• KSBB Journal
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• v.13 no.1
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• pp.96-100
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• 1998

Two promoters (trc and P$\rho$) were inserted in PHA operon derived from Alcaligenes eutrophus to obtain high chain molecules of polyhydroxybutyrate (PHB) in recombinant Escherichia coli. Newly designed PHA operon was used to control the gene expressions of hydroxybutyric CoA and PHA polymerization, separately. Plasmids containing new synthetic operon was transformed into E. coli DH5$\alpha$ and analyzed for PHB production. Without induction of the PHA biosynthetic operon, PHA synthase which has low activity might supply low concentration of initiator during the polymerization reaction, resulting very high molecular weight of polymer. An increase of PHB average molecular weight was observed with decreased IPTG (isopropyl $\beta$ -Dithiogalactosidase) concentration. When no IPTG was added to the culture of E. coli DH5$\alpha$ /$\rho$ SJS1 which contained two promoters in PHA operon, high chain polymer having an average molecular weight of $2.5{\times}10^7$ was achieved. Analysis of the enzyme activities of PHA biosynthetic enzymes suggests that PHA synthase, the enzyme responsible for polymerizing 3-hydroxybutyric CoA, controls the molecular weight of PHB produced in vivo.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.4
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• pp.243-249
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• 2010

Tissue engineered bone (TEB) can replace an autogenous bone graft requiring an secondary operation site as well as avoid complications like inflammation or infection from xenogenic or synthetic bone graft. Adult mesenchymal stem cells (MSC) for TEB are considered to have various ranges of differentiation capacity or multipotency by the donor site and age. This study examined the effect of age on proliferation capacity, differentiation capacity and bone morphogenetic protein-2 (BMP-2) responsiveness of human bone marrow stromal cells (hBMSC) according to the age. In addition, to evaluate the effect on enhancement for osteoblast differentiation, the hBMSC were treated with Trichostatin A (TSA) and 5-Azacitidine (5-AZC) which was HDAC inhibitors and methyltransferase inhibitors respectively affecting chromatin remodeling temporarily and reversibly. The young and old group of hBMSC obtained from the iliac crest from total 9 healthy patients, showed similar proliferation capacity. Cell surface markers such as CD34, CD45, CD90 and CD105 showed uniform expression regardless of age. However, the young group showed more prominent transdifferentiation capacity with adipogenic differentiation. The osteoblast differentiation capacity or BMP responsiveness was low and similar between young and old group. TSA and 5-AZC showed potential for enhancing the BMP effect on osteoblast differentiation by increasing the expression level of osteogenic master gene, such as DLX5, ALP. More study will be needed to determine the positive effect of the reversible function of HDAC inhibitors or methyltransferase inhibitors on enhancing the low osteoblast differentiation capacity of hBMSC.

• Journal of Plant Biotechnology
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• v.45 no.3
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• pp.257-265
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• 2018

Sweetener is one of the additives that makes you feel sweet. Artificial sweeteners and sugar are typical examples, and sweetness proteins with sweetness characteristics have been widely studied. These studies elucidated the transformation lettuce cells with Agrobacterium method for stable production of natural sweet proteins, brazzein, thaumatin, and miraculin. In this paper, we report use of a plant expression system for production of sweet proteins. A synthetic gene encoding sweet proteins was placed under the control of constitutive promoters and transferred to lettuce. High and genetically stable expression of sweetener was confirmed in leaves by RT-PCR and Western blot analysis. Sweet proteins expressed in transgenic lettuce had sweetness-inducing activity. Results demonstrate recombinant sweet proteins correctly processed in transgenic lettuce plants, and that this production system could be a viable alternative to production from the native plant.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.2
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• pp.677-682
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• 2014

Resveratrol has been examined in several model systems for potential effects against cancer. Adenosine monophosphate-activated protein kinase (AMPK) is reported to suppress proliferation in most eukaryocyte cells. Whether resveratrol via AMPK inhibits proliferation of oesophageal adenocarcinoma cells (OAC) is unknown. The aim of this study was to determine the roles of AMPK in the protective effects of resveratrol in OAC proliferation and to elucidate the underlying mechanisms. Treatment of cultured OAC derived from human subjects or cell lines with resveratrol resulted in decreased cell proliferation. Further, inhibition of AMPK by pharmacological reagent or genetical approach abolished resveratrol-suppressed OAC proliferation, reduced the level of $p27^{Kip1}$, a cyclin-dependent kinase inhibitor, and increased the levels of S-phase kinase-associated protein 2 (Skp2) of $p27^{Kip1}$-E3 ubiquitin ligase and 26S proteasome activity reduced by resveratrol. Furthermore, gene silencing of $p27^{Kip1}$ reversed resveratrol-suppressed OAC proliferation. In conclusion, these findings indicate that resveratrol inhibits Skp2-mediated ubiquitylation and 26S proteasome-dependent degradation of $p27^{Kip1}$ via AMPK activation to suppress OAC proliferation.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.24
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• pp.10819-10824
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• 2015

Aims: To explore the effect and probable mechanism of a synthetic retinoid 4-amino-2-tri-fluoromethylphenyl ester (ATPR) on apoptosis of MDA-MB-231 breast cancer cells. Materials and Methods: MTT assays were performed to measure the proliferation of MDA-MB-231 cells treated with different concentrations of all-trans retinoic acid (ATRA) and ATPR. Morphologic changes were observed by microscopy. The apoptosis rates and cell cycling of MDA-MB-231 cells treated with ATRA or ATPR were assessed using flow cytometry analysis. Expression of retinoic acid receptor and phosphorylation of ERK, JNK, p38 proteins were detected by Western blotting. Results: Treatment of the cells with the addition of $15{\mu}mol/L$ ATPR for 48 h clearly demonstrated reduced cell numbers and deformed cells, whereas no changes in the number and morphology were observed after treatment with ATRA. The apoptosis rate was 33.2% after breast cancer MDA-MB-231 cells were treated by ATPR ($15{\mu}mol/L$) whereas ATRA ($15{\mu}mol/L$) had no apoptotic effect. ATPR inhibited the phosphorylation of ERK, JNK, and p38 while ATRA had no significant effect. ATPR inhibited the expression of BiP and increased the expression of Chop at the protein level compared with control groups, ATRA and ATPR both decreased the protein expression of $RXR{\alpha}$, ATPR reduced the protein expression of $RAR{\beta}$ and $RXR{\beta}$ while ATRA did not decrease $RAR{\beta}$ or $RXR{\beta}$. Conclusions: ATPR could induce apoptosis of breast cancer MDA-MB-231 cells, possible mechanisms being binding to $RAR{\beta}/RXR{\beta}$ heterodimers, then activation of ER stress involving the MAPK pathway.

• Korean Journal of Environmental Biology
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• v.18 no.3
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• pp.291-298
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• 2000

Vitellogenesis, an important reproductive process in oviparous animals, includes the processes of hormonally regulated synthesis of yolk precursor protein, vitellogenin (Vg), and their deposition in ovarian oocytes as a vitellin which is an important energy source as well as buoyancy regulator of the egg. Vg genes consist of a gene family that encompasses a large number of lipoproteins and produce different Mr. Vg proteins in liver. The expression of Vg is largely dependent on the estrogen, and both reproductive cycle and temperature also influence Vg synthesis. Synthetic estrogens or estrogenic pollutants was sufficient to induce Vg in both sexes of oviparous vertebrates. Therefore, the estrogenic induction of vitellogenesis in male has been used for biological marker in the screening of estrogenicity of certain endocrine disrupting compounds and the monitoring the world-wide contamination of estrogenic compounds in wild life. In the studies on the biological hazard and influence of endocrine disrupting chemicals using the Vg induction in oviparous males, it is important to consider the reproductive cycle, zoogeography and biodiversity of the wild life animals in Korea.

• Molecules and Cells
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• v.41 no.10
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• pp.900-908
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• 2018

Insulin resistance is closely associated with metabolic diseases such as type 2 diabetes, dyslipidemia, hypertension and atherosclerosis. Thiazolidinediones (TZDs) have been developed to ameliorate insulin resistance by activation of peroxisome proliferator-activated receptor (PPAR) ${\gamma}$. Although TZDs are synthetic ligands for $PPAR{\gamma}$, metabolic outcomes of each TZD are different. Moreover, there are lack of head-to-head comparative studies among TZDs in the aspect of metabolic outcomes. In this study, we analyzed the effects of three TZDs, including lobeglitazone (Lobe), rosiglitazone (Rosi), and pioglitazone (Pio) on metabolic and thermogenic regulation. In adipocytes, Lobe more potently stimulated adipogenesis and insulin-dependent glucose uptake than Rosi and Pio. In the presence of pro-inflammatory stimuli, Lobe efficiently suppressed expressions of pro-inflammatory genes in macrophages and adipocytes. In obese and diabetic db/db mice, Lobe effectively promoted insulin-stimulated glucose uptake and suppressed pro-inflammatory responses in epididymal white adipose tissue (EAT), leading to improve glucose intolerance. Compared to other two TZDs, Lobe enhanced beige adipocyte formation and thermogenic gene expression in inguinal white adipose tissue (IAT) of lean mice, which would be attributable to cold-induced thermogenesis. Collectively, these comparison data suggest that Lobe could relieve insulin resistance and enhance thermogenesis at low-concentration conditions where Rosi and Pio are less effective.

• Proceedings of the Korean Society of Life Science Conference
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• 2005.04a
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• pp.23-36
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• 2005

Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces cell death when expressed In yeast. To investigate whether .Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various orgarusms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs In detail. PBIl is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorhodamine123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Bax-induced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower lovels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. H$_{2O2}$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of H2O2 treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased In the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MBP phosphorylation activity of AtMPK3 i'n vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation In situ. Thus, AtNDPK2 appears to play a novel regulatory role in H2O2-mediated MAPK signaling in plants.

• Applied Biological Chemistry
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• v.31 no.3
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• pp.274-283
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• 1988

Aqualysin I is an alkaline serine protease which is secretet into the culture medium by Thermus aquaticus YT-1, an extreme thermophile. Aqualysin I was purified, and its partial amino acid sequence was determined. The gene encoding aqualysin I was cloned into E. coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequenc, agreed with the determid amino acid sequences, including the $NH_2-$ and COOH terminal sequence of the tryptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin type serine protease, and 43% identity with proteinase K, 37-30% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. Aqualysin I contains two disulfide bonds, Cys67-Cys99 and Cys163-Cys194, and these disulfide bonds seem to contribute to the heat stability of the enzyme. The determined positions of the twe disulfide bonds of aqualysin I agreed with those predicted previously on the basis of computer graphics of the crystallographic data for subtilisin BPN'. Therefore, these findings sugests that the three-dimensional structure of aqualysin I is similar to that of subtilisin BPN' Aqualysin I is produced as a lage precursor, which contains $NH_2-$ and COOH- terminal portions besides the mature protease sequence.

• Molecular & Cellular Toxicology
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• v.2 no.3
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• pp.151-158
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• 2006

Khal was a synthetic congener of halocidin, a heterodimeric peptide consisting of 19 and 15 amino acid residues detected in Halocynthia aurantium. This compound was considered a candidate for the development of a novel peptide antibiotic. The genotoxicity of Khal was subjected to high throughput toxicity screening (HTTS) because they revealed strong antibacterial effects. Mouse lymphoma thymidine kinase ($tk^{+/-}$) gene assay (MOLY), single cell gel electrophoresis (Comet) assay and chromosomal aberration assay in mammalian cells and Ames reverse mutation assay in bacterial system were used as simplified, inexpensive, short-term in vitro screening tests in our laboratory. These compounds are not mutagenic in S. typhimurium TA98 and TA100 strains both in the presence and absence of metabolic activation. Before performing the comet assay, $IC_{20}$ of Khal was determined the concentration of $25.51\;{\mu}/mL\;and\;21.99\;{\mu}g/mL$ with and without S-9, respectively. In the comet assay, Khal was not induced DNA damage in mouse lymphoma cell line. Also, the mutation frequencies in the Khal-treated cultures were similar to the vehicle controls. It is suggests that Khal is non-mutagenic in MOLY assay. And no clastogenicity was observed in Khal-treated Chinese hamster lung cells. The results of this battery of assays indicate that Khal has no genotoxic potential in bacterial or mammalian cell systems. Therefore, we suggest that Khal, as the optimal candidates with both no genotoxic potential and antibacterial effects must be chosen.