• 제목/요약/키워드: Limiting amino acid

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Isolation and Characterization of ACC Synthase Gene Family in Mung Bean (Vigna radiata L.): Differential Expression of the Three ACC Synthase enes in Response to Auxin and Brassinosteroid

  • Sunjoo Joo;Kim, Woo-Taek
    • Journal of Plant Biotechnology
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    • 제2권2호
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    • pp.61-71
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    • 2000
  • By screening a cDNA library of auxin-treated mung bean (Vigna radiata L.) hypocotyls, we have isolated two full-length cDNA clones, pVR-ACS6 and pVR-ACS7, for 1-aminocyclopropane-1-carboxylate (ACC) synthase, the rate-limiting enzyme in the ethylene biosynthetic pathway. While PVR-ACS6 corresponds to the previously identified PCR fragment pMBA1, pVR-ACS7 is a new cDNA clone. A comparison of deduced amino acid sequences among auxin-induced ACC synthases reveal that these enzymes share a high degree of homology (65-75%) to VR-ACS6 and VR-ACS7 polypeptides, but only about 50% to VR-ACS1 polypeptide. ACS6 and ACS7 are specifically induced by auxin, while ACS1 is induced by cycloheximide, and to lesser extent by excision and auxin treatment. Results from nuclear run-on transcription assay and RNA gel blot studies revealed that all three genes were transcriptionally active displaying unique patterns of induction by IAA and various hormones in etiolated hypocotyls. Particularly, 24-epibrassinolide (BR), an active brassinosteroid, specifically enhanced the expression of VR-ACS7 by distinct temporal induction mechanism compared to that of IAA. In addition, BR synergistically increased the IAA-induced VR-ACS6 and VR-ACS7 transcript levels, while it effectively abolished both the IAA- and kinetin-induced accumulation of VR-ACS1 mRNA. In light-grown plants, VR-ACS1 was induced by IAA in roots, whereas W-ACS6 in epicotyls. IAA- and BR-treatments were not able to increase the VR-ACS7 transcript in the light-grown tissues. These results indicate that the expression of ACC synthase multigene family is regulated by complex hormonal and developmental networks in a gene- and tissue-specific manner in mung bean plants. The VR-ACS7 gene was isolated, and chimeric fusion between the 2.4 kb 5'-upstream region and the $\beta$-glucuronidase (GUS) reporter gene was constructed and introduced into Nicotiana tobacum. Analysis of transgenic tobacco plants revealed the VR-ACS7 promoter-driven GUS activity at a highly localized region of the hypocotyl-root junction of control seedlings, while a marked induction of GUS activity was detected only in the hypocotyl region of the IAA-treated transgenic seedlings where rapid cell elongation occurs. Although there was a modest synergistic effect of BR on the IAA-induced GUS activity, BR alone failed to increase the GUS activity, suggesting that induction of VR-ACS7 occurs via separate signaling pathways in response to IAA and BR.

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Kocuria gwangalliensis 유래 phytoene desaturase 유전자의 cloning과 특성 연구 (Molecular Cloning and Characterization of the Gene Encoding Phytoene Desaturase from Kocuria gwangalliensis)

  • 서용배;최성석;남수완;김군도
    • 한국미생물·생명공학회지
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    • 제45권3호
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    • pp.226-235
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    • 2017
  • Phytoene, lycopene, ${\beta}-carotene$과 같은 카로티노이드는 식품의 착색제나 영양보조제, 사료첨가제, 화장품의 원료로 사용된다. 이전 연구에서 본 연구진은 분홍색의 색소를 생산하는 새로운 해양 세균인 K. gwangalliensis를 분리 동정하였다. Phytoene desaturase (CrtI) 효소는 crtI 유전자에 암호화되어 있으며, phytoene을 lycopene으로 전환하며, 카로티노이드 합성 초기 단계에 있어서 필수적이다. CrtI는 카로티노이드 생합성 조절의 주요 효소 중 하나이며, 다양한 카로티노이드를 생합성하는 생물들의 카로티노이드 생합성 경로에 있어서 속도 조절 단계에 관련이 있다. 본 논문에서는 K. gwangalliensis로부터 lycopene 생합성을 담당하는 crtI 유전자를 클로닝 하였으며, 이 유전자는 1,584개의 염기서열을 가지며, 527개의 아미노산을 암호화하고 있다. crtI 유전자의 염기 서열을 Kocuria rhizophila와 Myxococcus xanthus를 포함한 다른 종의 염기 서열과 비교한 결과, 진화 과정에서 잘 보존되어 있음을 확인하였다. crtI 유전자를 포함하는 발현 플라스미드를 구축하여 발현시킨 결과, 이 플라스미드를 함유하는 대장균은 약 57 kDa의 재조합 단백질을 생산화였으며, 이는 phytoene desaturase의 분자량에 해당한다. lycopene의 생합성은 lycopene 생합성에 필요한 crtE, crtB 유전자를 포함한 pRScrtEB plasmid를 E. coli에 형질전환 했을 때, Escherichia coli에서 합성되는 것을 확인하였다. 이 연구의 결과는 분자 수준에서 K. gwangalliensis CrtI의 1차 구조에 대한 폭 넓은 지식 기반을 제공할 것이다.

유청단백질 및 Lactobacillus spp. 추출물이 전립선 세포 내 항산화 활성에 미치는 영향 (Effect of Whey Protein Isolate and Lactobacillus spp. Cell Extracts on Intracellular Antioxidative Activities in Human Prostate Epitherial Cells)

  • 변정열;윤영호
    • Journal of Animal Science and Technology
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    • 제48권5호
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    • pp.719-726
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    • 2006
  • 본 실험은 가수분해 시킨 유청단백질과 Lb. casei HY2782의 세포 추출물을 인간 전립선 세포에처리하여 세포 내 glutathione 농도 상승효과와 산화제 tbutyl hydroperoxide (TBHP)에 의한핵산 손상 방지 효과를 알아보기 위한 실험으로 전립선 세포 RWPE1 cells와 PC3MMM2 cells에 hydrolyzed WPI(500g/m)를 48시간 동안 처리 시, 가수분해 시키지 않은 WPI 보다 각각 28.2%, 38.4%씩 GSH 농도가 증가하는 것을 유의적으로 확인할 수 있었고(p<0.05), 카제인의 경우 RWPE1 cells and PC3MMM2 cells에 처리 시 가수분해 시킨 카제인과의 유의차가 보이지 않았으며 (p<0.05), glutathione 합성저해제인 Buthionine sulfoximin (BSO) 처리 시 Glutathione 농도가 현저하게 낮아지는 것을 확인할 수가 있었다(Anderson, 1998). 전립선 세포 RWPE1 cells 와 PC3MMM2 cells에 Lb. casei HY2782 세포 추출물 처리 시 PC3MMM2 cell에서는 GSH의 농도가 유의적으로 증가하는 것을 확인 할 수 있었고(p<0.05), RWPE1 cell에서는 증가 성향을 확인 할 수가 있었다.가수분해 시킨 유청단백질과 Lb. casei cell 세포추출물 처리 시 산화제에 의한 세포의 DNA 손상 억제정도와 사멸율의 변화를 확인 한 결과, 가수분해 WPI를 처리 후 Oxidant tbutyl hydroperoxide (TBHP)(500mM)를 이용하여 PC3MMM2 세포에 산화적인 스트레스를 주었을 시, TBHP만 처리한 구에서는 62.0%의 생존율을 나타내었고, glutathione 합성저해제인 BSO를 첨가시킨 처리구에서는 33.7%의 생존율을 나타내었다. 가수분해 시킨 WPI와 카제인 처리구에서는 각각 86.7%, 63.6%의 생존율을 나타내었고, WPI와 BSO를 함께 처리한 구에서는 71.5%의 생존율을 나타내었다. 이는 BSO에 의해 GSH의 생성이 저해되어 나타난 결과로 보여지며(Anderson, 1988), 가수분해시킨 WPI 가 가수분해 시킨 카제인보다는 높은 생존율을 나타내는 것을 유의적으로 확인할 수가 있었다. (p<0.05) Lb. casei 세포 추출물처리에 의한 생존율에서는 가수분해시킨 WPI와 유사한 수준의 82.4%의 생존율을 보였다. 가수분해시킨 유청단백질과 Lb. casei cell 세포추출물에 의한 세포에서의 glutathione 농도 증가에 의해 산화제에 의한 DNA의 손상이 적었던 것으로 사료되어지고, 이로 인해 생존율이 높았던 것으로 판단된다.

Analyses of Inter-cultivar Variation for Salinity Tolerance in Six Korean Rapeseed Cultivars

  • Lee, Yong-Hwa;Lee, Tae-Sung;Kim, Kwang-Soo;Jang, Young-Seok;Nam, Sang-Sik;Park, Kwang-Geun
    • 원예과학기술지
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    • 제30권4호
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    • pp.417-425
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    • 2012
  • Salinity stress is one of the most serious factors limiting the productivity of agricultural crops. The aim of this study was to assess inter-cultivar (intraspecific) variation for salinity tolerance in six Korean rapeseed (Brassica napus L.) cultivars at the seedling stage. The effect of three different salinity stress levels (EC 4, 8, and 16 $dS{\cdot}m^{-1}$) on seedlings of six cultivars was investigated through leaf size, leaf dry weight, and leaf chlorosis. At the highest salinity level (16 $dS{\cdot}m^{-1}$), the mean decrease of leaf dry weight in 'Sunmang', 'Tammi', 'Tamla', 'Naehan', 'Youngsan', and 'Halla' was about 56.2, 56.9, 78.4, 79.3, 77.4, and 80.9%, respectively. 'Tammi' and 'Sunmang' showed much less reduction in leaf dry weight than all the other cultivars. In addition, diluted seawater treatments increased the occurrence of leaf chlorosis in six cultivars. At EC 8 and 16 $dS{\cdot}m^{-1}$, 'Naehan', 'Youngsan', and 'Halla' showed a higher level of leaf chlorosis than 'Tammi' 'Sunmang', and 'Tamla'. On the basis of these results, six cultivars were placed into salinity-tolerant and sensitive groups. 'Tammi' and 'Sunmang' were the salinity-tolerant cultivars, while 'Naehan', 'Halla', 'Youngsan', and 'Tamla' were the salinity-sensitive cultivars. 'Tammi' and 'Naehan' rated as the most tolerant and most sensitive cultivar, respectively. To further analyze protein expression profiles in 'Tammi' and 'Naehan', 2-D proteomic analysis was performed using the plants grown under diluted seawater treatments. We identified eight differentially displayed proteins that participate in photosynthesis, carbon assimilation, starch and sucrose metabolism, amino acid metabolism, cold and oxidative stress, and calcium signaling. The differential protein expressions in 'Tammi' and 'Naehan' are likely to correlate with the differential growth responses of both cultivars to salinity stress. These data suggest that 'Tammi' is better adapted to salinity stressed environments than 'Naehan'.

A Novel Pyruvate Kinase M2 Activator Compound that Suppresses Lung Cancer Cell Viability under Hypoxia

  • Kim, Dong Joon;Park, Young Soo;Kim, Nam Doo;Min, Sang Hyun;You, Yeon-Mi;Jung, Yuri;Koo, Han;Noh, Hanmi;Kim, Jung-Ae;Park, Kyung Chan;Yeom, Young Il
    • Molecules and Cells
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    • 제38권4호
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    • pp.373-379
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    • 2015
  • Pyruvate kinase M2 isoform (PKM2), a rate-limiting enzyme in the final step of glycolysis, is known to be associated with the metabolic rewiring of cancer cells, and considered an important cancer therapeutic target. Herein, we report a novel PKM2 activator, PA-12, which was identified via the molecular docking-based virtual screening. We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of $4.92{\mu}M$, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium. In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia. We also verified that the effects of PA-12 were dependent on PKM2 expression in cancer cells, demonstrating the specificity of PA-12 for PKM2 protein. Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.

해조의 식용분말화에 관한 연구 (A Study on the Nutritive Value and Utilization of Powdered Seaweeds)

  • 유정열;이기열;김숙희
    • Journal of Nutrition and Health
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    • 제8권1호
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    • pp.15-37
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    • 1975
  • I. Subject of the study A study on the nutritive value and utilization of powdered seaweeds. II. Purpose and Importance of the study A. In Korea the shortage of food will be inevitable by the rapidly growing population. It will be very important study to develop a new food from the seaweeds which were not used hitherto for human consumption. B. The several kinds of seaweeds have been used by man in Korea mainly as side-dishes. However, a properly powdered seaweed will enable itself to be a good supplement or mixture to certain cereal flours. C. By adding the powdered seaweed to any cereals which have long been staple foods in this country the two fold benefits; saving of cereals and change of dietary pattern, will be secured. III. Objects and scope of the study A. Objects of the study The objects will come under four items. 1. To develop a powdered seaweed as a new food from the seaweeds which have been not used for human consumption. 2. To evaluate the nutritional quality of the products the analysis for chemical composition and animal feeding experiment will be conducted. 3. Experimental cocking and accepability test will be conducted for the powdered products to evaluate the value as food stuff. 4. Sanitary test and also economical analysis will be conducted for the powdered products. B. Scope of the study 1. Production of seaweed powders Sargassum fulvellum growing in eastern coast and Sargassum patens C.A. in southern coast were used as the material for the powders. These algae, which have been not used for human consumption, were pulverized through the processes of washing, drying, pulverization, etc. 2. Nutritional experiments a. Chemical composition Proximate components (water, protein, fat, cellulose, sugar, ash, salt), minerals (calcium, phosphorus, iron, iodine), vitamins (A, $B_1,\;B_2$ niacin, C) and amino acids were analyzed for the seaweed powders. b. Animal feeding experiment Weaning 160 rats (80 male and 80 female rats) were used as experimental animals, dividing them into 16 groups, 10 rats each group. Each group was fed for 12 weeks on cereal diet (Wheat flour, rice powder, barley powder, potato powder, corn flour) with the supplementary levels of 5%, 10%, 15%, 20% and 30% of the seaweed powder. After the feeding the growth, feed efficiency ratio, protain efficiency ratio and ,organs weights were checked and urine analysis, feces analysis and serum analysis were also conducted. 3. Experimental cooking and acceptability test a. Several basic studies were conducted to find the characteristics of the seaweed powder. b. 17 kinds of Korean dishes and 9 kinds of foreign dishes were prepared with cereal flours (wheat, rice, barley, potato, corn) with the supplementary levels of 5%, 10%, 15%, 20% and 30% of the seaweed powder. c. Acceptability test for the dishes was conducted according to plank's Form. 4. Sanitary test The heavy metals (Cd, Pb, As, Hg) in the seaweed powders were determined. 5. Economical analysis The retail price of the seaweed powder was compared with those of other cereals in the market. And also economical analysis was made from the nutritional point of view, calculating the body weight gained in grams per unit price of each feeding diet. IV. Results of the study and the suggestion for application A. Chemical composition 1. There is no any big difference in proximate components between powders of Sargassum fulvellum in eastern coast and Sargassum patens C.A. in southern coast. Seasonal difference is also not significant. Higher levels of protein, cellulose, ash and salt were found in the powders compared with common cereal foods. 2. The levels of calcium (Ca) and iron (Fe) in the powders were significantly higher than common cereal foods and also rich in iodine (I). Existence of vitamin A and vitamin C in the Powders is different point from cereal foods. Vitamin $B_1\;and\;B_2$ are also relatively rich in the powders.'Vitamin A in ·Sargassum fulvellum is high and the levels of some minerals and vitamins are seemed4 to be some influenced by seasons. 3. In the amino acid composition methionine, isoleucine, Iysine and valine are limiting amino acids. The protein qualities of Sargassum fulvellum and Sargassum patens C.A. are seemed to be .almost same and generally ·good. Seasonal difference in amino acid composition was found. B. Animal feeding experiment 1. The best growth was found at.10% supplemental level of the seaweed Powder and lower growth rate was shown at 30% level. 2. It was shown that 15% supplemental level of the Seaweed powder seems to fulfil, to some extent the mineral requirement of the animals. 3. No any changes were found in organs development except that, in kidney, there found decreasing in weight by increasing the supplemental level of the seaweed powder. 4. There is no any significant changes in nitrogen retention, serum cholesterol, serum calcium and urinary calcium in each supplemental level of the seaweed powder. 5. In animal feeding experiment it was concluded that $5%{\sim}15%$ levels supplementation of the seaweed powder are possible. C. Experimental cooking and acceptability test 1. The seaweed powder showed to be utilized more excellently in foreign cookings than in Korean cookings. Higher supplemental level of seaweed was passible in foreign cookings. 2. Hae-Jo-Kang and Jeon-Byung were more excellent than Song-Pyun, wheat cake, Soo-Je-Bee and wheat noodle. Hae-Je-Kang was excellent in its quality even as high as 5% supplemental level. 3. The higher levels of supplementation were used the more sticky cooking products were obtained. Song-Pyun and wheat cake were palatable and lustrous in 2% supplementation level. 4. In drop cookie the higher levels of supplementation, the more crisp product was obtained, compared with other cookies. 5. Corn cake, thin rice gruel, rice gruel and potato Jeon-Byung were more excellent in their quality than potato Man-Doo and potato noodle. Corn cake, thin rice gruel and rice gruel were excellent even as high as 5% supplementation level. 6. In several cooking Porducts some seaweed-oder was perceived in case of 3% or more levels of supplementation. This may be much diminished by the use of proper condiments. D. Sanitary test It seems that there is no any heavy metals (Cd, Pb, As, Hg) problem in these seaweed Powders in case these Powders are used as supplements to any cereal flours E. Economical analysis The price of the seaweed powder is lower than those of other cereals and that may be more lowered when mass production of the seaweed powder is made in future. The supplement of the seaweed powder to any cereals is also economical with the criterion of animal growth rate. F. It is recommended that these seaweed powders should be developed and used as supplement to any cereal flours or used as other food material. By doing so, both saving of cereals and improvement of individual's nutrition will greatly be achieved. It is also recommended that the feeding experiment for men would be conducted in future.

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