• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.367-372
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• 2019

Deactivation of aminoglycosides by their modifying enzymes, including a number of aminoglycoside O-phosphotransferases, is the most ubiquitous resistance mechanism in aminoglycoside-resistant pathogens. Nonetheless, in a couple of biosynthetic pathways for gentamicins, fortimicins, and istamycins, phosphorylation of aminoglycosides seems to be a unique and initial step for the creation of a natural defensive structural feature such as a 3',4'-dideoxy scaffold. Our aim was to elucidate the biochemical details on the beginning of these C3',4'-dideoxygenation biosynthetic steps for aminoglycosides. The biosynthesis of istamycins must surely involve these 3',4'-didehydroxylation steps, but much less has been reported in terms of characterization of istamycin biosynthetic genes, especially about the phosphotransferase-encoding gene. In the disruption and complementation experiments pointing to a putative gene, istP, in the genome of wild-type Streptomyces tenjimariensis, the function of the istP gene was proved here to be a phosphotransferase. Next, an in-frame deletion of a known phosphotransferase-encoding gene forP from the genome of wild-type Micromonospora olivasterospora resulted in the appearance of a hitherto unidentified fortimicin shunt product, namely 3-O-methyl-FOR-KK1, whereas complementation of forP restored the natural fortimicin metabolite profiles. The bilateral complementation of an istP gene (or forP) in the ${\Delta}forP$ mutant (or ${\Delta}istP$ mutant strain) successfully restored the biosynthesis of 3',4'-dideoxy fortimicins and istamycins, thus clearly indicating that they are interchangeable launchers of the biosynthesis of 3',4'-dideoxy types of 1,4-diaminocyclitol antibiotics.

• Reproductive and Developmental Biology
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• 제38권2호
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• pp.71-77
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• 2014

The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with ${\alpha}1,3$-galactosyltransferase gene (${\alpha}1,3$-GT gene), DT-A/pGT5'/neo/pGT3', DT-A/NLS/pGT5'/neo/pGT3' and pGT5'/neo/ pGT3'/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5' recombination arm (pGT5') and a 1.9-kb fragment as the 3' recombination arm (pGT3'). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of ${\alpha}1,3$-GT locus. DT-A/pGT5'/neo/pGT3' vector contain only positive-negative selection marker with conventional targeting vector. DT-A/NLS/pGT5'/neo/pGT3' vector contain positive-negative selection marker and NLS sequences in upstream of 5' recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5'/neo/pGT3'/NLS vector contain only positive selection marker and NLS sequence in downstream of 3' recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with $300{\mu}g/ml$ G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for ${\alpha}1,3$-GT gene disruption in 3' PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5'/neo/pGT3' knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.

• 한국응용곤충학회지
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• 제47권1호
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• pp.37-44
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• 2008

복숭아순나방(Grapholita molesta)은 사과에 주요 해충이다. 이 해충을 환경친화형 방법으로 방제하기 위해 성페로몬을 이용한 교미교란제 처리 기술이 개발되고 있다. 광범위한 영역에 대한 교미교란제의 처리는 복숭아순나방 방제에 효과적이나 이러한 광범위영역에 대한 구체적 크기를 결정하는 방법은 알려지지 않았다. 이를 결정하기 위해서는 복숭아순나방의 교미행동 범위를 결정할 수 있는 방법이 개발되어야 하며, 이를 위해 복숭아순나방의 이동을 추적할 수 있는 분자지표의 개발이 필요하게 되었다. 본 연구는 RAPD (random amplified polymorphic DNA) 기술을 이용하여 효과적인 두 개의 분자지표를 개발하였다. 상이한 지역과 시기에 따라 구분되는 야외 복숭아순나방 집단들에 대해서 이들 분자지표를 이용하여 집단 유전 분석이 실시되었다 이들 분자지표들은 특정지역에서 복숭아순나방 집단들이 시기적으로 유전적 조성에 뚜렷한 차이를 보여 주었다. 또한 서로 다른 지역의 복숭아순나방 집단들은 거리에 따라 상대적으로 차이가 증가하였지만, 계절이 진행함에 따라 그 차이가 감소하였다.

• Journal of Korean Biological Nursing Science
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• 제18권4호
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• pp.305-317
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• 2016

Purpose: Recent studies demonstrated disruption of the circadian clock gene is associated with the development of obesity and metabolic syndrome. Obesity is often caused by the high calorie intake, In addition, the chronic stress tends to contribute to the increased risk for obesity. To evaluate the molecular mechanisms, we examined the expression of circadian clock genes in high fat diet-induced mice models with the chronic stress. Methods: C57BL/6J mice were fed with a 45% or 60% high fat diet for 8 weeks. Daily immobilization stress was applied to mice fed with a 45% high fat for 16 weeks. We compared body weight, food consumption, hormone levels and metabolic variables in blood. mRNA expression levels of metabolic and circadian clock genes in both fat and liver were determined by quantitative RT-PCR. Results: The higher fat content induced more severe hyperglycemia, hyperlipidemia and hyperinsulinemia, and these results correlated with their relevant gene expressions in fat and liver tissues. Chronic stress had only minimal effects on metabolic variables, but it altered the expression patterns of metabolic and circadian clock genes. Conclusion: These results suggest that the fat metabolism regulates the function of the circadian clock genes in peripheral tissues, and stress hormones may contribute to its regulation.

• Molecules and Cells
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• 제45권8호
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• pp.537-549
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• 2022

Preproenkephalin (PPE) is a precursor molecule for multiple endogenous opioid peptides Leu-enkephalin (ENK) and Met-ENK, which are involved in a wide variety of modulatory functions in the nervous system. Despite the functional importance of ENK in the brain, the effect of brain-derived factor(s) on PPE expression is unknown. We report the dual effect of neural epidermal growth factor (EGF)-like-like 2 (NELL2) on PPE gene expression. In cultured NIH3T3 cells, transfection of NELL2 expression vectors induced an inhibition of PPE transcription intracellularly, in parallel with downregulation of protein kinase C signaling pathways and extracellular signal-regulated kinase. Interestingly, these phenomena were reversed when synthetic NELL2 was administered extracellularly. The in vivo disruption of NELL2 synthesis resulted in an increase in PPE mRNA level in the rat brain, suggesting that the inhibitory action of intracellular NELL2 predominates the activation effect of extracellular NELL2 on PPE gene expression in the brain. Biochemical and molecular studies with mutant NELL2 structures further demonstrated the critical role of EGF-like repeat domains in NELL2 for regulation of PPE transcription. These are the first results to reveal the spatio-specific role of NELL2 in the homeostatic regulation of PPE gene expression.

• BMB Reports
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• 제42권2호
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• pp.101-105
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• 2009

The homozygous T-DNA mutant of the AMY1 gene in Arabidopsis was identified and importantly, shown to cause an early flowering phenotype. We found that the disruption of AMY1 enhanced expression of CO and FT. The expression analyses of genes related to starch metabolism revealed that expression of the AGPase small subunit APS1 in the wild type was higher than in the amy1 mutant. However, there were no significant differences in expression levels of the AGPase large subunit genes ApL1, AMY2, or AMY3 between wild type and the amy1 mutant. Expression profiling showed that AMY1 was highly expressed in leaves, stems, and flowers, and expressed less in leafstalks and roots. Furthermore, the level of AMY1 mRNA was highly elevated with age and in senescing leaves. RT-PCR analyses showed that the expression of AMY1 was induced by heat shock, GA, and ABA, while salt stress had no apparent effect on its expression.

• Molecular & Cellular Toxicology
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• 제1권2호
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• pp.78-86
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• 2005

Dioxins, especially 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin), are ubiquitous environmental contaminants. TCDD is known that it has toxic effects in animals and humans, including chloracne, immune, reproductive and developmental toxicities, carcinogenicity, wasting syndrome and death. TCDD induces a broad spectrum of biological responses, including disruption of normal hormone signaling pathways, reproductive and developmental defects, immunotoxicity, liver damage, wasting syndrome and cancer. Many researches showed that TCDD induces gene expression of transcriptional factors related cell proliferation, signal transduction, immune system and cell cycle arrest at molecular and cellular levels. These toxic actions of TCDD are usually mediated with AhR (receptor, resulted from cell culture, animal and clinical studies). cDNA microarray can be used as a highly sensitive and informative marker for toxicity. Additionally, microarray analysis of dioxin-toxicity is able to provide an opportunity for the development of candidate bridging biomarkers of dioxin-toxicity. Through microarray technology, it is possible to understand the therapeutic effects of agonists within the context of toxic effects, classify new chemicals as to their complete effects on biological systems, and identify environmental factors that may influence safety.

• Molecular & Cellular Toxicology
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• 제3권3호
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• pp.185-189
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• 2007

Gene expression levels of choriogenin, vitellogenin and estrogen receptor were determined using Reverse transcription (RT)-PCR technique after exposure to estrogenic chemical bisphenol A in the Korean wild medaka (Oryzias sinensis). These genes have been known to be induced in male test fish when the fish are exposed to estrogenic chemicals. Therefore they can be suggested as a possible biomarker of endocrine disruption in fish, however, relatively little has been known about these genes expression by estrogenic chemicals in Korean wild fish. Mature male Oryzias sinensis were treated with bisphenol A at nominal concentrations of 0.02, 0.2 and 2 mg/L for 6 days and total RNA was extracted from the livers of treated fish for RT-PCR. When the five biomarker genes were amplified by RT-PCR in the same condition, mRNA induction level of each gene was elevated with different sensitivities. Conclusively, the results of this work indicated that measurement of vitellogenin and choriogenin using RT-PCR is effective as a simple tool for the screening of estrogenic chemicals and suggested that O. sinensis would be a suitable model fish for the environmental risk assessment of potential endocrine disruptors.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2002년도 추계학술대회
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• pp.27-30
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• 2002

Ralstonia eutrupha JMP134 is a well-known soil bacterium which can metabolite diverse aromatic compounds and xenobiotics, such as phenol, 2,4-dichlorophenoxy acetic acid (2, 4-D), and trichloroethylene (TCE), etc. Phenol is degraded through chromosomally encoded phenol degradation pathway. Phenol is first metabolized into catechol by a multicomponent phenol hydroxylase, which is further metabolized to TCA cycle intermediates via a meta-cleavage pathway. The nucleotide sequences of the genes for the phenol hydroxylase have previously been determined, and found to composed of eight genes phlKLMNOPRX in an operon structure. The phlR, whose gene product is a NtrC-like transcriptional activator, was found to be located at the internal region of the structural genes, which is not the case in most bacteria where the regulatory genes lie near the structural genes. In addition to this regulatory gene, we found other regulatory genes, the phlA and phlR2, downstream of the phlX. These genes were found to be overlapped and hence likely to be co-transcribed. The protein similarity analysis has revealed that the PhlA belongs to the GntR family, which are known to be negative regulators, whereas the PhlR2 shares high homology with the NtrC-type family of transcriptional activators like the PhlR. Disruption of the phlA by insertional mutation has led to the constitutive expression of the activity of phenol hydroxylase in JMP134, indicating that PhlA is a negative regulator. Possible regulatory mechanisms of phenol metabolism in R. eutropha JMP134 has been discussed.

• Journal of Microbiology
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• 제43권2호
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• pp.191-195
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• 2005

In this study, we attempted to characterize the Synechococcus sp. pee 7942 mutant resultant from a disruption in the gene encoding UDP-glucose: tetrahydrobiopterin a-glucosyltransferase (BGluT). 2D­PAGE followed by MALDI-TOF mass spectrometry revealed that phycocyanin rod linker protein 33K was one of the proteins expressed at lower level in the BGluT mutant. BGluT mutant cells were also determined to be more sensitive to high light stress. This is because photosynthetic O$_2$ exchange rates were significantly decreased, due to the reduced number of functional PSIs relative to the wild type cells. These results suggested that, in Synechococcus sp. pee 7942, BH4-glucoside might be involved in photosynthetic photoprotection.