• Proceedings of the Membrane Society of Korea Conference
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• 2005.05a
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• pp.131-135
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• 2005

• Molecules and Cells
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• v.39 no.5
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• pp.426-438
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• 2016

Plant disease resistance occurs as a hypersensitive response (HR) at the site of attempted pathogen invasion. This specific event is initiated in response to recognition of pathogen-associated molecular pattern (PAMP) and subsequent PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Both PTI and ETI mechanisms are tightly connected with reactive oxygen species (ROS) production and disease resistance that involves distinct biphasic ROS production as one of its pivotal plant immune responses. This unique oxidative burst is strongly dependent on the resistant cultivars because a monophasic ROS burst is a hallmark of the susceptible cultivars. However, the cause of the differential ROS burst remains unknown. In the study here, we revealed the plausible underlying mechanism of the differential ROS burst through functional understanding of the Magnaporthe oryzae (M. oryzae) AVR effector, AVR-Pii. We performed yeast two-hybrid (Y2H) screening using AVR-Pii as bait and isolated rice NADP-malic enzyme2 (Os-NADP-ME2) as the rice target protein. To our surprise, deletion of the rice Os-NADP-ME2 gene in a resistant rice cultivar disrupted innate immunity against the rice blast fungus. Malic enzyme activity and inhibition studies demonstrated that AVR-Pii proteins specifically inhibit in vitro NADP-ME activity. Overall, we demonstrate that rice blast fungus, M. oryzae attenuates the host ROS burst via AVR-Pii-mediated inhibition of Os-NADP-ME2, which is indispensable in ROS metabolism for the innate immunity of rice. This characterization of the regulation of the host oxidative burst will help to elucidate how the products of AVR genes function associated with virulence of the pathogen.

• Korean Journal of Microbiology
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• v.35 no.4
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• pp.315-321
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• 1999

This study has been performed to deveope a yeast strain having high ${\alpha}$-amylase production ability using nuclear transfer method. Hybrids formed between the strains of Saccharomyces fiburigera KCTC 7393 and Saccharomyces cerevisiae KCTC 7049 (tyr-, ura-)were obtained by nuclear transfer technique. Nuclei isolated from the wild type S. fiburigera strain were transfered into auxotrophic mutants S. cerevisiae and selected the hybrids showing an increased starch degrading capability were selected (MN-16). This transformant grew best and produced maximal ${\alpha}$-amylase activity on the medium containing 2% (V/V) soluble starch. ${\alpha}$-Amylase from MN-16 was purified electrophoretically homogenety and its properties were investigated. The enzyme was purified about 10.6 fold with an overall yield 9.7% from the culture medium by ammonium sulfate fractionation. DEAE-Sephacel column chromatography, and Sephacryl S-200 column chromatography. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis. The molecular weight of the ${\alpha}$-amylase was estimated to be 53,000 daltons by SDS-PAGE and by gel permeation chromatography on Sephacryl S-200. The purified enzyme showed the maximum activity at pH 5.5 and 40${\circ}C$. The km value for soluble starch was 2.5㎎/㎖. The enzyme activity increased in the presence of $Ca^{2+}, Co^{2+}, EDTA, Mg^{2+}, Mn^{2+}, Zn^{2+}$, but inhibited by $Cu^{2+}, Fe^{2+}$, and $Ni^{2+}$

• Korean Journal of Medicinal Crop Science
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• v.15 no.6
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• pp.391-397
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• 2007

To develop new high valuable Gugija (Lycium chinensis), biofunctionalities of Gugija standard species and its hybrids were investigated and compared with each water extracts and methanol extracts from Lycii Fructus, Lycii Folium and buds and Lycii Cortex Radicis. Among various biofunctionalities of Gugija standard species, antioxidant activity was showed the highest in methanol extracts from buds of Cheongwoon species (93%) and antihypertensive angiotensin I-converting enzyme (ACE) inhibitory activity was 84.1% in the water extracts from Lycii Cortex Radicis of Cheongyang NO.7. Futhermore, methanol extracts from Lycii Cortex Radicis of Myungan A-2 hybrid showed 93.1% of antioxidant activity and 96.9% of ACE inhibitory activity was also showed in the methanol extracts from Lycii Fructus of DO148-72(A11) hybrid. However, fibrinolytic activity and anticholesteromia HMG-CoA reductase inhibitory activity were weak or not detected in almost of Gugija standard species and its hybrids. Therefore, we finally selected Cheongwoon Gugija standard species (buds) and Myungan A-2 hybrid (Lycii Cortex Radicis) as good antioxidant sources and also DO148-72 (A11) hybrid (Lycii Fructus) as excellent antihypertensive ACE inhibitior sources for manufacturing functional food product.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1054-1059
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• 2005

An extracellular protease was identified from Bacillus subtilis KCCM 10257 by N-terminal sequencing and mass spectral analysis. The molecular mass of the extracellular protease was estimated to be 28 kDa by SDS-PAGE. Sequencing of the N-terminal of the protease revealed the sequence of A(G,S,R)QXVPYG(A)V(P,L)SQ. The N-terminal sequence exhibited close similarity to the sequence of other proteases from Bacillus sp. A mass list of the monoisotopic peaks in the MALDI-TOF spectrum was searched after peptide fragmentation of the protease. Six peptide sequences exhibiting monoisotopic masses of 1,276.61, 1,513.67, 1,652.81, 1,661.83, 1,252.61, and 1,033.46 were observed from the fragmented protease. These monisotopic masses corresponded to the lytic enzyme L27 from Bacillus subtilis 168, and the Mowse score was found to be 75. A doubly charged Top product (MS) at a m/z of 517.3 exhibiting a molecular mass of 1034.6 was further analyzed by de novo sequencing using a PE Sciex QSTAR Hybrid Quadropole-TOF (MS/MS) mass spectrometer. MS/MS spectra of the Top product (MS) at a m/z of 517.3 obtained from the fragmented peptide mixture of protease with Q-star contained the b-ion series of 114.2, 171.2, 286.2, 357.2, 504.2, 667.4, 830.1, and 887.1 and y-ion series of 147.5, 204.2, 367.2, 530.3, 677.4, 748.4, 863.4, and 920.5. The sequence of analyzed peptide ion was identified as LGDAFYYG from the b- and y-ion series by de novo sequencing and corresponded to the results from the MALDI-TOF spectrum. From these results the extracellular protease from Bacillus subtilis KCCM 10257 was successfully identified with the lytic enzyme L27 from Bacillus subtilis 168.

• Microbiology and Biotechnology Letters
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• v.24 no.1
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• pp.50-58
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• 1996

Interspecific hybrids between Aspergillus oryzae var oryzae and A. nidulans 514 were obtained by nuclear transfer technique. Several autotrophic mutants isolated from conidiospores of the two strains were mutagenized with ultraviolet and N-methyl-N-nitrosoguanidine. Optimal conditions for formation of intergeneric hybrids were investigated. Frequencies of hybrid formation by nuclear transfer were $3{\times}10^{-5}{\sim}1{\times}10^{-5}$. From observation of genetic stability, conidial size, DNA content, and nuclear stain, it was suggested that their karyptypes are aneuploid. The hybrids showed 1.1~1.4 fold higher xylanase activities than parental strains did. The xylanase of Aspergiilus sp. TAVD514-3 was purified and some of it's enzymatc characteristics were investigated. The enzyme was purified about 85 fold with an overall yield of 17% from the culture medium by ammonium sulfate fractionation, Sephadex G-75 gel permeation chromatography, and CM-sephadex A-50 ion exchange chromatography. The purified enzyme functions optimally at pH 9.0 and 80$^{\circ}C$. The enzymatic activity was increased by the presence of $Mg^{2+}$ and $Mn^2$ ions.

• Journal of Microbiology and Biotechnology
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• v.1 no.1
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• pp.22-30
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• 1991

In order to secrete the Bacillus subtilis cytidine deaminase (CDase, cytidine/2'-deoxycytidine deaminase) encoded by the B. subtilis cdd gene in E. coli by the aid of signal sequences, the cdd gene was fused in-frame to either amyE or penP signal sequences and the gene expression and CDase localization were examined. For the penP signal sequence::cdd fusion, the cdd gene with 9 amino acids truncated from the 5'-terminus was fused in-frame to the signal sequence, then the $cdd^{+}$ colonies were not occurred from the minimal plate by cdd complementation. The result suggests that 9 amino acids on the $NH_2-terminal$ of CDase have an essential function in the enzyme activity. The hybrid protein obtained by fused gene amyE signal sequence::cdd structural gene gave $cdd^{+}$ phenotype and about half of the total CDase activity was found to be secreted in the periplasm of E. coli transformant JF611/pSO202. The periplasmic CDase activity of JF611 harboring pSO52 containing the intact cdd gene was considerablely lower than that of the cells harboring pSO202 carrying the hybrid cdd gene. This suggests that the CDase was secreted to the periplasm through the cytoplasmic membrane by the aid of the amyE signal sequence in the E. coli transformant.

• Development and Reproduction
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• v.16 no.2
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• pp.129-135
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• 2012

Previously, we identified that the overexpression of IEX-1 induces apoptosis in ovarian cancer cells. Herein we report a new binding partner of IEX-1, CATHEPSIN B, as a lysosomal enzyme which contributes to the various apoptotic signaling in tumor cells. To investigate how IEX-1 regulates cellular survival and death event, we performed yeast two-hybrid screening of rat ovarian cDNA library using IEX-1 as the bait and found CATHEPSIN B. In the present study, CATHEPSIN B and IEX-1 proteins were overexpressed in 293T cells and their specific association was determined by immunoprecipitation and immunoblot analysis. In addition, the endogenous interaction between CATHEPSIN B and IEX-1 was confirmed in HeLa cells. The current finding of lysosomal CATHEPSIN B as the IEX-1-binding partner implies that IEX-1 may involve in lysosome-mediated apoptotic signaling pathways.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.2
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• pp.165-170
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• 2006

Using the somatic cell hybrid panel (SCHP) and radiation hybrid (IMpRH) panel, porcine SOCS2 gene was mapped at SSC5 (1/2) q21-q24 and closely linked with SW1383 marker (47 cR in distance), while SOCS3 gene was assigned to SSC12p11-(2/3p13) and closely linked with SW2490 (43 cR). The reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to detect the expression of these two genes in the different tissues and the results showed that both SOCS2 and SOCS3 genes were widely expressed in tissues investigated (heart, liver, spleen, lung, kidney skeletal muscle, fat and brain), although some tissues showed lower gene expression. Moreover, SOCS2 and SOCS3 genes had different expression levels at different stages, in different tissues and in different breeds. A G/A substitution, which can be recognized by restriction enzyme of Cfr421, was observed in 5' untranslated region (5'-UTR) of SOCS2 gene. The allele frequencies was investigated by PCR-restriction fragment length polymorphism (PCR-RFLP) method and it showed that the allele frequency among Dahuabai, Erhualian, Yushan, Qingping, Large white and Landrace tested were different. Association analysis in a cross experimental populations revealed no significant association between the SOCS2 gene polymorphism and the economic traits investigated. The full-length coding regions (CDs) of porcine SOCS3 gene was obtained by RT-PCR.

• Animal cells and systems
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• v.6 no.3
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• pp.277-282
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• 2002

Topoisomearse II is an essential enzyme in all organisms with several independent roles in DNA metabolism. Recently, it has been demonstrated that the C-terminal region of topoisomerases II is associated with hetero-logous protein-protein interactions in human and yeast. In this study, we identified that RTP1, a rat homologue of EIA binding protein BS69, is another topoisomerae II interacting protein by yeast two-hybrid screening. RTP1 has an E1A-binding domain and a MYND motif, which are known to be required for transcriptional regulation by binding to other proteins and interaction with the leucine zipper motif of topoisomerase II. The physical interaction between RTP1 and topoisomerase ll$\alpha$ was examined by GST pull-down assay in vitro. The expression level of RTP1 peaks in S phase as that of topoisomerase ll$\alpha$. These results suggest that the interaction between topoisomerase ll$\alpha$ and RTP1 might play an important role in regulating the transcription of genes involved in DNA metabolism in higher eukaryotes.