• Journal of Life Science
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• v.19 no.9
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• pp.1218-1225
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• 2009

Difractose anhydrides (DFAs) is studied as a sweetener for diabetics because of its structural property. DFAs have four types: DFA I, III, IV (degradation of levan) and V (degradation of inulin). Especially, DFA IV has been shown to enhance the absorption of calcium in experiments using rats. Levan fructotransferase is an enzyme for producing di-d-fructose-2,6':6,2-dianhydride (DFA IV). To identify structural characterization, we purified wild-type and mutants (D63A, D195N and N85S) of levan fructotransferase (LFTase) from Microbacterium sp. AL-210. These proteins were purified to apparent homogeneity by Ni-NTA affinity column, Q-sepharose ion exchange and gel filtration chromatography and detected by SDS-PAGE. They were also analyzed by circular dichroism (CD) measurements, JNET secondary structure prediction, activity measurements at various temperatures, and pH analysis. The optimum pH for the enzyme-catalyzed reaction was pH 7.5 and optimum temperature was observed at $55^{\circ}C$. Along with wild-type LFTase, mutants were analyzed by CD measurement, fluorescence analysis and differential scanning calorimetry (DSC). N85S showed less $\alpha$-helix and more $\beta$ strand than others. Also, N85S showed almost the same curve as wild-type in their steady-state fluorescence spectra, whereas mutant D63A and D195N showed higher intensity than wild-type. The amino acid sequence of wild-type LFTase was compared to the sequences of exo-inulinase from Aspergillus awamori, a plant fructan 1-exohydrolase from Cichorium intybus, and Thermotogo maritime (Tm) invertase and showed a high identity with Exo-inulinase from Aspergillus awamori.

• Journal of Microbiology and Biotechnology
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• v.16 no.4
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• pp.576-583
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• 2006

The alkali-soluble glucan of the yeast cell wall contains $\beta-(1,3)-$ and (1,6)-D-linkages and is known to systemically enhance the immune system. In the previous study [6], in order to isolate cell wall mutants, a wild-type strain was mutagenized by exposure to ultraviolet light, and the mutants were then selected via treatment with laminarinase $(endo-\beta-(1,3)-D-glucanase)$. The mass of alkali- and water-soluble glucans produced by the mutant was measured to be 33.8 mg/g of the dry mass of the yeast cell. Our results showed that the mutants generated the amount of alkali-soluble glucan 10-fold higher than that generated by the wild-type. Structural analysis showed that the alkali-soluble glucan from the mutants was associated with a higher degree of $\beta-(1,6)-D-linkage$ than was observed in conjunction with the wild-type. Yeast cell wall $\beta-glucan$ was shown to interact with macrophages via receptors, thereby inducing the release of tumor necrosis factor alpha $(TNF-\alpha)$ and nitric oxide. Alkali-soluble $\beta-glucans$, both from water-soluble and water-insoluble glucan, exhibited a higher degree of macrophage activity with regard to both the secretion of tumor necrosis factor alpha $(TNF-\alpha)$ and nitric oxide and direct phagocytosis, than did the positive control ($1{\mu}g$ of lipopolysaccharide).

• Korean Journal of Plant Resources
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• v.23 no.1
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• pp.25-30
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• 2010

This study was carried out to understand the effect of low temperature($4^{\circ}C$), heat shock($37^{\circ}C$) and drought stresses on the growth and gene expression of Arabidopsis ATSIZ3(at1g08910) mutants. The seedling growth of SIZ3-mutants were markedly inhibited by the treatment of heat shock or chilling stresses. However, there was no significant differences between wild type and SIZ3-mutants in seeding fresh weight. As compared to wild type plants, SIZ3-mutants showed 63.9% inhibition of seedling fresh weight by the treatment of 10 days drought stress, suggesting that SIZ3 is involved in the resistance of Arabidopsis to drought stress. Base on RT-PCR analysis, expression of SIZ3 mRNA in the wild type showed 20% inhibition by chilling stress, 3.7 and 4.5 fold increase by the treatment of heat shock or drought stresses, respectively.

• Journal of Plant Biology
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• v.39 no.1
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• pp.31-40
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• 1996

The physiological and biochemical characterizations of the ethylene-related mutants in Arabidopsis thaliana - ethylene overproducing mutant (eto1-l) and ethylene insensitive mutants (etrl-3, ein2-l) - were detailed in this studies. Two or three week.old mature rosette leaves (before bolting) were used as the plant materials. Ethylene productions of eto1-l, etrl-3, and ein2-l mutants were about 200%, 400%, and 450% compared to that of wild type, respectively. ACC synthase and ACC oxidase activities of eto1-l mutant were similar to those of wild type. ACC content and ACC N-malonyltransferase activity, however, were 4.5 times and 3 times higher than those of wild type, respectively. SAM synthetase activity increased by 50% in eto1-l mutant plant. These results indicated that the alteration in the eto1-l mutant occured before the step of the conversion of SAM to ACe. In etrl-3 and ein2-l mutants, ACC synthase activities increased, but ACC oxidase activities decreased. ACC content and ACC N-malonyltransfcrase activity were 2 times higher than those of wild type. SAM synthetase activity in etrl-3 is similar to those of wild type, while it increased by 73% in ein2-l. These results showed that the block in ethylene action affected the autoregulation of ethylene biosynthesis, so that ACC synthase activity was not autoinhibited and ACC oxidase activity was not auto stimulated by ethylene. When the leaf tissues were used for in vitro kinase assay, a cytosolic protein (approximately 36 kDa) was phosphorylated only in eto1-l and ein2-l mutants.utants.

• Journal of Plant Biology
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• v.39 no.4
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• pp.273-278
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• 1996

Arabidopsis mutants deficient in flavonoid and sinapate ester (tt4 and fah1-7, respectively) were evaluated in vivo and in vitro to study the possible role of flavonoid compounds in pollen tube growth. In vivo, we investigated pollen tube growth in the pistils of the mutants and wild type(Ler). The growth of pollen tubes was significantly different among the three genotypes. In the fal1-7 pistils, the tubes grew to a greater length relative to those of the wild type or tt4. To examine in vitro pollen tube growth, a solid medium was devised for pollen germination and subsequent growth. In vitro, the identical result was obtained; fahl-7 pollens developed the longest tubes and elongated most rapidly. Therefore, the growth response of pollen tubes to phenolic compounds was examined by adding quercetin or sinapate ester in various concentrations to the media. Quercetin enhanced both germination rate and tube growth in the pollens of the mutants and the wild type, especially in tt4. In contrast, sinapate ester inhibits pollen germination and pollen tube growth in three genotypes. These results suggest that flavonoids and related phenolic compounds have physiological role in the plant reproductive system.

• The Plant Pathology Journal
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• v.15 no.1
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• pp.38-43
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• 1999

Mutants of the monopartite geminivirus beet curly top virus (BCTV) have been screened for infectivity, systemic movement, replication and symptom development in Arabidopsis thaliana. As known by coding for coat protein, R1 mutant was not infectious and did not move systemically. R2, R3 and L2/L3 mutants produced milder symptoms compared to wild type BCTV but the infectivity was reduced by 40% to 60%. R2 ORF is thought to be involved in the regulation of ssDNA and dsDNA accumulation because only dsDNA was accumulated on R2-infected organs. Disruption of ORF L4 resulted in reduced infections, but the viral DNA was accumulated in infected organs from roots to shoot tips as much as wild type BCTV on Sei-O. In addition, 4 mutants did not produce callus-like tissues on infected organs, suggesting that L4 ORF may play a role in the induction of host cell divisions by virus infection. This result was supported by the patterns of mRNA expression and promoter analysis of the cell cycle marker gene, cycl, on Arabidopsis. cycl mRNA was accumulated on symptomatic organs by wild type BCTV infections but not by L4 mutant. We conclude that the BCTV L4 ORF is essential for symptom developments, specially callus-like formation on infected organs.

• Journal of Microbiology and Biotechnology
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• v.28 no.10
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• pp.1736-1748
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• 2018

Brucella abortus can survive and replicate within host macrophages, and great efforts have been made to demonstrate the genes involved in pathogenicity, such as internalization, in Brucella research. Here, intracellular responses were compared between THP-1 macrophage cells stimulated with B. abortus wild-type and four mutants (C1, C10, C27, and C32) using microarray to demonstrate the role of genes related to intracellular survival and replication. These mutants were generated by deleting genes encoding BAB_RS13225 (4-hydrobenzoate 3-monooxygenase, PHBH), BAB_RS00455 (heme exporter protein cytochrome C, CcmC), BAB_RS03675 (exopolyphosphatase, PPX), and BAB_RS13225 (peptidase M24). The results showed that mutants C1 and C10 induced significant suppression of survival levels and cytokine expression relative to wild-type in the THP-1 macrophage cells. These findings suggest that the BAB_RS13225 and BAB_RS00455 genes play important roles in survival within human macrophages. Conversely, mutants C27 and C32 induced significantly higher survival level than wild-type in the cells inhibiting cellular signal transduction. It is assumed that the BAB_RS03675 and BAB_RS13225 genes play a role in cellular resistance to B. abortus. Therefore, the disrupted genes are involved in B. abortus intracellular growth, and especially in its survival, and they could be effective targets for understanding the intracellular bacterium, B. abortus.

• Rapid Communication in Photoscience
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• v.3 no.3
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• pp.53-55
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• 2014

The shade avoidance syndrome is a morphological and physiological response when plants are exposed to shade. Recent work in Arabidopsis had begun to define the molecular components of the shade avoidance syndrome in dicotyledonous model plant. However, little is known about the shade avoidance response networks in agriculturally important monocotyledon crops such as rice. Here, we found that the degree of bending at the lamina joint is inversely proportional to the R:FR ratio. To elucidate which phytochrome is involved in this response, we did lamina joint inclination assay with the rice phytochrome-deficient mutants (osphyA, osphyB, and osphyC) and the wild type plants. Whereas the osphyA and osphyC knockout mutants bent at the lamina joint in the far-red rich condition as the wild type plants, the osphyB knockout mutants no longer bent at the lamina joint in the far-red rich condition. These results suggest that PHYB acts as a sole photoreceptor in the lamina joint inclination response in rice.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1308-1315
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• 2007

Lipase B from Candida antarctica (CalB) displayed on the cell surface of H. polymorpha has been functionally improved for catalytic activity by molecular evolution. CalB was displayed on the cell surface by fusing to a cell-wall anchor motif (CwpF). A library of CalB mutants was constructed by in vivo recombination in H. polymorpha. Several mutants with increased whole-cell CalB activity were acquired from screening seven thousand transformants. The two independent mutants CalB 10 and CalB 14 showed an approximately 5 times greater whole-cell activity than the wild-type. When these mutants were made as a soluble form, CalB 10 showed 6 times greater activity and CalB 14 showed an 11 times greater activity compared with the wild-type. Sequence analyses of mutant CALB genes revealed amino acid substitutions of $Leu^{278}Pro$ in CalB10 and $Leu^{278}Pro/Leu^{219}Gln$ in CalB14. The substituted $Pro^{278}$ in both mutants was located near the proline site of the ${\alpha}$10 helix. This mutation was assumed to induce a conformational change in the ${\alpha}$10 helix and increased the $k_{cat}$ value of mutant CalB approximately 6 times. Site-directed mutagenized CalB, LQ ($Leu^{219}Gln$) was secreted into the culture supernatant at an amount of approximately 3 times more without an increase in the CalB transcript level, compared with the wild-type.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.67.2-68
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• 2003

A rice cultivar, Taebaegbyeo, is highly resistant to rice blast and moderately resistant to bacterial leaf blight (BLB) caused by Magnaporthe grisea and Xanthomonas oryzae pv. oryzae, respectively. To study the rice disease resistance mechanism, we generated rice deletion M3 mutants by gamma-ray irradiation. Blast and BLB responses of 16,000 M3 mutants were screened by inoculating mixtures of 4 races (KJ-201, H-1113a, KI-313, KI-409) of M. grisea and 3 Korean races of X. oryzae pv. oryzae. We selected so far 21 M3 mutants of Taebaegbyeo showing high susceptibility to the diseases. One of the mutants, KCT-6417, was susceptible to KI-1113a race of M. grisea, suggesting the deletion of a race-specific blast resistance gene in the mutant. To isolate rice genes involved in blast resistance and defense response, we take a PCR-based suppression subtractive hybridization approach using cDNAs of blast-inoculated wild type and the KCT-6417 as a tester and a driver, respectively. Genes specifically expressed in the wild type will be presented. The selected genes would give us a clue to understand mechanism for the race specific resistance and defense responses against M. grisea H-1113a in Taebaegbyeo.