• Journal of Microbiology and Biotechnology
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• 제33권9호
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• pp.1228-1237
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• 2023

The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/㎍ RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.

• 대한약침학회지
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• 제26권4호
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• pp.319-326
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• 2023

Objectives: The study's goal was to find out whether Chrysanthemum rubellum extract has anti-diabetic properties by concentrating on α-glucosidase and the PTP-1B signaling pathway. C. rubellum flowers were used for extraction using Methanol/water (80/20) as solvent. Methods: LC-MS techniques was used to check the presence of phytoconstituents present in C. rubellum extract. In vitro antidiabetic activity was evaluated using α-glucosidase inhibitory activity and PTP-1B signaling pathway. On Streptozotocin (STZ)-induced rats with diabetes, the in vivo antidiabetic efficacy was assessed using a test for oral glucose tolerance. Results: The phytoconstituents identified in the extract of C. rubellum were apigenin, diosmin, myricetin, luteolin, luteolin-7-glucoside, and Quercitrin as compound 1-6, respectively. Results showed that diosmin exhibited highest α-glucosidase inhibitory activity i.e. 90.39%. The protein level of PTP-1B was lowered and the insulin signalling activity was directly increased by compounds 1-6. The maximum blood glucose levels were seen in all groups' OGTT findings at 30 minutes following glucose delivery, followed by gradual drops. In comparison to the control group, the extract's glucose levels were 141 mg/dL at 30 minutes before falling to 104 mg/dL after 120 minutes. The current study has demonstrated, in summary, that extract with phytoconstituents reduce blood sugar levels in rats. Conclusion: This finding suggests that extract may reduce the chance of insulin resistance and shield against disorders like hyperglycemia.

• 접착 및 계면
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• 제24권4호
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• pp.113-119
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• 2023

홍합의 수중 접착능력은 도파(DOPA)와 라이신(Lysine), 히스티딘(Histidine)과 같은 홍합접착단백질의 아미노산 잔기가 중요한 역할을 한다고 보고되었고, 이에 따라, 카테콜과 아민기를 동시에 갖는 접착성 카테콜아민(Catecholamine) 물질을 기반으로 다양한 의공학적 연구가 진행되고 있다. 카테콜기가 도입된 키토산은 아민이 풍부한 키토산에 카테콜기를 도입한 카테콜아민으로, 이를 이용하여 조직접착제나 창상치유제, 지혈제, 약물전달체 및 조직공학용 담체 등 다양한 의공학적 적용이 가능하다. 특히, 키토산-카테콜 물질은 지혈제로 미국 및 한국 식품의약품안전처의 승인을 받아, 연구개발에서부터 제품개발까지 이루어진 홍합 모사 물질이다. 이에 본 총설에서는 지혈제로써의 키토산-카테콜 물질에 대한 연구 동향을 살펴보고자 한다. 이를 위해, 카테콜기가 도입됨에 따라 나타나는 키토산-카테콜의 특성, 지혈 메커니즘, 다양한 제형에 대하여 다루고자 한다.

• Animal Bioscience
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• 제37권7호
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• pp.1196-1203
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• 2024

Objective: A study was conducted to quantify the performance differences of the near-infrared spectroscopy (NIRS) calibration models developed with different degrees of hay sample preparations. Methods: A total of 227 imported alfalfa (Medicago sativa L.) and another 360 imported timothy (Phleum pratense L.) hay samples were used to develop calibration models for nutrient value parameters such as moisture, neutral detergent fiber, acid detergent fiber, crude protein, and in vitro dry matter digestibility. Spectral data of hay samples prepared by milling into 1-mm particle size or unground were separately regressed against the wet chemistry results of the abovementioned parameters. Results: The performance of the developed NIRS calibration models was evaluated based on R², standard error, and ratio percentage deviation (RPD). The models developed with ground hay were more robust and accurate than those with unground hay based on calibration model performance indexes such as R² (coefficient of determination), standard error, and RPD. Although the R² of calibration models was mainly greater than 0.90 across the feed value indexes, the R² of cross-validations was much lower. The R² of cross-validation varies depending on feed value indexes, which ranged from 0.61 to 0.81 in alfalfa, and from 0.62 to 0.95 in timothy. Estimation of feed values in imported hay can be achievable by the calibrated NIRS. However, the NIRS calibration models must be improved by including a broader range of imported hay samples in the modeling. Conclusion: Although the analysis accuracy of NIRS was substantially higher when calibration models were developed with ground samples, less sample preparation will be more advantageous for achieving rapid delivery of hay sample analysis results. Therefore, further research warrants investigating the level of sample preparations compromising analysis accuracy by NIRS.

• 한국식생활문화학회지
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• 제35권4호
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• pp.333-341
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• 2020

This study evaluated the perception, consumption behavior, and optional attributes of Korean, Chinese, and U.S. consumers of energy bar products. Data were compared and analyzed by surveying 300 consumers in each country. Significant differences were observed in preference for energy bars according to their nationality, in the order China, the U.S., and Korea. Perception of taste, types and dietary suitability of the products ranked lower for Korean consumers, as compared to consumers of the United States and China. The order dietary fiber, protein, and calcium were the sought-after nutritional requirements of the products. The demand for protein was significantly higher in the U.S. Calcium demand was low in the United States and China, but was very high in Korea, which could be attributed to the low calcium intake of Koreans. Other optional attributes which were closely associated with the purchase and re-purchase decision, included price, taste and delivery period. All three factors were recognized as important options in Korea, whereas awareness of packaging/appearance and brand was not. The taste, nutrients and price in the U.S. ranked high as important optional attributes, while the packaging, external and expiration dates were recognized as low. Unlike Korea and the U.S., important optional attributes for Chinese consumers were determined in the order expiration date, taste, and nutrients, and showed low perception for packaging, appearance, weight, counts, and prices. Evaluating the preference for the main and secondary ingredients, Koreans preferred nuts over grains, Americans preferred dried fruits over nuts, and Chinese preferred nuts and grains; both Korean and American consumers had low preference for dried vegetables. The preference for chocolate was low in Korea and China, whereas preference for jelly was high in China as compared to Korea and the U.S. The intention of purchasing energy bars was significantly lower in Korea than in the U.S. and China. A variety of nutritious functional bars have recently been distributed and sold in Korea, but they are mostly produced in the U.S., which is the largest producer and consumer worldwide. Taken together, results of this study indicate that the demand for nutritional enhancement and preferred materials vary according to the nationality. Hence, it is necessary to develop products that reflect these criteria. Further research is required to analyze the relationship between preference and consumption behavior for each material product developed in the future.

• Journal of Periodontal and Implant Science
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• 제37권3호
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• pp.511-522
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• 2007

Introduction : The purpose of this study was to evaluate the possibility of the acellular dermal matrix (ADM) as a barrier membrane for bone regeneration, and to evaluate the osteogenic effect of ADM as a carrier system for rhBMP-2 in the rat calvarial defect model. Materials and Methods: An 8-mm, calvarial, critical-size osteotomy defect was created in each of 60 male Spraque-Dawley rats(weight $250{\sim}300g$). Three groups of 20 animals, each received either rhBMP-2(0.025mg/ml) in an ADM carrier, ADM only, or negative surgical control. And each group was divided into 2- and 8-weeks healing intervals. The groups were evaluated by histologic and histomorphometric parameters(10 animals/group/healing intervals). Data were expressed as $means{\pm}standard$ deviations($m{\pm}SD$). Comparisons between experimental and control groups were made using two-way ANOVA and post hoc t-test. Comparisons between 2 weeks and 8 weeks were made using paired t-test. The level of statistical difference was defined as P< 0.05. Results : The ADM group and rhBMP-2/ADM group results in enhanced local bone formation in the rat calvarial defect at both 2 and 8 weeks. The amount of defect closure and new bone formation were significantly greater in the rhBMP-2/ADM group relative to ADM group(P<0.05). At 8 weeks, the majority of ADM in the defect was contracted, and integrated with surrounding host tissues. In addition, host cell infiltration and neovascularization of the ADM in the absence of an inflammatory response were observed, and the newly formed bone around ADM showed a continuous remodeling and consolidation. Conclusion : The results of the present study indicated that ADM may be used as a barrier membrane for bone regeneration and that may be employed as a delivery system for BMPs.

• 한국미생물·생명공학회지
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• 제39권4호
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• pp.337-344
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• 2011

이전 연구에서, bacteriophage ${\Phi}FC1$이 Enterococcus faecalis KBL703에서 UV induction을 통해 분리 동정되었으며, ${\Phi}FC1$은 phage attachment site인 attP와 bacterial attachment site인 attB 사이에서 site-specific integration을 촉매하는 integrase를 가지고 있다는 것을 밝혀냈으며 이를 MJ1이라 명명하였다. 이 연구에서는 이를 바탕으로 MJ1에 의한 site-specific integration의 효율을 Escherichia coli와 NIH3T3 cell에서 확인 하기 위해 attP, attB, MJ1을 각각의 벡터에 삽입하였다. MJ1 인테그라제에 의한 재조합을 수행하기 위해서 기질 벡터 pABLP를 $DH5{\alpha}$에 형질전환시킨 후, LB 배지에서 $37^{\circ}C$ 1시간 배양한 후 암피실린(ampicillin)과 테트라싸이클린(tetracycline) 항생제 플레이트로 pGMJ1과 pABLP 같이 가지고 있는 colony 들을 선별하여, LacZ 유전자가 불활성화 된 흰색 콜로니 개수를 세고 통계를 낸 결과 integration의 frequency가 99% 이상인 것으로 나타났다. 또한, 실제로 재조합이 일어났는 지를 확인하기 위해서 콜로니 PCR을 수행하여 재조합의 산물인 attL 150 bp을 확인하였다. PCR 산물은 염기서열분석을 통해 정확한 site-specific integration이 일어났음을 확인하였다. MJ1에 의한 integration을 보이기 위해 attP와 attB를 가지고 있는 vector를 MJ1 expression vector와 함께 NIH3T3 cell에 cotransfection 했으며 GFP를 reporter로 사용해 그 activity를 관찰하였다. NIH3T3 cell에서 GFP의 발현을 형광 현미경을 통해 알아본 결과, MJ1에 의한 sitespecific integration이 다른 accessory protein의 도움 없이 일어난다는 것을 볼 수 있었다. 마찬가지 방법으로, attR과 attL 간의 excision을 GFP로 알아본 결과, GFP는 발현하지 않았으며, 이는 MJ1에 의한 excision이 일어나지 않았음을 보여주었다. 이와 같은 결과로 볼 때, MJ1의 host만이 아니라 넓은 범위안에서도 integration을 수행할 수 있다는 것을 보여주었다. 따라서 MJ1을 이용한 site-specific integration system의 개발은 gene therapy를 위한 gene delivery system의 구축에 있어서 좋은 시작이 될 수 있다.

• Journal of Nutrition and Health
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• 제36권5호
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• pp.459-469
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• 2003

We examined the effects of calcium intake levels on mineral metabolism during pregnancy using calcium-deficient young adult rats. Five week-old female Sprague-Dawley rats were fed normal Ca (NCa, 0.5%) and low Ca (LCa, 0.15%) diets for five weeks (pre-pregnancy). The low Ca intake group was then divided into three groups and fed low Ca (0.15%), normal Ca (0.5%) and high Ca (1.5%) diets for 3 weeks (pregnancy). All of the rats were mated with normal male rats. The control group was fed a consistently normal Ca (0.5%) diet during pre-pregnancy and pregnancy. On the day after delivery, dams and their pups were sacrificed. We measured total protein, albumin, alkaline phosphatase and mineral content in serum and weight, length, breaking force, ash and mineral content in the femur and lumbar (L2-L4) of the dams. Whole body mineral content was measured in the pups. There was no difference in weight gain and food intake among the groups. Serum total protein and albumin were in the normal range but a little lower during pregnancy. High Ca supplementation decreased serum Mg and Fe during pregnancy. Weight, ash and Ca of the femur and lumbar significantly decreased in rats fed a chronically low Ca diet during pre-pregnancy and pregnancy. Calcium supplementation levels were above normal during pregnancy and increased the bone weight and breaking force of rats fed the low Ca diet during pre-pregnancy. However, Ca supplementation did not increase the ash and Ca contents in the bones. High Ca supplementation during pregnancy significantly decreased Mg in the bones and increased Ca and P in the kidneys. Ash content of pups from dams fed the chronically low Ca diet decreased but there was no difference in whole body Ca among the groups. Mg and Fe in the whole body decreased in pups from dams fed the high Ca diet. Pregnancy performance was reduced in dams fed the low Ca diet. These results suggest that above normal Ca supplementation levels during pregnancy restored maternal bone status to some extent in rats fed the chronically low Ca diet. The same could not be said for mineral content. Also, high Ca supplementation during pregnancy may deteriorate mineral composition in bones and other tissues. Therefore, more detailed research is needed to facilitate sound recommendations on appropriate calcium intake during pregnancy. (Korean J Nutrition 36(5): 459∼469, 2003)

• 생명과학회지
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• 제18권12호
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• pp.1637-1643
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• 2008

HSP90에 노출된 혈관평활근세포에서 IL-6 transcript가 증가하고, IL-6 단백질의 분비가 증가하며, 또한 IL-6 유전자의 promote가 활성화되었다. HSP90에 의한 IL-6 유전자의 promoter 활성화는 dominant negative 형태의 TLR-4와 MyD88에 의하여 크게 감소되었지만, dominant negative 형태의 TLR-3와 TRIF의 영향을 받지 않았다. 그리고 TLR-4의 이합체화(dimerization)를 저해하는 curcumin은 HSP90에 의한 IL-6의 분비 및 IL-6 유전자 promoter 활성화를 억제하였다. 그리고 IL-6 유전자의 promoter의 NF-${\kappa}B$- 또는 C/EBP-binding sequence에 변이는 HSP90에 의한 IL-6 유전자의 promoter 활성화 억제하였다. 이러한 결과는 혈관평활근세포에서 HSP90에 의한 IL-6 유전자 활성화에 TLR-4와 NF-${\kappa}B$B가 관여함을 의미한다.

• Molecules and Cells
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• 제20권3호
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• pp.401-408
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• 2005

Reactive oxygen species (ROS) contribute to the development of various human diseases. Cu,Zn-superoxide dismutase (SOD) is one of the major means by which cells counteract the deleterious effects of ROS. SOD activity is dependent upon bound copper ions supplied by its partner metallochaperone protein, copper chaperone for SOD (CCS). In the present study, we investigated the protective effects of PEP-1-CCS against neuronal cell death and ischemic insults. When PEP-1-CCS was added to the culture medium of neuronal cells, it rapidly entered the cells and protected them against paraquat-induced cell death. Moreover, transduced PEP-1-CCS markedly increased endogenous SOD activity in the cells. Immunohistochemical analysis revealed that it prevented neuronal cell death in the hippocampus in response to transient forebrain ischemia. These results suggest that CCS is essential to activate SOD, and that transduction of PEP-1-CCS provides a potential strategy for therapeutic delivery in various human diseases including stroke related to SOD or ROS.