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Engineering of Flavonoid O-Methyltransferase for a Novel Regioselectivity
Eun Ji Joe,김봉규,Byoung-Chan An,정유훈,안중훈 한국분자세포생물학회 2010 Molecules and cells Vol.30 No.2
An O-methyltransferase isolated from poplar, POMT7, was identified as a flavone 7-O-methyltransferase. In order to generate a mutant of POMT-7 having a novel regioselectivity,we conducted an error-prone polymerase chain reaction. More than 100 mutants were screened and one of the mutants (POMT-M1) Asp257Gly, methylated the 3-hydroxyl group of flavonols in addition to 7-hydrdoxyl group. The mutation changed asparagine residue at the position of 257into glycine. The kinetic parameters showed that the wild type POMT7 was better activity toward kaempferol and quercetin than the POMT7-M1. Using E. coli transformant expressing POMT7-M1, 58 μM of 3, 7-O-dimethylquercetin and 70 μM of 3, 7-O-dimethylkaempferol from 100 μM of corresponding substrate were synthesized successfully.
Engineering of Flavonoid O-Methyltransferase for a Novel Regioselectivity
Joe, Eun-Ji,Kim, Bong-Gyu,An, Byoung-Chan,Chong, You-Hoon,Ahn, Joong-Hoon Korean Society for Molecular and Cellular Biology 2010 Molecules and cells Vol.30 No.2
An O-methyltransferase isolated from poplar, POMT7, was identified as a flavone 7-O-methyltransferase. In order to generate a mutant of POMT-7 having a novel regioselectivity, we conducted an error-prone polymerase chain reaction. More than 100 mutants were screened and one of the mutants (POMT-M1) Asp257Gly, methylated the 3-hydroxyl group of flavonols in addition to 7-hydrdoxyl group. The mutation changed asparagine residue at the position of 257 into glycine. The kinetic parameters showed that the wild type POMT7 was better activity toward kaempferol and quercetin than the POMT7-M1. Using E. coli transformant expressing POMT7-M1, 58 ${\mu}m$ of 3, 7-O-dimethylquercetin and 70 ${\mu}m$ of 3, 7-O-dimethylkaempferol from 100 ${\mu}m$ of corresponding substrate were synthesized successfully.
Comparison of serological tests for bovine brucellosis in South Korea
Ah-Ryeong Joe, Jin Ju Lee, Eun Ji Yum, Yoon-Jeong Seo, So-Ra Sung, Jeong-Soo Choi, Soon-Seek Yoon 한국예방수의학회 2023 예방수의학회지 Vol.47 No.3
Regarding to diagnosis for bovine brucellosis, more than one serological test should be conducted to confirm the infection by Brucella with a reliable result due to various factors including false positive serological reactions. In this study, we compared confirmatory serological tests to determine the appropriate way to detect and confirm the Brucella infection in South Korea. Several serological tests, including serum agglutination test (SAT), indirect (I)- and competitive (C)-ELISA, and fluorescence polarization assay (FPA), for detection of bovine brucellosis were performed with sera from 537 cattle. In addition, comparison of diagnostic efficacy was performed with bacterial isolation represented true positive. Of 537 serum samples, 426 (79.3% of prevalence), 433 (80.6%), 414 (77.1%), and 409 (76.2%) sera were positive for SAT, C-ELISA, I-ELISA, and FPA respectively. Based on the results of serology, the correlation among the serological tests revealed observed agreements of more than 92% with kappa (k) value of more than 0.77. The correlation between serological tests with bacterial isolation appeared observed agreements of between 79.9% and 84.7% with k value of between 0.42 and 0.59. Particularly, FPA recorded almost perfect agreements with C-ELISA and I-ELISA as well as the highest correlation with bacterial isolation. Accordingly, this investigation presented the comparison of correlation and diagnostic efficacy of serological tests for bovine brucellosis in South Korea. We suggest this finding will be a useful data to re-establish the potential serological diagnostic methods that can apply to maintain the low prevalence.
Ji, Kyung-Ae,Yang, Myung-Soon,Jeong, Hey-Kyeong,Min, Kyoung-Jin,Kang, Seung-Hee,Jou, Ilo,Joe, Eun-Hye John Wiley & Sons, Inc. 2007 Glia Vol.55 No.15
<P>Generally, it has been accepted that microglia play important roles in brain inflammation. However, recently several studies suggested possible infiltration of blood neutrophils and monocytes into the brain. To understand contribution of microglia and blood inflammatory cells to brain inflammation, the behavior of microglia, neutrophils, and monocytes was investigated in LPS (lipopolysaccharide)-injected substantia nigra pars compacta, cortex, and hippocampus of normal and/or leukopenic rats using specific markers of neutrophils (myeloperoxidase, MPO), and microglia and monocytes (ionized calcium binding adaptor molecule-1, Iba-1), as well as a general marker for these inflammatory cells (CD11b). CD11b-immunopositive (CD11b<SUP>+</SUP>) cells and Iba-1<SUP>+</SUP> cells displayed similar behavior in intact and LPS-injected brain at 6 h after the injection. Interestingly, however, CD11b<SUP>+</SUP> cells and Iba-1<SUP>+</SUP> cells displayed significantly different behavior at 12 h: Iba-1<SUP>+</SUP> cells disappeared while CD11b<SUP>+</SUP> cells became round in shape. We found that CD11b/Iba-1-double positive (CD11b<SUP>+</SUP>/Iba-1<SUP>+</SUP>) ramified microglia died within 6 h after LPS injection. The round CD11b<SUP>+</SUP> cells detected at 12 h were MPO<SUP>+</SUP>. These CD11b<SUP>+</SUP>/MPO<SUP>+</SUP> cells were not found in leukopenic rats, suggestive of neutrophil infiltration. MPO<SUP>+</SUP> neutrophils expressed inducible nitric oxide synthase, interleukin-1β, cyclooxygenase-2, and monocyte chemoattractant protein-1, but died within 18 h. CD11b<SUP>+</SUP> cells detected at 24 h appeared to be infiltrated monocytes, since these cells were once labeled with Iba-1 and were not found in leukopenic rats. Furthermore, transplanted monocytes were detectable in LPS-injected brain. These results suggest that at least a part of neutrophils and monocytes could have been misinterpreted as activated microglia in inflamed brain. © 2007 Wiley-Liss, Inc.</P>
Analysis of glucocorticoid-induced <i>MYOC</i> expression in human trabecular meshwork cells
Joe, Myung Kuk,Sohn, Seongsoo,Kim, Tae Eun,Im, Ji-eun,Choi, Young Ran,Kee, Changwon Elsevier 2011 VISION RESEARCH - Vol.51 No.9
<P><B>Research highlights</B></P><P><I>► MYOC</I> is a delayed secondary glucocorticoid-response gene. ► A region between −2548 and −1541 on the <I>MYOC</I> promoter is important for the glucocorticoid response. <I>► MYOC</I> mRNA is very stable and its level is increased in proportion to the time of exposure to glucocorticoid. <I>► MYOC</I> expression is tissue-specific and may require tissue-specific factor(s).</P> <P><B>Abstract</B></P><P>To understand the regulatory mechanisms governing glucocorticoid-mediated <I>MYOC</I> induction in <I>human trabecular meshwork</I> (HTM) cells, the expression and degradation of <I>MYOC</I> mRNA were quantified in HTM cells by Northern blot analysis, and the transcriptional activity of constructs containing variable lengths of putative <I>MYOC</I> promoters was assessed by luciferase reporter assay. Here, we confirmed that <I>MYOC</I> is a delayed secondary glucocorticoid-responsive gene by demonstrating that its transcription was not initiated immediately by the addition of dexamethasone (DEX) and was completely inhibited by treatment with cycloheximide. In addition, we demonstrated that <I>MYOC</I> mRNA is degraded very slowly, with approximately half persisting for at least 4days, suggesting that its mRNA is intrinsically quite stable. Promoter analysis of up to 5271 base pairs upstream of <I>MYOC</I> revealed that luciferase induction by DEX was increased by 280±34% in HTM cells. Moreover, DEX induction required the region between base pairs −2548 and −1541. However, the putative regulatory element exhibited little activity in other cell lines, including TM-5, 293A, SH-SY5Y, and <I>human retinal pigment epithelium</I> (RPE) cells. To our knowledge, this study provides the first evidence for the presence of a cis-acting region for secondary glucocorticoid responsiveness in the 5′-flanking sequences of <I>MYOC</I>. It will be a major step towards understanding the expression pattern of <I>MYOC</I> in HTM cells and TM tissue.</P>
Ji, Kyung-Ae,Eu, Mi Young,Kang, Seung-Hee,Gwag, Byoung Joo,Jou, Ilo,Joe, Eun-Hye Wiley Subscription Services, Inc., A Wiley Company 2008 Glia Vol.56 No.10
<P>Brain inflammation is a suggested risk factor for neurodegenerative disease. Interestingly, severe inflammation in the substantia nigra pars compacta (SNpc) accelerates the onset and progression of Parkinson's disease. In this study, we examined the underlying mechanisms of severe inflammation in the SNpc by comparing the inflammatory process with that in the cortex. In intact brain, the densities of CD11b<SUP>+</SUP> microglia were similar in the SNpc and cortex. However, lipopolysaccharide injection enhanced the CD11b<SUP>+</SUP> cell number in the SNpc, but not in the cortex. Previously, we reported that CD11b and myeloperoxidase (MPO) double-positive neutrophils infiltrate the SNpc following LPS injection (GLIA 55:1577–88). Notably, the MPO<SUP>+</SUP> neutrophil number increased dramatically in the SNpc, but only slightly in the cortex. The extent of neutrophil infiltration appeared to correlate with neuronal damage. We confirmed that loss of neurons in the SNpc was significantly reduced in neutropenic rats versus normal rats following LPS injection. In addition, the densities of astrocytes were much lower in the intact SNpc, compared with the cortex. Furthermore, after LPS injection, damage of endothelial cells and astrocytes, and blood–brain barrier (BBB) permeability was more pronounced in the SNpc. These results collectively suggest that excessive neutrophil infiltration and environmental factors, such as lower astrocyte density and higher BBB permeability, contribute to severe inflammation and neuronal death in the SNpc. © 2008 Wiley-Liss, Inc.</P>