• BMB Reports
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• v.51 no.7
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• pp.350-355
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• 2018

HnRNPK is a multifunctional protein that participates in chromatin remodeling, transcription, RNA splicing, mRNA stability and translation. Here, we uncovered the function of hnRNPK in regulating the proliferation and differentiation of myoblasts. hnRNPK was mutated in the C2C12 myoblast cell line using the CRISPR/Cas9 system. A decreased proliferation rate was observed in hnRNPK-mutated cells, suggesting an impaired proliferation phenotype. Furthermore, increased G2/M phase, decreased S phase and increased sub-G1 phase cells were detected in the hnRNPK-mutated cell lines. The expression analysis of key cell cycle regulators indicated mRNA of Cyclin A2 was significantly increased in the mutant myoblasts compared to the control cells, while Cyclin B1, Cdc25b and Cdc25c were decreased sharply. In addition to the myoblast proliferation defect, the mutant cells exhibited defect in myotube formation. The myotube formation marker, myosin heavy chain (MHC), was decreased sharply in hnRNPK-mutated cells compared to control myoblasts during differentiation. The deficiency in hnRNPK also resulted in the repression of Myog expression, a key myogenic regulator during differentiation. Together, our data demonstrate that hnRNPK is required for myoblast proliferation and differentiation and may be an essential regulator of myoblast function.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.18
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• pp.8467-8471
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• 2016

Background: One of the major mechanisms for drug resistance is associated with altered anticancer drug transport, mediated by the human-adenosine triphosphate binding cassette (ABC) transporter superfamily proteins. The overexpression of adenosine triphosphate binding cassette, sub-family B, member 1 (ABCB1) by multidrug-resistant cancer cells is a serious impediment to chemotherapy. In our study we have studied the possibility that structural single-nucleotide polymorphisms (SNP) are the mechanism of ABCB1 overexpression. Materials and Methods: A total of 101 gastric cancer multidrug resistant cases and 100 controls were genotyped with sequence-specific primed PCR (SSP-PCR). Gene expression was evaluated for 70 multidrug resistant cases and 54 controls by real time PCR. The correlation between the two groups was based on secondary structures of RNA predicted by bioinformatics tool. Results: The results of genotyping showed that among 3 studied SNPs, rs28381943 and rs2032586 had significant differences between patient and control groups but there were no differences in the two groups for C3435T. The results of real time PCR showed over-expression of ABCB1 when we compared our data with each of the genotypes in average mode. Prediction of secondary structures in the existence of 2 related SNPs (rs28381943 and rs2032586) showed that the amount of ${\Delta}G$ for original mRNA is higher than the amount of ${\Delta}G$ for the two mentioned SNPs. Conclusions: We have observed that 2 of our studied SNPs (rs283821943 and rs2032586) may elevate the expression of ABCB1 gene, through increase in mRNA stability, while this was not the case for C3435T.

• Journal of Yeungnam Medical Science
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• v.15 no.1
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• pp.47-54
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• 1998

Interleukin-10(IL-10) inhibits production of a wide range of cytokines in various cell types and transcriptionally inhibits lipopolysaccharide (LPS)-induced expression of proinflammatory mediators. Cytokine expression by macrophages is an important aspect to ochestrate inflammatory responses. As an approach to identify mechanistic targets of IL-10, it was examined the time course for expression of KC(murine homologue of Gro) gene in murine peritoneal macrophages stimulated with LPS with or without IL-10. The effect of IL-10 on LPS induced KC mRNA expression was delayed and only seen after 1 hour treatment. Pretreatment with IL-10 did not eliminate the delayed inhibitory response nor increase the magnitude of suppression. These effects did not depend upon time of IL-10 treatment but the time of LPS treatment. LPS-induced KC mRNA expression by inhibitory action of IL-10 was not controlled at the level of transcription. The result indicates that IL-10 acts late in the process of KC gene expression and that the prominant site of action may be mRNA stability or translation.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.4
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• pp.457-465
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• 2018

The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.

• Korean Journal of Microbiology
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• v.38 no.2
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• pp.96-102
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• 2002

The S-phase checkpoint mechanisms response to DNA damage or inhibition of DNA replication for maintenance of genetic stability in eukaryotic cells. These roles include cell cycle control arrest at S-phase and Iranscriptional induction of repair genes. To characterize the defects of dpbll mutant for both these responses, we examined the over-expression effect of DPBll gene, the sensitivity to HU, MMS, and the transcriptional pattern by DNA damage agent for RNRS mRNA. RNRS transcript is induced in response to a wide variety of agents that either damage D7A directly through chemical modification or induce stress by blocking DNA synthesis. As results, dpbll-1 cells are sensitive to DNA damage agents and the level of RNR3 mRNA is reduced approximately 40% than wild type cells. Moreover, we found the same results in dpb2-1 cells. Therefore, we propose that DPB2 and DPBll act as a sensor of replication that coordinates the transcriptional and cell cycle responses to replication blocks.

• Journal of Life Science
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• v.10 no.2
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• pp.150-156
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• 2000

This experiment was performed to evaluate the effect of TSH (thyroid-stimulating) on the ERp29 (endoplasmic reticulum resident 29 kDa protein) gene expression in the rat thyrocytes of FRTL-5 cells. Although ERp29 mRNA was constantly expressed, its expression began to increase remarkably from 10-9 M TSH. and its maximum expression was at 5×10-9 M TSH (about 3.5 fold). On the other hand, the effect of TSH on the abundance of ERp29 mRNA started within 6 h, and peaked at 8 h (about 2.5 fold). Actinomycin D (transcription inhibitor) strongly blocked this effect while cycloheximide (translation inhibitor) did not. The half-life of ERp29 mRNA was about 4.5 h in the presence or absence of TSH that was not affected by the stability of ERp29 mRNA. The effect of TSH on the ERp29 gene expression was specific, while other growth factors (transfferin, insulin, and hydrocortisone) did not alter its expression. Our data indicate for the first time that the expression of ERp29 is regulated transcriptionally by TSH in the thyrocytes.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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• pp.242-244
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• 2004

• 한국균학회소식:학술대회논문집
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• 2015.11a
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• pp.37-37
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• 2015

dsRNA was found in malformed cultures of Lentinula edodes strain FMRI0339, one of the three most popular sawdust cultivated commercial strains of shiitake, and was also found in healthy-looking fruiting bodies and actively growing mycelia. Cloning of the partial genome of the dsRNA revealed the presence of the RdRp sequence of a novel L. edodes mycovirus (LeV), and sequence comparison of the cloned amplicon showed an identical sequence to known RdRp genes of LeV found in strain HKA. The meiotic stability of dsRNA was examined by measuring the ratio of the presence of dsRNA among sexual monokaryotic progeny. More than 40% of the monokaryotic progeny still contained the dsRNA, indicating the persistence of dsRNA during sexual reproduction. Comparing the mycelia growth of monokaryotic progeny suggested that, although variations in the growth rate existed among progeny and virus infection was observed in highly actively growing progeny, there appeared to be a tendency toward a lower frequency of virus incidence in actively growing progeny. This study attempted to cure the edible mushroom L. edodes strain FMRI0339 of the L. edodes mycovirus (LeV) in order to obtain an isogenic virus-free fungal strain as well as a virus-infected strain for comparison. Mycelial fragmentation, followed by being spread on a plate with serial dilutions resulted in a virus-free colony. Viral absence was confirmed with gel electrophoresis after dsRNA-specific virus purification, Northern blot analysis, and PCR using reverse transcriptase (RT-PCR). Once cured, all of fungal cultures remained virus-free over the next two years. Interestingly, the viral titer of LeV varied depending on the culture condition. The titer from the plate culture showed at least a 20-fold higher concentration than that grown in the liquid culture. However, the reduced virus titer in the liquid culture was recovered by transferring the mycelia to a plate containing the same medium. In addition, oxygen-depleted culture conditions resulted in a significant decrease of viral concentration, but not to the extent seen in the submerged liquid culture. Although no $discernable phenotypic changes in colony morphology were observed, virus-cured strains showed significantly higher growth rates and mycelial mass than virus-infected strains. We were also explored effects of LeV on fruiting body formation and mushroom yield. The fruiting body formation yield of virus-free L. edodes was larger than virus-infected L. edodes. These results indicate that LeV infection has a deleterious effect on mycelial growth and fruiting body formation. In addition, we have been investigated host-parasite interaction between L. edodes and its mycovirus interaction to study viral mechanism by establishment of proteomics.

• Korean Journal of Environmental Agriculture
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• v.40 no.3
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• pp.194-202
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• 2021

BACKGROUND: Towards achievement of sustainable agriculture, using microbial inoculants may present promising alternatives without adverse environmental effects; however, there are challenging issues that should be addressed in terms of effectiveness and ecology. Viability and stability of the bacterial inoculants would be one of the major issues in effectiveness of microbial pesticide uses, and the changes within the indigenous microbial communities by the inoculants would be an important factor influencing soil ecology. Here we investigated the stability of the introduced bacterial strains in the soils planted with barley and its effect on the diversity shifts of the rhizosphere soil bacteria. METHODS AND RESULTS: Two different types of bacterial strains of Bacillus thuringiensis and Shewanella oneidensis MR-1 were inoculated to the soils planted with barley. To monitor the stability of the inoculated bacterial strains, genes specific to the strains (XRE and mtrA) were quantified by qPCR. In addition, bacterial community analyses were performed using v3-v4 regions of 16S rRNA gene sequences from the barley rhizosphere soils, which were analyzed using Illumina MiSeq system and Mothur. Alpha- and beta-diversity analyses indicated that the inoculated rhizosphere soils were grouped apart from the uninoculated soil, and plant growth also may have affected the soil bacterial diversity. CONCLUSION: Regardless of the survival of the introduced non-native microbes, non-indigenous bacteria may influence the soil microbial community and diversity.

• Molecules and Cells
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• v.27 no.1
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• pp.15-19
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• 2009

Notch signaling is controlled at multiple levels. In particular, stabilized Notch receptor activation directly affects the transcriptional activations of Notch target genes. Although some progress has been made in terms of defining the regulatory mechanism that alters Notch stability, it has not been determined whether Notch1/NICD stability is regulated by $GSK-3{\alpha}$. Here, we show that Notch1/NICD levels are significantly regulated by $GSK-3{\beta}$ and by $GSK-3{\alpha}$. Treatment with LiCl (a specific GSK-3 inhibitor) or the overexpression of the kinase-inactive forms of $GSK-3{\alpha}/{\beta}$ significantly increased Notch1/NICD levels. Endogenous NICD levels were also increased by either $GSK-3{\alpha}/{\beta}$- or $GSK-3{\alpha}$-specific siRNA. Furthermore, it was found that $GSK-3{\alpha}$ binds to Notch1. Deletion analysis showed that at least three Thr residues in Notch1 (Thr-1851, 2123, and 2125) are critical for its response to LiCl, which increased not only the transcriptional activity of endogenous NICD but also Hes1 mRNA levels. Taken together, our results indicate that $GSK-3{\alpha}$ is a negative regulator of Notch1/NICD.