• Journal of Life Science
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• v.17 no.1 s.81
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• pp.1-5
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• 2007

The Precesses for leaf development in dicotyledonous plants are surprisingly complex, while the mechanism of controlling and coordinating them is poorly understood. To characterize the fundamental features of the leaf development of Arabidopsis, we first attempted to isolate mutants that alter leaf morphology. Here, leaf morphological mutant of Arabidopsis, lanceolatal (lan1) which has small and narrow leaves have isolated and characterized. To clarify the function of LAN1 in organ development, we characterized lan1-7 mutant using an anatomical and genetic approach. The lan1-7 mutant had reduced size of foliage leaves and reduced dimensions of stems. A reduction both in cell size and in cell number was evident at the cellular level in the lan1 mutant, revealing that LAN1 gene appears to affect cell division at an earlier stage and cell elongation throughout the development of leaf primordia. from the analysis of heterogeneous plant with lan1 mutation and 35S-AG transgenic plant, AG gene is revealed to regulate leaf morphology under the control of 35S promoter. Thus, MADS-box gene was revealed to have some relationship to that of LAN1 gene at certain stage in leaf development processes.

• Journal of Life Science
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• v.28 no.10
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• pp.1140-1146
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• 2018

Dual-specificity phosphatases (DUSPs) play a role in cell growth and differentiation by modulating mitogen-activated protein kinases. DUSPs are considered targets for drugs against cancers, diabetes, immune diseases, and neuronal diseases. Part of the DUSP family, DUSP19 modulates c-Jun N-terminal kinase activity and is involved in osteoarthritis pathogenesis. Here, we report screening of cavity-creating mutants and the crystal structure of a cavity-creating L75A mutant of DUSP19 which has significantly enhanced enzyme activity in comparison to the wild-type protein. The crystal structure reveals a well-formed cavity due to the absent Leu75 side chain and a rotation of the active site-bound sulfate ion. Despite the cavity creation, residues surrounding the cavity did not rearrange significantly. Instead, a tightened hydrophobic interaction by a remote tryptophan residue was observed, indicating that the protein folding of the L75A mutant is stabilized by global folding energy minimization, not by local rearrangements in the cavity region. Conformation of the rotated active site sulfate ion resembles that of the phosphor-tyrosine substrate, indicating that cavity creation induces an optimal active site conformation. The activity enhancement by an internal cavity and its structural information provide insight on allosteric modulation of DUSP19 activity and development of therapeutics.

• Korean Journal of Medicinal Crop Science
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• v.3 no.1
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• pp.40-44
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• 1995

This study was aimed to mutant induction in Gentiana scabra BUNGE through the tissue culture techniques and comparison of gentiopicroside content in each mutant plants derived from tissue culture. In order to induce the mutants of Gentiam scabra, seed, apical and lateral bud were soaked in $NaN_3$, EMS and MNU solutions and cultured on MS basal medium containing 0.5mg/l NAA, BA $NaN_3$, MNU-treated buds survived about 50% but not survived in EMS treatment. Seed germination rate was extremely low in EMS and MNU treatments but various types of mutant plantlets were obtained by those treatments. 63% of elongated type and 37% of dwarf type were obtained from tissue culture treated by various mutagen. Gentiopicroside contents of regenerated plantlets was analyzed. Root of dwarf type contained more gentiopicroside(1.383%) than that of elongation type (0.083%).

• Journal of Microbiology
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• v.44 no.3
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• pp.301-310
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• 2006

Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr =166,208). The EMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.

• Korean Journal of Microbiology
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• v.30 no.5
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• pp.371-376
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• 1992

Plasmid pSL4 of plasmid pKM 101 mutant have high protection effects and mutagenecity for UV and methyl methanesulfonate, The mucA gene and a pan of mucE gene of pKM 101 and pSL4 were sucloned onto lacZ' fusion vector pMC874 and the hybrid plasmids pBH31 and pBH30 were selected. These plsmids were intrduced into $recA^{+}lexA^{-}$, $recA^{-}와lexA^{+}$ strains and determined the activity of $\beta$-galactosidase for UV. In $recA^{+}lexA^{+}$ strain.$\beta$-galactosidase activity of pBH30 included mue region of pSL4 was higher thall pBH31 inclued muc region of pKM 10 I and the tf-galactosidase of two plasmids was not induced in reeA and leeA mutants with or without UV illumination. Without UV illumination. the .$\beta$-galactosidasc of pBH30 was expressed a little higher level than that of pBH3L We suggest that the functional difference of pKM 10l and pSL4 are due to the variety of mue regulatory region. Also. a plasmid pBH 100 earring umuC' -lacZ' gene fusion was constructed in vitro to study the regulation of the umu operon. It was shown that the umu operon is induced by UV and is regulated by the reeA and lexA genes.

• Applied Biological Chemistry
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• v.38 no.5
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• pp.387-392
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• 1995

To study the regulatory expression mechanism of soybean glycinin gone, Gy2, the 5' upstream region of the gene was searched for the presence of putative regulatory elements by nucleotide sequencing. It revealed various kinds of regulatory sequence elements commonly found in plant storage protein genes. There were canonical promoter sequences, TATA box (TATAAT) and AGGA box (GAAT) which are common in the 5' upstream region of the plant genes. The embryo factor binding sequence, RY repeat, CACA sequences, ${\alpha}$-conglycinin enhancer-like sequences were also found. To delineate the function of these sequences, 5' upstream deletion mutants of Gy2 were prepared and fused to the ${\alpha}$-glucuronidase (GUS) gene. Each chimeric construct was transferred into soybean protoplasts for transient assay, which led to the identification of the sequences between -281 and -223, -170 and -122, of Gy2 promoter as negative regulatory elements, and the sequences between -223 and -170, -122 and -16 as positive regulatory elements. These results are consistent in transformed tobacco plants as well. The serially deleted promoter fragments fused to the GUS were transformed into Nicotiana tabacum by Agrobacterium tumefaciens using the binary vector system. GUS activity of Gy2 promoter deletion constructs was detected only in seeds but not in leaves with different levels of expression as in transient assay. These results suggest that the glycinin Gy2 promoter drives a tissue-specific expression in transgenic tobacco plants.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.1
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• pp.9-14
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• 2003

Recent studies indicated that cancer cells become resistant to ionizing radiation (IR) and chemotherapy drugs by enhanced DNA repair of the lesions. Therefore, it is expected to increase the killing of cancer cells and reduce drug resistance by inhibiting DNA repair pathways that tumor cells rely on to escape chemotherapy. There are a number of key human DNA repair pathways which depend on multimeric polypeptide activities. For example, Ku heterodimer regulatory DNA binding subunits (Ku70/Ku80) on binding to double strand DNA breaks (DSBs) are able to interact with 470-kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and are essential for DNA-dependent protein kinase (DNA-PK) activity. It has been known that DNA-PK is an important factor for DNA repair and also is a sensor-transmitting damage signal to downstream targets, leading to cell cycles arrest. Our ultimate goal is to develop a treatment of breast tumors by targeting proteins involved in damage-signaling pathway and/or DNA repair. This would greatly facilitate tumor cell cytotoxic activity and programmed cell death through DNA damaging drug treatment. Therefore, we designed a domain of Ku80 mutants that binds to Ku70 but not DNA end binding activity and used the peptide in co-therapy strategy to see whether the targeted inhibition of DNA-PK activity sensitized breast cancer cells to irradiation or chemotherapy drug. We observed that the synthesized peptide (HNI-38) prevented DNA-PKcs from binding to Ku70/Ku80, thus resulting in inactivation of DNA-PK activity. Consequently, the peptide treated cells exhibited poor to no DNA repair, and became highly sensitive to IR or chemotherapy drugs, and the growth of breast cancer cells was inhibited. Additionally, the results obtained in the present study also support the physiological role of resistance of cancer cells to IR or chemotherapy.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.150-155
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• 1988

In order to produce L-lysine from cellulosic substrates by the intergeneric protoplast fusion between cellulolytic bacteria, Cellulomonas flavigena KFCC31221 and amino acid producing bacteria, Brevibacterium flavum ATCC14067, Corynebacteriurn glutamicum ATCC13032, conditions for protoplast formation and regeneration of these strains were investigated. After the strains were mutated with 500$\mu\textrm{g}$/$m\ell$ N-methyl-N'-nitro N-nitrosoguanidine for 30 min and the mutants were enriched by treating 300$\mu\textrm{g}$/$m\ell$ penicillin-G for 2 hrs, B. flavum Hse- Str$^{r}$ , C. glutamicum Met$^{-}$Thr$^{-}$ Rif$^{r}$ and Cellulomonas flavigena Thr$^{-}$Val$^{-}$Kan$^{r}$ were isolated. The rate of protoplast formation ranged from 95 to 98% when strains were treated at the concentration of 500$\mu\textrm{g}$/$m\ell$ of lysozyme, pH 6.5, 33$^{\circ}C$, for 6 hrs. in Tris- malate buffer supplemented with 0.4M sucrose as osmotic stabilizer. Approximately 30-33% protoplast was regenerated on the regeneration complete medium(RCM) containing 1.5% agar and 0.5M sodium succinate overlaid with the same medium except 0.7% agar.

• Microbiology and Biotechnology Letters
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• v.26 no.2
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• pp.179-186
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• 1998

Dextransucrase hyper-producing Leuconostoc mesenteroides B-512FMCM and dextransucrase constitutive mutants B-742CB and B-1355C catalyzed the transfer of glucose from sucrose to other carbohydrates which were present or were added to the reaction digests. When the acceptor was a maltose, gentiobiose, lactose or raffinose, there was produced a series of oligosaccharide acceptor products or single product based on the kinds of enzymes and reaction conditions. To obtain the quantitative information about the yield and the distribution of acceptor products and dextran two experimental parameters were studied: a) the ratio of acceptor to sucrose and b) the amount of enzyme at constant carbohydrate concentration (100 mM). As the amount of enzyme increased, the synthesis of acceptor products (of maltose or gentiobiose) increased, and the formation of dextran decreased. As the ratio of acceptor to sucrose increased, the amount of dextran and the number of acceptor-products decreased and the amount of acceptor-products increased. When maltose or gentiobiose was an acceptor, the glucose from sucrose was transferred to the C-6 hydroxyl group of the nonreducing-end glucose residue of accepters to give a homologous series of isomaltosyl dextrins. In case of lactose or raffinose, there was produced only one acceptor product from B-512FMCM dextransucrase reaction. In the lactose acceptor reaction, the glucose from sucrose was transferred to the C-2 hydroxyl of the reducing end glucose residue of lactose. To get a series of oligosaccharides from lactose or raffinose acceptor reaction we used B-742CB dextransucrase or B-1355C alternansucrase with 500 mM sucrose in reaction digest.

• Journal of Life Science
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• v.17 no.11
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• pp.1497-1504
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• 2007

To investigate the function and secretion of human thrombopoietin (TPO) in mammalian cells, hTPO cDNA was cloned using human liver cDNA, and recombinant hTPO (rec-hTPO) was produced in CHO cell lines. In addition, six N-linked glycosylation sites were substituted for Ala to elucidate the role of each carbohydrate chain. To analyze the biological activity, rec-hTPO protein was injected subcutaneously. Blood was withdrawn for platelet determination. The metabolic clearance rate (MCR) was also analyzed at the 1, 4, 10 and 24 hr after tail vein injection. Wild-type TPO (WT) was efficiently secreted into the medium. However, a hTPO mutant with 116 deleted nucleotides detected by PCR cloning was not secreted. The N-linked glycosylation sites had nearly the same expression quantity as rec-hTPO WT apart from mutants 3 and 4. The glycosylation site of mutant 4 appeared to be an indispensable site for hTPO secretion. Also characterized was the biological activity through an injection with rec-hTPO (10 ng) to ICR mice (7 weeks). The result of the blood analysis showed a considerable increase in the platelet number six days after He injection. To analyze the pharmacokinetics, rec-hTPO was injected into the tail vein (5 ng). The result was 200 pg/ml 1hr after this injection. Following this, it dramatically decreased and virtually disappeared 10 hours after the injection. Thus, rec-hTPO may be a treatment for thrombopenia by the production of the high active rec-hTPO. In addition, hTPO can permit the development of potent new analogues that stimulate the platelet value.