• Asian Pacific Journal of Cancer Prevention
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• 제13권5호
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• pp.1809-1817
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• 2012

Background: Gastric cancer is frequently lethal despite aggressive multimodal therapies, and new treatment approaches are therefore needed. Retinoids are potential candidate drugs: they prevent cell differentiation, proliferation and malignant transformation in gastric cancer cell lines. They interact with nuclear retinoid receptors (the retinoic acid receptors [RARs] and retinoid X receptors [RXRs]), which function as transcription factors, each with three subclasses, ${\alpha}$, ${\beta}$ and ${\gamma}$. At present, little is known about retinoid expression and influence on prognosis in gastric cancers. Patients and Methods: We retrospectively analyzed the expression of the subtypes RARa, $RAR{\beta}$, $RAR{\gamma}$, RXRa, $RXR{\beta}$, $RXR{\gamma}$ by immunohistochemistry in 147 gastric cancers and 51 normal gastric epithelium tissues for whom clinical follow-up data were available and correlated the results with clinical characteristics. In addition, we quantified the expression of retinoid receptor mRNA using real-time PCR (RT-PCR) in another 6 gastric adenocarcinoma and 3 normal gastric tissues. From 2008 to 2010, 80 patients with gastric cancers were enrolled onto therapy with all-trans-retinoic acid (ATRA). Results: RARa, $RAR{\beta}$, $RAR{\gamma}$ and $RXR{\gamma}$ positively correlated with each other (p < 0.001) and demonstrated significantly lower levels in the carcinoma tissue sections (p < 0.01), with lower $RAR{\beta}$, $RAR{\gamma}$ and RXRa expression significantly related to advanced stages (p < =0.01). Tumors with poor histopathologic grade had lower levels of RARa and $RAR{\beta}$ in different histological types of gastric carcinoma (p < 0.01). Patients whose tumors exhibited low levels of RARa expression had significantly lower overall survival compared with patients who had higher expression levels of this receptor (p < 0.001, HR=0.42, 95.0% CI 0.24-0.73), and patients undergoing ATRA treatment had significantly longer median survival times (p = 0.007, HR=0.41, 95.0% CI 0.21-0.80). Conclusions: Retinoic acid receptors are frequently expressed in epithelial gastric cancer with a decreased tendency of expression and RARa may be an indicator of a positive prognosis. This study provides a molecular basis for the therapeutic use of retinoids against gastric cancer.

• Biomolecules & Therapeutics
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• 제17권1호
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• pp.92-97
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• 2009

As nanomaterials might enter into cells and have high reactivity with intracellular structures, it is necessary to assay possible genotoxic risk of them. One of these approaches, we investigated possible genotoxic potential of gold nanoparticle (AuNP) using L5178Y cells. Four different sizes of AuNP (4, 15, 100 or 200 nm) were synthesized and the sizes and structures of AuNP were analyzed using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and stability was analyzed by a UV/Vis. Spectrophotometer. Cytotoxicity was assessed by direct cell counting, and cellular location was detected by dark field microscope at 6, 24 and 48 h after treatment of AuNP. Comet assay was conducted to examine DNA damage and tumor necrosis factor (TNF)-${\alpha}$ mRNA level was assay by real-time reverse transcription polymerase chain reaction. Synthetic AuNP (4, 50, 100 and 200 nm size) had constant characteristics and stability confirmed by TEM, SEM and spectrophotometer for 10 days, respectively. Dark field microscope revealed the location of AuNP in the cytoplasm at 6, 24 and 48 h. Treatment of 4 nm AuNP induced dose and time dependent cytotoxicity, while other sizes of AuNP did not. However, Comet assay represented that treatment of 100 nm and 200 nm AuNP significantly increased DNA damage compared to vehicle control (p <0.01). Treatment of 100 nm and 200 nm AuNP significantly increased TNF-${\alpha}$ mRNA expression compared to vehicle control (p<0.05, p<0.01, respectively). Taken together, AuNP induced DNA damage in L5178Y cell, associated with induction of oxidative stress.

• 대한약침학회지
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• 제4권1호
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• pp.123-124
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• 2001

Purpose. Free radicals are implicated in the pathophysiology of aging, ischemic injury and neurodegenerative disorders. To deform]no whether Hominis Placenta extract prevents $H_2O_2$-induced apoptosis, we have performed morphological and biochemical analyses for the detection of apoptotic phenomena in the pineal tumor cell line $PGT-{\beta}$ We have also peformed cytochemical and immunocytochemical analyses for the detection of changes in nitric oxide synthase (NOS) activity and estimated the expression . of apoptotic genes using reverse transcription-polymerase chain reaction (RT-PCR) Methods. $PGT-{\beta}\;cells$ were pretreated with Hominis Placenta extracts $(0,\;10^{-2}\;{\mu}g/ml)$ for 2 hours and then exposed to $H_2O_2\;(0,\;50\;{\mu}M)$ for 3 hours. Appearance of apoptotic characteristics were monitored using 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) staining assay, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay and flow cytometric analysis. NOS activity was measured by NADPH-diaphorase cytochemistry. Expression of inducible NOS (iNOS) and nuclear factor kappa B (NF k B) was assessed via immunocytochemistry. The expression of apoptotic genes was examined by RT-PCR. Results. After 3 flours of exposure to $H_2O_2$, it was shown that $PGT-{\beta}\;cells$ treated with $H_2O_2(50\;{\mu}M)$ exhibit classical apoptotic features and increases in NOS activity and caspase-3 expression. Treatment with Hominis Placenta extract resulted in a reduced occurrence of apoptotic features. DAPI staining, TUNEL and flow cytometric assays revealed decreases in the occurrence of nuclear fragmentation and in the sub-Gl fraction in the $PGT-{\beta}\;cells$ treated with Hominis Placenta extract. Cells treated with Hominis Placenta extract also showed lower activity of NADPH-diaphorase and immunoreactivities of both iNOS and NF k B than those of $H_2O_2$-treated cells which were not treated with Hominis Placenta extract. By RT-PCR, it was shown that the level of caspase-3 mRNA was derreased In the cells treated with Hominis Placenta . extract. Conclusions. This study shows that Hominis Placenta extract prevents $H_2O_2$-induced apoptosis in $PGT-{\beta}\;cells$; inhibitions of iNOS and caspnse-3 are possible mechanisms of the protection against apoptosis.

• Asian-Australasian Journal of Animal Sciences
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• 제31권8호
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• pp.1366-1372
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• 2018

Objective: A disintegrin and metallopeptidase with thrombospondin motifs type 8 (ADAMTS8) is crucial for diverse physiological processes, such as inflammation, tissue morphogenesis, and tumorigenesis. The chicken ADAMTS8 (chADAMTS8) gene was differentially expressed in the kidney following exposure to different calcium concentrations, suggesting a pathological role of this protein in metabolic diseases. We aimed to examine the molecular characteristics of chADAMTS8 and analyze the gene-expression differences in response to toll-like receptor 3 (TLR3) stimulation. Methods: The ADAMTS8 mRNA and amino acid sequences of various species (chicken, duck, cow, mouse, rat, human, chimpanzee, pig, and horse) were retrieved from the Ensembl database and subjected to bioinformatics analyses. Reverse-transcription polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) experiments were performed with various chicken tissues and the chicken fibroblast DF-1 cell line, which was stimulated with polyinosinic-polycytidylic acid (poly[I:C]; a TLR3 ligand). Results: The chADAMTS8 gene was predicted to contain three thrombospondin type 1 (TSP1) domains, whose amino acid sequences shared homology among the different species, whereas sequences outside the TSP1 domains (especially the amino-terminal region) were very dif­ferent. Phylogenetic analysis revealed that chADAMTS8 is evolutionarily clustered in the same clade with that of the duck. chADAMTS8 mRNA was broadly expressed in chicken tissues, and the expression was significantly up-regulated in the DF-1 cells in response to poly(I:C) stimulation (p<0.05). These results showed that chADAMTS8 may be a target gene for TLR3 signaling. Conclusion: In this report, the genetic information of chADAMTS8 gene, its expression in chicken tissues, and chicken DF-1 cells under the stimulation of TLR3 were shown. The result suggests that chADAMTS8 expression may be induced by viral infection and correlated with TLR3-mediated signaling pathway. Further study of the function of chADAMTS8 during TLR3-dependent inflammation (which represents RNA viral infection) is needed and it will also be important to examine the molecular mechanisms during different regulation, depending on innate immune receptor activation.

• Journal of Microbiology and Biotechnology
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• 제26권2호
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• pp.421-431
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• 2016

Recently, poly-γ-glutamic acid synthetase A (pgsA) has been applied to display exogenous proteins on the surface of Lactobacillus casei or Lactococcus lactis, which results in a surface-displayed component of bacteria. However, the ability of carrying genes encoded by plasmids and the expression efficiency of recombinant bacteria can be somewhat affected by the longer gene length of pgsA (1,143 bp); therefore, a truncated gene, pgsA, was generated based on the characteristics of pgsA by computational analysis. Using murine IL-10 as an exogenous gene, recombinant Lactobacillus plantarum was constructed and the capacity of the surface-displayed protein and functional differences between exogenous proteins expressed by these strains were evaluated. Surface expression of IL-10 on both recombinant bacteria with anchorins and the higher expression levels in L. plantarum-pgsA'-IL-10 were confirmed by western blot assay. Most importantly, up-regulation of IL-1β, IL-6, TNF-α, IFN-γ, and the nuclear transcription factor NF-κB p65 in RAW264.7 cells after stimulation with Poly(I:C) or LPS was exacerbated after co-culture with L. plantarum-pgsA. By contrast, IL-10 expressed by these recombinant strains could reduce these factors, and the expression of these factors was associated with recombinant strains that expressed anchorin (especially in L. plantarum-pgsA'-IL-10) and was significantly lower compared with the anchorin-free strains. These findings indicated that exogenous proteins could be successfully displayed on the surface of L. plantarum by pgsA or pgsA', and the expression of recombinant bacteria with pgsA' was superior compared with bacteria with pgsA.

• 농업과학연구
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• 제28권2호
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• pp.78-84
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• 2001

본 연구는 한국 재래산양의 ${\beta}$-lactoglobulin(${\beta}$-LG) 유전자 발현조절부위의 특성을 구명하기 위하여 PCR기법으로 specific primer를 이용하여 ${\beta}$-LG 발현조절부위를 증폭한 후 염기서열을 분석하여 Sheep종의 ${\beta}$-LG유전자 발현조절부위의 염기서열상의 차이를 분석하였다. 한국 재래산양의 genomic DNA로부터 PCR기법을 이용하여 ${\beta}$-LG 유전자 발현조절부위를 증폭한 결과 Sheep종에서 보고된 바와 같이 1,077bp의 단편크기로 증폭되었음을 확인할 수 있었다. Sheep종에서 보고된 ${\beta}$-LG 발현조절 부위의 염기서열과 한국재래산양에서 분석된 염기서열간의 차이는 총 897개의 염기중 46개의 nucleotide에서 차이를 나타내어 94.9%의 상동성을 보였다. 특히 한국재래산양과 Sheep종간 염기서열상의 차이는 Sheep종의 T염기가 재래산양의 C염기로 치환되었거나 반대로 Sheep종의 C염기가 재래산양에서 T염기로 치환되어 있음을 확인할 수 있었다. 이상의 결과에서 ${\beta}$-LG유전자 발현조절부위의 염기서열은 재래산양과 Sheep 종간에 높은 상동성을 보였으나 이들 종간의 발현조절부위의 염기서열상의 차이에 대한 유전자 발현조절 관계와 전사요소는 앞으로 연구가 더 진행되어야 할 것으로 사료된다.

• 생명과학회지
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• 제27권12호
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• pp.1393-1402
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• 2017

식물에서, 칼슘-의존적 단백질 카이네즈(CDPKs)는 $Ca^{2+}$ 신호전달에서 중요한 $Ca^{2+}$ 수용체이다. 벼(Oryza sativa L.)의 CDPKs인 3개의 OsCPKs는 생물정보에 대한 분석이 이루어졌으나, OsCPK11 유전자는 연구가 완전히 수행되지 않았다. 다양한 조직에서 OsCPK11 유전자가 전사수준에서 발현한다는 것은 알려져 있으나, 단백질 수준에서 발현과 생화학적인 특징은 잘 알려져 있지 않다. 이 연구는 OsCPK11의 몇 가지 생화학적 특징을 알아보기 위해 이루어졌다. 먼저 in vitro에서 E. coli를 이용하여 GST-OsCPK11를 발현시키고, 카이네즈 활성 측정과 칼슘-의존적 단백질 카이네즈로서 OsCPK11의 생화학적 분석도 수행하였다. OsCPK11은 스스로 자가인산화하며, $Ca^{2+}$의 존재 하에서 기질로서 histone III-s와 MBP로 인산기 전달 작용을 수행한다. 재조합 OsCPK11의 활성은 $Mg^{2+}$에 의해 영향을 받으며, pH 7.0-7.5에서 최적의 활성을 보인다. 또한 OsCPK11의 활성은 높은 수준의 $Ca^{2+}$가 존재하는 조건에서는 $Mg^{2+}$, $Mn^{2+}$, $Na^+$의 영향을 받지 않는다. 또한 OsCPK11의 자가인산화는 OsCPK11의 $Ca^{2+}$ 민감도를 감소시키는 것으로 밝혀졌다. 마지막으로, OsCPK11의 N-말단 다양화 지역으로 토끼 항체를 만들었고, immunoblot을 기초로 polyclonal antibody는 95.5 kD의 GST-OsCPK11를 인식하는 것으로 나타났다. 이 결과는 벼의 $Ca^{2+}$ 매개 신호전달에서 OsCPK11의 기능을 더 잘 이해하는데 도움을 줄 것이며, 심화 연구를 위해 다양한 OsCPKs의 단백질 정보를 결정하는 것이 필요할 것이다.

• 생명과학회지
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• 제27권12호
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• pp.1415-1420
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• 2017

고구마 현탁배양세포의 EST library에 높은 빈도로 존재하는 metallothionein (MT) 유전자를 선별하였다(IbMT3). MT 유전자는 세포와 조직의 스트레스 조절과 연관되어 있다고 알려져 있다. 본 연구에서 IbMT3 cDNA의 전장을 확보하여 염기서열 분석을 한 결과, IbMT3 유전자는 구조적으로 유형 3에 속하는 MT 단백질을 암호화하고 있었다. 식물에서 유형 3에 속하는 MT 단백질의 기능은 명확히 알려지지 않다. Northern blot 분석 결과, IbMT3 유전자는 고구마 식물체 잎보다 현탁배양 세포에서 매우 강하게 발현되었다. 일반적으로 세포배양은 세포에 산화 스트레스 상태를 부과하는 것으로 알려져 있다. 이에, 고구마 식물체에 산화 스트레스를 처리하여 IbMT3 유전자의 발현이 어떻게 조절되는지 조사하였다. 제초제인 methyl viologen (MV)을 6, 12, 24시간 동안 처리했을 때, IbMT3 유전자의 발현은 6시간 후에 아주 강하게 유도되었고 그 이후에는 감소함을 알 수 있었다. 저온 스트레스($15^{\circ}C$)를 24, 48시간 동안 처리했을 때, IbMT3 유전자는 처리시간이 경과함에 따라 발현이 더 많이 유도되었다. 이로써, IbMT3 유전자는 환경 및 산화 스트레스에 반응하여 발현이 조절되는 유전자임을 알 수 있었다. IbMT3 isoform은 고구마 식물체 내에서 항산화제로써 작용할 가능성이 있을 뿐 아니라, 스트레스 하에서의 세포 적응 메커니즘에 중요한 기능을 할 것으로 사료된다.

• KSBB Journal
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• 제21권2호
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• pp.144-150
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• 2006

본 연구는 메타게놈 유전자 특성과 E. coli에서 정상적으로 발현되는 유전자 특성을 생물정보학 기법으로 비교 분석하고 그 결과를 메타게놈 선별 연구에 활용하고자 하는데 그 목적을 두었다. 이를 위하여 메타게놈 유래의 URF 와 숙주세포로 이용되는 E. coli이 ORF에 대한 염기구조, 발현되는 단백질의 크기 및 분자량, 아미노산의 구성 및 코돈사용은 물론 전사와 번역에 관여하는 프로모터 부위와 리보솜 결합부위의 보존서열 특성을 비교 분석하였다. 메타게놈과 E. coli가 합성하는 단백질의 크기와 분자량은 매우 비슷한 경향을 보였으나, 아미노산의 조성비, G+C 함량 및 코돈사용에서는 매우 다른 경향을 나타내었다. 특히 전사와 번역에 직접적으로 관여하는 프로모터와 RBS 영역에서의 DNA 보존서열이 상당부분 부합되지 않아 E. coli에서 메타게놈의 발현율이 현저히 낮을 것으로 예측할 수 있었다. RBS와 같이 유전자 발현에 필수적인 조절인자가 메타게놈과 E. coli에서 큰 차이를 나타내는 문제점은 메타게놈으로부터 유용한 유전자원을 탐색하는 연구에서 심도있게 개선하여야 할 사항이다. 부분적으로는 라이브러리 구축에 사용되는 벡터 및 숙주의 개량을 통하여 위의 문제를 극복할 수도 있을 것이다.

• 한국수정란이식학회지
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• 제32권3호
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• pp.209-220
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• 2017

The estrogen-mediated effect of mesenchymal stem cells (MSCs) is a highly critical factor for the clinical application of MSCs. However, the present study is conducted on MSCs derived from adult donors, which have different physiological status with steroid hormonal changes. Therefore, we explores the important role of $17{\beta}$-estradiol (E2) in MSCs derived from female and male newborn piglets (NF- and NM-pBMSCs), which are non-sexually matured donors with steroid hormones. The results revealed that in vitro treatment of MSCs with E2 improved cell proliferation, but the rates varied according to the gender of the newborn donors. Following in vitro treatment of newborn MSCs with E2, mRNA levels of Oct3/4 and Sox2 increased in both genders of MSCs and they may be correlated with both estrogen receptor ${\alpha}$ ($ER{\alpha}$) and $ER{\beta}$ in NF-pBMSCs, but NM-pBMSCs were only correlated with $ER{\alpha}$. Moreover, E2-treated NF-pBMSCs decreased in ${\beta}$-galactosidase activity but no influence on NM-pBMSCs. In E2-mediated differentiation capacity, E2 induced an increase in the osteogenic and chondrogenic abilities of both pBMSCs, but adipogenic ability may increased only in NF-pBMSCs. These results demonstrate that E2 could affect both genders of newborn donor-derived MSCs, but the regulatory role of E2 varies depending on gender-dependent characteristics even though the original newborn donors had not been affected by functional steroid hormones.