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Proteome Analysis of Escherichia coli after High-dose Radiation
임상용,이미송,조민호,송현파,김동호 한국방사선산업학회 2011 방사선산업학회지 Vol.5 No.1
Since proteomics can be employed to compare changes in the expression levels of manyproteins under particular genetic and environmental conditions, using mass spectrometry to establishradiation stimulon, we performed two-dimensional gel electrophoresis and identified E. coliproteins whose expressions are affected by high dose of ionizing radiation. After exposure to 3 kGy,it was found that 6 proteins involved in carbon and energy metabolism were reduced. Although 4of 7 protein spots showing a significant increase in expression level were neither identified norclassified, uridine phosphorylase (Udp), superoxide dismutase (SodB), and thioredoxin-dependentthiol peroxidase (Bcp) were proven to be up-regulated after irradiation. This suggests that E. colisubjected to high doses of radiation (3 kGy) may operate a defense system that is able to detoxifyreactive oxygen species and stimulate the salvage pathway of nucleotide synthesis to replenish damagedDNA.
Eun Kyung Cho(조은경),Young Ju Choi(최영주) 한국생명과학회 2008 생명과학회지 Vol.18 No.5
고온에 노출된 식물의 단백질 보호에 있어서 HSP70의 효과를 연구하기 위해 2차 전기영동 법이 시행되었다. 본 논문에서는 고온에서 유도되는 담배 HSP70인 NtHSP70-1 (AY372069)에 대한 식물 HSP70의 고온내성을 분석하였다. 우선, NtHSP70-1이 항상 발현되는 형질전환 식물과 vector만을 지닌 형질전환 식물, 그리고 antisense 형태의 형질전환 식물이 제조되었다. 이들 세 종류의 형질전환체들은 western blot 분석법을 사용하여 확인되었고, NtHSP70-1에 대한 sense형태의 형질전환체들은 고온에 대한 내성이 있는 것으로 보였다. 45도에서 2시간 동안 고온처리 후 26도에서 5일 동안 유지한 세 종류의 형질전환체들과 고온처리 하지 않은 이들 형질전환체들에 대한 2차 전기영동 분석에서 잠정적으로 28개의 단백질들에 대해 비교한 결과 이들 단백질의 발현량에 차이가 있음을 확인 할 수 있었다. NtHSP70-1이 항상 발현되는 형질전환체들에서는 이들 단백질들이 유지되거나 향상된 반면, 다른 종류의 형질전환체들에서는 이들 단백질들이 감소되거나 사라졌다. 이 결과들은 NtHSP70-1이 고온에서 단백질을 보호하는 분자 chaperone으로써 기능을 함으로써 식물의 고온내성에 중요한 역할을 할 것으로 시사하고 있다. The effect of heat shock protein 70 (HSP70) on soluble proteins extracted from seedlings exposed to high temperature was examined by two-dimensional gel electrophoresis (2-DE). NtHSP70-1 (AY372069), which is an HSP70 that is induced by heat stress in Nicotiana tabacum, was studied to analyze the protective role of plant HSP70. First, transgenic tobacco plants that constitutively expressed elevated levels of the tobacco HSP70, NtHSP70-1, and transgenic plants containing either the vector alone or the NtHSP70-1 gene in the antisense orientation were constructed. Plants with altered NtHSP70-1 levels were subjected to immunoblotting, and seedlings with enhanced levels of NtHSP70-1 in their transgenic sense lines were observed to show tolerance to heat stress. In the analysis using 2-DE, 28 putative proteins were differentially expressed in the comparison of three kinds of transgenic seedlings after high temperature/recovery treatment (45℃, 2 hr; 26℃, 5 day) with the seedlings under normal condition (26℃). In transgenic sense seedlings, the proteins were maintained or increased, whereas proteins in the transgenic seedlings, containing either the vector alone or the NtHSP70-1 in the antisense orientation, were present in decreased amounts or had disappeared. These results suggested that NtHSP70-1 as molecular chaperone protecting proteins from aggregation induced by heat stress may play an important role in acquisition of thermotolerance.
Kimata, Junko,Shigeri, Yasushi,Yoshida, Yasukazu,Niki, Etsuo,Kinumi, Tomoya Korean Society for Mass Spectrometry 2012 Mass spectrometry letters Vol.3 No.1
Artificially oxidized cysteine residues in peroxiredoxin 6 (Prx6) were detected by electrospray interface capillary liquid chromatography-linear ion trap mass spectrometry after the preparation of two-dimensional gel electrophoresis (2D-GE). We used Prx6 as a model protein because it possesses only two cysteine residues at the 47th and 91st positions. The spot of Prx6 on 2D-GE undergoes a basic (isoelectric point, pI 6.6) to acidic (pI 6.2) shift by exposure to peroxide due to selective overoxidation of the active-site cysteine Cys-47 but not of Cys-91. However, we detected a tryptic peptide containing cysteine sulfonic acid at the 47th position from the basic spot and a peptide containing both oxidized Cys-47 and oxidized Cys-91 from the acidic spot of Prx6 after the separation by 2D-GE. We prepared two types of oxidized Prx6s: carrying oxidized Cys-47 (single oxidized Prx6), and other carrying both oxidized Cys-47 and Cys-91 (double oxidized Prx6). Using these oxidized Prx6s, the single oxidized Prx6 and double oxidized Prx6 migrated to pIs at 6.2 and 5.9, respectively. These results suggest that oxidized Cys-47 from the basic spot and oxidized Cys-91 from the acidic spot are generated by artificial oxidation during sample handling processes after isoelectric focusing of 2D-GE. Therefore, it is important to make sure of the origin of cysteine oxidation, if it is physiological or artificial, when an oxidized cysteine residue(s) is identified.
( Junko Kimata ),( Yasushi Shigeri ),( Yasukazu Yoshida ),( Etsuo Niki ),( Tomoya Kinumi ) 한국질량분석학회 2012 Mass spectrometry letters Vol.3 No.1
Artificially oxidized cysteine residues in peroxiredoxin 6 (Prx6) were detected by electrospray interface capillary liquid chromatography-linear ion trap mass spectrometry after the preparation of two-dimensional gel electrophoresis (2D-GE). We used Prx6 as a model protein because it possesses only two cysteine residues at the 47th and 91st positions. The spot of Prx6 on 2D-GE undergoes a basic (isoelectric point, pI 6.6) to acidic (pI 6.2) shift by exposure to peroxide due to selective overoxidation of the active-site cysteine Cys-47 but not of Cys-91. However, we detected a tryptic peptide containing cysteine sulfonic acid at the 47th position from the basic spot and a peptide containing both oxidized Cys-47 and oxidized Cys-91 from the acidic spot of Prx6 after the separation by 2D-GE. We prepared two types of oxidized Prx6s: carrying oxidized Cys-47 (single oxidized Prx6), and other carrying both oxidized Cys-47 and Cys-91 (double oxidized Prx6). Using these oxidized Prx6s, the single oxidized Prx6 and double oxidized Prx6 migrated to pIs at 6.2 and 5.9, respectively. These results suggest that oxidized Cys-47 from the basic spot and oxidized Cys-91 from the acidic spot are generated by artificial oxidation during sample handling processes after isoelectric focusing of 2D-GE. Therefore, it is important to make sure of the origin of cysteine oxidation, if it is physiological or artificial, when an oxidized cysteine residue(s) is identified.
Her, Erk,Zor, Uriel 大韓免疫學會 1995 大韓免疫學會誌 Vol.17 No.3
항원에 자극된 호염기구에 있어, 인조 glucocorticoid 중의 하나인 hydrocortisone (HC)에 의한leukotriene C4(LTC4)생산억제 호과는 적어토 2-5시간정도의 지연시간이 필요했다. 이러한 HC의 지연효과는 cycloheximide와 eC recepfo. 길항제에 의해 억 제되 었다 ffer and Zor, submitted). 이 러 한 실험결과들은 班C는 단백질생산에 의한 간접작용을 한다는 것을 암시했다. 그래서 HC에 의해 조절생산펀 단백질이 어떠한 단백질인지 알기위해 호염기구 암세포를 등위원소 i놀eoni緖을 례벨했다. 1'높Tmethionine을 레벨하는 동안,다행이도 HC가 이 동위원소의 축적이나 아미노산에 결합하는것을 억제하지는 않았다. One-dimensional SDS-PAGE을 이용한 단백질분리법에서는 HC유무에 아무런 차이가 없었다. 그래서 단백질분리가 쉬운 two곯mensional gel기법을 사용했다- 이 실험기법에서, HC에 의해 9개의 단백질생살 증감이 있었는데 이 중 8개의 단백질 acidic prole구 1 basicole조) 생산이 증가했으며, 한개의 acidic protein 생산이 감소했다. %5이 9개 단백질 중 5개 단백질(363kDa/pI 5.6; 38kDa/pI 5.9; 40kDa/p16.0; 42kDa/p16.1; 43kD리pl 5.2)은불용성 단백질이 였고,4개의 단백질(51kDa/pl 6.0; 52kDa/pI pl 6.8; 53kDa/pI 4.5: 71kDa/pl 8.4)은 불용성단백 질이 였다. 수용성,불용성 단백질 구분은 시료를 1關,Oeoxg속도하에서 1시간동안 원심분리한 후 상총액부위에 있는 단백질을 수용성 단백질이라 하고,절랫부위에 있는 단백질을 불용성 단백질이라 했다. 이 9게 단백질 중 분자량이 36-42tDa단백질이 phospholipaseA,억제물질이라고 밝혀진 lipocortin의분자량과 유사하다 .