• Journal of Life Science
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• v.14 no.4
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• pp.670-675
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• 2004

We have examined the effects of S-nitroso-N-acetylpenicillamine (SNAP), prostaglandin $E_2$ (PG $E_2$) and dibutric cyclic AMP (dbcAMP) on the methylation of interferon- ${\gamma}$ (IFN- ${\gamma}$ ) gene in human Jurkat T cells. The CpG dinucleotide which is critical for promoter function of IFN- ${\gamma}$ gene was methylated by treatment with SNAP, PG $E_2$ and dbcAMP, respectively. The DNA methylation induced by PG $E_2$ was suppressed by the addition of 2',5'-dideoxyadenosine (DDA), an inhibitor of adenylyl cyclase, but the suppression was not observed in SNAP treated cells. The NO production was not enhanced in PG $E_2$ or dbcAMP treated cells. The methylation induced by PG $E_2$ and dbcAMP was not suppressed by the addition of $N^{G}$-methyl-L-arginine (L-NMMA), NO synthase inhibitor. In conclusion, the inhibition of INF- ${\gamma}$ gene expression by PG $E_2$ was associated with the methylation of INF- ${\gamma}$ gene by elevation of intracellular cAMP in human Jurkat T cells. However, the methylation induced by PG $E_2$ might not be mediated through the NO production.rough the NO production.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.16 no.11
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• pp.7575-7581
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• 2015

Corticotropin-releasing factor(CRF), one of the stress driven neuropeptides, was widely proposed to influence hair loss and re-growth. For the development of receptor antagonists, the screening system based on intracellular calcium signal process was developed and optimized. The aequorin parental cells were transfected with CRF1 receptor and alpha 16 promiscuous G protein cDNA to establish HEK293a16/hCRF1, a stable cell line for the human CRF1 receptor. In HEK293a16/hCRF1 cells, the range of sauvagine dose response was 12-fold higher($EC_{50}:15.21{\pm}1.83nM$) than in the transiently expressed cells, hence essential conditions for the antagonist screening experiments such as the robust signals and high solvent tolerance were secured. The standard antagonists for the CRF1 receptor, antalarmin and CP154526, resulted $IC_{50}$ values of $414.1{\pm}5.5$ and $290.7{\pm}1.9nM$, respectively. Similar results were presented with frozen HEK293a16/hCRF1 cells. Finally, our HEK293a16/hCRF1 cells with the aequorin based cellular functional assay can be a model system for the development of functional cosmetics and modulators that can have a clinical efficacy on hair re-growth.

• Journal of Microbiology and Biotechnology
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• v.20 no.6
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• pp.1022-1026
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• 2010

The different cleavage patterns of pYBamy59 plasmid isolated from E. coli $DH5{\alpha}$ and B. longum MG1 by the cell extract of B. longum MG1 suggested that the main reason for its low transformation efficiency was related to the restriction modification (R-M) system. To confirm the correlation between the R-M system and transformation efficiency, in vitro methylation and site-directed mutagenesis were performed in pYBamy59. Sequence analysis of pYBamy59 fragments digested by the cell extract of B. longum MG1 revealed that all fragments were generated by restriction of the sequence recognized by SacII endonuclease. When pYBamy59 from E. coli was methylated in vitro by CpG or GpC methyltransferase, it was protected from SacII digestion. Site-directed mutagenesis, which removed SacII sites from pYBamy59, or in vitro methylation of pYBamy59 showed 8- to 15-fold increases in the transformation efficiency over intact pYBamy59. Modification of the SacII-related R-M system in B. longum MG1 and in vitro methylation in pYBamy 59 can improve the transformation efficiency in this strain. The results showed that the R-M system is a factor to limit introduction of exogenous DNA, and in vitro modification is a convenient method to overcome the barrier of the R-M system for transformation.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.5 no.1
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• pp.39-50
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• 2002

Hepatitis B viral infection which affect about 10% of Korean population manifests asymptomatic carrier, chronic hepatitis and liver cirrhosis and even associates with hepatocellular carcinoma. Clinical manifestations induced by hepatitis B virus vary depending on the degree of immune response by cytotoxic T cells against viral epitope-presenting liver cells. Since hepatitis B virus presents high rate of mutaton that might change the presented epitope and eventually alter immune response, viral mutations, especially in promoters and enhancers, have an important implication in hepatic inflammation and viral replication. To identify mutations related to the hepatic inflammation, we investigated sequence variations of hepatitis B viral promotor regions in the presence or absence of symptoms in hepatitis B carriers. For this, sera from persistently hepatitis B virus-infected mother-child pairs were collected. After PCR amplifiation of all hepatitis B viral promoters (C promoter, S1 promoter, S2/S promoter, X promoter) using serum DNA from each pair, viral promotors were sequenced by automatic sequencer and then sequence data were analyzed by ClustalW. In most cases, the dominant type of maternal virus was transmitted to the child. However, in some children, some new host specific viral variants could be observed in Cp, S1p and S2/Sp. The mutations in C promoter did not seem to be vertically transmitted but arose in new host independently after the wild type had been transmitted. Enhancer I containing X promoter revealed high host specific variations as has been reported before. Two S promoters, S1p and S2/Sp, have shown some point mutations in children, but no deletion mutations were detected as in chronic hepatitis patients in whom deletion mutations are frequently found. In conclusion, the children with the vertically transmitted hepatitis B virus mostly retain the dominant type virus that had been transmitted. However, host specific variants tended to accumulate over time, possibly as clinical symptoms develop.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.13
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• pp.5449-5454
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• 2014

Although genetic characteristics are considered to be a factor influencing the geographic variation in the prevalence of gallbladder cancer (GBC), they have not been well studied in Bolivia, which has a high prevalence rate of GBC. The purpose of this study was to examine the frequency of TP53 and K-ras mutations in Bolivian patients with GBC and to compare them with our previous data obtained in other high-GBC-prevalence countries, namely Japan, Chile, and Hungary. DNA was extracted from cancer sites in paraffin-embedded tissue from 36 patients using a microdissection technique. TP53 mutations at exons 5 to 8 and K-ras mutations at codons 12, 13 and 61 were examined using direct sequencing techniques. The data obtained were compared with those in the other high-GBC-prevalence countries. Of the 36 patients, 18 (50.0%) had a TP53 mutation (one mutation in each of 17 patients and three mutations in one patient), and only one (2.8%) had a K-ras mutation. Of the 20 TP53 mutations, 12 were of the transition type (60.0%). This rate was significantly lower than that in Chile (12/12, P<0.05). In addition, three mutations were of the CpG transition type (15.0%), which is a feature of endogenous mutation. All three were found in the hot spot region of the TP53 gene. In contrast, G:C to T:A transversion was found in Bolivia, suggesting the presence of exogenous carcinogens. Our findings suggest that the development of GBC in Bolivia is associated with both exogenous carcinogens and endogenous mechanisms. The identification of an environmental risk factor for GBC is needed to confirm these findings.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.81-89
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• 2004

Tumor necrosis factor-$\alpha$ (TNF-$\alpha$) and lymphotoxin-$\alpha$ (LT-$\alpha$, TNF-$\beta$) can initiate and perpetuate human diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), and insulin-dependent diabetes mellitus (IDDM). TNFs can be blocked by the use of soluble TNF receptors. However, since monomeric soluble receptors generally exhibit low affinity or function as agonists, the use of monomeric soluble receptors has been limited in the case of cytokines such as TNF-$\alpha$, TNF-$\alpha$, interleukin (IL)-1, IL-4, IL-6, and IL-13, which have adapted to a multi component receptor system. For these reasons, very high-affinity inhibitors were created for the purpose of a TNFs antagonist to bind the TNFR and trigger cellular signal by using the multistep polymerase chain reaction method. First, recombinant simple TNFR-Ig fusion proteins were constructed from the cDNA sequences encoding the extracellular domain of the human p55 TNFR (CD120a) and the human p75 TNFR (CD120b), which were linked to hinge and constant regions of human $IgG_1$ heavy chain, respectively using complementary primers (CP) encoding the complementary sequences. Then, concatameric TNFR-Ig fusion proteins were constructed using recombinant PCR and a complementary primer base of recombinant simple TNFR-Ig fusion proteins. For high level expression of recombinant fusion proteins, Chinese hamster ovary (CHO) cells were used with a retroviral expression system. The transfected cells produced the simple concatameric TNFR-Ig fusion proteins capable of binding TNF and inactivating it. These soluble versions of simple concantameric TNFR-Ig fusion proteins gave rise to multiple forms such as simple dimers and concatameric homodimers. Simple TNFR-1g fusion proteins were shown to have much more reduced TNF inhibitory activity than concatameric TNFR-Ig fusion proteins. Concatameric TNFR-Ig fusion proteins showed higher affinity than simple TNFR-Ig fusion proteins in a receptor inhibitor binding assay (RIBA). Additionally, concatameric TNFR-Ig fusion proteins were shown to have a progressive effect as a TNF inhibitor compared to the simple TNFR-Ig fusion proteins and conventional TNFR-Fc in cytotoxicity assays, and showed the same results for collagen induced arthritis (CIA) in mice in vivo.