• Journal of Microbiology
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• 제39권2호
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• pp.95-101
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• 2001

In an effort to develop an efficient expression and secretion system for heterologous proteins in Aspergilius nidulans, the PCR-amplified coding sequence for alkaline pretense (AlpA) of A. oryzae was cloned into a fungal expression vector downstream of A. nidulans aicA (alcohol dehydrogenase) promoter to yield pRAAlp. Transformation of A. nidulans with pRAAlp gave stable transformants harboring various copy numbers (3 to 10) of integrated alpA gene, from among which 6 representatives were selected. On a medium containing 0.8% ammonium sulfate that represses the expression of the host's own pretense, the alcA prumoter-controlled AlpA expression was strongly induced by threonine but repressed by glucose. The level of AlpA secretion was highest (approximately 666 mU/ml) in transformant ALP6 containing the largest copy number integrated alpA. However, the level of AlpA secretion was not necessarily proportional to the copy numbers of the integrated alpA genes. The N-terminal sequence or the secreted mature AlpA was determined to be Gly-Leu-Thr-Thr-Gln-Lys-Ser and its molecular mass to be approximately 34 kDa, indicating that AlpA is properly processed by the removal of 121 N-terminal amino acids.

• 미생물학회지
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• 제33권2호
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• pp.92-96
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• 1997

Pseudomonas sp. strain DJ77로부터 클로닝한 catechol 분해와 관련된 phnDEFG 유전자들이 존재하는 pHENX7에서 phnF 유전자의 염기서열을 밝혔다. Extradiol dioxygenase 유전자인 phnE와, 2-hydroxymuconic semialdehyde dehydrogenase를 생산하는 phnG 유전자 사이에 존재하는 유전자 phnF는 432 bps로 된 하나의 open reading frame(ORF)으로 존재하였고, 여기서 유추한 아미노산은 143개로 분자량 13,859 dalton의 polypeptide를 만들어 내고 있다. phnF 유전자는 Sphingomonas sp. strain HV3 catE 유전자 부위와 sphingomonas yanoikuyae B1의 xylE와 xylG 사이에 존재하는 ORF 부위의 염기서열과 각각 99%, 68.6%의 상동성을 가지고 있었다. 또한 PhnF 단백질의 아미노산서열은 citrobacter freundii DSM30040의 orfY 부위의 아미노산서열과 62.3%의 상동성이 있었다.

• IMMUNE NETWORK
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• 제11권6호
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• pp.348-357
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• 2011

Background: N-myc downstream-regulated gene 2 (NDRG2), a member of a newly described family of differentiation-related genes, has been characterized as a regulator of dendritic cells. However, the role of NDRG2 on the expression and activation of transcription factors in blood cells remains poorly understood. In this study, we investigated the effects of NDRG2 overexpression on GATA-1 expression in PMAstimulated U937 cells. Methods: We generated NDRG2-overexpressing U937 cell line (U937-NDRG2) and treated the cells with PMA to investigate the role of NDRG2 on GATA-1 expression. Results: NDRG2 overexpression in U937 cells significantly induced GATA-1 expression in response to PMA stimulation. Interestingly, JAK2/STAT and BMP-4/Smad pathways associated with the induction of GATA-1 were activated in PMA-stimulated U937-NDRG2 cells. We found that the inhibition of JAK2 activation, but not of BMP-4/Smad signaling, can elicit a decrease of PMA-induced GATA-1 expression in U937-NDRG2 cells. Conclusion: The results reveal that NDRG2 promotes the expression of GATA-1 through activation of the JAK2/STAT pathway, but not through the regulation of the BMP-4/Smad pathway in U937 cells. Our findings further suggest that NDRG2 may play a role as a regulator of erythrocyte and megakaryocyte differentiation during hematopoiesis.

• Molecules and Cells
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• 제28권5호
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• pp.455-461
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• 2009

Calcineurin is a $Ca^{2+}$/Calmodulin activated Ser/Thr phosphatase that is well conserved from yeast to human. It is composed of catalytic subunit A (CnA) and regulatory subunit B (CnB). C. elegans homolog of CnA and CnB has been annotated to tax-6 and cnb-1, respectively and in vivo function of both genes has been intensively studied. In C. elegans, calcineurin play roles in various signaling pathways such as fertility, movement, body size regulation and serotonin-mediated egg laying. In order to understand additional signaling pathway(s) in which calcineurin functions, we screened for binding proteins of TAX-6 and found a novel binding protein, HLH-11. The HLH-11, a member of basic helix-loop-helix (bHLH) proteins, is a putative counterpart of human AP4 transcription factor. Previously bHLH transcription factors have been implicated to regulate many developmental processes such as cell proliferation and differentiation, sex determination and myogenesis. However, the in vivo function of hlh-11 is largely unknown. Here, we show that hlh-11 is expressed in pharynx, intestine, nerve cords, anal depressor and vuvla muscles where calcineurin is also expressed. Mutant analyses reveal that hlh-11 may have role(s) in regulating body size and reproduction. More interestingly, genetic epistasis suggests that hlh-11 may function to regulate serotoninmediated egg laying at the downstream of tax-6.

• 대한핵의학회지
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• 제37권1호
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• pp.43-52
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• 2003

Microarray technology allows the simultaneous analysis of gene expression patterns of thousands of genes, in a systematic fashion, under a similar set of experimental conditions, thus making the data highly comparable. In some cases arrays are used simply as a primary screen loading to downstream molecular characterization of individual gene candidates. In other cases, the goal of expression profiling is to begin to identify complex regulatory networks underlying developmental processes and disease states. Microarrays were originally used with ceil lines or other simple model systems. More recently, microarrays have been used in the analysis of more complex biological tissues including neural systems and the brain. The application of cDNA arrays in neuropsychiatry has lagged behind other fields for a number of reasons. These include a requirement for a large amount of input probe RNA In fluorescent-glass based array systems and the cellular complexity introduced by multicellular brain and neural tissues. An additional factor that impacts the general use of microarrays in neuropsychiatry is the lack of availability of sequenced clone sets from model systems. While human cDNA clones have been widely available, high qualify rat, mouse, and drosophilae, among others are just becoming widely available. A final factor in the application of cDNA microarrays in neuropsychiatry is cost of commercial arrays. As academic microarray facilitates become more commonplace custom made arrays will become more widely available at a lower cost allowing more widespread applications. in summary, microarray technology is rapidly having an impact on many areas of biomedical research. Radioisotope-nylon based microarrays offer alternatives that may in some cases be more sensitive, flexible, inexpensive, and universal as compared to other array formats, such as fluorescent-glass arrays. In some situations of limited RNA or exotic species, radioactive membrane microarrays may be the most practical experimental approach in studying psychiatric and neurodegenerative disorders, and other complex questions in the brain.

• 원예과학기술지
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• 제29권1호
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• pp.53-60
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• 2011

Cytoplasmic male sterility (CMS) induced by mutant mitochondria genome, has been used for commercial seed production of $F_1$ hybrid cultivars in diverse crops. In pepper (Capsicum annuum L.), two sterile cytoplasm specific gene organization, atp6-2 and coxII were identified. An open reading frame, orf456 nearby coxII gene has been speculated to induce male sterility (MS) by mutagenic analysis. Moreover, molecular markers for atp6-2 and coxII of mitochondrial genotype (mitotype) were developed. However, the Cytoplasmic MS specific markers, atp6SCAR and coxIISCAR markers appeared in both N and S cytoplasms when polymerase chain reaction (PCR) cycles prolonged more than 40 cycles. Since the reported molecular markers were dominant markers, the presence of the faint sterile-specific band in normal cytoplasm may lead to the mis-classification of pepper breeding lines. To solve this problem, one common forward primer and two different reverse primers specific to normal coxII and sterile orf456 genes were designed after analyzing their gene organizations. By using these three primers, N and S coxII specific bands were co-amplified in male-sterile lines, but only normal coxII specific band was amplified in maintainer lines. Since the reverse primer for sterile coxII was specifically designed 275 bp downstream of orf456, relatively stable PCR amplification patterns were observed regardless of the number of PCR cycles. These primer sets easily identified different mitotypes among the divergent breeding lines, commercial cultivars and diverse germplasms.

• Nutrition Research and Practice
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• 제7권4호
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• pp.267-272
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• 2013

The anti-obesity effects of a hot water extract from wasabi (Wasabia japonica Matsum.) leaves (WLE), without its specific pungent constituents, such as allyl-isothiocyanate, were investigated in high fat-diet induced mice. C57J/BL mice were fed a high-fat diet (control group) or a high-fat diet supplemented with 5% WLE (WLE group). Physical parameters and blood profiles were determined. Gene expression associated with lipid metabolism in liver and white adipose tissue were analyzed. After 120 days of feeding, significantly lower body weight gain, liver weight and epididymal white adipose tissue weight was observed in the WLE group compared to the control group. In liver gene expression within the WLE group, PPAR${\alpha}$ was significantly enhanced and SREBP-1c was significantly suppressed. Subsequent downstream genes controlled by these regulators were significantly suppressed. In epididymal white adipose tissue of the WLE group, expression of leptin, PPAR${\gamma}$, and C/EBP${\alpha}$ were significantly suppressed and adiponectin was significantly enhanced. Acox, related to fatty acid oxidization in adipocytes, was also enhanced. Our results demonstrate that the WLE dietary supplement induces mild suppression of obesity in a high-fat diet induced mice, possibly due to suppression of lipid accumulation in liver and white adipose tissue.

• Journal of Applied Biological Chemistry
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• 제64권3호
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• pp.225-236
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• 2021

The epipyrone (EPN) biosynthetic gene cluster of Epicoccum nigrum is composed of epnC, epnB, and epnA, which encode cytochrome P450 oxidase, glycosyltransferase, and highly reducing polyketide synthase, respectively. Gene inactivation mutants for epnA, epnB, and epnC were previously generated, and it was found that all of them were incapable of producing EPN and any of its related compounds. It was also reported that epnB inactivation abolished epnA transcription, generating ΔepnAB. This study shows that the introduction of native epnC readily restored EPN production in ΔepnC, suggesting that epnC is essential for polyketide release from EpnA and implies that EpnC works during the polyketide chain assembly of EpnA. Introduction of epnC promoter-epnA restored EPN production in ΔepnA. The ΔepnB genotype was prepared by introducing the epnA expression vector into ΔepnAB, and it was found that the resulting recombinant strain did not produce any EPN-related compounds. A canonical epnB inactivation strain was also generated by deleting its 5'-end. At the deletion point, an Aspergllus nidulans gpdA promoter was inserted to ensure the transcription of epnA, which is located downstream of epnB. Examination of the metabolite profile of the resulting ΔepnB mutant via LC-mass spectrometry verified that no EPN-related compound was produced in this strain. This substantiates that C-glycosylation by EpnB is a prerequisite for the release of EpnA-tethered product. In conclusion, it is proposed that cytochrome P450 oxidase and glycosyltransferase work in concert with polyketide synthase to generate EPN without the occurrence of any free intermediates.

• 한국육종학회지
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• 제42권4호
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• pp.365-373
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• 2010

TILLING (Targeting Induced Local Lesions IN Genomes) is broadly regarded as an excellent methodology for reverse genetics applications. Approximately 15,000 $M_3$ TILLING lines have been developed via the application of gamma-ray irradiation to rice seeds (cv. Donganbyeo), followed by subsequent selections. In an effort to evaluate the genetic diversity of the TILLING population, we have employed the AFLP multiple dominant marker technique. A total of 96 (0.64%) TILLING lines as well as Donganbyeo were selected randomly and their genetic diversity was assessed based on AFLP marker polymorphisms using 5 primer combinations. An average of 100.4 loci in a range of 97 to 106 was detected using these primer combinations, yielding a total of 158 (31.4%) polymorphic loci between Donganbyeo and each of the 96 lines. A broad range of similarity from 80% to 96% with an average of 89.4% between Donganbyeo and each of the 96 lines was also observed, reflecting the genetic diversity of the TILLING population. Approximately 28 polymorphic loci have been cloned and their sequences were BLAST-searched against rice whole genome sequences, resulting in 20 matches to each of the gene bodies including exon, intron, 1 kb upstream and 1 kb downstream regions. Six polymorphic loci evidenced changes in the coding regions of genes as compared to the rice pseudomolecules, 4 loci of which exhibited missense mutations and 2 loci of which exhibited silent mutations. Therefore, the results of our study show that the TILLING rice population should prove to be a useful genetic material pool for functional genomics as well as mutation breeding applications.

• Journal of Ginseng Research
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• 제43권3호
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• pp.452-459
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• 2019

Background: Ginsenoside compound K(C-K), a major metabolite of ginsenoside, exhibits anticancer activity in various cancer cells and animal models. A cell signaling study has shown that C-K inhibited nuclear factor-kappa B ($NF-{\kappa}B$) pathway in human astroglial cells and liver cancer cells. However, the molecular targets of C-K and the initiating events were not elucidated. Methods: Interaction between C-K and Annexin A2 was determined by molecular docking and thermal shift assay. HepG2 cells were treated with C-K, followed by a luciferase reporter assay for $NF-{\kappa}B$, immunofluorescence imaging for the subcellular localization of Annexin A2 and $NF-{\kappa}B$ p50 subunit, coimmunoprecipitation of Annexin A2 and $NF-{\kappa}B$ p50 subunit, and both cell viability assay and plate clone formation assay to determine the cell viability. Results: Both molecular docking and thermal shift assay positively confirmed the interaction between Annexin A2 and C-K. This interaction prevented the interaction between Annexin A2 and $NF-{\kappa}B$ p50 subunit and their nuclear colocalization, which attenuated the activation of $NF-{\kappa}B$ and the expression of its downstream genes, followed by the activation of caspase 9 and 3. In addition, the overexpression of Annexin A2-K320A, a C-K binding-deficient mutant of Annexin A2, rendered cells to resist C-K treatment, indicating that C-K exerts its cytotoxic activity mainly by targeting Annexin A2. Conclusion: This study for the first time revealed a cellular target of C-K and the molecular mechanism for its anticancer activity.