• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2003년도 추계국제학술대회
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• pp.177-177
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• 2003

Methylmercury (MeHg) is a well-known neurotoxicant that causes severe damage to the central nervous system in humans. Many reports have shown that MeHg is poisonous to human body through contaminated foods and has released into the environment. Despite many studies on the pathogenesis of MeHg-induced central neuropathy, no useful mechanism of toxicity has been established so far. In this study, suppressive subtractive hybridization (SSH) was performed to identify differentially expressed genes on human neuroblastoma cell line, SH-SY5Y treated with DMSO and MeHg (6.25 uM) for 6 hr. Differentially expressed cDNA clones were sequenced and were screened by dot blot to eliminate false positive clones. 13 of 35 screened genes were confirmed using real time RT-PCR. These genes include EB1,90-kDa heat-shock protein, chromosome condensation-related SMC-associated protein and brain peptide Al, etc. Analysis of these genes may provide an insight into the neurotoxic effects of MeHg in human neuronal cells and a possibility to develop more efficient and exact monitoring system of heavy metals as ubiquitous environmental pollutants.

• 대한바이러스학회지
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• 제29권1호
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• pp.33-38
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• 1999

Retroviral vector provide a highly efficient method for gene transfer into eukaryotic cells. This vector system can be divided into two components; the retroviral vector itself and the retroviral packaging cell line. The key improvement in the design of these two components are, focused on two aspects; the reduction of helper virus production and high titer-virus. We used PA317 for retrovirus packaging cell line, for its high producibility of viral titer. To test the ability of the vectors to generate high titer-virus, we have chosen four different retroviral vectors; LN, LNSX, LNCX and LXSN. To test easily the viral titer, we have made recombinant construction with CD4 and CD8, checked their viral titer and stained their surface expression. LXSN which contain SV40 early promoter in front of neo gene showed best results in viral transient transfection assay, dot blot assay and surface expression. In addition, recombinant containing CD8 generally showed much higher viral titration and surface expression than CD4.

• The Plant Pathology Journal
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• 제17권3호
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• pp.164-168
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• 2001

Rice black-streaked dwarf virus (RBSDV) and Maize rough dwarf virus (MRDV) are closely related viruses. Since the two viruses produce identical symptoms on maize, barley, and wheat, diagnosis of infected plants is difficult. Previously, we reported that partial cDNA clones of RBSDV S5 and S6 from the Japanese isolate (RBSDV-H) have lower sequence homology to MRDV than do cDNA clones from other genomic segments. In order to test whether cDNA clones of RBSDV-H S5 and S6 can be used for molecular diagnosis, RBSDV field isolates from Korea and from Hokkaido, Japan were tested in dot blot hybridizations probed with RBSDV-H S5 and S6 cDNA colnes. Hybridization with these probes was more intense against the RBSDV genome than against the MRDV genome. Therefore, RBSDV-H S5 and S6 cDNA clones can be used to differentiate between the two viruses. Furthermore, RBSDV-H S5 and S6 clones reacted more strongly against the viruses from stunted maize plants from Korean fields than to MRDV, indicating that RBSDV may be the causal disease agent in maize plants in Korea.

• 대한수의학회지
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• 제36권1호
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• pp.187-193
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• 1996

The T sergenti DNA fragments used as probes of KTS1(2.4kb) and KTS3(1.5kb) were labeled with digoxigenin-11-dUTP for the Southern hybridization. T sergenti DNAs from different geographic locations(Korea; Chonbuk, Kyungbuk, Chungnam, Kangwon, Cheju island, Japan; Shintoku, Shintoku 9209, Shintoku 9201, Shintoku 9202, Shintoku 9102) which had been digested with Pst I and EcoR I were probed by the digoxigenin-11-dUTP-labeled KTS1 and KTS3. As the results, the samples from Chonbuk, Kyungbuk, Cheju island in Korea and Shintoku, Shintoku 9209, Shintoku 9201, Shintoku 9102 in Japan were positively reacted, but the others from the other locations not reacted. In the comformation test of T sergenti DNA from different geographic locations, all of the samples were positively detected by PCR amplification.

• 한국식품과학회지
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• 제52권3호
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• pp.244-248
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• 2020

To retain valuable resources, organisms adopt several strategies including coprophagy. Cells covering the outer skin and internal digestive lumen are actively recycled to maintain their integrity. In present study, we suggested that the small intestine can consume dead cells in a manner similar to how it consumes protein from the diet. We examined the eluates from five segments of the mouse small intestine and cecum and 2 segments of the large intestine and small intestine tissue, and detected immunoreactivity with eukaryotic caveolin-1 and β-actin antibodies only in the cecum and 2 segments from the large intestine. Bacterial agitation of the mouse intestine with Shigella disrupted the architecture and absorptive function of the small intestine. Small intestine eluates were immunoreactive with murine caveolin-1 and contained heme as determined by dot blot analysis. We concluded that the body conserves resources in the small intestine by disposing of and recycling shedded cells.

• 한국식물병리학회지
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• 제14권2호
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• pp.164-170
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• 1998

This study was carried out to develop DNA probe for specific detection of Erwinia carotovora subsp. carotovora. Universal rice primer (URP, 20 mer) developed from repetitive sequence of rice was applied for producing PCR DNA fingerprints of Erwinis spp. In E. carotovora subsp. carotovora strains, primer URP2F amplyfied polymorphic bands which are distinguisable from other Erwinia spp. A PCR band of 0.6 kb selected from PCr polymorphic bands of E. carotovora subsp. carotovora strains was cloned and evaluated as a diagnostic DNA probe. Among 28 bacterial strains including 22 Erwinia spp, the probe (pECC2F) only hybridized to total DNAs from e. carotovora subsp. carotovora strains and E. carotovora subsp. wasabiae, but sizes of hybridized bands were different between these subspecies, 10.0 kb and 3.5 kb respectively. In dot blot assays using probe pECC2F, as few as 103 colony forming units (CFU) of E. carotovora subsp. carotovora could be detected in a suspension containing about 1$\times$103 CFU of soil bacteria.

• Molecular & Cellular Toxicology
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• 제2권1호
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• pp.60-66
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• 2006

Methylmercury (MeHg), one of the heavy metal compounds, can cause severe damage to the central nervous system in humans. Many reports have shown that MeHg is poisonous to human body through contaminated foods and has released into the environment. Despite many studies on the pathogenesis of MeHg-induced central neuropathy, no useful mechanism of toxicity has been established so far. This study, using of suppression subtractive hybridization (SSH) method, was peformed to identify differentially expressed genes by MeHg in SH-SY5Y human neuroblastoma cell line. We prepared to total RNA from SH-SY5Y cells treated with solvent (DMSO) and $6.25\;{\mu}M\;(IC_{50})$ MeHg and performed forward and reverse SSH. Differentially expressed cDNA clones were screened by dot blot, sequenced and confirmed that individual clones indeed represent differentially expressed genes with real time RT-PCR. These sequences were identified by BLAST homology search to known genes or expressed sequence tags (ESTs). Analysis of these sequences may provide an insight into the biological effects of MeHg in the pathogenesis of neurodegenerative disease and a possibility to develop more efficient and exact monitoring system of heavy metals as ubiquitous environmental pollutants.

• Archives of Pharmacal Research
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• 제23권3호
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• pp.252-256
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• 2000

A thymic stromal cell line, TFGD, was established from a thymic tumor mass developed spontaneously in p53 knock out mouse, and was found to produce cytokines that could induce bone marrow hematopoietic stem cells (HSCs) to differentiate into macrophages. The cytokines produced by the TFGD line were assessed by immunoassays. High level of macrophage-colony stimulating factor (M-CSF) and interleukin (IL)-6 was detected in the TFGD-culture supernatant, whereas granulocyte/macrophage-colony stimulating factor (GM-CSF), IL-3, IL-4, IL-5, IL-13, or interferon (IFN)-$\gamma$ was undetectable. Blocking experiments showed that anti-M-CSF monoclonal antibody could neutralize the differentiation-inducing activity shown by the TFGD-culture supernatant. Dot blot analysis of the total RNA isolated from the cultured fetal thymic stromal cells showed that M-CSF transcripts were expressed in the normal thymus. These observations, together with the earlier finding that M-CSF plus IL-6 is the optimal combination of cytokines for the induction of macrophage differentiation from HSCs in vitro, may indicate that thymic macrophages could be generated within the thymus by cytokines involving M-CSF.

• 한국미생물·생명공학회지
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• 제30권1호
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• pp.15-20
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• 2002

Diacylglycerol Kinase (DGK)는 E. coli 및 진핵세포에서의 신호전달에 관여하며, 또한 미생물에서 생리적 자극에 따라 다른 발현 양상을 보이는 것으로 밝혀져 있다. Bacillus subtilis에서 이 유전자에 대한 환경에서의 자극 신호들, 즉 pH 변화, 삼투압의 변화 및 온도의 변화에 따른 발현양상을 연구하였다. 이미 동정된 dgk locus의 KpnI-HindIII의 0.45 kb의 DNA fragment를 probe로 하여 Dot blot, Northern blot analysis를 통해 발현량을 조사해 본 결과 dgk 유전자는 pH변화, 삼투압의 변화 및 온도의 변화에 대응하여 발현되는 유전자임을 알 수 있었다. 특히 낮은 pH, 고 삼투압, 및 저온에서 dgk 유전자의 발현량이 많아짐을 확인 할 수 있었다. Northern hybridization에서 약 2.5kb의 mRNA가 관찰되었다. dgk gene의 ORF size는 약 0.4 kb로 관찰된 transcript size와는 일치하지 않았다. 따라서 Streptococcus mutans의 dgk gene과 마찬가지로 B. subtilis의 dgk 유전자도 polycistronic mRNA로 발현되는 것을 추정할 수 있었으며, 염색체상의 dgk gene에서 상류의 ORF2까지의 크기가 약 2.5 kb로 관찰된 mRNA size와 동일하였다. dgk gene 상류의 ORF2영역의 0.6 kb의 DNA fragment를 probe로 하여 northern blot hybridization을 수행한 결과, 2.5 kb의 mRNA가 관찰되었으며 발현되는 형태도 dgk probe를 이용한 결과와 동일하였다. 따라서 dgk gene은 상류부위의 ORF2 gene과 operon을 형성하여 polycistronic mRNA로 전사되는 것으로 판단된다.

• BMB Reports
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• 제37권6호
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• pp.720-725
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• 2004

A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-${\gamma}1$ has two putative PH domains, an $NH_2$-terminal (PH1) and a split PH domain ($nPH_2$ and $cPH_2$). We previously reported that the split PH domain of PLC-${\gamma}1$ binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)$P_2$) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)$P_2$, we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-${\gamma}1$ $nPH_2$ domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-${\gamma}1$ nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-${\gamma}1$ molecules showed reduced PI(4,5)$P_2$ hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both $PH_1$ and $nPH_2$ domains are responsible for membrane-targeted translocation of PLC-${\gamma}1$ upon serum stimulation. Together, our data reveal that the amino acid residues $Pro^{500}$ and $His^{503}$ are critical for binding of PLC-${\gamma}1$ to one of its substrates, PI(4,5)$P_2$ in the membrane.