• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제36권6호
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• pp.481-489
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• 2010

Introduction: TLR-5, a member of the toll-like receptor (TLR) family, is a element of the type I transmembrane receptors, which are characterized by an intracellular signaling domain homolog to the interleukin-1 receptor. These receptors recognize microbial components, particularly bacterial flagellin. All-trans retinoic acid (atRA, tretinoin), a natural metabolite of vitamin A, acts as a growth and differentiation factor in many tissues, and is also needed for immune functions. In this study, THP-1 human macrophage-monocytes were used to examine the mechanisms by which atRA regulated the expression of TLR-5. Because the molecular mechanism underlying this regulation at the transcriptional level is also unclear, this study examined which putative transcription factors are responsible for TLR-5 expression by atRA in immune cells. Materials and Methods: This study examined whether atRA induces the expression of TLR-5 in THP-1 cells using reverse transcription-polymerase chain reaction (RT-PCR), and which transcription factors are involved in regulating the TLR-5 promoter in RAW264.7 cells using a reporter assay system. Western blot analysis was used to determine which signal pathway is involved in the expression of TLR-5 in atRA-treated THP-1 cells. Results: atRA at a concentration of 10 nM greatly induced the expression of TLR-5 in THP-1 cells. Human TLR-5 promoter contains three Sp-1/GC binding sites around -50 bp and two NF-kB binding sites at -380 bp and -160 bp from the transcriptional start site of the TLR-5 gene. Sp-1/GC is primarily responsible for the constitutive TLR-5 expression, and may also contribute to NF-kB at -160 bp to induce TLR-5 after atRA stimulation in THP-1 cells. The role of NF-kB in TLR-5 expression was further confirmed by inhibitor pyrrolidine dithiocarbamate (PDTC) experiments, which greatly reduced the TLR-5 transcription by 70-80%. Conclusion: atRA induces the expression of the human TLR-5 gene and NF-kB is a critical transcription factor for the atRA-induced expression of TLR-5. Accordingly, it is conceivable that retinoids are required for adequate innate and adaptive immune responses to agents of infectious diseases. atRA and various synthetic retinoids have been used therapeutically in human diseases, such as leukemia and other cancers due to the antiproliferative and apoptosis inducing effects of retinoids. Therefore, understanding the molecular regulatory mechanism of TLR-5 may assist in the design of alternative strategies for the treatment of infectious diseases, leukemia and cancers.

• 한국환경성돌연변이발암원학회지
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• 제24권3호
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• pp.121-127
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• 2004

Cytochrome P4501B1(CYP1B1) is known to be inducible by xenobiotic compounds such as policyclic aromatic hydrocarbon(PAH) and dioxins such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin(TCDD). And these induction of CYP1B1 is also regulated by many categories of chemicals. In order to investigate the effects of several chemicals on CYP1B1 gene expression in Hepa-I and MCF-7 cells, 5' flanking DNA of human CYP1B1 was cloned into pGL3 basic vector containing luciferase gene, and then transfected into these cells. After treatment of chemicals, the luciferase activity was measured. CYP1B1 enzyme metabolize PAHs and estradiol. CYP1B1 metabolize estradiol to 4-hydrozyestradiol that is considered as carcinogenic metabolite. Luciferase activity was induced about 20 folds over that control by 1 nM TCDD (2,3,7,8-tetrachloto-p-dioxin). Recent industrialized society, human has been widely been exposed to widespread environmental contaminants such as PAHs(polycyclic aromatic hydrocarbon) that are originated from the imcomplete combustion of hydrocarbons. PAHs are known to be ligands of the AhR(aryl hydrocarbon receptor). Induction of cytochrome P4501B1(CYP1B1) in cell culture is widely used as a biomarker for PAHs. Therefore we have studied the effect of PAHs in the human breast cancer cells MCF-7 to evaluate bioactivity of PAHs. We have used the United State of America EPA selected 13 different PAHs, PAHs mixtures and extracts from environmental samples to evaluate the bioassay system. We examined effects of PAHs on the CYP1B1-luciferase reporter gene and CYP1B1 mRNA level. Benzo(k)fluoranthene and dibenzo(a, h)anthracene showed strong response to CYP1B1 promoter activity stimulation, and also CYP1B1 mRNAs increase in MCF-7 cells in a concentration-dependent manner. Acenaphthene, anthracene, benzo(b)fluoranthene, fluorene, fluoranthene, anphthanlene, pyrene, phenanthrene and carbazole were weak responders in MCF-7 cells. RT-PCR analysis indicated that PAHs significantly up-regulate the level of CYP1B1 mRNA.

• 한국작물학회지
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• 제65권4호
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• pp.274-283
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• 2020

본 연구는 조평과 조운의 벼 출수 후 적산온도와 검정온도에 따른 수발아 발생 정도를 조사하고, RNA-sequencing 방법을 사용하여 수발아 발생 원인을 찾았다. 품종간 수발아성에 관여하는 생리적, 유전학적 요인을 구명하고자 수행하였으며 분석한 결과는 다음과 같다. 1. 출수 후 적산온도가 높아질수록, 검정온도가 높아질수록 수발아 처리시 수발아율이 높았고, 조운벼가 내수발아성이 강하고, 조평벼가 수발아성이 높은것으로 나타났다. 2. 수발아성이 높은 조평벼를 대상으로 한 RNA-sequencing 결과 ABA 생합성에 관여하는 OsNCEDs의 발현이 감소하고, ABA 분해에 관여하는 OsCYP707As의 발현이 증가하였다. 3. 조평과 조운의 OsNCEDs와 OsCYPY707As의 Quantitation Real-Time PCR 결과 조평보다 조운에서 OsNCEDs의 발현이 높게 나타나 수발성과 상관관계를 보였으나, OsCYP707As는 수발아성과 상관관계를 보이지 않았다. 4. 조운벼는 등숙기간중 종실내 ABA함량이 조평보다 높으며 수발아 처리시 남아있는 ABA함량이 높아 내수발아성이 상대적으로 강하게 나타났다.

• 한국수정란이식학회지
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• 제33권3호
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• pp.129-137
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• 2018

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig ($GalT^{-MCP/-MCP}$). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain $GalT^{-MCP/-MCP}/CD39/CD73$ pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from $GalT^{-MCP/-MCP}$ pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate $GalT^{-MCP/-MCP}/CD39/CD73$ pig expressing CD39 and CD73 at endothelial cells.

• The Plant Pathology Journal
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• 제33권5호
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• pp.499-507
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• 2017

In an attempt to develop a biological control agent against mycotoxigenic Fusarium species, we isolated Bacillus amyloliquefaciens strain DA12 from soil and explored its antimicrobial activities. DA12 was active against the growth of mycotoxigenic F. asiaticum, F. graminearum, F. proliferatum, and F. verticillioides both in vitro and in planta (maize). Further screening using dual culture extended the activity range of strain DA12 against other fungal pathogens including Botrytis cinerea, Colletotrichum coccodes, Endothia parasitica, Fusarium oxysporum, Raffaelea quercus-mongolicae, and Rhizoctonia solani. The butanol extract of the culture filtrate of B. amyloliquefaciens DA12 highly inhibited the germination of F. graminearum macroconidia with inhibition rate 83% at a concentration of $31.3{\mu}g/ml$ and 100% at a concentration of $250{\mu}g/ml$. The antifungal metabolite from the butanol extract was identified as iturin A by thin layer chromatography-bioautography. In addition, volatile organic compounds produced by DA12 were able to inhibit mycelial growth of various phytopathogenic fungi. The volatile compounds were identified as 2-heptanone, 5-methyl heptanone and 6-methyl heptanone by gas chromatography-mass spectrometry (GC-MS) analysis. These results indicate that the antagonistic activity of Bacillus amyloliquefaciens DA12 was attributable to iturin A and volatile heptanones, and the strain could be used as a biocontrol agent to reduce the development of Fusarium diseases and mycotoxin contamination of crops.

• 한국환경성돌연변이발암원학회지
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• 제22권4호
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• pp.303-311
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• 2002

This study evaluated the concentrations of urinary metabolites of polycyclic aromatic hydrocarbons (PAHs) in industrial waste incineration workers. The effect of genetic polymorphisms of xenobiotic metabolism enzymes on urinary concentration of PAH metabolites was assessed. And, aromatic DNA adduct levels were also determined in total white blood cells. Fifty employees were recruited from a company handling industrial wastes located in Ansan, Korea: non-exposed group (n=21), exposed group (n=29). Sixteen ambient PAHs were determined by GC/MSD (NIOSH method) from personal breathing zone samples of nine subjects near incinerators. Urinary 1-hydroxypyrene glucuronide (1-OHPG), a major pyrene metabolite, was assayed by synchronous fluorescence spectroscopy after immunoaffinity purification using monoclonal antibody 8E11 (SFS/IAC). Multiplex PCR was used for genotyping for GSTMI/TI and PCR-RFLP for genotyping of CYP1A1 (MspI and Ile/Val). PAH-DNA adducts in peripheral blood WBC were measured by the nuclease P1-enhanced postlabeling assay. Smoking habit, demographic and occupational information were collected by self-administered questionnaire. The range of total ambient PAH levels were 0.00-7.00 mg/㎥ (mean 3.31). Urinary 1-OHPG levels were significantly higher in workers handling industrial wastes than in those with presumed lower exposure to PAHs (p=0.006, by Kruskal-Wallis test). There was a statistically significant dose-response increase in 1-OHPG levels with the number of cigarettes consumed per day (Pearson correlation coefficient=0.686, p<0.001). Urinary 1-OHPG levels in occupationally exposed smoking workers were highest compared with non-occupationally exposed smokers (p=0.053, by Kruskal-Wallis test). Smoking and GSTMI genotype were significant predictors for log-transformed 1-OHPG by multiple regression analysis (overall model R²=0.565, p<0.001), whereas smoking was the only significant predictor for log-transformed aromatic DNA adducts (overall model R²=0.249, p=0.201). Aromatic DNA adducts was also a significantly correlation between log transferred urinary 1-OHPG levels (pearson's correlation coefficient=0.307, p=0.04). However, the partial correlation coefficient adjusting for Age, Sex, and cigarette consumption was not significant (r=0.154, p=0.169). The significant association exists only in individuals with the GSTMI null genotype (pearsons correlation coefficient=0.516, p=0.010; partial correlation coefficient adjusting for age, sex, and cigarette consumption, r=0.363, p=0.038). Our results suggest that the significant increase in urinary 1-OHPG in the exposed workers is due to higher prevalence of smokers among them, and that the association between urinary PAH metabolites and aromatic DNA adducts in workers of industrial waste handling may be modulated by GSTMI genotype. There results remain to be confirmed in future larger studies.

• 한국산림과학회지
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• 제103권2호
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• pp.211-217
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• 2014

잣나무의 주요생장관련 대사물질 인자를 구명하기 위하여 3개 지역에 풍매차대 가계로 조성한 29년생 잣나무의 대사물질을 GC/MS을 이용하여 대사체분석을 실시하였다. 분리한 110종의 대사 물질 중 62종이 우수가계와 저조가계 간에 유의적 차이(p<0.05)가 있었으며 모두 상대적으로 우수가계에서 대사물질 함량을 높게 함유하고 있는 것으로 나타났다. Phosphoric acid, alanine, glycine, malic acid, sucrose, d-turanose, succinic acid 등 22종의 물질은 생장 저조 가계에 비해 생장 우수 가계에서 1.5배 이상 높게 함유되어 있었다. 한편 생장특성과 대사물질간의 상관관계를 분석한 결과 생장 우수가계와 생장 저조가계 그룹 간에 유의적 차이가 있었던 alanine, malic acid, sucrose, d-turanose, succinic acid 등 15종에서 고도의 정의 상관관계(p<0.01)가 있었다. 또한 생장과 유의적 상관관계(p<0.05)가 있으면서 지역간에 변이가 적어 환경에 영향을 적게 받고 있는 대사물질로는 acetic acid, succinic acid, butanoic acid, glutamic acid 및 inositol로 분석되었다. 이들 물질은 잣나무 생장 우수개체에 유의적으로 높게 함유하고 있는 대사물질 인자로 추정되었다.

• 한국산림과학회지
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• 제102권1호
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• pp.129-135
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• 2013

낙엽송(Larix kaempferi) 채종원의 종자생산을 증진하기 위하여 접목 32년생의 채종목에 환상박피 처리를 하였다. 환상박피처리에 의하여 무처리보다 4배에 이르는 착과량 증가 효과가 있어 환상박피 처리의 효과가 뚜렷하게 나타났다. 환상박피 처리에 의한 낙엽송 주간 수피조직내의 대사물질의 변화를 IR-MS, GC-MS 및 HPLC로 분석하여 무처리와 비교한 결과 전질소 함량, sucrose 함량 및 전체 유리 아미노산의 함량이 무처리에 비해 유의적(p<0.05)으로 증가되었다. 또한 유리아미노산의 경우 화아원기가 형성되는 8월의 aspartic acid, glutamic acd, glycine, serine, cysteine, threonine 및 alanine이 환상박피 처리에서 무처리에 비해 유의적으로 높게 함유된 것으로 나타났다. 대사물질과 착과량간에 상관관계를 분석한 결과 착과량과 수피조직내의 전질소 함량(r=0.765, p<0.01)과 전체 유리 아미노산 함량(r=0.802, p<0.01)간에 고도의 정의 상관관계가 있었다. 특히, 화아원기 형성기인 7월 및 8월의 수피조직내의 aspartic acid, glutamic acd, glycine, serine 및 cysteine이 착과량과 정의 상관관계(p<0.05)가 있어, 아미노산의 질소화합물 인자가 낙엽송 화아원기 유도에 직, 간접적으로 관련이 있을 것으로 추정되었다.

• Journal of Plant Biotechnology
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• 제31권3호
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• pp.231-237
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• 2004

Oxycarotenoids는 녹색식물, 곰팡이, 효모, 버섯 및 세균 등이 만들어 내는 황색, 적색 또는 자색의 polyene계 색소로 분자내에 산소를 함유하며 생체내에서 중요한 역할을 담당하고 있다. 본 실험에서는 Oxycarotenoids의 생합성 경로상에 존재하는 $\beta$-carotene hydroxylase 유전자 (Chyb)가 재조합된 Ti-plasmid (pGCHYB)를 A. tumerfacience GV3101에 의해 Arabidopsis thaliana (cv. Columbia)에 형질전환하였다. 50 mg/L hygromycin 함유한 MS 배지에서 선발된 개체를 이용하여 Chyb 유전자의 도입여부를 PCR로 분석한 결과, 대조구에서는 Chyb 유전자의 증폭 되지 않았으나 형질전환체에서는 증폭 산물을 확인 할 수 있었다. 또한 형질전환체의 발현여부를 RT-PCR분석한 결과 도입된 Chyb 유전자가 안정적으로 발현되었다. 형질전환체의 carotenoids를 HPLC 분석한 결과 xanthophyll cycle carotenoids (violaxanthin과 zeaxanthin)의 함량 및 $\beta$-carotene 함량은 감소되었으며, 대조구 Arabidopsis에는 생합성되지 않는 astaxanthin이 생합성되었다. 따라서 본 실험에서 육성된 형질전환체를 이용하여 oxycarotenoids 생합성 과정상의 중간대사물질의 표지, 관여된 transcript 및 metabolite 분석 등을 통해 carotenoids 대사계의 연구소재로 활용 할 수 있을 것으로 기대한다.

• 한국미생물·생명공학회지
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• 제37권1호
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• pp.55-61
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• 2009

인삼 근권에 존재하는 토양 미생물중 esculin agar법을 이용하여 $\beta$-glucosidase를 생산하는 균주를 분리하고, 다시 ginsenoside $Rb_1$을 선택적으로 분해하는 균주 Gsoil 235를 선발 및 동정하였다. 16S rRNA 염기서열을 sequencing한 후, genebank에서 가장 가까운 type strain을 결정하여 유연 관계를 분석한 결과 Cellulosimicrobium 속의 funkei ATCC BAA-$886^T$(AY501364)와 99.7% 일치하는 균주임을 확인하였다. TSB, LB, NB등 3종류의 배지에서 균의 생장은 접종 후 12-24 시간에서 가장 잘 자라며, TSB>LB>NB의 순으로 잘 자라는 것을 알 수 있었다. ginsenoside $Rb_1$과 8, 24, 48시간 동안 반응시킨 후 TLC로 분석한 결과 NB>LB>TSB순으로 $Rb_1$ 분해 활성이 뛰어나 배지의 생장과 대조적인 결과를 얻었다. 반응시간이 증가할수록 Rd를 포함한 minor ginsenoside의 생성이 증가하였으며, 특히 다른 배지에 비해 균주 생장속도가 상대적으로 낮은 NB는 48시간 후 $Rb_1$을 거의 분해하여 강한 효소 활성을 확인할 수 있었다.