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Cloning, Over-expression, and Characterization of YjgA, a Novel ppGpp-binding Protein

  • Gnanasekaran, Gopalsamy;Pan, SangO;Jung, Wontae;Jeong, Kwangjoon;Jeong, Jae-Ho;Rhee, Joon Haeng;Choy, Hyon E.;Jung, Che-Hun
    • Bulletin of the Korean Chemical Society
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    • v.34 no.8
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    • pp.2419-2424
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    • 2013
  • Guanosine-5'-diphosphate 3'-diphosphate (ppGpp) serves as alarmone in bacterial stringent responses. In this study, an affinity column was constructed by immobilizing ppGpp to NHS-Sepharose for isolating ppGpp-binding proteins. A novel ppGpp-binding protein, YjgA, was isolated and characterized by MALDI-TOF MS (matrix-assisted laser desorption ionization-time-of-flight mass spectrometry) coupled with two-dimensional gel electrophoresis. YjgA and truncated forms of YjgA were cloned and over-expressed in BL21 (DE3). The binding affinity of YjgA to ppGpp was determined by equilibrium dialysis. The interaction of YjgA with ppGpp was very specific, considering that the dissociation constant of YjgA with ppGpp was measured as $5.2{\pm}2.0{\mu}M$, while the affinities to GTP and GDP were about 60 and 30 times weaker than ppGpp. Expression of yjgA gene in Escherichia coli K-12 MG1655 was examined by reverse transcription polymerase chain reaction (RT-PCR). RT-PCR results revealed that yjgA was expressed from early to late stationary phase. The yjgA deletion mutant exhibited decreased cell number at stationary phase compared to parent strain and the over-expression of YjgA increased the cell number. These results suggested that YjgA might stimulate cell division under stationary phase. In most prokaryotic genome, about half of the protein candidates are hypothetical, that are expected to be expressed but there is no experimental report on their functions. The approach utilized in this study may serve as an effective mean to probe the functions of hypothetical proteins.

Interorder Hybridization between Pleurotus ostreatus and Elfvingia applanata by Protoplast Fusion (원형질체(原形質體) 융합(融合)에 의한 느타리버섯과 잔나비걸상버섯의 이목간(異目間) 교잡(交雜))

  • Yoo, Young-Bok
    • The Korean Journal of Mycology
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    • v.22 no.1
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    • pp.107-116
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    • 1994
  • Interorder somatic hybrids were obtained by protoplast fusion between Pleurotus ostreatus in the order Agaricales and Elfvingia applanata in the order Aphyllophorales. The fusants were classified into stable heterokaryons and spontaneously segregated heterokaryons. Hyphae of all fusion products except two strains did not form clamp connections. Out of them, two clamped and three clampless fusants produced mature fruiting bodies by light-dark cycle on sawdust rice bran medium. All of these basidiocarps had clamp connections. Three fusants were analysed with the distribution of progenies and segregation of genetic characters by random spore analyses. The genetic markers were shown to segregate and recombine in the first generation of monospores isolated from basidiocarps. Phenotypes of a large number of auxotrophic progenies were not detected in the two clamped fusants. The aberration ratio of segregants indicated the gene interaction resulting from different genome structure between distantly related species. The polymerase chain reaction (PCR) was adopted for the detection of somatic hybrids nuclear DNA. Four fusants showed a positive results in three kinds of primers. The prominent reaction products are represented by new bands in primer # 87 and # 125. Out of four fusants, two somatic hybrids had non-parental mtDNA patterns when digested with EcoR1 and HindIII. Comparison of somatic hybrids, tissue culture isolates(TC) and multispore germination isolates(MS) were made using esterase isozyme analysis. It is apparent that somatic hybrids had a minor banding patterns which are quite different from those of parents.

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Implementation of Improved Frame Slotted ALOHA Algorithm for Fast Tag Collection in an Active RFID System (고속 태그 수집을 위한 개선된 능동형 RFID 시스템용 프레임 Slotted ALOHA 알고리즘 구현)

  • Kim, Ji-Tae;Kang, Byeong-Gwon;Lee, Kang-Won
    • The Journal of Korean Institute of Communications and Information Sciences
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    • v.39B no.9
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    • pp.598-605
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    • 2014
  • In this paper, we suggest a modified slotted ALOHA algorithm for fast tag collection in active RFID system and implement the reader and tag operation using CC2530 chip of Texas Instruments Co. to prove the performance of the proposed algorithm. In the present international standard related with active RFID including ISO/IEC 18000-7 the reader sends sleep command to each tag after successful obtaining tag's information. Meanwhile, in this paper, the tags decide to sleep after checking the second command from the reader resulting in enormously decreased tag collection time. We tested the proposed algorithm with 30 tags over the range of 0-3m and the results showed that the tag collection process was completed in 400msec at average. And 30 tags are collected in one second with 99.7% and the collection rate is 100% in 2m distance between reader and tag. The collection rates are 99.94% and 99.7% for distance 2.5m and 3m, respectively. The average collection rate is 99.91% over all range and it is concluded that the proposed algorithm is enough to apply to real fields.

Optimization of Post-Activation Systems to Improve the Embryonic Development in Porcine Parthenogenesis and Somatic Cell Nuclear Transfer

  • Roy, Pantu Kumar;Kim, Ghangyong;Fang, Xun;Hassan, Bahia MS;Soysa, Mahanama De;Shin, Sang Tae;Cho, Jong Ki
    • Journal of Embryo Transfer
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    • v.32 no.3
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    • pp.95-104
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    • 2017
  • This study was conducted to establish the optimal chemical post-activation conditions in porcine embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) using 4 different chemical compositions (cytochalasin B (CB), cyclohexamide (CHX), demecolcine (DC), 6-dimethylaminopurine (DMAP). Porcine embryos were produced by PA and SCNT and then, cultured for post-activation with CB ($7.5{\mu}g/mL$), CB ($7.5{\mu}g/mL$) + CHX ($10{\mu}g/mL$), CB ($7.5{\mu}g/mL$) +DC ($0.4{\mu}g/mL$), and CB ($7.5{\mu}g/mL$) + DMAP (2 mM). In PA embryonic development, cleavage rates have been significantly higher in CB group (94.7%) and CB+DMAP group (94.1%) than that of CB+CHX and CB+DC group (88.1 and 84.3%, respectively). There have been no significant differences in blastocyst formation rates among the four groups. In cell number of blastocyst was shown in CB group (42.3%) significantly higher than CB+CHX and CB+DC group (40.6 and 40.6%, respectively). In SCNT embryonic development, CB+DMAP group (89.7%) significant differences were found on embryo cleavage rates when compared with other three groups. Blastocyst formation rates in CB+DMAP group (26.9%) were significantly higher when compared with CB, CB+CHX, and CB+DC groups (25.5, 20.2, and 22.1%, respectively). In blastocyst cell number, CB+DMAP group (41.4%) was found higher significant difference compared with other three groups. Additionally, we have investigated survivin expression in early development stages of porcine SCNT embryos for more confirmation. Our results establish that CB group and CB+DMAP group for 4 h during post-activation improves pre-implantation improvement of PA and SCNT embryos.

Nematicidal Activity of Kojic Acid Produced by Aspergillus oryzae against Meloidogyne incognita

  • Kim, Tae Yoon;Jang, Ja Yeong;Jeon, Sun Jeong;Lee, Hye Won;Bae, Chang-Hwan;Yeo, Joo Hong;Lee, Hyang Burm;Kim, In Seon;Park, Hae Woong;Kim, Jin-Cheol
    • Journal of Microbiology and Biotechnology
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    • v.26 no.8
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    • pp.1383-1391
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    • 2016
  • The fungal strain EML-DML3PNa1 isolated from leaf of white dogwood (Cornus alba L.) showed strong nematicidal activity with juvenile mortality of 87.6% at a concentration of 20% fermentation broth filtrate at 3 days after treatment. The active fungal strain was identified as Aspergillus oryzae, which belongs to section Flavi, based on the morphological characteristics and sequence analysis of the ITS rDNA, calmodulin (CaM), and β-tubulin (BenA) genes. The strain reduced the pH value to 5.62 after 7 days of incubation. Organic acid analysis revealed the presence of citric acid (515.0 mg/kg), malic acid (506.6 mg/kg), and fumaric acid (21.7 mg/kg). The three organic acids showed moderate nematicidal activities, but the mixture of citric acid, malic acid, and fumaric acid did not exhibit the full nematicidal activity of the culture filtrate of EML- DML3PNa1. Bioassay-guided fractionation coupled with 1H- and 13C-NMR and EI-MS analyses led to identification of kojic acid as the major nematicidal metabolite. Kojic acid exhibited dose-dependent mortality and inhibited the hatchability of M. incognita, showing EC50 values of 195.2 μg/ml and 238.3 μg/ml, respectively, at 72 h post-exposure. These results suggest that A. oryzae EML-DML3PNa1 and kojic acid have potential as a biological control agent against M. incognita.

Estimation of Allowable Drop Height for Oriental Pears by Impact Tests (충격시험에 따른 배의 허용낙하높이 추정)

  • Kim, M. S.;Jung, H. M.;Seo, R.;Park, I. K.;Hwang, Y. S.
    • Journal of Biosystems Engineering
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    • v.26 no.5
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    • pp.461-468
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    • 2001
  • Impact between fruits and other materials is a major cause of product damage in harvesting and handling systems. The oriental pears are more susceptible to bruising than other fruits such as European pears and apples, and are required more careful handling. The interest in the handling of the pears for the processing systems has raised the question of the allowable drop height to which pears can be dropped without causing objectionable damage. Drop tests on pears were conducted using an impact device developed by authors to estimate the allowable drop height without bruising. The impact device was constructed to hold in a selected orientation and to release a fruit by vacuum for dropping on to a force transducer. The drop height was adjustable for zero to 60 cm to achieve the desired distance between the bottom of the fruits and the top of the impact force transducer. The transducer was secured to 150 kg$\sub$f/ concrete block. The transducer signal was sampled every 0.17 ms with a strain gage measurement board in the micro computer where it was digitaly stored for later analysis. The selected sample fruit was Niitaka cultivar of pears which is one of the most promising fruit for export in Korea. The pears were harvested during the 1998 harvest season from an orchard in Daejeon. The sample fruit was selected from two groups which were stored for 3 months and 5 months respectively by the method of current commercial practice. The pears were allowed to stabilize at environmental condition(18$^{\circ}C$, 65% rh) of the experimental room. One hundred fifty six pears were tested from the heights of 5, 7.5. 10 and 12.5 cm while measurement were made of impact peak force, contact time, time to peak force, dwell time, pear diameter and mass. The bioyield strength and modulus of elasticity were measured using UTM immediately after each drop test. The allowable drop height was estimated on the base of bioyield strength of the pears in two ways. One was assumed the peak force during impact test increasing linearly with time, and the other was based on the actual drop test results. The computer program was developed for measuring the impact characteristics of the pears and analyzing the data obtained in the study. The peak force increased while contact times decreased with increasing drop height and contact times of the sample from the hard tissue group. The allowable drop height increased with increasing bioyield strength and contact times, and also varied with Poisson\`s ratio, mass and equilibrium radius of the pears. The allowable drop height calculated by a theoretical method was in the range from 1 to 4 cm, meanwhile, the estimated drop height considering the result of the impact test was in the range from 1 to 6 cm. Since the physical properties of fruits affected significantly the allowable drop height, the physical properties of the fruits should be considered when estimating the allowable drop height.

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The Long-Term Effects of High-Frequency Transcutaneous Electrical Nerve Stimulation(TENS) on the Lower Limb Spasticity and the Balance in the Chronic Stroke Patients (장기간 고빈도 경피신경전기자극이 뇌졸중 환자의 하지 경직 및 균형에 미치는 영향)

  • In, Tae-Sung;Cho, Hwi-Young;Lee, Sun-Hyun;Lee, Dong-Yeop;Lee, Jae-Kuck;Song, Chang-Ho
    • Journal of the Korea Academia-Industrial cooperation Society
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    • v.12 no.4
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    • pp.1740-1748
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    • 2011
  • The purpose of this study was to investigate effects of the long-term high-frequency transcutaneous electrical nerve stimulation(TENS) on the spasticity and the balance in chronic stroke patients. Twenty-six subjects with spasticity over lower limbs were allocated randomly into two groups under standard rehabilitation: (1) TENS group, (2) placebo-TENS group. TENS stimulation was applied on the both the gastrocnemius for 30 minutes, 5 days a week for 4 weeks(100 Hz, 0.25 ms, 2 times sensory threshold). The Modified Ashworth Scale(MAS) and Hand-held manual muscle tester were used to assess the ankle plantarflexor spasticity. Balance function under three conditions was measured by using force-plate and the amount of postural sway was assessed; in (1) the condition of standing with eyes opened, (2) with eyes closed and (3) the condition of standing on unstable surface with eyes opened. Both groups showed significant improvement in spasticity and balance function after treatment for 4 weeks(p<.05). Especially, TENS group showed a significant reduction of spasticity compared to placebo-TENS group(p<.05). These results suggested that additional stimulation of a long-term high-frequency TENS to standard rehabilitation induced an improved balance function and a spasticity reduction. The long-term application of high-frequency TENS will be an effective intervention for reducing spasticity and increasing balance ability in the chronic stroke patients.

Physicochemical properties of rice-distilled spirit matured in oak and stainless steel containers (숙성용기를 달리한 전통 쌀 증류식 소주의 숙성 중 이화학 특성 및 향기성분의 변화)

  • Kang, Sun-Hee;Kim, Jae-Ho;Lee, Ae-Ran;Kim, A-Ra;Kim, Tae-Wan
    • Korean Journal of Food Science and Technology
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    • v.49 no.4
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    • pp.369-376
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    • 2017
  • Maturation of distilled spirit can generate diverse flavors and tastes. Rice Soju was matured in oak casks (MSO) and stainless steel containers (MSS) for one year at ambient temperature. Ipguk (Aspergillus luchuensis) and Saccharomyces cerevisiae Y88-4 were used, and reduced pressure ($110{\pm}20Torr$) distillation was applied to brew Soju. Acidity and conductivity were increased in both MSO and MSS. MSO reduced alcohol content (from 43 to 40%) and volume (from 18,000 to 12,730 mL), and significantly altered yellowness (from 0.2 to 30.2). Furthermore, MSO increased the isoamyl alcohol (from 276.7 to $339.2{\mu}g/mL$) and isobutyl alcohol (from 122.3 to $144.2{\mu}g/mL$) content. Gas chromatography-mass spectrometry was used to detect volatile compounds in Soju, which included 20 esters, 7 alcohols, 2 acids, and 5 miscellaneous compounds. Oak lactone was detected only in MSO and was considered as a specific flavor component associated with oak maturation. Thus, maturation materials contribute to the physicochemical property of distilled spirits.

Influence of Microwave Pretreatment on the Formation of Heterocyclic Amines in Fried Beef Patties (Microwave를 이용한 예열 처리가 조리한 쇠고기 패티에서의 Heterocyclic Amines 형성에 미치는 영향)

  • Jung, Kyung-Hee;Shin, Han-Seung
    • Food Science of Animal Resources
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    • v.29 no.6
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    • pp.719-725
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    • 2009
  • Heterocyclic aromatic amines (HCAs) are potent mutagens and possible human carcinogens that are formed during the heating of protein-rich foods. The effects of preheating treatment of beef patties using a microwave prior to frying at $220^{\circ}C$ for 10 min on each side on the reduction of HCAs (amino-carbolines and amino-imidazo-azaarenes) were evaluated. The amount of HCAs was then evaluated by solid-phase extraction and high pressure liquid chromatography (HPLC) analysis. The beef patties were treated by microwaving for various times (0, 1, 1.5, 2, or 3 min) before pan-frying. The results revealed the presence of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 3-amino-1,4-dimethyl-5H-pyrido-[4,3-b] indole (Trp-P-1), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), 9H-pyrido[3,4-b]indole (Norharman), 1-methyl-9H-pyrido[3,4-b]indole (Harman), 2-amino-9H-pyrido[2,3-b] indole ($A{\alpha}C$), 2-amino-3,8 dimethylimidazo[4,5-f]-quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP) in all samples. However, microwave pretreatment for 1 min inhibited the formation of these HCAs by up to 90% when compared to the control.

RF 플라즈마 아킹의 PM-tube를 이용한 광량 측정

  • Kim, Yong-Hun;Jang, Hong-Yeong
    • Proceedings of the Korean Vacuum Society Conference
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    • 2010.08a
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    • pp.125-126
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    • 2010
  • 플라즈마 아킹은 PECVD, 플라즈마 식각 그리고 토카막과 같은 플라즈마를 이용하는 여러 공정과 연구 분야에서 문제점을 야기시켜왔다. 하지만, 이에 대한 연구는 아킹 현상의 불규칙성과 과도적인 행동으로 인해 미비한 상태이다. 특히, RF 방전에서의 아킹 연구는 DC 방전에서의 아킹 연구에 비해 많이 부족한 것이 현실이다. 플라즈마 아킹은 집단전자방출(collective electron emission)에 의한 스파크 방전(spark discharge)현상이다. 집단전자방출은 전계방출(field emission)이나 플라즈마와 쉬스를 두고 인접한 표면위에서의 유전분극(dielec emission)에 의해 발생한다. 이렇게 방출된 집단 전자들은 쉬스에서 가속되어 에너지를 얻게 되고 원자와의 충돌로 전자 아발란체를 일으킨다. 이렇게 배가된 전자들은 아킹 스트리머(arcing streamer)를 형성하게 되고 아킹 발생 시 높은 전류와 공정 실패의 원인이 된다. 우리는 $30cm{\times}20cm$ 크기의 사각 전극을 위 아래로 가진 챔버에서 Ar 가스를 RF(13.56 MHz)파워를 이용해 방전시켰다. 방전 전압과 전류는 파워 전극 압단에서 High voltage probe (Tektronix P6015A)와 Current probe (TCPA300 + TCP312)를 이용해 측정했다. 플라즈마 아킹시 변하는 플라즈마 플로팅 포텐셜은 챔버 중앙에 위치한 랑뮈프 프로브에 의해 측정되고 챔버 옆의 뷰포트 앞에 위치한 PM-tube를 이용해 아킹시 변하는 광량을 측정한다. RF 방전에서의 플라즈마 아킹은 아킹시 플로팅 포텐셜의 변화에 의해 크게 세부분으로 나눌 수 있다. 아킹 발생과 동시에 급격히 감소하는 감소부분 (약 2us) 그리고 감소한 포텐셜이 유지되는 유지부분 (약 0~10ms) 그리고 감소했던 포텐셜이 서서히 원래 상태로 회복되는 회복부분(약 100 us)이다. 아킹 초기시 방출된 집단 전자들과 원자들간의 충돌에 의해 형성된 아킹 스트리머는 플라즈마 전체를 단락시키게 되고 이로 인해 플로팅 포텐셜은 급격히 감소하게 된다. 이렇게 감소한 플로팅 포텐셜은 아킹 스트리머가 유지되는 한 계속 감소한 상태를 유지하게 된다. 그리고 플라즈마를 섭동했던 아킹 스트리머가 중단되면 플라즈마는 섭동전의 원래 상태로 돌아가려 하기 때문에 플로팅 포텐셜은 서서히 증가하면서 원래 상태로 회복된다. 플라즈마 아킹 발생시 생성되는 아킹 스트리머는 순간적으로 많은 전자들을 국소적으로 생성하게 되고 이 전자들에 의해 광량이 순간적으로 증가하게 된다. PM-tube (750.4 nm)에 의해 측정된 아킹시 광량은 정상방전 상태의 두배 가량이 된다. 그리고 이 순간적으로 증가된 광량은 시간이 지남에 따라 감소하게 되고 정상방전 일때의 광량이 된다. 광량이 증가한 후 정상방전 상태의 광량에 이르는 부분은 플로팅 포텐셜이 감소한 상태에서 유지되는 부분과 일치하고 이는 플로팅 포텐셜의 유지부분동안 아킹 스트리머가 발생하고 있다는 간접적인 증거가 된다. 그리고 정상 방전 상태 일때의 광량이 되면 아킹 스트리머가 중단되었다는 것이므로 그 시점부터 플로팅 포텐셜은 정산 방전상태 일 때의 포텐셜로 복구되기 시작한다. 이처럼 PM-tube를 이용한 아킹 광량 측정은 아킹 스트리머를 간접적으로 측정하게 하고 아킹 스트리머를 이용해 아킹시의 플로팅 포텐셜의 변화를 설명하게 해 준다. 응용적인 측면에서 아킹 광량 측정을 이용한 아킹 판독은 방전 전류와 방전 전압과 같은 전기적 신호를 이용한 아킹 판독에 비해 여러가지 장점을 가진다. 우선, 전기적 신호를 이용한 아킹 판독처럼 매칭 회로나 플라즈마를 섭동시키지 않는다. 그리고 원하는 부분의 아킹만을 판독하는 것도 가능하며 photo-diode를 이용할 경우 전기적 신호를 이용하는 것에 비해 경제적으로 유리하다.

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