• 한국미생물·생명공학회지
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• 제47권4호
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• pp.662-666
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• 2019

본 연구에서는 Saccharomyces cerevisiae를 이용해서 neoagarobiose hydrolase (NABH)를 효율적으로 생산하기 위한 NABH558 유전자 발현시스템을 구축하였다. ADH1 promoter와 GAL10 promoter 하류에 NABH558 유전자를 가진 pAMFα-NABH plasmid와 pGMFα-NABH plasmid는 S. cerevisiae 2805 균주에 형질전환되었다. 2805/pAMFα-NABH 균주는 YPD (2% dextrose) 배지에서 가장 높은 NABH 효소 활성(0.069 unit/ml/DCW)을 보였고, 2805/pGMFα-NABH 균주의 경우는 배지의 조성과 상관없이 비슷한 수준의 NABH 활성(0.02-0.027 unit/ml/DCW)을 보였다. RT-PCR을 통한 NABH558 유전자의 transcription level은 NABH 활성 증가에 따라 비슷한 수준으로 증가되었음을 확인할 수 있었다. 또한 재조합균주에서 생산된 NABH는 agarose를 galactose와 AHG로 분해하였다. 따라서 NABH558 유전자의 발현에는 ADH1 promoter를 사용하는 것이 더 효율적이며 GAL10 promoter와 비교해서 최대 3배정도 높은 활성의 재조합 NABH를 생산할 수 있음을 알 수 있었다.

• The Korean Journal of Physiology and Pharmacology
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• 제14권6호
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• pp.353-358
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• 2010

This study demonstrates the ability of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, to inhibit LPS-induced expression of iNOS gene and activation of NF-${\kappa}B$/Rel in RAW 264.7 cells. Immunohisto-chemical staining of iNOS and Western blot analysis showed magnolol to inhibit iNOS gene expression. Reporter gene assay and electrophoretic mobility shift assay showed that magnolol inhibited NF-${\kappa}B$/Rel transcriptional activation and DNA binding, respectively. Since p38 is important in the regulation of iNOS gene expression, we investigated the possibility that magnolol to target p38 for its anti-inflammatory effects. A molecular modeling study proposed a binding position for magnolol that targets the ATP binding site of p38 kinase (3GC7). Direct interaction of magnolol and p38 was further confirmed by pull down assay using magnolol conjugated to Sepharose 4B beads. The specific p38 inhibitor SB203580 abrogated the LPS-induced NF-${\kappa}B$/Rel activation, whereas the selective MEK-1 inhibitor PD98059 did not affect the NF-${\kappa}B$/Rel. Collectively, the results of the series of experiments indicate that magnolol inhibits iNOS gene expression by blocking NF-${\kappa}B$/Rel and p38 kinase signaling.

• Asian Pacific Journal of Cancer Prevention
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• 제14권2호
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• pp.651-658
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• 2013

Uterine leiomyomas (UL) are extremely common neoplasms in women of reproductive age, and are associated with a variety of characteristic choromosomal aberrations (CAs). The p53 gene has been reported to play a crucial role in suppressing the growth of a variety of cancer cells. Therefore, the present study investigated the effects of CAs and the p53 gene on ULs. We performed cytogenetic analysis by G-banding in 10 cases undergoing myomectomy or hysterectomy. Fluorescence in situ hybridization (FISH) with a p53 gene probe was also used on interphase nuclei to screen for deletions. In patients, CAs were found in 23.4% of 500 cells analysed, significantly more frequent than in the control group (p<0.001). In the patients, 76% of the abnormalities were structural aberrations (deletions, translocations and breaks), and only 24% were numerical. Deletions were the most common structural aberration observed in CAs. Among these CAs, specific changes in five loci 1q11, 1q42, 2p23, 5q31 and Xp22 have been found in our patients and these changes were not reported previously in UL. The chromosome breaks were more frequent in cases, from high to low, 1, 2, 6, 9, 3, 5, 10 and 12. Chromosome 22, X, 3, 17 and 18 aneuploidy was observed to be the most frequent among all numerical aberrations. We observed a low frequency of p53 losses (2-11%) in our cases. The increased incidence of autosomal deletions, translocations, chromatid breaks and aneuploidy, could contribute to the progression of the disease along with other chromosomal alterations.

• Bulletin of the Korean Chemical Society
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• 제30권4호
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• pp.897-901
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• 2009

Heme oxygenase-1 (HO-1) is an anti-inflammatory and anti-apoptotic protein and has been applied to various gene therapy researches. However, constitutive expression of HO-1 may induce deleterious side effects. In this research, hypoxia inducible HO-1 expression plasmid, pEpo-SV-HO-1, was constructed with the erythropoietin (epo) enhancer and simian virus 40 (SV40) promoter to avoid these unwanted side effects. Dexamethasone conjugated polyethylenimine (PEI-Dexa) was used as a gene carrier. It was previously reported that dexamethasone protected cardiomyocytes from apoptosis under hypoxia. In this research, PEI-Dexa reduced the caspase-3 level in hypoxic H9C2 cardiomyocytes as a derivative of dexamethasone, suggesting that PEI-Dexa is an anti-apoptotic reagent as well as a gene carrier. pEpo-SV-HO-1 was transfected to H9C2 cardiomyocytes using PEI-Dexa and the cells were incubated under normoxia or hypoxia. HO-1 expression was induced in the pEpo-SV-HO-1 transfected cells under hypoxia. In addition, cell viability under hypoxia was higher in the pEpo-SV-HO-1 transfected cells than the pEpo-SV-Luc transfected cells. Also, caspase-3 level was reduced in the pEpo-SV-HO-1 transfected cells under hypoxia. In addition to the anti-apoptotic effect of PEI-Dexa, hypoxia inducible HO-1 expression by pEpo-SVHO- 1 may be helpful to protect cardiomyocytes under hypoxia. Therefore, pEpo-SV-HO-1/PEI-Dexa complex may be useful for ischemic heart disease gene therapy.

• International Journal of Industrial Entomology and Biomaterials
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• 제9권2호
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• pp.235-239
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• 2004

Drosophila melanogaster heat shock protein 70 gene promoter (Dhsp70p) is widely used in transgenic insect to drive exogenous gene, and the homologous region 3 from Bombyx mori nucleopolyhedrovirus (BmNPVhr3) functions as an enhancer for several promoters. To test whether BmNPVhr3 can enhance the Dhsp70ps transcriptional activity, the reporter plasmids, which contain the Dhsp70p, the reporter $\beta$-galactosidase gene with SV40 terminator and BmNPVhr3 fragment, are constructed and transfected into the insect cell lines (Bm-N cells and Sf-21 cells) by lipofectin-mediated method. The results from the transient expression assay show that BmNPVhr3 significantly increases transcriptional activity of Dhsp70p both under the normal condition and under the heat-shock treatment, although the effects are significantly different between in Bm-N cells and in sf-21 cells. The enhancing behavior of BmNPVhr3 on the Dhsp70p is in an orientation-independent manner. Meanwhile, the effects of heat-shock treatment on Dhsp70p alone or Dhsp70p/BmNPVhr3 combination present no significant difference, indicating that BmNPVhr3 only enhances the transcriptional activity of Dhsp70p, but cant alter its characteristic of the response to the heat-shock stress. The above results suggest that the Dhsp70p/BmNPVhr3 combination is more effective one to drive exogenous gene for transgene or stable cell expression system in insects.

• Asian-Australasian Journal of Animal Sciences
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• 제31권6호
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• pp.775-783
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• 2018

Objective: The purpose of this study was to investigate the genetic effects of six keratin (KRT) genes on the wool traits of 418 Chinese Merino (Xinjiang type) (CMXT) individuals. Methods: To explore the effects and association of six KRT genes on sheep wool traits, The polymerase chain reaction-based single-strand conformation polymorphism (PCR-SSCP), DNA sequencing, and the gene pyramiding effect methods were used. Results: We report 20 mutation sites (single-nucleotide polymorphisms) within the six KRT genes, in which twelve induced silent mutations; five induced missense mutations and resulted in $Ile{\rightarrow}Thr$, $Glu{\rightarrow}Asp$, $Gly{\rightarrow}Ala$, $Ala{\rightarrow}Ser$, $Se{\rightarrow}His$; two were nonsense mutations and one was a same-sense mutation. Association analysis showed that two genotypes of the KRT31 gene were significantly associated with fiber diameter (p<0.05); three genotypes of the KRT36 gene were significantly associated with wool fineness score and fiber diameter (p<0.05), three genotypes of the KRT38 gene were significantly associated with the number of crimps (p<0.05); and three genotypes of the KRT85 gene were significantly associated with wool crimps score, body size, and fiber diameter (p<0.05). Analysis of the gene pyramiding effect between the different genotypes of the gene loci KRT36, KRT38, and KRT85, each genotype in a gene locus was combined with all the genotypes of another two gene loci and formed the different three loci combinations, indicated a total of 26 types of possible combined genotypes in the analyzed population. Compared with the other combined genotypes, the combinations CC-GG-II, CC-HH-IJ, CC-HH-JJ, DD-HH-JJ, CC-GH-IJ, and CC-GH-JJ at gene loci KRT36, KRT38, and KRT85, respectively, had a greater effect on wool traits (p<0.05). Conclusion: Our results indicate that the mutation loci of KRT31, KRT36, KRT38, and KRT85 genes, as well as the combinations at gene loci KRT36, KRT38, and KRT85 in CMXT have significant effects on wool traits, suggesting that these genes are important candidate genes for wool traits, which will contribute to sheep breeding and provide a molecular basis for improved wool quality in sheep.

• Journal of Animal Science and Technology
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• 제56권8호
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• pp.27.1-27.6
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• 2014

The fat mass and obesity associated (FTO) gene plays an important role in the regulation of energy homeostasis, fat deposition and obesity. For this reason, the FTO gene is a physiological and functional candidate gene for carcass and meat quality traits in beef cattle. The objectives of this study were to identify SNPs in the exonic regions of FTO gene and to evaluate the association of these SNPs with carcass traits in Hanwoo (Korean cattle). In this study, we newly identified two exonic SNPs in Hanwoo population. The g.125550A > T SNP was located in exon 3 and the g.175675C > T SNP was located in exon 6. Genotyping of the two SNP markers was carried out using PCR-RFLP analysis in Hanwoo steers to evaluate their association with carcass traits. As a result, g.125550A > T SNP genotype was significantly associated with effects on marbling score. Animals with the AA and TT homozygous genotypes had a significantly higher marbling score (p < 0.001) than those with AT heterozygous genotype, and this was significant after Bonferroni correction of the significance threshold (p = 0.003). Dominance effect was also observed for the marbling score (P < 0.05) with higher marbling score of homozygous animals. However, no significant associations with meat quality traits were observed for the g.175675C > T SNP. Our results suggest that the exonic SNP g.125550A > T in the FTO gene may be used as a DNA marker for the selection of Hanwoo with higher marbling.

• Genomics & Informatics
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• 제1권2호
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• pp.113-118
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• 2003

Among a number of antigens characterized in M leprae, an etiological agent of Leprosy, the 18 kDa antigen, is unique to M leprae. We have previously determined a sequence specific element in the 18 kDa gene of M leprae, which confers transcriptional repression. In this report, we have examined if the element could be applied to genes other than the 18 kDa gene of M leprae. To identify the roles of the regulatory sequence in heterologous promoter, we have constructed pB3 vector series, which contains BCG hsp65 promoter and the M leprae 18 kDa transcription repression responsive element in tandem using LacZ gene as a reporter gene. Cloning of hsp65 promoters of M bovis BCG or M smegmatis in front of LacZ gene resulted in normal $\beta$­galactosidase activity as expected. However, when the sequence element was placed between the promoter and the LacZ gene, $\beta$-galactosidase activity was reduced 10-fold less. Also we have examined with pB3(-) vector, that harbors the transcription repression responsive element in a reversed orientation, the $\beta$-galactosidase activity was found to be similar to pB3(+) vector. Thus, these results further confirm that M leprae 18 kDa transcription repression responsive element could regulate BCG hsp65 heterologous promoter and that the element could act as an operator for the transcription of mycobacteria.

• 대한골관절종양학회지
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• 제3권1호
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• pp.9-17
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• 1997

Resistance to combination chemotherapy remains challenge in the treatment of osteosarcoma. One of the mechanisms of multiple drug resistance is an increased expression of the multidrug resistance gene(mdr1). Expression of the P-glycoprotein(mdr-1 gene product) was studied immunohistochemically and the mdr-1 gene by in situ hybridization in 33 osteosarcomas relating to various prognostic factors. Thirty cases out of 33 osteosarcomas(90.9%) showed positive cytoplasmic reactions with P-glycoprotein and nineteen instances(57.6%) were strong positive(2+). The older(>20 years) and female patients revealed more intense immunohistochemical reactions rather than those of the younger and male patients. Osteoblastic and chondroblastic osteosarcomas revealed more strong immunohistochemical reactions compared to fibroblastic types. There were no significant staining differences between the type of bony involvement, Broder's grade and the presence of necrosis. On follow-up, the mean survival rate was decreased in the strong positive group, however, this was not statistically significant. In situ hybridization for mdr-1 gene revealed positive signals in 22 cases out of 29 osteosarcomas(75.9%). Chemotherapy was done in 15 cases out of 28 patients(53.6%). The results of immunohistochemistry and in situ hybridization were not correlated with the protocols for chemotherapy. However, this result should be confirmed by a larger scale study about mdr1 mRNA expression.

• Asian Pacific Journal of Cancer Prevention
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• 제14권2호
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• pp.695-700
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• 2013

Background: Mutations affecting the epidermal growth factor receptor (EGFR) are good predictors of clinical efficacy of EGFR tyrosine kinase inhibitors (TKI) in patients with non-small cell lung cancer. Serum carcinoembryonic antigen (CEA) levels are also regarded as predictive for the efficacy of EGFR-TKI and EGFR gene mutations. This study analyzed the association between EGFR gene mutations and clinical features, including serum tumor marker levels in lung adenocarcinomas patients. Patients and Methods: A total of 70 lung adenocarcinoma patients with complete clinical data and pathological specimens were investigated. EGFR gene mutations at exons 19 and 21 were assessed. Serum tumor markers were detected by protein chip-chemiluminescence at the corresponding time, and correlations were analyzed. Results: Mutations of the EGFR gene were detected in 27 of the 70 patients and the serum CEA and CA242 concentrations were found to be significantly associated with the incidence of EGFR gene mutations (P<0.05). The AUCs for CEA and CA242 were 0.724 (95% CI: 0.598~0.850, P<0.05) and 0.769 (95% CI: 0.523~0.800, P<0.05) respectively. Conclusions: Serum CEA and CA242 levels are associated with mutations of the EGFR gene in patients with lung adenocarcinomas.