• The Plant Pathology Journal
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• v.26 no.4
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• pp.386-393
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• 2010

In order to study disease resistance mechanisms in rice against the rice blast fungus Magnaporthe grisea, we screened fungal elicitor-responsive genes from rice suspension-cultured cells treated with fungal elicitors employing differential hybridization (DH). By DH screening, 31 distinct rice clones were isolated and a majority of them were full-length cDNAs encoding pathogenesisrelated (PR) genes. Sixteen of the 31 genes were upregulated at 4, 8, and 12 h following fungal elicitor treatment. To elucidate the effect of signal molecules and biotic elicitors on the regulation of rice defense genes, we further characterized the transcriptional expression patterns of representative isolated PR genes; OsGlu1, OsGlu2, OsTLP, OsRLK, and OsPR-10, following treatment with fungal elicitor, phytohormones, cycloheximide, and inhibitors of protein phosphorylation. Jasmonic acid (JA) induced transcriptional expression of OsGlu1, OsTLP, and OsRLK, but not of OsGlu2 and OsPR-10 at any of the tested time points. Salicylic acid (SA) and abscisic acid weakly induced the expression of OsTLP and OsRLK. SA showed an antagonistic effect with fungal elicitor and JA. Cycloheximide suppressed all these genes upon elicitor treatment, except for OsGlu2. Staurosporine only induced the expression of OsRLK. Application of calyculin A strongly induced OsRLK expression, but suppressed the expression of OsGlu2. Our study yielded a number of PR genes that play a role in defense mechanisms against the rice blast fungus, as well as contribute towards the elucidation of crosstalk between phytohormones and other modifications during defense signaling.

• Microbiology and Biotechnology Letters
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• v.31 no.3
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• pp.216-223
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• 2003

A 1.3-kb of chitosanase gene (choA) encoding 45-kDa polypeptide was cloned, expressed, and characterized from a newly isolated Bacillus cereus H-1. The chitosanase protein (ChoA) of B. cereus H-l was purified to homogeneity by ammonium sulfate precipitation and CM-sephadex column chromatography. Optimum pH was around 7, and stable pH range in the incubation at 50 C was 4-11. Optimum temperature was around 50 C, and enzyme activity was relatively stable below 45 C. ChoA showed the activities toward carboxymethyl cellulose (CMC) in addition to soluble or glycol chitosan. Based on MALDI-TOF MS analysis of purified ChoA, the entire amino acid sequence of ChoA was interpreted by database searching of previously known Bacillus chitosanases. A 1.6 kb of PCR product of corresponding chitosanase gene was obtained and its DNA sequence was determined. The deduced amino acid of choA revealed that ChoA have a 98% homology with those of Bacillus sp. No.7-M strain and Bacillus sp. KCTC0377BP. The recombinant ChoA protein was expressed in E. coli DH5$\alpha$. Deduced amino acid comparison of choA with other chitosanases suggested that it belongs to family 8 microbial endo-chitosanase with chitosanase-cellulase activity.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.117-123
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• 2003

For screening of autoregulator receptor gene from Saccharopolyspora erythraea, PCR was performed with primers of receptor gene designed on the basis of amino acid sequences of autoregulator receptor proteins with known function. PCR products were subcloned into the BamHI site of pUC19 and transformed into the E. coli DH5$\alpha$. The isolated plasmid from transformant contained the fragment of 120 bp, which was detected on 2% gel after BamHI treatment. The insert, 120 bp PCR product, was confirmed as the expected internal segment of gene encoding autoregulator receptor protein by sequencing. Southern and colony hybridization using Saccha. erythraea chromosomal DNA were performed with the insert as probe. The plasmid (pEsg) having 3.2 kbp SacI DNA fragment from Saccha. erythraea is obtained. The 3.2 kbp SacI DNA fragment was sequenced by the dye terminator sequencing. The nucleotide sequence data was analyzed with GENETYX-WIN (ver 3.2) computer program and DNA database. frame analyses of the nucleotide sequence revealed a gene encoding autoregulator receptor protein which is a region including KpnI and SalI sites on 3.2 kbp SacI DNA fragment. The autoregulator receptor protein consisting of 205 amino acid was named EsgR by author. In comparison with known autoregulator receptor proteins, homology of EsgR showed above 30%.

• Food Science of Animal Resources
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• v.30 no.6
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• pp.923-929
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• 2010

Colostral whey prepared from colostrum (pooled from first six post-partum milkings) was heated for 10 min at $100^{\circ}C$ Heated colostral whey was incubated with 1% enzymes (protein equivalent basis) for 15, 30, 60, 90, and 120 min at $50^{\circ}C$. Papain, pepsin, trypsin, and alcalase produced different degrees of hydrolysis (DH), 10.66%, 12.42%, 10.83%, and 25.31%, respectively, at an incubation time of 120 min. The SDS-PAGE reveals that significant amounts of bovine serum albumin (BSA), ${\beta}$-lactoglobulin (${\beta}$-LG), and ${\alpha}$-lactalbumin (${\alpha}$-LA) survived papain digestion. In contrast, pepsin completely removed BSA but not ${\beta}$-LG present in heated colostral whey. Alcalase completely eliminated BSA, ${\beta}$-LG, and ${\alpha}$-LA. This differential hydrolysis was confirmed by reversed-phase HPLC analysis. Using ion-exchange chromatography, fraction-1 (F-1) was obtained from alcalase hydrolysate at a NaCl gradient concentration of 0.25 M. Reversed-phase HPLC chromatograms of alcalase F-1 showed numerous small peaks, which probably indicate that a variety of new peptides were produced. Iron content of alcalase F-1 was 28.94 ppm, which was the highest among all enzyme fractions, whereas iron content of colostral whey was 36.56 ppm. Main amino acids contained in alcalase F-1 were Thr (15.45%), Glu (14.12%), and Ser (10.39%). Therefore, alcalase can be used to generate good iron-binding peptides in heated colostral whey, and the resulting iron-binding peptides could be suitable as a value-added food ingredient for food supplements.

• Journal of Life Science
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• v.18 no.11
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• pp.1592-1599
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• 2008

The $\beta$-lactamase gene was cloned into E. coli DH5$\alpha$ from Bacillus sp. J105 with strong resistance against $\beta$-lactam antibiotics. The chromosomal DNA was partially digested with Sau3AI and ligated to BamHI digested pLAFR3. $\beta$-Lactamase positive clones were obtained by using in vitro packaging kit. The pKL11-${\Delta}4.6$ with $\beta$-lactamase activity was obtained by subcloning of the recombinant plasmid ($\beta$-lac +). The 6.5 kb fragment in the subcloned plasmid was sequenced. The DNA fragment that contains the $\beta$-lactamase gene encodes 309 amino acids. The 0.17 kb upstream region was similar to those of B. thuringinesis and B. cereus with 97% identity. The deduced amino acids sequence was also similar to those of $\beta$-lactamase from B. thuringinesis and B. cereus with 97% and 94% identity, respectively. The phylogenetic tree also showed the relationships of the $\beta$-lactamase gene of Bacillus sp. J105 to genetically related that of other Bacillus strains. Analysis of expression pattern of the pKL11-${\Delta}4.6$ in E. coli, revealed that the secretion efficiency of $\beta$-lactamase was $4{\sim}5%$ and the molecular weight was as same as that of original $\beta$-lactamase (31 kDa) from Bacillus sp. J105.

• Korean Journal of Environment and Ecology
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• v.26 no.4
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• pp.528-536
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• 2012

This study has compared the different types of forest fires(unburned, crown fire, ground fire) and the degree of vegetation recovery at Samcheuk-si, Gangwon-do by assessing the biomass and net primary production from July 2007 through July 2010. The research showed that the average biomass of unburned site(Un), crown fire site(C-1), crown fire site(C-3), ground fire site(G-2) were $181.20{\pm}5.39$, $62.04{\pm}4.38$, $131.09{\pm}14.38$, $63.39{\pm}2.72ton{\cdot}ha^{-1}$, respectively. And the research showed that the average net primary production of unburned site(Un), crown fire site(C-1), crown fire site(C-3), ground fire site(G-2) were $4.17{\pm}0.56$, $3.27{\pm}1.56$, $11.51{\pm}0.53$, $2.10{\pm}0.31ton{\cdot}ha^{-1}{\cdot}yr^{-1}$, respectively. Quercus mongolica $DH_{10}$(Diameter at the 10cm tree height) growth rate at each plot was compared to the crown fire site(C-1) in the annual average $1.21{\pm}0.55mm{\cdot}yr^{-1}$ at the speed of the fastest growth follows; showed crown fire site(C-3), ground fire site(G-2), unburned site(Un) appeared in the order. And that showed the growth rate of height was highest in the $15.43{\pm}4.57cm{\cdot}yr^{-1}$ at crown fire site(C-3), then the crown fire site(C-1), and ground fire site(G-2), and lowest in the unburned site(Un).

• Journal of the Korean Society of Food Science and Nutrition
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• v.31 no.1
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• pp.92-97
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• 2002

The effects of Pueraria flos (PF) and Pueraria radix (PR) water extract on the hepatic alcohol metabolic enzyme activities were examined in rats that were orally administered ethanol (25% v/v, 5g/kg body weight/day) for 5 weeks. The PF and PR water extract were supplemented in a diet, based on 1.2 g or 2.4 g of raw PF or PR/kg body weight/body. Alcohol administration without the PF or PR supplementation significantly decreased net weight gain, feed intake and feed efficiency ratio. However. both dose of the PF of PR supplementation resulted in significant enhancement of growth and suppression of increased relative weight of liver, brain and heart by alcohol administration. Activities of hepatic alcohol dehydrogenase and microsomal ethanol oxidizing system were higher in the alcohol treated group than in the normal group, while aldehyde dehydrogenase activity was significantly lowered in the alcohol treated group. The hepatic metabolic enzyme activities altered by alcohol administration were normalized by both doses of PF or PR supplement. Hepatic monoamine oxidase activity and hydrogen peroxide, which were significantly higher in the alcohol treated group than in the normal group, were also decreased by the supplementation with either PF or PR. These results indicate that low-or high-supplementation of either water extract PF or PR may alleviate ethanol-induced hepatotoxicity by altering alcohol metabolic enzyme activities.

• Applied Biological Chemistry
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• v.46 no.2
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• pp.130-133
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• 2003

A method for separating heme-iron from hemoglobin (Hgb) hydrolysate by dialysis was developed. Recovery of heme-iron increased with increasing Hgb concentration, whereas rejection of peptide and separation effciency expressed by HP ratio (heme-iron/peptide) did not show significant differences. HP ratio increased with increases in the degree of hydrolysis of Hgb and $KH_2PO_4$ concentrations of dialysis solution. Recovery of heme-iron decreased with increase in the pH of dialysis solution due to wash-out of heme-iron across the dialysis membrane caused by increase in solubility of heme-iron. Rejections of peptide were 74.5 and 87.5% (2 and 5 kDa of cut off size, respectively), whereas recovery of heme-iron decreased from 86.5 (2 kDa) to 63.1% (25 kDa). Amounts of heme-iron and peptide of dried heme-iron product were 21.7 and 77.0%, and HP ratio and production yield were 28.2 and 6.5%, respectively.

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.330-338
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• 2019

Chronic infection with intracellular Brucella abortus (B. abortus) in livestock remains as a major problem worldwide. Thus, the search for an ideal vaccine is still ongoing. In this study, we evaluated the protective efficacy of a combination of B. abortus recombinant proteins; superoxide dismutase (rSodC), riboflavin synthase subunit beta (rRibH), nucleoside diphosphate kinase (rNdk), 50S ribosomal protein (rL7/L12) and malate dehydrogenase (rMDH), cloned and expressed into a pMal vector system and $DH5{\alpha}$, respectively, and further purified and applied intraperitoneally into BALB/c mice. After first immunization and two boosters, mice were infected intraperitoneally (IP) with $5{\times}10^4CFU$ of virulent B. abortus 544. Spleens were harvested and bacterial loads were evaluated at two weeks post-infection. Results revealed that this combination showed significant reduction in bacterial colonization in the spleen with a log protection unit of 1.31, which is comparable to the average protection conferred by the widely used live attenuated vaccine RB51. Cytokine analysis exhibited enhancement of cell-mediated immune response as IFN-${\gamma}$ is significantly elevated while IL-10, which is considered beneficial to the pathogen's survival, was reduced compared to control group. Furthermore, both titers of IgG1 and IgG2a were significantly elevated at three and four-week time points from first immunization. In summary, our in vivo data revealed that vaccination with a combination of five different proteins conferred a heightened host response to Brucella infection through cell-mediated immunity which is desirable in the control of intracellular pathogens. Thus, this combination might be considered for further improvement as a potential candidate vaccine against Brucella infection.

• Applied Biological Chemistry
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• v.39 no.6
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• pp.443-448
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• 1996

In order to investigate the function of xylA promoter(Pxyl) as regulatory region Pxyl-lacZ fusion gene was constructed by the insertion of xylA promoter to the multiple cloning site of upstream of lacZ gene in a multicopy numbered plasmid pMC1403 containing promoterless lac operon, which was designated pMCX191, and Pxyl-lacZ fragment from pMCX191 was inserted to low copy numbered plasmid pLG339, designated pLGX191. The expressions of ${\beta}-galactosidase$ in these recombinant plasmids containing Pxyl-lacZ fusion gene were induced strongly by the addition of xylose, repressed by the addition of 0.2% glucose in the presence of xylose. The catabolite repressions were derepressed by the addition of 1 mM cAMP as same as native xylA gene. The fragment of xylA promoter was partially deleted from the upstream of xylA promoter by exonuclease III to investigate the regulation site of xylA promoter and the degrees of deletion derivatives of xylA promoter were analyzed by the DNA base sequencing. By the investigations of the induction by xylose, repression by glucose and derepression by cAMP on xylose isomerase production, the regulation site of xylA promoter may be located in segment between -165 and -59 bp upstream from the initiation site of xylA translation.