• Journal of Life Science
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• v.11 no.2
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• pp.181-189
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• 2001

Cytokines are hormone-like proteins which mediate and regulast inflammatory and immune responses. Transforming growth factor -$\beta$1(TGF-$\beta$) plays an important role in the control of the immune response and wound healing, and in the development o various tissues and organs, Nitric oxide(NO) is major messenger molecule regulating immune function and blood vessel dilation and serving as a neurotransmitter in the brain and peripheral nervous system. Also, NO is to be a potent mutagen that cause mutation in the p53 tumor suppressor gene in early phases of human gastric carcinogenesis. The purpose of this study was to investigate the effect of Helicobacter phlori lystes, lipopolysaccharide (LPS), and Staphylococcus enterotoxin B(SEB) on production of TGF-$\beta$1 and NO by human fibroblasts. Primary cultured human fibroblasts were incubated with H. pylori lysates(Hp), LPs, SEB, Hp+LPS, Hp+SEB, Hp+LPS+SEB. Cultured supernatants that were collected at 24, 48 and 72 hr were assessed for TGF-$\beta$1 by enzyme-linked immunosorbent assay and NO production by quantification of nitrite ion. TGF-$\beta$1 production in fibroblasts exposed with Hp, LPS or SEB for 48 hrs was enhanced, but for 72 hrs inhibited. Its production by doble exposure such as Hp+LPS, Hp+SEB, Hp+LPS+SEB was lowered in comparison with single exposure of Hp in cases of 24 and 48 hrs incubation, but for 72 hrs decreased in Hp vaculoating toxin(+), increased in Hp vacuolating toxin(-). No production in fibroblasts increaed at all doses of LPS. But its production by exposure of SEB increased or decreased according to dose and incubation time. Also, NO production by Hp vacuolating toxin(+) increased at all doses, but its production by Hp vacuolating toxin(-) decreased. Its production by doble exposure such as Hp+LPS, Hp+SEB, Hp+LPS+SEB decreased in comparison with single exposure Hp Therefore, quantities pf TGB-$\beta$1 and NO released by human fibroblasts shows differences according to kinds of stimulants. Also, in care stimulated with same kinds of stimulants, its productions exhibit quantitative differences according to exposure times. These results suggest that the decreased of TGF-$\beta$1 in fibroblasts by mixed exposure with Hp producing vacuolating toxin and bacterial toxins such as LPS and SEB may effect negatively in healing of host tissue and increased of NO by infection oh H. pylori may related to the increased susceptibility for human gastric carcinogenesis.

• Journal of Life Science
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• v.28 no.1
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• pp.37-42
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• 2018

Non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) is a transforming growth factor beta (TGF-${\beta}$) superfamily gene associated with pro-apoptotic and anti-tumorigenic activities. In the present study, we investigated if caffeic acid phenethyl ester (CAPE) derived from propolis could induce the expression of anti-tumorigenic gene NAG-1. Our results indicate that CAPE significantly induced NAG-1 expression in a time- and concentration-dependent manner in HCT116 cells. We also found that CAPE induced NAG-1 expression in a concentration-dependent manner in another human colorectal cancer cell line, LOVO. In addition, CAPE triggered apoptosis, which was detected with Western blot analysis using poly-(ADP-ribose) polymerase antibody. NAG-1 induction by CAPE was not dependent on transcription factor p53, which was confirmed with Western blot analysis using p53 null HCT116 cells. The luciferase assay results indicated that the new cis-elements candidates were located between -474 and -1,086 of the NAG-1 gene promoter. CAPE dramatically induced activating transcription factor 3 (ATF3) expression, but not cAMP response element-binding protein (CREB), which shares the same binding sites with ATF3. The co-transfection experiment with pCG-ATF3 and pCREB showed that only ATF3 was associated with NAG-1 up-regulation by CAPE, whereas CREB had no effect. In conclusion, the results suggest that CAPE could induce the expression of anti-tumorigenic gene NAG-1 mainly through ATF3.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.11
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• pp.1529-1539
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• 2017

Objective: The objective of this study was to compare the DNA methylation profile in the longissimus dorsi muscle between Small Tailed Han and Dorper${\times}$Small Tailed Han crossbred sheep which were known to exhibit significant difference in meat-production. Methods: Six samples (three in each group) were subjected to the methylated DNA immunoprecipitation sequencing (MeDIP-seq) and subsequent bioinformatics analyses to detect differentially methylated regions (DMRs) between the two groups. Results: 23.08 Gb clean data from six samples were generated and 808 DMRs were identified in gene body or their neighboring up/downstream regions. Compared with Small Tailed Han sheep, we observed a tendency toward a global loss of DNA methylation in these DMRs in the crossbred group. Gene ontology enrichment analysis found several gene sets which were hypomethylated in gene-body region, including nucleoside binding, motor activity, phospholipid binding and cell junction. Numerous genes were found to be differentially methylated between the two groups with several genes significantly differentially methylated, including transforming growth factor beta 3 (TGFB3), acyl-CoA synthetase long chain family member 1 (ACSL1), ryanodine receptor 1 (RYR1), acyl-CoA oxidase 2 (ACOX2), peroxisome proliferator activated receptor-gamma2 (PPARG2), netrin 1 (NTN1), ras and rab interactor 2 (RIN2), microtubule associated protein RP/EB family member 1 (MAPRE1), ADAM metallopeptidase with thrombospondin type 1 motif 2 (ADAMTS2), myomesin 1 (MYOM1), zinc finger, DHHC type containing 13 (ZDHHC13), and SH3 and PX domains 2B (SH3PXD2B). The real-time quantitative polymerase chain reaction validation showed that the 12 genes are differentially expressed between the two groups. Conclusion: In the current study, a tendency to a global loss of DNA methylation in these DMRs in the crossbred group was found. Twelve genes, TGFB3, ACSL1, RYR1, ACOX2, PPARG2, NTN1, RIN2, MAPRE1, ADAMTS2, MYOM1, ZDHHC13, and SH3PXD2B, were found to be differentially methylated between the two groups by gene ontology enrichment analysis. There are differences in the expression of 12 genes, of which ACSL1, RIN2, and ADAMTS2 have a negative correlation with methylation levels and the data suggest that DNA methylation levels in DMRs of the 3 genes may have an influence on the expression. These results will serve as a valuable resource for DNA methylation investigations on screening candidate genes which might be related to meat production in sheep.

• Journal of Korean Academy of Oral and Maxillofacial Radiology
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• v.28 no.1
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• pp.245-259
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• 1998

Cellular transforming genes have been identified in a number of different tumor cell lines and tumor types. A significant number of these oncogenes belong to the ras gene family. The ras gene family consists of three closely related genes:H-ras, K-ras and N-ras which code for a related 21 kDa protein. Mutations in codon 12, 13 and 61 of one of the three ras genes convert these genes into acute oncogenes. The presence of H-ras gene mutations has important prognostic implications in various tumors. Each genomic DNA was isolated from tumors induced by implantation with DMBA, or by treatment with DMBA -implantation/irradiation. When genome DNA was transfected into NIH 3T3 cells and investigated by two-step PCR-RFLP, the fOllowing results were concluded: 1. Transformation foci developed in two groups when the genome DNA of two experimental groups were transfected into NIH 3T3 cells. 2. Transformation efficiency was 0.01-0.02 foci/㎍DNA in the experimental group with the DMBA-implantation, 0.01-0.03 foci/㎍lgDNA in the experimental group with the DMBA-implantation/irradiation according to results of transfection assay. 3. When the point mutation of H-ras gene was investigated by a two-step PCR-RFLP, there was 13.9％ (5/36) in the experimental group with the DMBA implantation, 15.4 ％ (6/39) in the experimental group with the DMBA -implantation/irradiation. 4. The point mutation in codon 12 and 61 of H-ras was 5.6％(2/36) and 8.3％(3/36) in the experimental group with the DMBA implantation. 5. The point mutation in codon 12 and 61 of H-ras gene was 7.7％(3/39) in the experimental group with the DMBA -implantation/irradiation.

• Proceedings of the Korean Society of Life Science Conference
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• 2001.06a
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• pp.67-86
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• 2001

A strain producing strongly fibrinolytic enzyme was isolated from soil and was identified to be Bacillus subtilis by biochemical and physiological characterization. The optimal culture conditions for the production of fibrinolytic enzyme was determined to be 1.0% tryptone, 1.5% soluble starch, 0.5% Peptone, 0.5% NaCl, $(NH_{4})_{3}PO_4.3H_{2}O, and MgSO_{4}.7H_{2}O.$ Initial pH and temperature were pH 8.0 and $30^{\circ}C$ , respectively, The highest enzyme production was observed at 30 hours of cultivation at $30^{\circ}C$ The fibrinolytic enzyme was purified to homogeneity by DEAE Sephadex A-50 ion exchange column chromatography, 70% ammonium sulfate precipitation, Sephadex G-200 and G-75 gel filtration column chromatography. The molecular weight of the purified enzyme was 28,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A gene encoding the fibrinolytic enzyme was cloned into a plasmid vector pBluescript, transforming E.coli XL-1 Blue. The clone was able to degrade fibrin, This indicated that the gene could encode a fibrinolytic enzyme. The nucleotide sequence of the 2.7 kb insert was determined in both direction. One open reading frame composed of 1023 nucleotides was found to be a potential protein coding region. There was the putative Shine-Dalgano sequence and TATA box upstream of the open reading frame. The homology search data in the genome database showed that both the 2.7 kb insert and 1 kb open reading frame carried no significance in the nucleotide sequence of known fibrinolytic enzyme from Bacillus serovars. The recombinant cell harboring the novel gene involved in fibrinolysis was subjected to protein purification. The molecular mass of the purified fibrinolytic enzyme was determined to be 31864 Dalton, which was highly in accordance with the molecular mass(33 kDa) of the fibrinolytic gene deduced from the insert. The fibrinolytic enzyme was Purified 50.5 folds to homogeneity in overall yield of 10.7% by DEAE Sephadex A-50 ion exchange, 85% ammonium sulfate precipitation, Sephadex G-50, Superdex 75 HR FPLC gel filtration. In conclusion, a novel fibrinolytic gene from Bacillus subtilis was identified and characterized by cloning a genomic library of Bacillus subtilis into pBleuscript. For the soybean fermented by this strain, it is found that there increased assistant protein about 20% compared to the soybean not fermented and increased about 30% according to amino acid analysis and, in particular, essential amino acid increased about 40%. When keeping this fermented soybean powder at room temperature for about 70days, it showed very high stability maintaining almost perfect activity and, therefore, it gave us great suggestion its possibility of development as a new functional food.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.193-196
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• 2001

IL-18. formerly known as IGIF(interferon -gamma inducing factor), is structurally IL-l related but functionally IL-12 related pro-inflammatory cytokine. The human IL -18(hIL-lS), like IL-$1{\beta}$, is synthesized as a biologically inactive precursor of 24kDa lacking a signal peptide, and then cleaved into an active mature form by cystein protease IL-$1{\beta}$ converting enzyme (ICE: caspase- 1), We tested if the mature hIL -18 can be expressed and secreted into culture medium by transforming the forming gene construct consisting of a mature hIL-18 gene fused to signal peptide of rice amylase lA. Secondly, we were tested if the pro- IL-18 could be processed into a biologically active form by caspase-l like protease in plant. Cell suspension culture was established from the leaf-derived calli of transgenic tobacco plant. Southern and Northern blot analysis indicated the expression of both pro-hIL-18 and mature hIL-18 plant cells. Western blot analysis introduced the protein products of pro- hIL -18 and mhIL -18 were observed in transigenic cell lines. In addition, the molecular size of recombinant pro-hILl-18 and mhIL-18 were estimated to be 24kDa and 18kDa, respectively. ELISA revealed that the amount of pro- hIL -18 was 1.3ug per gram of fresh weight calli. Moreover, the presence of mhIL-18 was detected in the culture medium and it appeared to be 25ug/L.

• Molecules and Cells
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• v.41 no.4
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• pp.342-350
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• 2018

Flowering time is determined by florigens. These genes include, Heading date 3a (Hd3a) and Rice FT 1 (RFT1) in rice, which are specifically expressed in the vascular tissues of leaves at the floral transition stage. To study the cis-regulatory elements present in the promoter region of Hd3a, we generated transgenic plants carrying the 1.75-kb promoter fragment of Hd3a that was fused to the ${\beta}$-glucuronidase (GUS) reporter gene. Plants expressing this construct conferred a vascular cell-specific expression pattern for the reporter gene. However, GUS was expressed in leaves at all developmental stages, including the early seedling stage when Hd3a was not detected. Furthermore, the reporter was expressed in roots at all stages. This suggests that the 1.75-kb region lackings cis-elements that regulate leaf-specific expression at the appropriate developmental stages. Deletion analyses of the promoter region indicated that regulatory elements determining vascular cell-specific expression are present in the 200-bp region between -245 bp and -45 bp from the transcription initiation site. By transforming the Hd3a-GUS construct to rice cultivar 'Taichung 65' which is defective in Ehd1, we observed that Ehd1 is the major regulatory element that controls Hd3a promoter activity.

• International Journal of Industrial Entomology and Biomaterials
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• v.27 no.1
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• pp.159-165
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• 2013

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF-${\beta}$) superfamily and are involved in osteoblastic differentiation. The largest TGF-${\beta}$ superfamily subgroup shares genetic homology with human BMPs (hBMPs) and silkworm decapentaplegic (dpp). In addition, hBMPs are functionally interchangeable with Drosophila dpp. Bombyx mori dpp may induce bone formation in mammalian cells. To test this hypothesis, we synthesized the 1,285-base pairs cDNA of full-length B. mori dpp using total RNAs obtained from the fat body of 3-day-old of the $5^{th}$ instar larvae and cloned the cDNA into the pCEP4 mammalian expression vector. Next, B. mori dpp was expressed in C3H10T1/2 cells. The target cells transfected with the pCEP4-Bm dpp plasmid showed biological functions similar to those of osteogenic differentiation induction growth factors such as hBMPs. We determined the relative mRNA expression rates of Runt-related transcription factor 2 (RUNX2), osterix, osteocalcin, and alkaline phosphatase (ALP) to validate the osteoblast-specific differentiation effects of B. mori dpp by performing quantitative real-time RT-PCR. Interestingly, mRNA expression levels of the 3 marker genes except RUNX2, in cells expressing B. mori dpp were much higher than those in control cells and C3H10T1/2 cells transfected with pCEP4. These results suggested that B. mori dpp signaling regulates osterix expression during osteogenic differentiation via RUNX2-independent mechanisms.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.3
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• pp.153-159
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• 2009

We investigated whether deficiency of inducible nitric oxide synthase (iNOS) could prevent isoproterenol-induced cardiac hypertrophy in iNOS knockout (KO) mice. Isoproterenol was continuously infused subcutaneously (15 mg/kg/day) using an osmotic minipump. Isoproterenol reduced body weight and fat mass in both iNOS KO and wild-type mice compared with saline-infused wild-type mice. Isoproterenol increased the heart weight in both iNOS KO and wild-type mice but there was no difference between iNOS KO and wild-type mice. Posterior wall thickness of left ventricle showed the same tendency with heart weight. Protein level of iNOS in the left ventricle was increased in isoproterenol-infused wild-type mice. The gene expression of interleukin-6 (IL-6) and transforming growth factor-${\beta}$ (TGF-${\beta}$) in isoproterenol-infused wild-type was measured at 2, 4, 24, and 48-hour and isoproterenol increased both IL-6 (2, 4, 24, and 48-hour) and TGF-${\beta}$ (4 and 24-hour). Isoproterenol infusion for 7 days increased the mRNA level of IL-6 and TGF-${\beta}$ in iNOS KO mice, whereas the gene expression in wild-type mice was not increased. Phosphorylated form of extracellular signal-regulated kinases (pERK) was also increased by isoproterenol at 2 and 4-hour but was not increased at 7 days after infusion in wild-type mice. However, the increased pERK level in iNOS KO mice was maintained even at 7 days after isoproterenol infusion. These results suggest that deficiency of iNOS does not prevent isoproterenol-induced cardiac hypertrophy and may have potentially harmful effects on cardiac hypertrophy.

• Biomedical Science Letters
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• v.19 no.3
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• pp.195-205
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• 2013

Obesity may cause metabolic syndrome and adult diseases. This study was undertaken to investigate the ameliorative or useful effects of beanleaves on inflammation and liver damage in obese rat models. Rats were divided into three groups: a control group (normal diet, n=6), a fat diet group (45%-fat diet, n=7), and a bean leaf group (45%-fat+Korean bean leaves diet, n=7). Body weights in the bean leaf group were lower than those of the fat group (P<0.05). Serum tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) and prostaglandin $E_2$ ($PGE_2$) concentrations were lower in both the control and bean leaf groups than in the fat group (P<0.001). TNF-${\alpha}$ concentrations in the bean leaf group were slightly higher than in the control group but statistically significant (P<0.05). The bean leaf group histologically exhibited lower fatty degeneration, spotty necrosis, and leukocyte infiltrations in hepatic tissues than those of the fat group. In the homogenized liver tissues, the cyclooxygenase-2 (COX-2) gene was only expressed in the fat group. The gene expression levels of hepatic TNF-${\alpha}$, inducible nitric-oxide synthase, peroxiome proliferator-activated receptor-${\alpha}$ (PPAR-${\alpha}$), poly (ADP-ribose) polymerase (PARP), and transforming growth factor-${\beta}1$ (TGF-${\beta}1$) were weaker in the bean leaf group than in the fat group. These results suggest that adding bean-leaves to the diet may ameliorate obesity-induced systemic inflammation and liver damage and that bean leaves may be a useful food for preventing obesity and thereby metabolic syndrome and adult diseases.