• Korean Journal of Agricultural Science
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• v.44 no.2
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• pp.145-180
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• 2017

Alternanthera mosaic virus (AltMV) is a member of the genus Potexvirus which has been known for less than twenty years, and has been detected in Australasia, Europe, North and South America, and Asia. The natural host range to date includes species in at least twenty-four taxonomically diverse plant families, with species in at least four other families known to be infected experimentally. AltMV has been shown to differ from Potato virus X (PVX), the type member of the genus Potexvirus, in a number of ways, including the subcellular localization of the Triple Gene Block 3 (TGB3) protein and apparent absence of interactions between TGB3 and TGB2. Differences between AltMV variants have allowed identification of viral determinants of pathogenicity, and identification of residues involved in interactions with host proteins. Infectious clones of AltMV differing significantly in symptom severity and efficiency of RNA silencing suppression have been produced, suitable either for high level protein expression (with efficient RNA silencing suppression) or for Virus-Induced Gene Silencing (VIGS; with weaker RNA silencing suppression), demonstrating a range of utility not available with most other plant viral vectors. The difference in silencing suppression efficiency was shown to be due to a single amino acid residue substitution in TGB1, and to differences in subcellular localization of TGB1 to the nucleus and nucleolus. The current state of knowledge of AltMV biology, including host range, strain differentiation, host interactions, and utility as a plant viral vector for both protein expression and VIGS are summarized.

• Biomolecules & Therapeutics
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• v.31 no.1
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• pp.82-88
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• 2023

Genomic analysis indicated that the genome of Drosophila melanogaster contains more than 80 cytochrome P450 genes. To date, the enzymatic activity of these P450s has not been extensively studied. Here, the biochemical properties of CYP6A8 were characterized. CYP6A8 was cloned into the pCW vector, and its recombinant enzyme was expressed in Escherichia coli and purified using Ni²⁺-nitrilotriacetate affinity chromatography. Its expression level was approximately 130 nmol per liter of culture. Purified CYP6A8 exhibited a low-spin state in the absolute spectra of the ferric forms. Binding titration analysis indicated that lauric acid and capric acid produced type I spectral changes, with K_d values 28 ± 4 and 144 ± 20 µM, respectively. Ultra-performance liquid chromatography-mass spectrometry analysis showed that the oxidation reaction of lauric acid produced (ω-1)-hydroxylated lauric acid as a major product and ω-hydroxy-lauric acid as a minor product. Steady-state kinetic analysis of lauric acid hydroxylation yielded a k_cat value of 0.038 ± 0.002 min^-1 and a K_m value of 10 ± 2 µM. In addition, capric acid hydroxylation of CYP6A8 yielded kinetic parameters with a k_cat value of 0.135 ± 0.007 min^-1 and a K_m value of 21 ± 4 µM. Because of the importance of various lipids as carbon sources, the metabolic analysis of fatty acids using CYP6A8 in this study can provide an understanding of the biochemical roles of P450 enzymes in many insects, including Drosophila melanogaster.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.8
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• pp.4031-4036
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• 2012

Background: The negative signaling provided by interactions of the co-inhibitory molecule, programmed death-1 (PD-1), and its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), is a critical mechanism contributing to tumor evasion; blockade of this pathway has been proven to enhance cytotoxic activity and mediate antitumor therapy. Here we evaluated the anti-tumor efficacy of AAV-mediated delivery of the extracellular domain of murine PD-1 (sPD-1) to a tumor site. Material and Methods: An rAAV vector was constructed in which the expression of sPD-1, a known negative regulator of TCR signals, is driven by human cytomegalovirus immediate early promoter (CMV-P), using a triple plasmid transfection system. Tumor-bearing mice were then treated with the AAV/sPD1 construct and expression of sPD-1 in tumor tissues was determined by semi quantitative RT-PCR, and tumor weights and cytotoxic activity of splenocytes were measured. Results: Analysis of tumor homogenates revealed sPD-1 mRNA to be significantly overexpressed in rAAV/sPD-1 treated mice as compared with control levels. Its use for local gene therapy at the inoculation site of H22 hepatoma cells could inhibit tumor growth, also enhancing lysis of tumor cells by lymphocytes stimulated specifically with an antigen. In addition, PD-1 was also found expressed on the surfaces of activated CD8+ T cells. Conclusion: This study confirmed that expression of the soluble extracellular domain of PD-1 molecule could reduce tumor microenvironment inhibitory effects on T cells and enhance cytotoxicity. This suggests that it might be a potential target for development of therapies to augment T-cell responses in patients with malignancies.

• Journal of Life Science
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• v.15 no.1 s.68
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• pp.38-44
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• 2005

Infection with hepatitis B virus (HBV) is a major health problem worldwide. Although a tremendous amount has been known about HBV, there have been obstacles in the study of HBV due to the narrow host range of HBV limited to humans and primates. In the present study, we investigated the susceptibility to HBV infection of mouse hepatoma cell line, Hepa-1c1c7. In addition, based on that human hepatocytes infected by HBV increase the expression of the pro-inflammatory cytokine TNF-a, the inducibility of TNF-a expression by HBV in the cells was determined. HBV surface antigen (HBsAg) secretion was measured by the microparticle enzyme immunoassay and steady state mRNA expression was analyzed by quantitative competitive RT-PCR. Transient transfection of Hepa-1c1c7 cells with HBV expression vector resulted in a dose-dependent induction of TNF-a expression. Infection of Hepa-1c1c7 cells with the serum of HBV carrier also increased TNF-a mRNA expression. Both in the transfected and infected cells, HBV mRNA was expressed and significant HBsAg secretion was detected. There was no significant variation in $\beta-actin$ mRNA expression by HBV. These results demonstrate that HBV is infectious to Hepa-lc1c7 in vitro and the viral infection induces TNF-a expression, which suggests that Hepa-lc1c7, a mouse hepatoma cell line, may be a possible model system for analysis of various molecular aspects of HBV infection.

• The Journal of Internal Korean Medicine
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• v.27 no.1
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• pp.27-39
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• 2006

Objectives : Induction of CYP2E1 by ethanol is believed to be one of the major mechanism by which ethanol generate a state of oxidative stress. Previous studies showed that treatment with Chungganhaeju-tang prevents hepatic inflammation and apoptosis in alcoholic liver disease. The purpose of our study is to determine if Chungganhaeju-tang can also protect against alcohol-induced cytotoxicity in CYP2E1-transfected HepG2 cells. Materials and Methods : CYP2E1-transfected HepG2 cells and control vector-transfected HepG2 cells were exposed for isx hours to Chungganhaeju-tang, and then 50 mM of ethanol was added and left for two days. Results : Ethanol significantly decreased cell viability in CYP2E1-transfected HepG2 cells and increased apoptosis. These alterations were attenuated by Chungganhaeju-tang. This was accompanied by an improvement of NF-${\kappa}B$ and Akt activation. Conclusion : These results suggest that Chungganhaeju-tang exerts inhibitory effect against the cytotoxicity induced by alcohol in CYP2E1-transfected HepG2 cells, and that this is a protective action due, at least in part, to an activation of NF-${\kappa}B$ that plays a key role in the protection mechanism, and in reducing hepatotoxic cytokine gene expression.

• Mycobiology
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• v.49 no.5
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• pp.491-497
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• 2021

An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pretreatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.

• KIPS Transactions on Software and Data Engineering
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• v.12 no.8
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• pp.365-370
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• 2023

This paper aims to compare the performance of speech data classification into two groups, adult and elderly, based on the acoustic and linguistic characteristics that change due to aging, such as changes in respiratory patterns, phonation, pitch, frequency, and language expression ability. For acoustic features we used attributes related to the frequency, amplitude, and spectrum of speech voices. As for linguistic features, we extracted hidden state vector representations containing contextual information from the transcription of speech utterances using KoBERT, a Korean pre-trained language model that has shown excellent performance in natural language processing tasks. The classification performance of each model trained based on acoustic and linguistic features was evaluated, and the F1 scores of each model for the two classes, adult and elderly, were examined after address the class imbalance problem by down-sampling. The experimental results showed that using linguistic features provided better performance for classifying adult and elderly than using acoustic features, and even when the class proportions were equal, the classification performance for adult was higher than that for elderly.

• Environmental Mutagens and Carcinogens
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• v.24 no.3
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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.

• Journal of Plant Biotechnology
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• v.38 no.1
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• pp.42-48
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• 2011

Chloroplast genetic engineering of higher plants offers several unique advantages compared with nuclear genome transformation, such as high levels of transgene expression, a lack of position effect due to site-specific transgene integration by homologous recombination, multigene engineering in a single transformation event and reducing risks of gene flow via pollen due to maternal inheritance. We established a reproducible chloroplast transformation system of potato using a tobacco specific plastid transformation vector, pCtVG (trnI-Prrn-aadA-mgfp-TpsbA-trnA). Stable transgene integration into chloroplast genomes and the homoplasmic state of the transgenome were confirmed by PCR and Southern blot analyses. Northern, immunoblot analysis, and GFP fluorescence imaging revealed high expression and accumulation of GFP in the plastids of potato leaves. This system would provide new opportunities for genetic improvement and mass production of value added foreign proteins in this crop.

• Environmental Mutagens and Carcinogens
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• v.24 no.4
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• pp.198-205
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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. Recent industrialized 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. RT-PCR analysis indicated that PAHs significantly up-regulate the level of CYP1B1 mRNA. Some flavonoids such as genistein, daidzein, chrysin, naringenin and morin were also investigeted. These flavonoids decreased B(k)F infuced luciferase activity at low concentration. But, these flavonoids exhibited stimulatory effect at high concentration.