• Journal of Applied Biological Chemistry
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• v.61 no.4
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• pp.335-340
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• 2018

Hemoglobin (Hb) is a member of heme-protein that can perform catalytic non-specific chain reaction in the presence of hydrogen peroxide ($H_2O_2$). Catalytic ability of Hb to degrade pyrene was demonstrated using soil contaminated with $^{14}C$ pyrene and 10 mg pyrene /kg soil. The composition of soil was similar to previously used soil except that it had lower organic carbon content. Bench scale laboratory tests were conducted in the presence of buffer only, $H_2O_2$ only, or Hb with $H_2O_2$ for 24 h. After 24 h reaction, 0.1 and 1.3% of $^{14}C$ pyrene in contaminated soil were mineralized with $H_2O_2$ only or Hb plus $H_2O_2$. No mineralization to $^{14}CO_2$ was detected with buffer only. Approximately 12.2% of pyrene was degraded in the presence of $H_2O_2$ only while 44.0% of pyrene was degraded in the presence of Hb plus $H_2O_2$ during 24 h of catalytic reaction. When degradation intermediate products were examined, two chemicals were observed in the presence of $H_2O_2$ only while 25 chemicals were found in the presence of Hb plus $H_2O_2$. While most degradation products were simple hydrocarbons, four of the 27 chemicals had aromatic rings. However, none of these four chemicals was structurally related to pyrene. These results suggest that Hb catalytic system could be used to treat pyrene-contaminated soil as an efficient and speedy remediation technology. In addition, intermediate products generated by this system are not greatly affected by composition change in soil organic matter content.

• Laboraroty Animal Research
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• v.34 no.4
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• pp.279-287
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• 2018

Placenta specific 8 (PLAC8, also known as ONZIN) is a multi-functional protein that is highly expressed in the intestine, lung, spleen, and innate immune cells, and is involved in various diseases, including cancers, obesity, and innate immune deficiency. Here, we generated a Plac8 knockout mouse using the CRISPR/Cas9 system. The Cas9 mRNA and two single guide RNAs targeting a region near the translation start codon at Plac8 exon 2 were microinjected into mouse zygotes. This successfully eliminated the conventional translation start site, as confirmed by Sanger sequencing and PCR genotyping analysis. Unlike the previous Plac8 deficient models displaying increased adipose tissue and body weights, our male Plac8 knockout mice showed rather lower body weight than sex-matched littermate controls, though the only difference between these two mouse models is genetic context. Differently from the previously constructed embryonic stem cell-derived Plac8 knockout mouse that contains a neomycin resistance cassette, this knockout mouse model is free from a negative selection marker or other external insertions, which will be useful in future studies aimed at elucidating the multi-functional and physiological roles of PLAC8 in various diseases, without interference from exogenous foreign DNA.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2022.09a
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• pp.112-112
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• 2022

This study aims to confirm the possibility of using the invasive alien plants in Jeju as a functional biomaterial. To achieve this purpose, 70% ethanol extract and solvent fractions were prepared for five invasive alien plants (Hypochaeris radicata, Rumex acetosella, Humulus japonicus Siebold & Zucc., Solanum viarum, Lactuca scariolar) and their antioxidant, antibacterial anti-inflammatory and anti-obesity effects were investigated. The DPPH radical scavenging activity of ethanol extract from invasive alien plants was shown in the order of Rumex acetosella > Hypochaeris radicata > Humulus japonicus. Antimicrobial activity of ethanol extract against food poisoning bacteria (4 species) and oral cavity-induced microorganisms (6 species) was measured. As a result, the extract of Humulus japonicus showed high antibacterial effects against food poisoning bacteria (E. coli, V. parahaemolyticus) and oral microbes (L. casei, S. epidermidis, E. faecalis). In LPS-induced RAW 264.7 cells, the anti-inflammatory effect of ethanol extract from invasive alien plants was investigated. As a result, the NO production inhibition activity was highest in the Rumex acetosella and the Humulus japonicus Siebold & Zucc. ethanol extract, and the NO production inhibition activity was concentration-dependent. In addition, the Rumex acetosella and the Humulus japonicus Siebold & Zucc. ethanol extract showed a concentration-dependent inhibitory effect on cytokine (IL-6) production. These extracts also showed inhibitory activity of COX-2, an inflammatory protein. This suggests that NO production inhibition activity by the extract of invasive alien plants is the result of inhibition of iNOS and COX-2 expression. Currently, organic solvent fractions of crude extract are manufactured and the investigation of active ingredients is continuing along with evaluation of biological activity such as anti-inflammatory. These results are expected to be a major data for the study on the separation and utilization of active ingredients with antioxidant, antibacterial and anti-inflammatory effects using foreign plant crude extract and solvent fractions, and are highly likely to be applied to the development of functional food and cosmetics materials.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.3
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• pp.99-108
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• 2023

Background: Efficient gene editing technology is needed for successful knock-in. Homologous recombination (HR) is a major double-strand break repair pathway that can be utilized for accurately inserting foreign genes into the genome. HR occurs during the S/G2 phase, and the DNA mismatch repair (MMR) pathway is inextricably linked to HR to maintain HR fidelity. This study was conducted to investigate the effect of inhibiting MMR-related genes using CdCl₂, an MMR-related gene inhibitor, on HR efficiency in HC11 cells. Methods: The mRNA and protein expression levels of MMR-related genes (Msh2, Msh3, Msh6, Mlh1, Pms2), the HR-related gene Rad51, and the NHEJ-related gene DNA Ligase IV were assessed in HC11 cells treated with 10 μM of CdCl₂ for 48 hours. In addition, HC11 cells were transfected with a CRISPR/sgRNA expression vector and a knock-in vector targeting Exon3 of the mouse-beta casein locus, and treated with 10 μM cadmium for 48 hours. The knock-in efficiency was monitored through PCR. Results: The treatment of HC11 cells with a high-dose of CdCl₂ decreased the mRNA expression of the HR-related gene Rad51 in HC11 cells. In addition, the inhibition of MMR-related genes through CdCl₂ treatment did not lead to an increase in knock-in efficiency. Conclusions: The inhibition of MMR-related gene expression through high-dose CdCl₂ treatment reduces the expression of the HR-related gene Rad51, which is active during recombination. Therefore, it was determined that CdCl₂ is an inappropriate compound for improving HR efficiency.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.5
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• pp.366-374
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• 2010

Introduction: This study evaluated the capability of silk fibroin (SF) and recombinant human bone morphogenetic protein-2 loaded SF (SF-BMP) as a bone defect replacement matrix when grafted in a calvarial bone defect of rats in vivo. Materials and Methods: A total 70 calvarial critical size defects (5.0 mm in diameter) made on 35 adult female Sprague-Dawley rats were used in this study. The defects were transplanted with (1) rhBMP-2 loaded silk fibroin graft (SF-BMP: 0.8+$10\;{\mu}g$), (2) Silk fibroin (SF: $10\;{\mu}g$), and (3) no graft material (Raw). The samples were evaluated with soft x-rays, alkaline phosphatase activity, calcium/phosphate quantification, histological and histomorphometric analysis at postoperative 4 and 8 weeks. Results: The SF-BMP group ($48.86{\pm}14.92%$) had a significantly higher mean percentage bone area than the SF group ($24.96{\pm}11.01%$) at postoperative 4 weeks.(P<0.05) In addition, the SF-BMP group ($40.01{\pm}12.43%$) had a higher % bone area at postoperative 8 weeks than the SF group ($33.26{\pm}5.15%$). The mean ratio of gray scale levels to the host bone showed that the SF-BMP group ($0.67{\pm}0.08$) had a higher mean ratio level than the SF group ($0.61{\pm}0.09$) at postoperative 8 weeks. These differences were not statistically significant.(P=0.168 and P=0.243, respectively) The ratio of the calcium and phosphate contents of the SF-BMP ($0.93{\pm}0.22$) group was lower than that of the SF ($1.90{\pm}1.42$) group at postoperative 4 weeks. However, the SF-BMP group ($0.75{\pm}0.31$) had a higher Ca/$PO_4$ ratio than the SF ($0.68{\pm}0.04$) at postoperative 8 weeks. These differences were not statistically significant.(P=0.126 and P=0.627, respectively) For the bone-specific alkaline phosphatase (ALP) activity, which is recognized as a reliable indicator of the osteoblast function, the SF-BMP ($23.71{\pm}8.60\;U/L$) groups had a significantly higher value than the SF group ($12.65{\pm}6.47\;U/L$) at postoperative 4 weeks.(P<0.05) At postoperative 8 weeks, the SF-BMP ($21.65{\pm}10.02\;U/L$) group had a lower bone-specific ALP activity than the SF group ($16.72{\pm}7.35\;U/L$). This difference was not statistically significant.(P=0.263) For the histological evaluation, the SF-BMP group revealed less inflammation, lower foreign body reactions and higher bone healing than the SF group at postoperative 4 and 8 weeks. The SF group revealed more foreign body reactions at postoperative 4 weeks. However, this immunogenic reaction decreased and the remnant of grafted material was observed at postoperative 8 weeks. For histomorphometric analysis, the SF-BMP group had a significantly longer bone length to total length ratio than those of the SF group at postoperative 4 and 8 weeks.(P<0.05) Conclusion: The rhBMP-2 loaded silk fibroin graft revealed fewer immunoreactions and inflammation as well as more new bone formation than the pure silk fibroin graft. Therefore, silk fibroin may be a candidate scaffold for tissue engineered bone regeneration.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.34 no.1
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• pp.1-11
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• 2012

Purpose: The purpose of this study was to evaluate the bone regeneration capacity of silk fibroin (SF) when combined with beta tricalcium phosphate (${\beta}$-tricalcium phosphate [TCP]) and rh-bone morphogenetic protein (BMP) in vivo by micro-computed tomography (CT), soft x-ray, and histological analysis. Methods: A total of 56 critical size defects formed by a trephine bur made on 28 adult female Spague-Dawley rats were used for this study and the defect size was 5.0 mm in diameter. The defects were transplanted with (1) no graft material (raw defect), (2) autogenous bone, (3) SF ($10{\mu}g$), (4) SF-BMP ($10{\mu}g$, $0.8{\mu}g$ each), and (5) SF+${\beta}$-TCP ($10{\mu}g$). At 4 and 8 weeks after operation, the experimental animals were sacrificed. Samples were evaluated with soft x-ray, histological examinations and 3-dimensional micro-CT analysis. Results: In the 3-dimensional micro-CT evaluation, bone volume and bone surface data were higher in the SF-BMP ($12.8{\pm}1.5$, $138.6{\pm}45.0$ each) (P<0.05) and SF-TCP ($12.3{\pm}1.5$, $144.9{\pm}30.9$ each) group than in the SF group ($6.1{\pm}3.3$, $77.2{\pm}37.3$ each) (P<0.05), except for the autogenous group ($15.0{\pm}3.0$, $190.7{\pm}41.4$ each) at 4 weeks. At 8 weeks, SF-BMP ($16.8{\pm}3.5$, $173.9{\pm}34.2$ each) still revealed higher (P<0.05) bone volum and surface, but SF-TCP ($11.3{\pm}1.5$, $1132.9{\pm}52.1$ each) (P=0.5, P=0.2) revealed the same or lower amount compared with the SF group ($13.8{\pm}2.7$, $127.5{\pm}44.8$ each). The % of bone area determined by radiodensity was higher in the SF-TCP ($31.4{\pm}9.1%$) and SF-BMP ($36.2{\pm}16.2%$) groups than in the SF ($19.0{\pm}10.4$) group at the period of 4 weeks. Also, in the histological evaluation, the SF-BMP group revealed lower inflammation reaction, lower foreign body reaction and higher bone healing than the SF group at postoperative 4 weeks and 8 weeks. The SF-TCP group revealed lower inflammation at 4 weeks, but accordingly, as the TCP membrane was absorbed, inflammatory and foreign body reaction are increased at 8 weeks. Conclusion: The current study provides evidence that the silk fibrin can be used as an effective grafted material for tissue engineering bone generation through a combination of growth factor or surface treatment.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.67 no.2
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• pp.111-120
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• 2022

Improving flour quality is one of the major targets of wheat breeding programs. This study determined the optimum high-molecular-weight glutenin subunits (HMW-GS) to improve flour quality, and analyzed the correlation between agronomic and quality traits in Korea. A total of 180 wheat varieties, including 55 Korean and 125 foreign cultivars, carrying various Glu-1 alleles, were evaluated for their quality and agronomic traits. Results indicated that Glu-A1b, Glu-B1b, and Glu-D1f were the most prevailing alleles for each Glu-1 locus for Korean wheat cultivars. Korean wheat cultivars recorded shorter days to heading (DTH) and longer days to maturity (DTM) compared to foreign cultivars. In addition, an interaction effect was found between Glu-A1 and Glu-B1 alleles on several quality parameters. The combination of Glu-A1c and Glu-B1i showed a higher protein content, dry gluten content, and higher sodium dodecyl sulfate (SDS) sedimentation value than other Glu-A1×Glu-B1 combinations. Cultivars carrying Glu-A1a or Glu-A1b, Glu-B1i or Glu-B1al, and Glu-D1d for each Glu-1 locus exhibited a longer mixing time and stronger mixing tolerance. The DTM positively correlated with the protein content, gluten index and SDS sedimentation value. However, a negative correlation was observed between DTH and quality traits. Owing to the above results, this study suggests that an increase in the frequency of Glu-B1i or Glu-B1al, Glu-D1d coupled with a short DTH and long DTM could significantly improve wheat quality properties.

• Journal of Animal Science and Technology
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• v.47 no.4
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• pp.513-524
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• 2005

This study was conducted to introduce recycling procedures of food waste(FW) as feed according to the dehydration, semi-dehydration fermentation and liquid fermentation methods through the on-site survey of companies related, to trace physico-chemical components and nutritional losses depending upon the processing stage for each method and finally to suggest more desirable methodology for the efficient utilization of FW as animal feed. For the dehydration method, dewatering of FW alone reduced(P<0.05) moisture(approximately 10%) and ether extract contents and increased(P<0.05) fiber contents. Dewatering and subsequent dehydration of FW decreased(P<0.05) contents of ether extract, limiting amino acids such as lysine, methionine and histidine, pepsin digestibility of protein by half, and NaCl content by 40%, increased(P<0.05) contents of fiber, crude ash, Ca and P, and did not alter(P>0.05) pH. The semi-dehydration fermentation method of FW did not affect(P>0.05) the chemical components, pepsin digestibility of protein, pH and NaCl content. For the liquid fermentation method, pasteurization and fermentation of FW decreased(P<0.05) contents of dry matter, ether extract, crude fiber, lysine and NaCl; however, it did not affect(P>0.05) other chemical components, pepsin digestibility of protein and pH. Among the processing methods, nutrient losses were highest for the dehydration method(25% of metabolizable energy loss, 12% of organic matter loss) and little for the semi-dehydration and liquid fermentation methods. The on-site survey of companies related revealed that the existence of foreign materials in FW products were problematic for all the three companies surveyed, thus it was necessary to develop a more efficient screener. Before feeding FW-containing diets to pigs, high quality of protein and energy feedstuffs needed to be fortified for the dehydration method. For the semi-dehydration fermentation method, the scientific diet formulation technology was required at the initial mixing stage. For the liquid fermentation method, possibly most energetic and proteinaceous feeds needed to be supplemented for the normal animal growth.

• Korean Journal of Organic Agriculture
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• v.16 no.2
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• pp.219-230
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• 2008

This trial was conducted to assess effects of cattle slurry application on productivity and feed values of barley and hairy vetch when they were influenced by single or mixed-sowed cultivation in paddy fields, and to obtain good quality of organic forage resources. The results summarized are as follows. For barley and hairy vetch, single-sowed cultivation was lower in annual dry matter (DM) and total digestible nutrients (TDN) yields than mixed-sowed (P<0.05). Although crude protein (CP) contents for the barley within single-sowed treatments were lowest as 6.5%, those of hairy vetch within the same sowed method were highest as 16.7%. However, mixed-sowed cultivation with barley and hairy vetch, showing 13.8% CP content, was significantly (P<0.05) higher than CP and relative feed value (RFV) of barley alone treatment. For barley alone treatment, cattle slurry application significantly increased annual DM and TDN yields in comparison with treatments of P+K fertilization as chemical fertilizers and no fertilizer as controls. Results from cattle slurry application showed 84% and 82% in contrast with chemical fertilizer for annual DM and TDN yields, respectively. For mixed-sowed cultivation with barley and hairy vetch, cattle slurry application showed 90% and 94% annual DM and TDN yields, respectively as compared with N+P+K fertilization as chemical fertilizers. Crude protein contents ($14.2{\sim}15.9%$) for cattle slurry application treatments were significantly (P<0.05) higher than those of other treatments. Moreover, cattle slurry application treatment had the highest TDN and RFV among treatments, showing $60.7{\sim}61.8%$ and $112.2{\sim}118.1$, respectively. For hairy vetch alone treatments, annual DM and TDN yields of cattle slurry alone application treatment were highest among fertilization treatments. Furthermore, CP, TDN and RFV of cattle slurry alone application treatments were significantly (P<0.05) higher than those of other treatments. The results showed that mixed-sowed cultivation rather than single-sowed for barley or hairy vetch improved their nutritive value and quality, and also within mixed-sowed cultivation, cattle slurry application increased production yield per ha and CP contents. In the application of above system to organic livestock farming, it would be expected that forages produced by cattle slurry application under mixed-sowed method might become a substitute for foreign organic grain as protein sources.

• Journal of Applied Biological Chemistry
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• v.53 no.1
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• pp.1-7
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• 2010

xylA promoter is a major promoter in xylose operon of Escherichia coli. xylA promoter is sufficient as the promoter for the construction of new expression vector because this promoter was tightly controlled and induced by the addition of xylose. For the construction of xylose-inducible expression vector, 600 bp of xylA promoter was ligated between AatII and HindIII of pUC18, named pXA600. In order to investigate the effect of XylR protein encoded by xylR gene on the xylA promoter, 1,988 bp of xylR gene including its promoter was ligated into downstream of multiple cloning site to the opposite direction of xylA promoter in pXA600, named pXAR600. For the measurement of expression level, 3,048 bp of lacZ structural gene was fused into xylA promoter in both plasmids pXA600 and pXAR600 as a reporter gene, named pXA600-lacZ and pXAR600-lacZ, respectively. The $\beta$-galactosidase activity of pXA600-lacZ and pXAR600-lacZ in E. coli JM109 was determined to be 1,641 and 2,304 unit by the induction with xylose in LB medium, respectively. The $\beta$-galactosidase activity of pXAR600-lacZ/JM109 was about 1.4 times higher by the induction with xylose than that of pXA600-lacZ/JM109. The $\beta$-galactosidase activity of pXA600-lacZ and pXAR600-lacZ in E.coli JM109 showed 6,282 and 9,320 unit by the induction with xylose in DM minimal medium, respectively. A regulator, xylR protein works as an activator for the gene expression by the addition of xylose in the xylose-inducible vectors because the level of gene expression in pXA600 is increased by the insertion of xylR gene into the same vector. The xynA gene of Streptomyces thermocyaneoviolaceus cloned in pXA600 and pXAR600 was successfully expressed in E. coli BLR(DE3). As a result, plasmids pXA600 and pXAR600 using xylA promoter are sufficient as new expression system to produce a foreign protein in E. coli.