• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.213-213
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• 2004

Exogeneous DNA can reproducibly be delivered by co-injected spermatozoa and this transgenesis method is very efficient protocol. But, mosaic patterns of transgenic embryos and offspring were shown frequently. Whole blastomere integration is important in transgenic animal economics. (omitted)

• BMB Reports
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• 제30권4호
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• pp.285-291
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• 1997

It is becoming increasingly evident that significant changes in gene expression occur during the course of neuronal differentiation. Thus, it should be possible to gain information about the biochemical events by identifying differentially expressed genes in neuronal differentiation The PC12 cell line is a useful model system to investigate the molecular mechanism underlying neuronal differentiation and has been used extensively for the study of the molecular events that underlie the biological actions of nerve growth factor (NGF). In this study, we report an application of the recently described mRNA differential display method to analyze differential gene expression during neuronal differentiation. Using this technique, we have identified several cDNA tags expressed differentially during neuronal differentiation. Interestingly, one of these clones was cytochrome c oxidase subunit I (COX I) gene. The differential expression of COX I gene was confirmed by Northern blot analysis as well as RT-PCR. Southern blot analysis of the genomic DNA of PC12 cells revealed that COX I is a single gene. Induction of the oxidative enzyme might reflect the energy requirement in neuronal differentiation.

• The Korean Journal of Physiology and Pharmacology
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• 제7권6호
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• pp.375-379
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• 2003

$Ca^{2+}$ influx appears to be important for triggering myoblast fusion. It remains, however, unclear how $Ca^{2+}$ influx rises prior to myoblast fusion. Recently, several studies suggested that NMDA receptors may be involved in $Ca^{2+}$ mobilization of muscle, and that $Ca^{2+}$ influx is mediated by NMDA receptors in C2C12 myoblasts. Here, we report that other types of ionotropic glutamate receptors, non-NMDA receptors (AMPA and KA receptors), are also involved in $Ca^{2+}$ influx in myoblasts. To explore which subtypes of non-NMDA receptors are expressed in C2C12 myogenic cells, RT-PCR was performed, and the results revealed that KA receptor subunits were expressed in both myoblasts and myotubes. However, AMPA receptor was not detected in myoblasts but expressed in myotubes. Using a $Ca^{2+}$ imaging system, $Ca^{2+}$ influx mediated by these receptors was directly measured in a single myoblast cell. Intracellular $Ca^{2+}$ level was increased by KA, but not by AMPA. These results were consistent with RT-PCR data. In addition, KA-induced intracellular $Ca^{2+}$ increase was completely suppressed by treatment of nifedifine, a L-type $Ca^{2+}$ channel blocker. Furthermore, KA stimulated myoblast fusion in a dose-dependent manner. CNQX inhibited not only KA-induced myoblast fusion but also spontaneous myoblast fusion. Therefore, these results suggest that KA receptors are involved in intracellular $Ca^{2+}$ increase in myoblasts and then may play an important role in myoblast fusion.

• Journal of Microbiology and Biotechnology
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• 제32권9호
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• pp.1110-1119
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• 2022

Fe-S clusters are versatile and essential cofactors that participate in multiple and fundamental biological processes. In Escherichia coli, the biogenesis of these cofactors requires either the housekeeping Isc pathway, or the stress-induced Suf pathway which plays a general role under conditions of oxidative stress or iron limitation. In the present work, the Fe-S cluster assembly Isc and Suf systems of acidophilic Bacteria and Archaea, which thrive in highly oxidative environments, were studied. This analysis revealed that acidophilic microorganisms have a complete set of genes encoding for a single system (either Suf or Isc). In acidophilic Proteobacteria and Nitrospirae, a complete set of isc genes (iscRSUAX-hscBA-fdx), but not genes coding for the Suf system, was detected. The activity of the Isc system was studied in Leptospirillum sp. CF-1 (Nitrospirae). RT-PCR experiments showed that eight candidate genes were co-transcribed and conform the isc operon in this strain. Additionally, RT-qPCR assays showed that the expression of the iscS gene was significantly up-regulated in cells exposed to oxidative stress imposed by 260 mM Fe₂(SO₄)₃ for 1 h or iron starvation for 3 h. The activity of cysteine desulfurase (IscS) in CF-1 cell extracts was also upregulated under such conditions. Thus, the Isc system from Leptospirillum sp. CF-1 seems to play an active role in stressful environments. These results contribute to a better understanding of the distribution and role of Fe-S cluster protein biogenesis systems in organisms that thrive in extreme environmental conditions.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2003년도 Annual Meeting of KSAP : International Symposium on Pharmaceutical and Biomedical Sciences on Obesity
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• pp.98-98
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• 2003

${\beta}$-(1,3)-D-Glucans have been known to exhibit antitumor and antimicrobial activities. The presence of dectin-1,${\alpha}$, ${\beta}$-glucan receptor of dendritic cell, on macrophage has been controvertial. RT-PCR analysis led to the detection of dectin-1${\alpha}$ and ${\beta}$ in murine macrophage Raw264.7 cell line. Among the various organs of mouse, dectin-1${\alpha}$ and ${\beta}$ were detected in the thymus, lung, spleen, stomach and intestine. To analyze gene expression modulated by ${\beta}$-glucan treated murine Raw264.7 macrophage, total mRNA was applied to cDNA microarray to interrogate the expression of 7,000 known genes. cDNA chip analysis showed that ${\beta}$-glucan of P. osteatus increased gene expressions of immunomodulating genes, membrane antigenic proteins, chemokine ligands, complements, cytokines, various kinases, lectin associated genes and oncogenes in Raw 264.7 cell line. When treated with ${\beta}$-glucan of P. osteatus and LPS, induction of gene expression of TNF-${\alpha}$ and IFN-R1 was confirmed by RT-PCR analysis. Induction of TNF-R type II expression was confirmed by FACS analysis. IL-6 expression was abolished by EDTA in ${\beta}$-glucan and LPS treated Raw264.7 cell line, indicating that ${\beta}$-glucan binds to dectin-l in a Ca$\^$＋＋/ -dependent manner. To increase antitumor efficacy of ${\beta}$-glucan, ginsenoside Rh2 (GRh2) was co-treated with ${\beta}$-glucan in vivo and in vitro tests. IC$\sub$50/ values of GRh2 were 20 and 25 $\mu\textrm{g}$/$m\ell$ in SNU-1 and B16 melanoma F10 cell line, respectively. Co-treatment with ${\beta}$-glucan and GRh2 showed synergistic antitumor activity with cisplatin and mitomycin C both in vitro and in vivo. Single or co-treatment with ${\beta}$-glucan and GRh2 increased tumor bearing mouse life span. Co-treatment with ${\beta}$-glucan and GRh2 showed more increased life span with mitomycin C than that with cisplatin. Antitumor activities were 67% and 72 % by co-injection with ${\beta}$-glucan and GRh2 in the absence or presence of mitomycin C, respectively.

• Asian Pacific Journal of Cancer Prevention
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• 제13권7호
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• pp.3203-3207
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• 2012

Backgrounds: To investigate the inhibiting effects of multi-target anti-microRNA antisense oligonucleotide (MTg-AMOs) on proliferation and migration of human gastric cancer cells. Methods: Single anti-microRNA antisense oligonucleotides (AMOs) and MTg-AMOs for miR-221, 21, and 106a were designed and transfected into SGC7901, a gastric cancer cell line, to target the activity of these miRNAs. Their expression was analyzed using stem-loop RT-PCR and effects of MTg-AMOs on human gastric cancer cells were determined using the following two assay methods: CCK8 for cell proliferation and transwells for migration. Results: In the CCK-8 cell proliferation assay, $0.6{\mu}mol/L$ was selected as the preferred concentration of MTg-AMOs and incubation time was 72 hours. Under these experimental conditions, MTg-AMOs demonstrated better suppression of the expression of miR-221, miR-106a, miR-21 in gastric cancer cells than that of single AMOs (P = 0.014, 0.024; 0.038, respectively). Migration activity was also clearly decreased as compared to those in randomized and blank control groups ($28{\pm}4$ Vs $54{\pm}3$, P <0.01; $28{\pm}4$ Vs $59{\pm}4$, P < 0.01). Conclusions: MTg-AMOs can specifically inhibit the expression of multiple miRNAs, and effectively antagonize proliferation and migration of gastric cancer cells promoted by oncomirs.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1229-1239
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• 2005

Human immunodeficiency virus-like particles (HIVVLPs) with native conformations similar to that of the wild-type virion could be valid candidates for vaccine development. To this end, we used a Semliki Forest Virus (SFV) expression system to produce HIV- VLPs containing high quantities of native envelope proteins. Here, we described a single SFV replicon containing the HIV gagpol and env genes under the control of separate subgenomic promoters. Mature VLPs incorporating the Gag and Env proteins were detected in the supernatant of replicon-expressing cells by Western blot analysis. The HIV-VLPs showed the expected molecular density (1.14-1.18 g/ml) on a $20-60\%$ sucrose gradient; the particles were 100-120 nm in diameter and Env proteins were observed on their surfaces by immunogold electron microscopy. RT-PCR analysis of VLP-associated RNAs in mature HIV-VLPs revealed two SF V-derived RNA species (full-length and subgenomic). Immunization studies in Balb/c mice showed that these HIV-VLPs were capable of inducing both HIV-specific antibodies and cell-mediated immune responses. Taken together, our results indicate that the SFV replicon system is useful for the production of HIV-VLPs, which may be valuable candidates for an HIV vaccine.

• 한국미생물·생명공학회지
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• 제38권1호
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• pp.70-76
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• 2010

The single-stranded large circular (LC)-sense DNA were utilized as probes for DNA chip experiments. The microarray experiment using LC-sense DNA probes found differentially expressed genes in A549 cells as compared to WI38VA13 cells, and microarray data were well-correlated with data acquired from quantitative real-time RT-PCR. A 5K LC-sense DNA microarray was prepared, and the repeated experiments and dye swap test showed consistent expression patterns. Subsequent functional analysis using LC-antisense library of overexpressed genes identified several genes involved in A549 cell growth. These experiments demonstrated proper feature of LC-sense molecules as probe DNA for microarray and the potential utility of the combination of LC-sense microarray and antisense libraries for an effective functional validation of genes.

• 한국산학기술학회논문지
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• 제16권3호
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• pp.1964-1971
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• 2015

사람의 골수 또는 제대정맥에서 유래된 중간엽 줄기 세포 (hBM-MSC 또는 hUC-MSC)는 임상적 치료 적용에 매우 유용한 세포유형으로 알려져 왔다. 우리는 이러한 세포에서 two-pore 도메인 포타슘 (K2P)채널을 조사하였다. K2P 채널은 다양한 세포유형들에서 안정막 전위를 형성하는데 중요한 역할을 한다. 그들 중 TREK1은 수소, 저산소증, 다불포화 지방산, 항우울제 및 신경전달물질들의 표적이다. 우리는 RT-PCR 분석과 팻취고정기법을 이용하여 hBM-MSCs와 hUC-MSC가 기능적인 TREK1 채널을 발현하는지 조사했다. hBM-MSCs와 hUC-MSCs에서 100 pS 단일 채널 전도도를 가진 포타슘채널이 발견되었고, 그 채널은 세포막 신전 (-5 mmHg ~ -15 mmHg), 아라키도닉산 ($10{\mu}M$), 세포내 산성화 (pH 6.0)에 의해 활성화 되었다. 이러한 전기생리학적 성질은 TREK1과 유사하였다. 우리의 결과는 안정막 전위에 기여하는 TREK1 채널이 hBM-MSC와 hUC-MSC에 기능적으로 존재하고 있음을 제시한다.

• International Journal of Oral Biology
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• 제31권4호
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• pp.141-148
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• 2006

The effects of adenosine triphosphate(ATP) on salivary glands have been recognized since 1982. The presence of purinergic recepetors(P2Rs) that mediate the effects of ATP in various tissues, including parotid and submandibular salivary gland, has been supported by the cloning of receptor cDNAs and the expression of the receptor proteins. P2Rs have many subtypes, and the activation of these receptor subtypes increase intracellular $Ca^{2+}$, a key ion in the regulation of the secretion in the salivary gland. The apical pores of taste buds in circumvallate and foliate papillae are surrounded by the saliva from von Ebner salivary gland(vEG). Thus, it is important how the secretion of vEG is controlled. This study was designed to elucidate the roles of P2Rs on salivary secretion of vEG. Male Sprague-Dawley rats (about 200 g) were used for this experiment. vEG-rich tissues were obtained from dissecting $500-1,000\;{\mu}m$ thick posterior tongue slices under stereomicroscope view. P2Rs mRNA in vEG acinar cells were identified with RT-PCR. To observe the change in intracellular $Ca^{2+}$ activity, we employed $Ca^{2+}-ion$ specific fluorescence analysis with fura-2. Single acinar cells and cell clusters were isolated by a sequential trypsin/collagenase treatment and were loaded with $10\;{\mu}M$ fura -2 AM for 60 minutes at room temperature. Several agonists and antagonists were used to test a receptor specificity. RT-PCR revealed that the mRNAs of $P2X_4$, $P2Y_1$, $P2Y_2$ and $P2Y_3$ are expressed in vEG acinar cells. The intracellular calcium activity was increased in response to $10\;{\mu}M$ ATP, a P2Rs agonist, and 2-MeSATP, a $P2Y_1$ and $P2Y_2R$ agonist. However, $300\;{\mu}M\;{\alpha}{\beta}-MeATP$, a $P2X_1$ and $P2X_3R$ agonist, did not elicit the response. The responses elicited by $10\;{\mu}M$ ATP and UTP, a $P2Y_2R$ agonists, were maintained when extracellular calcium was removed. $10\;{\mu}M$ suramin, a P2XR antagonist, and reactive blue 2, a P2YR antagonist, partially blocked ATP-induced response. However, when extracellular calciums were removed, suramin did not abolish the responses elicited by ATP. These results suggest that P2Rs play an important role in salivary secretion of vEG acinar cells and the effects of ATP on vEG salivary secretion may be mediated by $P2X_4$, $P2Y_1$, $P2Y_2$, and/or $P2Y_3$.