• Title/Summary/Keyword: $Ca^{2+}-induced\

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Electroporation Conditions for DNA Transfer into Somatic Embryogenic Cells of Zoysia japonica (들잔디 체세포 배발생 세포로의 DNA 전입을 위한 Electroporation 조건 구명)

  • 박건환;안병준
    • Korean Journal of Plant Tissue Culture
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    • v.25 no.1
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    • pp.13-19
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    • 1998
  • We have reported previously that intact embryogenic cells can be used instead of protoplasts for electroporation-mediated transformation of zoysiagrass and rice. In this study, conditions of the tissue electroporation were examined to optimize the procedures. Embryogenic cell suspensions were established in liquid MS medium containing 2 mg/L of 2,4-D with embryogenic calluses induced from mature embryos of Z. japonica. The suspension-cultured cell clumps were electroporated with 35S-gusA expression vector DNA, and degrees of DNA introduction into the cells were determined by histological expression rates of the gusA marker gene. DNA transfer into the cell clumps occurred in wide range of voltage (100-400 V) and capacitance (10-1980 $\mu\textrm{F}$), but more in the ranges of 200-300 V and 330-800 $\mu\textrm{F}$ DNA concentrations higher than 6 $\mu\textrm{g}$/mL were adequate for GUS expression of the electroporated cells. DNA transfers were confirmed in all three embryogenic cell lines but only in one out of eleven non-embryogenic lines. Positive GUS expressions occurred with DNAs added even 20-40 h after pulse treatments. As a promoter of gusA, Act1 and Ubi1 were effective 7 and 5 times than 35S respectively in number of GUS expression units on electroporated cell clumps. Embryogenic cell clumps survived and regenerated into plantlets after pulse treatments of wide range of conditions.

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Expression and Characterization of Calcium- and Zinc-Tolerant Xylose Isomerase from Anoxybacillus kamchatkensis G10

  • Park, Yeong-Jun;Jung, Byung Kwon;Hong, Sung-Jun;Park, Gun-Seok;Ibal, Jerald Conrad;Pham, Huy Quang;Shin, Jae-Ho
    • Journal of Microbiology and Biotechnology
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    • v.28 no.4
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    • pp.606-612
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    • 2018
  • The enzyme xylose isomerase (E.C. 5.3.1.5, XI) is responsible for the conversion of an aldose to ketose, especially xylose to xylulose. Owing to the ability of XI to isomerize glucose to fructose, this enzyme is used in the food industry to prepare high-fructose corn syrup. Therefore, we studied the characteristics of XI from Anoxybacillus kamchatkensis G10, a thermophilic bacterium. First, the gene coding for XI (xylA) was inserted into the pET-21a(+) expression vector and the construct was transformed into the Escherichia coli competent cell BL21 (DE3). The expression of recombinant XI was induced in the absence of isopropyl-thio-${\beta}$-galactopyranoside and purified using Ni-NTA affinity chromatography. The optimum temperature of recombinant XI was $80^{\circ}C$ and measurement of the heat stability indicated that 55% of residual activity was maintained after 2 h incubation at $60^{\circ}C$. The optimum pH was found to be 7.5 in sodium phosphate buffer. Magnesium, manganese, and cobalt ions were found to increase the enzyme activity; manganese was the most effective. Additionally, recombinant XI was resistant to the presence of $Ca^{2+}$ and $Zn^{2+}$ ions. The kinetic properties, $K_m$ and $V_{max}$, were calculated as 81.44 mM and $2.237{\mu}mol/min/mg$, respectively. Through redundancy analysis, XI of A. kamchatkensis G10 was classified into a family containing type II XIs produced by the genera Geobacillus, Bacillus, and Thermotoga. These results suggested that the thermostable nature of XI of A. kamchatkensis G10 may be advantageous in industrial applications and food processing.

Intergeneric Transfer of Isolated Nuclei from Lentinus edodes into Protoplasts of Pleurotus florida (사철느타리버섯 원형질체내에 표고 핵의 전이)

  • Yoo, Young-Bok;Shin, Pyung-Gyun
    • The Korean Journal of Mycology
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    • v.24 no.2 s.77
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    • pp.111-126
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    • 1996
  • Transfer of the isolated nuclei from Lentinus edodes into protoplasts of Pleurotus florida was induced with polyethlene glycol (PEG) and $CaCl_2$. The intergeneric transfer products were classified into nuclear hybrid, heterokaryon or synkaryon, and reconstituted cell. These progenies except nuclear hybrids formed mature fruiting bodies on sawdust rice bran medium. Formation of fruit bodies was influenced by several factors such as light, temperature, nutrition and physic state of the culture media. Most of fruiting body characters were similar to those of P. florida in synkaryon and L. edodes in reconstituted cell, respectively. All these basidiocarps had clamp connections though initial heterokaryon colonies were lacking. Isozyme patterns of intergeneric progenies were quite different from those of parents. DNA polymorphisms of transfer products were also compared by random amplified polymorphic DNAs (RAPD) analysis based on polymerase chain reaction. The RAPD patterns were different from those of donor and recipient. DNA fingerprints ranged in size from 0.25 to 4.0 Kb. On the basis of RAPD, the transfer products were classified into five groups. Two synkaryon were analysed with distribution of progenies and segregation of genetic markers by random spore analyses. The genetic markers were segregated into wild type and riboflavine requiring auxotrophs.

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Biological Control of Cotton Caterpillar, Palpita indica Saunder (Lepidoptera: Pyralidae) with Entomopathogenic Nematodes (곤충병원성 선충을 이용한 목화바둑명나방(Palpita indica Saunder)의 생물적방제)

  • 김형환;추호렬;박정규;이상명;추영무
    • Korean journal of applied entomology
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    • v.40 no.3
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    • pp.245-252
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    • 2001
  • Entomopathogenic nematodes were effective in the control of the cotton caterpillar, Palpita indica Saunder. P. indica mortality was significantly different depending on nematode species, treatment concentration, and instar. S. carpocapsae Pocheon strain was more effective against P. indica than S. glaseri Dongrae strain, S. longicaudum Nonsan strain, Heterorhabditis sp. Gyeongsan isolate, and H. bacteriophora Hamyang strain. When S. carpocapsae was treated with the rate of > 20 infective juveniles (ijs)/larva, mortality was ca. 100% at the 1st-4th instars of P. indica in 72h. $LC_{50}$ of entomopathogenic nematodes were significantly different depending on nematode species. The lowest $LC_{ 50}$ value was obtained by S. carpocapsae with 4.9-8.2 ijs in the 1st-prepupa while the highest $LC_{50}$ by Heterorhabditis sp. with 5.5-21.9 ijs, the shortest LT$_{50}$ value of P. indica was induced by S. carpocapsae when 20 ijs/larva were inoculated. The $LT_{50}$ s were 3.4-9.2h against the 1st-prepupa. The mortality of P. indica in fields was significantly different depending on field condition, nematode concentration, and leaf location. Mortality of P. indica by S. carpocapsae was higher at greenhouse,$ 3$\times$10^{9}$ ijs/ha and upper leaves than at field, 1$\times$10$^{9}$ ijs/ha and lower and middle leaves, respectively.

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Citrus platymamma inhibits the expression of pro-inflammatory cytokines, inducible nitric oxide synthase, and cyclooxygenase-2 in RAW264.7 macrophage (RAW264.7 대식세포에서 Citrus platymamma의 iNOS, COX-2, 염증성 사이토카인 발현 억제 효과)

  • Kim, Sang Suk;Park, Kyung Jin;An, Hyun Joo;Choi, Young Hun
    • Food Science and Preservation
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    • v.23 no.7
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    • pp.1026-1032
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    • 2016
  • Citrus platymamma hort. ex Tanaka is widely used in traditional Korean medicine because of its medicinal benefits including an anti-inflammatory effect. This study aimed to evaluate changes in the flavonoid content and anti-inflammatory activities of C. platymamma during its harvest period. Fruit peel samples were obtained between September 2015 and February 2016. The results indicate that C. platymamma peel extract (CPE) was an effective inhibitor of lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells. The inhibitory effects of CPE at $100{\mu}g/mL$ concentration included dose-dependent decreases in the expression of iNOS and COX-2 proteins. In addition, CPE decreased the expression of pro-inflammatory cytokines TNF-${\alpha}$, IL-$1{\beta}$, and IL-6. The highest anti-inflammatory activity and flavonoid content were observed in CPE of C. platymamma peel harvested during the immature fruit period in September. Further, to assess the suitability of CPE for cosmetic use, we performed MTT assays using HaCaT keratinocytes and observed that CPE did not exhibit any cytotoxicity. To test the potential application of CPE as a cosmetic material, we also performed primary skin irritation tests on normal skin of 30 volunteers and no adverse reactions were observed. The results of this study indicate that CPE may be considered as an anti-inflammatory candidate for inclusion in cosmetic materials.

Analysis of Nutritional Components and Cell-based Antioxidant Activity on Brassica juncea Cultivated in Jeongseon, South Korea (정선 갓의 영양성분 및 세포기반 항산화 활성 분석)

  • Kwon, Hee-Yeon;Choi, Sun-Il;Cho, Bong-Yeon;Choi, Seung-Hyun;Sim, Wan-Sup;Han, Xionggao;Jang, Gill-Woong;Choi, Ye-Eun;Yeo, Jin-Hui;Cho, Ju-Hyun;Lee, Ok-Hwan
    • The Korean Journal of Food And Nutrition
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    • v.32 no.5
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    • pp.462-472
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    • 2019
  • The purpose of this study was to demonstrate the quality characteristics of Brassica juncea cultivated in Jeongseon (BJJ), South Korea. We analyzed the nutritional components and antioxidant activity of BJJ. As a result of the free sugar analysis, the contents of glucose and fructose in BJJ were $0.29{\pm}0.02g/100g$ and $0.10{\pm}0.00g/100g$, respectively. The major fatty acids were palmitic acid, octadecenoic acid and stearic acid. The palmitic acid was the highest at 31.22% of all fatty acids. The major minerals were identified as Ca, P, K, Mg and Na. The contents of vitamin $B_1$, vitamin $B_2$, vitamin $B_6$, vitamin C and vitamin E in BJJ were $0.02{\pm}0.00mg/100g$, $0.087{\pm}0.01mg/100g$, $0.02{\pm}0.00mg/100g$, $0.56{\pm}0.06mg/100g$ and $0.20{\pm}0.03mg\;{\alpha}-TE/100g$, respectively. As a result of the free amino acid analysis, total amino acid contents in BJJ were $2,801.21{\pm}115.38mg/100g$. L-proline content was the highest ($744.30{\pm}119.06mg/100g$) in BJJ. BJJ extract inhibits reactive oxygen species production in 3T3-L1 adipocytes. Also, BJJ extract exhibits a protective effect on oxidative stress in $H_2O_2$-induced human dermal fibroblast. These results indicate that BJJ comprises various valuable nutrients which can be used as functional food ingredients.

Anti-inflammatory, Anti-bacterial and Anti-oxidant Activities for Subcritical Extract of Chamaecyparis obtusa (편백 잎 아임계 수 추출물의 항염, 항균 및 항산화 활성)

  • Jung Eun Kim;Min Jeong Kim;Ji Young Moon;Jeong Mi Kim;Tae Heon Oh;Nam Ho Lee
    • Journal of the Korean Applied Science and Technology
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    • v.41 no.3
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    • pp.543-554
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    • 2024
  • In this study, the anti-inflammatory, anti-bacterial and anti-oxidative activities of subcritical water extract from Chamaecyparis obtusa leaves was compared with hot water extract. The yield of subcritical water extract (165℃ and 180℃, 80 bar) was 39.4~48.5%, higher than 31.5% of hot water extract. In the anti-inflammatory tests using lipopolysaccaride (LPS)-induced RAW264.7 macrophages, the subcritical water extract concentration-dependently inhibited production of nitric oxide (NO) without causing cell toxicity. Upon the anti-bacterial studies using Cutibacterium acnes and Staphylococcus epidermidis, subcritical water extract showed the stronger activity than hot water extract. In addition, DPPH and ABTS cation radical scavenging activity experiments showed that the radical scavenging activity of subcritical water extract was similar to that of hot water extract. Moreover, in the study of cell protection effect using HaCaT keratinocytes damaged by hydrogen peroxide (H2O2), the subcritical water extract (165℃, 80 bar) indicated protective effect against oxidative stress. These results suggested that the subcritical water extract of C. obtusa leaves as natural ingredients could be used as anti-inflammatory, anti-bacterial and anti-oxidative ingredients in cosmetic formulations.

Quality Characteristics and Anti-Diabetic Effect of Yacon Vinegar (야콘 식초의 품질특성 및 항당뇨 효과)

  • Lee, Mi-Kyung;Choi, Sa-Ra;Lee, Jin;Choi, Yun-Hong;Lee, Ju-Hye;Park, Kyung-Uk;Kwon, Seung-Hyek;Seo, Kwon-Il
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.41 no.1
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    • pp.79-86
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    • 2012
  • This study was performed to investigate the physiochemical properties and anti-diabetic effect of yacon vinegar by two-step fermentation. Yacon was matured at room temperature for 20 days. The sugar content of yacon juice prepared from mature yacon was approximately $14^{\circ}Brix$. In the first stage, yacon wine was produced from the juice at $28^{\circ}C$ for 6 days. In the second stage, acetic acid fermentation was conducted at $30^{\circ}C$ and 200 rpm for 6 days to produce yacon vinegar with 4.75% acidity. The major free sugars of yacon vinegar were glucose and fructose at 2,072.12 mg% and 463.95 mg%, respectively. The acetic acid content was the highest of the major organic acids at 3,881.44 mg%. The total free amino acid content was 62.88 mg% with the main free amino acids being proline, ${\gamma}$-amino-n-butyric acid and ornithine. The major minerals of yacon vinegar were Ca, K and Mg. The in vivo anti-diabetic activity of yacon vinegar was investigated in high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice. Diabetic mice were administered orally with 10% yacon juice and two yacon vinegars (5% and 10%) at a dose of 7 mL/kg body weight once per day for 4 weeks. Five% yacon vinegar improved the fasting blood glucose levels and glucose tolerance test significantly compared to the diabetic control group (p<0.05). Yacon vinegar increased the pancreatic C-peptide concentration in a dose-dependent manner. These results show that 5% yacon vinegar has a more potent effect on ameliorating hyperglycemia than 10% yacon juice.

Triptolide-induced Transrepression of IL-8 NF-${\kappa}B$ in Lung Epithelial Cells (폐상피세포에서 Triptolide에 의한 NF-${\kappa}B$ 의존성 IL-8 유전자 전사활성 억제기전)

  • Jee, Young-Koo;Kim, Yoon-Seup;Yun, Se-Young;Kim, Yong-Ho;Choi, Eun-Kyoung;Park, Jae-Seuk;Kim, Keu-Youl;Chea, Gi-Nam;Kwak, Sahng-June;Lee, Kye-Young
    • Tuberculosis and Respiratory Diseases
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    • v.50 no.1
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    • pp.52-66
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    • 2001
  • Background : NF-${\kappa}B$ is the most important transcriptional factor in IL-8 gene expression. Triptolide is a new compound that recently has been shown to inhibit NF-${\kappa}B$ activation. The purpose of this study is to investigate how triptolide inhibits NF-${\kappa}B$-dependent IL-8 gene transcription in lung epithelial cells and to pilot the potential for the clinical application of triptolide in inflammatory lung diseases. Methods : A549 cells were used and triptolide was provided from Pharmagenesis Company (Palo Alto, CA). In order to examine NF-${\kappa}B$-dependent IL-8 transcriptional activity, we established stable A549 IL-8-NF-${\kappa}B$-luc. cells and performed luciferase assays. IL-8 gene expression was measured by RT-PCR and ELISA. A Western blot was done for the study of $I{\kappa}B{\alpha}$ degradation and an electromobility shift assay was done to analyze NF-${\kappa}B$ DNA binding. p65 specific transactivation was analyzed by a cotransfection study using a Gal4-p65 fusion protein expression system. To investigate the involvement of transcriptional coactivators, we perfomed a transfection study with CBP and SRC-1 expression vectors. Results : We observed that triptolide significantly suppresses NF-${\kappa}B$-dependent IL-8 transcriptional activity induced by IL-$1{\beta}$ and PMA. RT-PCR showed that triptolide represses both IL-$1{\beta}$ and PMA-induced IL-8 mRNA expression and ELISA confirmed this triptolide-mediated IL-8 suppression at the protein level. However, triptolide did not affect $I{\kappa}B{\alpha}$ degradation and NF-$_{\kappa}B$ DNA binding. In a p65-specific transactivation study, triptolide significantly suppressed Gal4-p65T Al and Gal4-p65T A2 activity suggesting that triptolide inhibits NF-${\kappa}B$ activation by inhibiting p65 transactivation. However, this triptolide-mediated inhibition of p65 transactivation was not rescued by the overexpression of CBP or SRC-1, thereby excluding the role of transcriptional coactivators. Conclusions : Triptolide is a new compound that inhibits NF-${\kappa}B$-dependent IL-8 transcriptional activation by inhibiting p65 transactivation, but not by an $I{\kappa}B{\alpha}$-dependent mechanism. This suggests that triptolide may have a therapeutic potential for inflammatory lung diseases.

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Physicochemical Changes in Tilapia Oreochromis niloticus Muscle Induced by Acclimation to Sea Water (해수순화에 따른 틸라피아 근육의 물리화학적 변화)

  • Hwang, Gyu-Chul;Yoon, Ho-Dong;Ji, Cheong-Il;Park, Jeong-Heum;Kim, Seong-Jun
    • Korean Journal of Food Science and Technology
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    • v.25 no.2
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    • pp.185-190
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    • 1993
  • Tilapia cultured in fresh water were acclimated in sea water with daily increase of $5%_{\circ}$ of salinity and it was completely terminated at the 7th day (0 week). Each three tilapia acclimated were examined weekly based from 0 week to elucidate changes of chloride cells in gill, mineral contents and physical properties in muscle and biochemical characteristics in myofibrils. Chloride cells existed in gills were gradually developed in number and size by acclimation to sea water and became to almost constant state at the third week. Shearing value, compressing strength and content of minerals such as Mg, Na and K in muscle were showed remarkable increase by acclimation to sea water in comparison to those of muscle from tilapia reared in fresh water. Myofibrillar $Mg^{2+}-,\;Ca^{2+}-$ and $K^+(EDTA)-ATPase$ activities of tilapia acclimated in sea water also increased showing significant statistic difference (p<0.01) from those of tilapia reared in fresh water However. thermostability of myofibrils was dropped by acclimation to sea water. The increase of shearing value and compressing strength in the muscle of tilapia by acclimation to sea water would be attributed to the increase of myofibrillar ATPase activities which act to accelerate the decomposition rate of ATP. Therefore, it is suggested that this phenomenon associated with muscle contraction could be brought an improvement of texture of tilapia acclimated in sea water.

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