분열유발인자에 의한 흰쥐 림프구 단백의 인산화

주일로(Ilo Jou),고성수(Sung Soo Ko),안영수(Young Soo Ahn) 대한약리학회 1993 대한약리학잡지 Vol.29 No.1

흰쥐 말초 T림프구에 분열유발 물질인 PMA와 Con A를 투여하여 인산화되는 단백을 확인하고, PKC 억제제인 H-7, CaM kinase 억제제인 W-7을 전처치한 후의 인산화 변동과 시간 경과에 따른 인산화 변동을 관찰하였다. 그 결과 흰쥐 T림프구를 PMA로 자극하면 5개의 인산화 단백이 새로이 나타나고 7개 단백의 인산화가 증가 되었으며, Con A자극으로는 1개의 단백이 새로이 인산화 되고 7개 단백의 인산화가 증가되었다. PMA 및 Con A자극으로 인산화 되는 13개 단백은 kinase억제제 전처치에 의하여 3군으로 각각 구분되며, H-7 전처치로 24 kDa/pI 7.1, 24/7.2, 26/6.1, 74/6.2 단백의, W-7 전처치로 14 kDa/pI5.9, 28/6.8, 29/6.9, 28/7.0, 44/6.8, 58/6.2 단백의 인산화가 현저히 감소 되었으며, 18 kDa/p1 5.4, 25/7.3 및 54/5.2단백은 두 억제제에 의해 영향을 받지 않았다. 이들 인산화 단백은 대부분 세포의 soluble fraction에서 확인되며 자극후 반응 초기에 인산화 된 후 인산화가 감소하나, 침전물에서 관찰되는 소수의 인산화 단백은 지속적인 인산화를 보였다. 한편 Kinase 억제제 처리에 의하여 구분된 3군에 속하는 단백들의 시간에 따른 인산화 양상을 관찰한 결과 각 군에 따른 인산화 양상에 상호 연관성이 없었다. 이상의 실험결과로 보아 림프구 활성의 초기 단계에서 인산화 되는 단백에는 PKC, CaM kinase 및 다른 kinase에 의해 인산화 되는 3종류의 단백이 존재하며, 3종류의 kinase의 활성은 단계적인 활성이 아니라 독립적 또는 상호 협동적으로 작용하여 림프구 활성을 유발시키는 것으로 생각된다. This study was done to classify the proteins involved in the specific phosphorylation using the rat peripheral blood lymphocytes (rPBL) stimulated with mitogens, phorbol 12-myristate 13-acetate (PMA) and concanavalin A (Con A). The lymphocytes were incubated with ³²P-orthophosphate before PMA or Con A stimulation. The migration patterns of the phosphorylated proteins of mitogen-treated rPBL in two dimensional electrophoretic fields were analyzed after autoradiography. The stimulation of the lymphocytes with PMA and Con A increased the phosphorylation of thirteen protein fractions. The phosphorylation intensities of the protein spots differ to the treatments of the cells with specific kinase inhibitors, H-7 and W-7. These protein fractions were grouped into 3 classes, namely, PKC-mediated, CaM kinase-mediated, and other kinase mediated proteins. The effect of the duration of the stimulation on the phosphorylated behaviors occurred concurrently, not sequentially, although each individual protein fraction had a different time for the peak phosphorylation during the stimulation period upto 30 minutes. The phosphoproteins found in the cytosolic soluble fraction were phosphorylated prior to those in the pellet, whose phosphorylations were sustained at a high level for over 10 minutes. The above results suggest that the early events in lymphocyte activation involve 3 different sets of proteins which are phosphorylated by CaM kinase, PKC and other kinases, and those kinases do not work sequentially, but rather, independently or cooperatively.

Curcumin이 microglia의 활성화에 미치는 영향

정기경(Ki Kyung Jung),이상진(Sang Jin Lee),이선우(Sun Woo Yi),강석연(Seog Young Kang),김태균(Tae Gyun Kim),강주혜(Ju Hye Kang),홍성렬(Sung Youl Hong),주일로(Ilo Jou),김승희(Seung Hee Kim),한형미(Hyung Mee Han) 대한약학회 2000 약학회지 Vol.44 No.5

Microglia, brain resident macrophages, play a central role in the inflammatory responses of the brain and are activated in brain injuries and several neurodegenerative diseases such as Alzheimer's and Parkinson's disease, thereby aggravating the course of these diseases. In this study, the effects of plant-derived compounds such as curcumin or gingerol on the microglial activation were examined. Microglial cultures were prepared from 2-3 week mixed primary glial cultures obtained from the cerebral cortex of 1-2 day old rats and identified by immunocytochemistry using microglial-specific antibody OX-42. Microglia were activated by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) and the effect of curcumin or 6-gingerol on the microglial activation was examined. Specific parameters measured to monitor microglial activation were nitric oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) release. Curcumin (1-10mcM) inhibited NO release induced by LPS and IFN-gamma in a dose-dependent manner whereas 6-gingerol (2-20mcM) did not have any effect on LPS/IFN-gamma-induced NO release. The levels of PGE2 and TNF-alpha induced by LPS and IFN-gamma were also inhibited by 1-10pM curcumin in a dose-dependent manner. These results showed that curcumin could modulate microglial activation.

흰쥐 말초 림프구의 분자량 22 kDa 인산화 단백질의 특성

주일로,권혁춘,안영수 아주대학교 의과학연구소 1996 아주의학 Vol.1 No.1

Lymphocytes were found to possess all of the enzymatic machinery needed to phosphorylate and dephosphorylate proteins. At least four groups of protein kinases participate in T cell activation and interact with each other in a complex and as yet unknown manner. Increased phosphorylation in lymphocytes following stimulation with mitogens or interleukin-2, and the pivotal role of protein kinase C(PKC) in the initial biochemical reaction have been reported. But the exact role of PKC in T cell activation and the substrate of PKC are not well known. This study was attempted to clarify the characteristics of 22 KDa phosphoprotein obtained from rat peripheral blood lymphocytes(rPBL) stimulated with phorbol 12-myristale 13-acetate(PMA), using various kinase inhibitors as well as kinase activators. The lymphocytes were incubated with ^(32)P orthophosphate before PMA stimulation. The migration pattern of the phosphorylated proteins of PMA-treated rPBL in the two dimensional electrophortic fields were analyzed after autoradiography. And the phosphorylation sites of 22 kDa protein were analyzed by high performance liquid chromatography(HPLC) and scintilation counting. The results are as follows: 1) Increased phosphorylation of 22 kDa protein was observed with PMA. 2) Forskolin, an activator of adenylyl cyclase, causes no significant change of phosphorylation of 22 kDa. 3) A 23187, a Ca^(2+) ionophore, has no noticed effect on the phosphorylation of 22 kDa protein. 4) Staurosporine, a potent PKC inhibitor, showed inhibitory action on PMA-stimulated phosphorylation of 22 kDa. 5) Calphostin C, specific PKC inhibitor inhibited the PMA-stimulated phosphorylation of 22 kDa. 6) Among trypic peptide fractions of 22 kDa by HPLC, one ^(32)P phosphopeptide peak was observed at about 20% acetonitrile. From the above results, it could be suggested that the 22 kDa phosphoprotein of rat peripheral blood lymphocytes would be a substrate of PKC, and has one phosphorylation on serine residue.

Effects of Curcumin on the Modulation of Microglial Activation(Ⅱ)

Jung, ki Kyung,Lee, Jin Ho,Han, Hyung Mee,Kim, Tae Gyun,Kang, Ju Hye,Jou, Ilo,Hong, Sungyoul,Kim, Seung Hee,Kang, Seog Youn 식품의약품안전청 2000 식품의약품안전청 연보 Vol.4 No.-

뇌손상 및 Alzheiirler's disease등 만성 염증을 동반하는 뇌질환을 악화시키는 원인이 된다고 알려진 microglia의 활성화를 카레의 주성분인 curcumin이 억제하는지 알아보고자 하였다. 쟁후 0일에서 2일된 rat의 대뇌피질로부터 rrlicroglial cell을 2~3주간 배양하여 얻은 다음 LPS와 fFN-r로 활성화를 유도하고 curcumin을 I~SrM 범위로 처리했을 패, NO와 PGEr 생성에 관여하는 iNOS꽈 COX-2발현이 용량의존적으로 억제되었으며 iNOS의 경우 세포단계에서도 용량의존적으로 억제되었다. 뿐만 아니라 curcumin은 I~SrM 범위에서 염증반응을 매개하는 IL-1 및 U-6의 생성을 용량의존적으로 억제하였다. 전사주즐인자인 NF-rB는 ploinffarnmatory cytokine인 TNF-α, a-1, IL-6 및 iNOS와 COX-2등의 발현을 조절하는 것으로 알려져 있는데, curcumin이 NF-rB의 활성화를 억제하는 것으로 보아 LPS 와 IFN-γ로 활성화된 microglia 모델에서 curcumin의 항염증효과는 NF-rB pathu~·ay를 통하여 나타나는 겄으로 사료된다. -MicrogEa, braf resident macrophages, play a central role in the inflammatory responses of the brain and are activated in brain injuries and several neurodegenerative6seases such as Alzhehner's and Huntington's disease, thereby aggravating the course o(these diseases. We previ()usly repofed that curcunBin (1 ~8#M) inhibited tlte release ofinflammatory mediators such as nitric oxide (NO), prostaglandin Ef (PGEa), Tumor necrosisfactor- α (TNF-o) in Ipopolysacchafde (LPS) plus interferon- γ (HN-γ)-stimulatedmicroglia. h thiE sttdy, we examined whether curcuBCn can inhibit the production ofFfoinflarnmatdry mecators by suppressing the activation of Nuclear factor- f B (NP~ rE).Curcunfn inRbited other inflammatory cytokines, InterBeukin-1 (TL-1) and Interleukin-6 (t-6)in a dose-dependent manner, and also suppressed the induction of both inducible nitricoxide synthase fHOS) and cyclooxygenase-2 (COX-2), which catalyses the conversion ofarachidonic acid to prostagtandins. In addition, curcumin inhibited the activation of NF- f B,which controls the expresslon of a wide variety of genes active in inflammation that includecytokines (e.g., U-1, T.fF- f , IL-5), enzymes (e.g., iNOS, COX-2). Antiinflammatory effectof curcumin in cuthlred m:icroglial model seems to be mediated through NF- f B pathway,suggesting that may havc a significant impact in the prevention of immune-mediatedneurodeg enerativ e disordors.