• 한국환경성돌연변이발암원학회:학술대회논문집
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• 한국환경성돌연변이발암원학회 2002년도 Current Trends in Toxicological Sciences
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• pp.25-35
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• 2002

Transcription factor Nrf2 is essential for the antioxidant responsive element (ARE)-mediated induction of phase II detoxifying and oxidative stress enzyme genes. Detailed analysis of differential Nrf2 activity displayed in transfected cell lines ultimately led to the identification of a new protein, which we named Keap1, that suppresses Nrf2 transcriptional activity by specific binding to its evolutionarily conserved amino-terminal regulatory domain. The closest homolog of Keap1 is a Drosophila actin-binding protein called Kelch, implying that Keap1 might be a Nrf2 cytoplasmic effector. We then showed that electrophilic agents antagonize Keap1 inhibition of Nrf2 activity in vivo, allowing Nrf2 to traverse from the cytoplasm to the nucleus and potentiate the ARE response. We postulate that Keap1 and Nrf2 constitute a crucial cellular sensor for oxidative stress, and together mediate a key step in the signaling pathway that leads to transcriptional activation by this novel Nrf2 nuclear shuttling mechanism. The activation of Nrf2 leads in turn to the induction of phase II enzyme and antioxidative stress genes in response to electrophiles and reactive oxygen species.

• BMB Reports
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• 제41권11호
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• pp.784-789
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• 2008

Mac-2BP is a ligand of the galectin family that has been suggested to affect tumor proliferation and metastasis formation. We assessed Mac-2BP expression at the transcriptional and translational levels to evaluate nerve growth factor (NGF)-induced Mac-2BP expression. A time kinetic analysis using reverse transcription-polymerase chain reaction showed that NGF-induced Mac-2BP transcript levels were 4-5 times higher than in controls. Mac-2BP enzyme-linked immunosorbent assay and immuno-fluorescence staining showed a 2-3-fold increase in intracellular and secreted Mac-2BP as a result of NGF stimulation. This increase was regulated by Akt activation and NF-${\kappa}B$ binding. p65 and p50-NF-${\kappa}B$ are major transcriptional factors in the Mac-2BP promoter region, and were shown to be regulated in accordance with the Akt activation states. Collectively, these results suggest that NGF induces Mac-2BP expression via the PI3K/Akt/NF-${\kappa}B$ pathway.

• Journal of Microbiology and Biotechnology
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• 제25권8호
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• pp.1246-1256
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• 2015

Chlamydophila psittaci is an important intracellular pathogen. Persistent infection is an important state of the host-parasite interaction in this chlamydial infection, which plays a significant role in spreading the organism within animal populations and in causing chronic chlamydiosis and serious sequelae. In this study, a C. psittaci persistent infection cell model was induced by penicillin G, and real-time quantitative PCR was used to study the transcriptional levels of 10 C. psittaci genes (dnaA, dnaK, ftsW, ftsY, grpE, rpsD, incC, omcB, CPSIT_0846, and CPSIT_0042) in acute and penicillin-G-induced persistent infection cultures. Compared with the acute cultures, the penicillin-G-treated cultures showed a reduced chlamydial inclusion size and a significantly decreased number of elementary body particles. Additionally, some enlarged aberrant reticulate body particles were present in the penicillin-G-treated cultures but not the acute ones. The expression levels of genes encoding products for cell division (FtsW, FtsY) and outer membrane protein E encoding gene (CPSIT_0042) were downregulated (p < 0.05) from 6 h post-infection onward in the persistent infection cultures. Also from 6 h post-infection, the expression levels of DnaA, DnaK, IncC, RpsD, GrpE, and CPSIT_0846 were upregulated (p < 0.05); however, the expression level of OmcB in the persistent infection was< almost the same as that in the acute infection (p > 0.05). These results provide new insight regarding molecular activities that accompany persistence of C. psittaci, which may play important roles in the pathogenesis of C. psittaci infection.

• 한국미생물·생명공학회지
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• 제49권3호
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• pp.305-315
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• 2021

The cotton leafworm, Spodoptera littoralis, is a major pest in Egypt and many countries worldwide, and causes heavy economic losses. As a result, management measures to control the spread of the worm are required. S. littoralis nucleopolyhedrovirus (SpliNPV) is one of the most promising bioagents for the efficient control of insect pests. In this study, a chitinase gene (chitA) of a 1.8 kb DNA fragment was cloned and fully characterized from SpliNPV-EG1, an Egyptian isolate. A sequence of 601 amino acids was deduced when the gene was completely sequenced with a predicted molecular mass of 67 kDa for the preprotein. Transcriptional analyses using reverse transcription polymerase chain reaction (RT-PCR) revealed that chitA transcripts were detected first at 12 h post infection (hpi) and remained detectable until 168 hpi, suggesting their transcriptional regulation from a putative late promoter motif. In addition, quantitative analysis using quantitative RT-PCR showed a steady increase of 7.86-fold at 12 hpi in chitA transcription levels, which increased up to 71.4-fold at 120 hpi. An approximately 50 kDa protein fragment with chitinolytic activity was purified from ChitA-induced bacterial culture and detected by western blotting with an anti-recombinant SpliNPV chitinase antibody. Moreover, purification of the expressed ChitA recombinant protein showed in vitro growth inhibition of two different fungi species, Fusarium solani and F. oxysporum, confirming that the enzyme assembly and activity was correct. The results supported the potential role and application of the SpliNPV-ChitA protein as a synergistic agent in agricultural fungal and pest control programs.

• Nutrition Research and Practice
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• 제15권5호
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• pp.579-590
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• 2021

BACKGROUND/OBJECTIVES: Petasites japonicus Maxim (P. japonicus) has been used as an edible and medicinal plant and contains many bioactive compounds. The purpose of this study is to investigate the effect of P. japonicus on osteogenesis. MATERIALS/METHODS: The leaves and stems of P. japonicus were separated and extracted with hot water or ethanol, respectively. The total phenolic compound and total polyphenol contents of each extract were measured, and alkaline phosphatase (ALP) activity of each extract was evaluated to determine their effect on bone metabolism. To investigate the effect on osteoblast differentiation of the aqueous extract of P. japonicus leaves (AL), which produced the highest ALP activity among the tested extracts, collagen content was measured using the Sirius Red staining method, mineralization using the Alizarin Red S staining method, and osteocalcin production through enzyme-linked immunosorbent assay analysis. Also, real-time reverse transcription polymerase chain reaction was performed to investigate the mRNA expression levels of Runt-related transcriptional factor 2 (Runx2) and Osterix. RESULTS: Among the 4 P. japonicus extracts, AL had the highest values in all of the following measures: total phenolic compounds, total polyphenols, and ALP activity, which is a major biomarker of osteoblast differentiation. The AL-treated MC3T3-E1 cells showed significant increases in induced osteoblast differentiation, collagen synthesis, mineralization, and osteocalcin production. In addition, mRNA expressions of Runx2 and Osterix, transcription factors that regulate osteoblast differentiation, were significantly increased. CONCLUSIONS: These results suggest that AL can regulate osteoblasts differentiation, at least in part through Runx2 and Osterix. Therefore, it is highly likely that P. japonicus will be useful as an alternate therapeutic for the prevention and treatment of osteoporosis.

• BMB Reports
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• 제38권4호
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• pp.457-467
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• 2005

Open reading frame (ORF) 3 on the Choristoneura fumiferana granulovirus (ChfuGV), located in the 11 kb fragment of the BamHI genomic bank encodes a predicted 32-kDa putative kinase protein. Bioinformatics analysis on the predicted amino acid sequence of ChfuGV PK-1 revealed the existence of 11 catalytic subdomains. Sequence analysis within the 5'-untranslated region (5'-UTR) of ChfuGV pk-1 indicates the presence of both putative early and late promoter motifs, indicating that pk-1 may be expressed throughout the infection cycle. Promoter sequence analysis reveals that pk-1 is deprived of a TATA box and appears instead to be regulated by other cis-acting transcriptional regulatory elements. Temporal transcription analysis by RT-PCR confirms the appearance of transcripts detected from 2 h p.i. until 72 h p.i. Northern blot hybridization characterizes pk-1 transcription as a 1.2 kb transcript. Homology comparisons reveal that ChfuGV PK-1 protein is most closely related to Phthorimaea operculalla granulovirus (PoGV) with 80% amino acid identity.

• 대한약침학회지
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• 제3권1호
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• pp.1-19
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• 2000

To study anti-cancer effect and molecular biological mechanism of bee venom for aqua-acupuncture, the effects of bee venom on cell viability and apoptosis were analyzed using MTT assay, tryphan blue assay, $[^3H]$thymidine release assay, flow cytometric analysis, and activity of caspase-3 protease activity assay. To explore whether anti-cancer effects of bee venom are associated with the transcriptional control of gene expression, quantitative RT-PCR analysis of apoptosis-related genes was performed. The obtained results are summarized as follows: 1. The MTT assay demonstrated that cell viability was decreased by bee venom in a dose-dependant manner. 2. Significant induction of apoptosis was identified using tryphan blue assay, $[^3H]$thymidine release assay, and flow cytomet1 ric analysis of sub $G_1$ fraction. 3. In analysis of caspase-3 protease activity, the activity had increased significantly, in a dose-dependant manner. 4. Quantitative RT-PCR analysis of the apoptosis-related genes showed that Bcl-2 and Bcl-$X_L$ were down-regulated whereas Bax was up-regulated by bee venom treatment.

• Journal of Plant Biotechnology
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• 제43권2호
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• pp.231-239
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• 2016

담배에서 후성유전관련 유전자의 발현연구를 위해 담배유래 시토신 DNA 탈메틸화 관련 NtROS2a 유전자를 과발현 및 RNAi 식물체를 육성하였다. 이들 형질전환체들은 고염 및 산화 스트레스하에서 내성이 증진되었으며, 다양한 표현형변이를 보였다(Lee et al. 2015). 본연구에서는 선발된 과발현 (OX1), RNAi 식물체(RNAi 13) 및 대조식물체(WT)를 이용하여 Agilent Tobacco 4 X 44K Oligo chip으로 microarray분석을 수행하였다. OX1과 RNAi13 계통을 이용하여 WT과 함께 비교 분석한 결과, 대부분 세포 내 이온 수송, 영양 공급 등과 같은 물질대사와 생물적 비생물적 스트레스 및 methylation과 관련되어 영향을 주는 유전자들에서 up-regulation 되었고, 물질대사관련 유전자와 세포 내 기능유전자의 역할을 담당하는 조효소, 그리고 다양한 스트레스 및 메틸레이션 관련 유전자군에서 또한 down-regulation되었다. 각각의 up-, down-regulation된 유전자들을 WT과 비교하여 qRT-PCR을 수행한 결과, KH domain-containing protein, MADS-box protein 및 Zinc phosphodiesterase ELAC protein 유전자들에서 발현이 높게 나타났으며, 반면에 pentatricopeptide (PPR) repeat-containing protein, histone deacetylase HDAC3 protein 및 protein kinase는 0.4 ~ 1.0-fold 발현양이 감소되었다. 따라서 DNA glycosylase를 암호화하는 NtROS2a 유전자는 demethylation과 관련되어 담배 식물체에서 다양한 전사레벨을 조절하는 것으로 판단된다.

• The Korean Journal of Physiology and Pharmacology
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• 제23권5호
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• pp.367-379
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• 2019

Although atopic dermatitis (AD) is known to be a representative skin disorder, it also affects the systemic immune response. In a recent study, myoblasts were shown to be involved in the immune regulation, but the roles of muscle cells in AD are poorly understood. We aimed to identify the relationship between mitochondria and atopy by genome-wide analysis of skeletal muscles in mice. We induced AD-like symptoms using house dust mite (HDM) extract in NC/Nga mice. The transcriptional profiles of the untreated group and HDM-induced AD-like group were analyzed and compared using microarray, differentially expressed gene and functional pathway analyses, and protein interaction network construction. Our microarray analysis demonstrated that immune response-, calcium handling-, and mitochondrial metabolism-related genes were differentially expressed. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology pathway analyses, immune response pathways involved in cytokine interaction, nuclear factor-kappa B, and T-cell receptor signaling, calcium handling pathways, and mitochondria metabolism pathways involved in the citrate cycle were significantly upregulated. In protein interaction network analysis, chemokine family-, muscle contraction process-, and immune response-related genes were identified as hub genes with many interactions. In addition, mitochondrial pathways involved in calcium signaling, cardiac muscle contraction, tricarboxylic acid cycle, oxidation-reduction process, and calcium-mediated signaling were significantly stimulated in KEGG and Gene Ontology analyses. Our results provide a comprehensive understanding of the genome-wide transcriptional changes of HDM-induced AD-like symptoms and the indicated genes that could be used as AD clinical biomarkers.

• Journal of Ginseng Research
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• 제47권5호
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• pp.662-671
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• 2023

Background: 20(S)-protopanaxadiol (PPD), a ginsenoside metabolite, has prominent benefits for the central nervous system, especially in improving learning and memory. However, its transcriptional targets in brain tissue remain unknown. Methods: In this study, we first used mass spectrometry-based drug affinity responsive target stability (DARTS) to identify the potential proteins of ginsenosides and intersected them with the transcription factor library. Second, the transcription factor PURA was confirmed as a target of PPD by biolayer interferometry (BLI) and molecular docking. Next, the effect of PPD on the transcriptional levels of target genes of PURA in brain tissues was determined by qRT-PCR. Finally, bioinformatics analysis was used to analyze the potential biological features of these target proteins. Results: The results showed three overlapping transcription factors between the proteomics of DARTS and transcription factor library. BLI analysis further showed that PPD had a higher direct interaction with PURA than parent ginsenosides. Subsequently, BLI kinetic analysis, molecular docking, and mutations in key amino acids of PURA indicated that PPD specifically bound to PURA. The results of qRT-PCR showed that PPD could increase the transcription levels of PURA target genes in brain. Finally, bioinformatics analysis showed that these target proteins were involved in learning and memory function. Conclusion: The above-mentioned findings indicate that PURA is a transcription target of PPD in brain, and PPD upregulate the transcription levels of target genes related to cognitive dysfunction by binding PURA, which could provide a chemical and biological basis for the study of treating cognitive impairment by targeting PURA.