through targeting TRAF6, suggesting that miR-361-3p might be a potential target for MM therapy.

• The Journal of Pediatrics of Korean Medicine
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• v.26 no.4
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• pp.10-23
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• 2012

Objectives: Gagamyanggyeoksan (G-YGS) has been used to suppress allergic reaction, however, the cellular target of G-YGS and its mode of action remain unclear. The present study was designed to investigate the effect of extracted G-YGS on the PMA and lonomycin (PI)-induced activation of RBL-2H3. Methods: For this investigation, We examined IL-4, IL-13 mRNA expression by Real-Time PCR, IL-4, IL-13 production by ELISA analysis and manifestations of GATA-1, GATA-2, NF-AT1, NF-AT2, AP-1 and NF-${\kappa}B$ p65 transcription factors by western blotting, OVA-specific IgE, IL-4, IL-13 by mouse be sensitive to OVA. Results: Here we showed that treatment of RBL-2H3 mast cells with G-YGS, suppressed PI-induced production of Th2 cytokines including IL-4 and IL-13 in a dose dependent manner. The mRNA expression of IL-4 were completely abolished by G-YGS at the concentration of $100{\mu}g/ml$. Data from a stable cell lines consistently expressing IL-4. And the mRNA expression of IL-13 were abolished by G-YGS at the $200{\mu}g/ml$. But there is no difference between the $50{\mu}g/ml$, the $100{\mu}g/ml$ and the comparison. Results from the western blot analysis of transcription factors involving IL-4 and IL-13 expression indicated that it prominently decreased the expression of mast cell specific transcricption factors including GATA-1, GATA-2, NF-AT2, c-Jun, NF-${\kappa}B$ p65 but not c-Fos. And G-YGS suppressed IgE, IL-4, IL-13 in mouse be sensitive to OVA. Conclusions We suggested the anti-allergic activities of G-YGS might be mediated by down-regulation of Th2 cytokines such as IL-4 and IL-13 through the regulation of transcription factors as GATA-1, GATA-2, NF-AT2, c-Jun, NF-${\kappa}B$ p65.

• Molecules and Cells
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• v.37 no.3
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• pp.213-219
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• 2014

MicroRNAs (miRNAs) represent a class of small non-coding regulatory RNAs that play important roles in normal hematopoiesis, including erythropoiesis. Although studies have identified several miRNAs that regulate erythroid commitment and differentiation, we do not understand the mechanism by which the crucial erythroid transcription factors, GATA-1and NF-E2 directly regulate and control differentiation via miRNA pathways. In this study, we identified miR-199b-5p as a key regulator of human erythropoiesis, and its expression was up-regulated during the erythroid differentiation of K562 cells. Furthermore, the increase of miR-199b-5p in erythroid cells occurred in a GATA-1- and NF-E2-dependent manner during erythrocyte maturation. Both GATA-1 and NF-E2 bound upstream of the miR-199b gene locus and activated its transcription. Forced expression of miRNA-199b-5p in K562 cells affected erythroid cell proliferation and maturation. Moreover, we identified c-Kit as a direct target of miR-199b-5p in erythroid cells. Taken together, our results establish a functional link among the erythroid transcription factors GATA-1/NF-E2, miR-199b-5p and c-Kit, and provide new insights into the coupling of transcription and post-transcription regulation in erythroid differentiation.

• Journal of Korean Medicine for Obesity Research
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• v.23 no.1
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• pp.1-9
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• 2023

Objectives: Noni fruit juice (NFJ) is liquor extracted from Morinda citrifolia (noni) fruit and has been used as an herbal remedy in many countries. However, the NFJ's anti-adipogenic and anti-inflammatory effects on adipocytes are poorly understood. The purpose of this study was to explore the commercially standardized NFJ effects on lipid accumulation throughout 3T3-L1 preadipocytes differentiation and interleukin-1β (IL-1β)-mediated inducible nitric oxide synthase (iNOS) expression in 3T3-L1 preadipocytes. Methods: Cellular lipid accumulation and triglyceride (TG) content in differentiating 3T3-L1 preadipocytes were assessed subsequently via the Oil Red O staining and AdipoRed assay. MTS assay was used to examine NFJ cytotoxicity in (differentiating) 3T3-L1 preadipocytes. Immunoblotting and reverse transcriptase polymerase chain reaction analysis were used to measure the expression levels of target protein and mRNA in (differentiating) 3T3-L1 preadipocytes, respectively. Results: NFJ treatment at 150 μL/mL led to a substantial reduction of fat accumulation and TG content during 3T3-L1 adipogenesis with no discernable impact on the cell viability. Of note, while NFJ treatment (150 μL/mL) largely inhibited the CCAAT/enhancer-binding protein-α (C/EBP-α) and peroxisome proliferator-activated receptor-β (PPAR-β) protein expressions, it did not influence PPAR-γ in differentiating 3T3-L1 preadipocytes. Of interest, treatment with IL-1β at 20 ng/mL for 4 hours elicited in firm induction of iNOS mRNA expression in 3T3-L1 preadipocytes. However, NFJ treatment at 100 or 200 μL/mL greatly attenuated the IL-1β-induced iNOS mRNA expression in 3T3-L1 preadipocytes. Conclusions: NFJ has anti-adipogenic and anti-inflammatory effects on (differentiating) 3T3-L1 preadipocytes which are in part intervened via control of the expression of C/EBP-α, PPAR-β, and iNOS.

• Biomolecules & Therapeutics
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• v.22 no.1
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• pp.17-26
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• 2014

${\alpha}$-Asarone exhibits a number of pharmacological actions including neuroprotective, anti-oxidative, anticonvulsive, and cognitive enhancing action. The present study investigated the effects of ${\alpha}$-asarone on pro-inflammatory cytokines mRNA, microglial activation, and neuronal damage in the hippocampus and on learning and memory deficits in systemic lipopolysaccharide (LPS)-treated C57BL/6 mice. Varying doses of ${\alpha}$-asarone was orally administered (7.5, 15, or 30 mg/kg) once a day for 3 days before the LPS (3 mg/kg) injection. ${\alpha}$-Asarone significantly reduced TNF-${\alpha}$ and IL-$1{\beta}$ mRNA at 4 and 24 hours after the LPS injection at dose of 30 mg/kg. At 24 hours after the LPS injection, the loss of CA1 neurons, the increase of TUNEL-labeled cells, and the up-regulation of BACE1 expression in the hippocampus were attenuated by 30 mg/kg of ${\alpha}$-asarone treatment. ${\alpha}$-Asarone significantly reduced Iba1 protein expression in the hippocampal tissue at a dose of 30 mg/kg. ${\alpha}$-Asarone did not reduce the number of Iba1-expressing microglia on immunohistochemistry but the average cell size and percentage areas of Iba1-expressing microglia in the hippocampus were significantly decreased by 30 mg/kg of ${\alpha}$-asarone treatment. In the Morris water maze test, ${\alpha}$-asarone significantly prolonged the swimming time spent in the target and peri-target zones. ${\alpha}$-Asarone also significantly increased the number of target heading and memory score in the Morris water maze. The results suggest that inhibition of pro-inflammatory cytokines and microglial activation in the hippocampus by ${\alpha}$-asarone may be one of the mechanisms for the ${\alpha}$-asarone-mediated ameliorating effect on memory deficits.

• Biomedical Science Letters
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• v.24 no.4
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• pp.430-434
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• 2018

Candida glabrata is the second most prevalent causative agent for candidiasis following C. albicans. The opportunistic yeast, C. glabrata, is able to cause the critical bloodstream infections in hospitalized patients. Conventional identification methods for yeasts are often time consuming and labor intensive. Therefore, recent studies on sequence-based identification have been conducted. Recently, sequencing the D1/D2 domain of the large subunit ribosomal RNA gene and the internal transcribed spacers (ITS) 1 and ITS2 regions of the ribosomal DNA has proven useful for DNA-based identification of most species of fungi. In the present study, therefore, fungal ITS and D1/D2 domain regions were targeted and analyzed by DNA sequencing for the accurate identification of C. glabrata clinical isolates. A total of 102 C. glabrata clinical isolates from various clinical samples including bloodstream, catheterized urine, bile and other body fluids were used in the study. The results of the DNA sequence analysis showed that the mean standard deviation of species identity percent score between ITS and D1/D2 domain regions was $97.8%{\pm}2.9$ and $99.7%{\pm}0.46$, respectively. These results revealed that the D1/D2 domain region might be a better target for identifying C. glabrata clinical isolates based on DNA sequences than the ITS1 and ITS2 regions. However, in order to evaluate the usefulness of D1/D2 domain region for species identification of all Candida species, other Candida species such as C. albicans, C. tropicalis, C. dubliniensis, and C. krusei should be verified in further studies additionally.

• Korean Journal of Environmental Agriculture
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• v.29 no.1
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• pp.77-85
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• 2010

Reactive oxygen species (ROS) are very harmful to living organisms due to the potential oxidation of membrane lipids, DNA, proteins, and carbohydrates. transformed E.coli strain QC 871, superoxide dismutase (SOD) double-mutant, with three sequence variant MnSOD1, MnSOD2, and MnSOD3 manganese superoxide dismutase (MnSOD) gene isolated from wheat. Although all QC 871 transformants grown at $37^{\circ}C$ expressed mRNA of MnSOD variants, only MnSOD2 transformant had functional SOD activity. MnSOD3 expressed active protein when grown at $22^{\circ}C$, however, MnSOD1 did not express functional protein at any growing and induction conditions. The sequence comparison of the wheat MnSOD variants revealed that the only amino acid difference between the sequence MnSOD2 and sequences MnSOD1 and 3 is phenylalanine/serine at position 58 amino acid. We made MnSOD2S58F gene, which was made by altering the phenylalaine to serine at position 58 in MnSOD2. The expressed MnSOD2S58F protein had functional SOD activity, even at higher levels than the original MnSOD2 at all observed temperatures. These data suggest that amino acid variation can result in highly active forms of MnSOD and the MnSOD2S58F gene can be an ideal target used for transforming crops to increase tolerance to environmental stresses.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.1
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• pp.9-15
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• 2005

We attempted to analyze the mechanism of polychlorinated biphenyl (PCB)-induced neurotoxicity and identify the target molecules in the neuronal cells for PCBs.Since the developing neuron is particularly sensitive to PCB-induced neurotoxicity, we isolated cerebellar granule cells derived from 7-day old Sprague Dawley (SD) rats and grew cells in culture for additional 7 days to mimic PND-14 conditions. Only non-coplanar PCBs at a high dose showed a significant increase of total protein kinase C (PKC) activity at phobol 12,13-dibutyrate ([$^3M$]PDBu) binding assay, indicating that non-coplanar PCBs are more neuroactive than coplanar PCBs in neuronal cells. PKC isozymes were immunoblotted with the selected monoclonal antibodies. PKC-${\alpha}$, ${\delta}$, and ε were activated with non-coplanar PCB exposure. Receptor for activated C kinase-1 (RACK-1), anchoring protein for activated PKC, was more induced with exposure to coplanar PCBs than non-coplanar PCBs. Reverse transcription PCR (RT-PCR) analysis showed induction of neurogranin (RC-3) and growth associated protein-43 (GAP-43) mRNA with non-coplanar PCBs. The results indicate that these factors may be useful biomarkers for differentiating non-coplanar PCBs from coplanar PCBs. The present study demonstrated that non-coplanar PCBs are more neuroactive congeners than coplanar PCBs.