• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.681-684
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• 2007

An immunoassay method was developed to quantitatively detect phosphinothricin-N-acetyltransferase (PAT) encoded by the Bialaphos resistance (bar) gene in genetically modified (GM) pepper. The histidine-tagged PAT was overexpressed in Escherichia coli M15 (pQE3l-bar) and efficiently purified by $Ni^{2+}$ affinity chromatography. A developed sandwich enzyme-linked immunosorbent assay (S-ELISA) method (detection limit: $0.01{\mu}g/ml$) was 100-fold more sensitive than a competitive indirect ELISA (CI-ELISA) method or Western blot analysis in detecting the recombinant PAT. In real sample tests, PAT in genetically modified herbicide-tolerant (GMHT) peppers was successfully quantified [$4.9{\pm}0.4{\mu}g/g$ of sample (n=6)] by the S-ELISA method. The S-ELISA method developed here could be applied to other GMHT crops and vegetables producing PAT.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.87-93
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• 2004

Dynamic modification of cytoplasmic and nuclear proteins by O-linked N-acetylglucosamine (O-GlcNAc) on Ser and Thr residues is ubiquitous in higher eukaryotes. And this modification may serve as a signaling mod-ification analogous to protein phosphorylation. Addition and cleavage of O-GlcNAc are catalyzed by O-linked GlcNAc transferase (OGT) and O-linked N-acety1glucosaminidase (O-GlcNAcase), respectively. Two types of human O-GlcNAcase gene were cloned and expressed as three fusion proteins in Escherichia coli. O-GlcNA-case activity showed in the order of thioredoxin fusion> $6{\times}His$ tag> GST fusion. O-GlcNAcase had enzy-matic activity against only ${\rho}$NP-GlcNAc of seven tested substrate analogs. Blast search revealed that O-GlcNAcase has two conserved domains, amino terminal hyaluronidase-like domain and carboxy terminal N-acetyltransferase domain. Extensive deletion studies were done to define catalytically important domains. The deletions of hyaluronidase-like domain and N-acetyltransferase domain abolished enzyme activity. But, N-ter-minal 55 amino acid deletion and C-terminal truncation showed lower activity. Based on deletion analysis, we suggest that hyaluronidase-like domain is essential for enzyme activity and carboxy terminal N-acetyltrans-ferase domain may be modulatory function.

• Animal cells and systems
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• v.16 no.1
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• pp.27-33
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• 2012

The suprachiasmatic nucleus (SCN) of the teleost hypothalamus contains a central circadian pacemaker, which adjusts circadian rhythms within the body to environmental light-dark cycles. It has been shown that exposure to darkness during the day causes phase shifts in circadian rhythms. In this study, we examined the effect of exposure to darkness on the mRNA expression levels of two circadian clock genes, namely, $Period2$ ($Per2$) and $Cryptochrome1$ ($Cry1$), and the rate-limiting enzyme in melatonin synthesis, arylalkylamine $N$-acetyltransferase-2 (Aanat2), in the pineal gland of olive flounder, $Paralichthys$ $olivaceus$. The expression of these genes showed circadian variations and was significantly higher during the dark phase. These changes may be involved in the mechanism of dark-induced phase shifts. Furthermore, this study suggests that olive flounder may be a teleost model to investigate the localization and function of circadian oscillators.

• Archives of Pharmacal Research
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• v.19 no.6
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• pp.502-506
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• 1996

Protein X is one of the subunits of pyruvate dehydrogenase complex. The biological role of this protein has not been fully elucidated, mainly because of the difficulty in its dissociation from the tightly bound dihydrolipoamide acetyltransferase-protein X subcomplex. We have found that the detachment of protein X from acetyltransferase subunit can be easily accomplished by the cycles of freezing and thawing proces. Several lines of evidence including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequence analysis and acetylation with $[2^{14}C]$ pyruvate confirmed that the purified protein is protein X. The purified intact form of protein X was acetylated by $[2^{14}C]$ pyruvate in the presence of py-ruvate dehydrogenase subunit.The acetylation efficiency of this protein was lower than that of acetyltransferase and was not affected by the presence of acetyltransferase.

• Biomolecules & Therapeutics
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• v.10 no.3
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• pp.193-199
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• 2002

The arylamine N-acetyltransferases (NATs) are a family of enzymes that N-acetylate mylhydrazines and arylamines through transfer of an acetyl group from acetyl coenzyme A. This activity was found to vary among individuals as a Mendalian trait and the basis of the genetic differences in human NAT activity is one of the best of the genetic studied examples of pharmacogenetic variation. The classical N-acetylation polymorphism is regulated at the NAT2 locus, which segregates individuals into rapid, intermediate, and slow acetylator phenotypes. In this study, the relationship between NAT2 activity phenotype using HPLC:UV assay for the determination of dapsone and monoacetyldapsone in plasma and NAT2 genotype by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) was investigated in the F2 hybrid (Fischer 344 vs Wistar-Kyoto) rats. Three Common mutant alleles at the NAT2 gene locus have been identified in the F2 generation progeny of Fischer 344 rats as raid acetylator and Wistar-Kyoto rats as slow acetylator segregated into three modes (low, intermediates, and high) with simple Mendelian inheritance. The metabolic activity of NAT2 of the intermediate and rapid acetylators is significant1y greater than slow acetylator, but the metabolic activity of rapid acetylator is not significantly different from Intermediate type. Therefore, we could observe that complete trimodal NAT2 genotypic alleles and incomplete trimodal NAT2 metabolic phenotypic distribution in tile F2 hybrid rats. These observations suggest that the relationships between NAT2 genotype and metabolic phenotype exists and F2 hybrid (Fischer 344: Wistar-Kyoto) animal models about NAT2 polymorphism might be applied in the toxicity and pharmacogenetic studies of arylamine drugs and carcinogens.

• Korean Journal of Head & Neck Oncology
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• v.17 no.2
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• pp.131-138
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• 2001

Objectives: Alcohol intake has been reported to be a risk factor of laryngeal cancer. Since the aldehyde dehydrogenase 2 (ALDH2) genotype is a major determinant of personal alcohol drinking habit, there is a possibility that ALDH2 genotype would be a risk factor for laryngeal cancer. N-Acetyltransferase 2 (NAT2) is a detoxifying enzyme and its polymorphism has been reported to be related to the risk of many environmental cancers. However, studies on the associations between these two genotypes and laryngeal cancer risk are scarce. We have assessed the effects of alcohol intake and the genotype of ALDH2 and NAT2 on the risk of laryngeal cancer in Koreans. Materials and Methods: Eighty-four pathologically proven laryngeal cancer patients and 168 age matched controls were included as the study subjects. Information about alcohol intake and smoking habit was collected using a self administered questionnaire. ALDH2 and NAT2 genotypes were analyzed using PCR-RFLP methods. Results: Alcohol intake was significant as a risk factor for laryngeal cancer (OR : 2.58, 95% CI : 1.24, 5.36), especially for supraglottic laryngeal cancer (OR : 3.24, 95% CI : 1.02, 10.31). Personal drinking habit was closely related with personal smoking habit, which was a potent risk factor of laryngeal cancer. In a stratified analysis according to the level of cumulative smoking amount, drinking was significant neither in light smokers (equal or less than 30 pack-years) nor in heavy smoker (over 30 pack-years). The ALDH2 genotype was significantly associated with the risk of laryngeal cancer in a univariate analysis. The statistical significance, however, disappeared after adjusting alcohol intake using a multiple conditional logistic model. The NAT2 genotype was not significant as a risk factor for laryngeal cancer. Conclusion: Alcohol drinking and ALDH2 genotype would have indirect effects on laryngeal cancer by their correlations with cigarette smoking or with alcohol drinking. It is less likely that the NAT2 genotype would be a potent risk factor of laryngeal cancer.

• Journal of Preventive Medicine and Public Health
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• v.39 no.2
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• pp.135-140
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• 2006

Objectives : Polymorphisms of genes from glutathione Stransferases (GSTs) and N-acetyltransferase 2 (NAT2) have been associated with increased susceptibility to various cancers. Previous results showed that East Asians such as Koreans, Japanese and Chinese have a much higher frequency of the GSTM1 and GSTT1 null genotypes and NAT2 rapid acetylator type. Therefore, we investigated the association between the polymorphic types of GSTs (GSTM1, GSTT1, GSTP1) and NAT2 and the incidence of gastric cancer which is one of the most prevalent cancers among the East Asians. Methods : It was performed in a case-control study consisting of 238 healthy subjects and 108 cancer patients (54 distal and 54 proximal carcinomas). We also evaluated the association between GSTs and NAT2 and the risk factors for gastric cancer such as alcohol consumption, smoking, H. pylori infection, family history of gastric cancer, and tumor location. Results : In our study, the percentage of cases whose hometown was rural was higher than those of controls (odds ratio (OR) =2.88; 95% CI=1.72-4.76), and the frequency of the lower socio-economic status increased significantly in patients (OR=2.53; 95% CI=1.59-4.02). There was no significant difference in the GST polymorphic types between the cases and controls. However, NAT2 rapid or intermediate acetylator types were frequently detected in the cases with family history of gastric cancer (OR=1.92; 95% CI=1.79-26.0). Conclusions : These results suggest that the hometown and socio-economic status are important environmental factors for gastric carcinogenesis, and NAT2 polymorphic types could be associated with familial gastric carcinoma.

• 대한임상약리학회:학술대회논문집
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• 2001.12a
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• pp.44-45
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• 2001

• Proceedings of the Korean Biophysical Society Conference
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• 2001.06a
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• pp.48-48
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• 2001

Gene expression and protein levels of serotonin N-acetyltransferase (AA-NAT, EC2.3.1.87) directly control the diurnal production of the melatonin and correlate with enzyme activity. Effects of protein phosphorylation on the AA-NAT activity were investigated in rat pineal glands and COS-7 cells transiently transfected with AA-NAT cDNA.(omitted)

• Archives of Pharmacal Research
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• v.16 no.2
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• pp.155-157
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• 1993

DNA transfection conditions were investigated by calcium phosphate-DNA co-precipitation in SK-N-BE(2)C human neuroblastoma cells. The DNA plasmid of TH2400CAT was used in which rat tyrosine hydroxylase gene was inserted into chloramphenicol acetyltransferase reporter gent. The transfection efficiency was 25-30% and the method was simple and reproducible. So, the method will be a good tool for transient transfection analysis.