cAMP Dependent Protein Kinase에 의한 Adenylate Cyclase의 억제

許圭晶 梨花女子大學校 韓國生活科學硏究院 1992 韓國生活科學硏究院 論叢 Vol.50 No.-

To figure out possible roles of protein kinase A in the process of adenylate cyclase-desensitization, Ca^2+/calmodulin-sensitive adenylate cyclase was isolated from the membranes of lens fiber cells using calmodulin affinity column. The isolated adenylate cyclase which is free from GTP binding preteins, was gradually activated by various concentrations of calmodulin. However, the activity of adenylate cyclase was dramatically decreased in the presence of protein kinase A. Especially, stimulation of the activity of adenylate cyclase by calmodulin was almost abolished in the presence of protein kinase A. These results suggest that protein kinase A phosphorylates catalytic subunit of adenylate cyclase, and then inactivates enzyme activity.

"지황 1호"를 이용한 숙지황 제조기술 연구

박남규,김선림,허한순,박충현 한국국제농업개발학회 2002 韓國國際農業開發學會誌 Vol.14 No.1

재래종 지황과 신육성품종 지황 1호를 이용하여 절단 및 원형으로 숙지황을 제조한 결과는 다음과 같다. 1. 가압에 의한 숙지황 제조시간이 재래종은 254∼264시간, 제품수율이 71.42∼71.48%였고, 지황 1호는 제조시간이 260∼276시간 소요되었고, 수율은 71.50∼71.51%였으며 중량은 감소하는 경향이었다. 2. 상압에 의한 숙지황 제조에서도 가압에 의한 제조와 비슷한 경향을 보였다. 3. 가압과 상압에 의한 숙지황 제조 후 유리당 조성은 재래종, 지황 1호 전처리구에 63∼65% 이상이 fructose였으며, 30% 이상이 glucose였고, sucrose, maltose가 함유되었다. 4. 가압 및 상압에 의한 숙지황 제조시 catalpol 함량 변화는 종자회수가 많아질수록 caltalpol 함량이 낮아지는 경향이었으나 완전히 없어지지는 않았다. 5. 숙지황 제조 후 포장재별로 포장하여 상온에 저장한 결과 제품에 곰팡이 발생이 없어 저장에 문제가 없었다. New processing method for R. Radix preparata was developed by using the landrace variety as a control and "Jiwhang 1" (Rehmannia glutinosa Liboschitiz). Sliced Rehmanna glutionsa Liboschitiz was steam heated by normal and high pressure conditions for the round-shaped R. Radix preparata processing. About 254 to 264 hours were required for the processing of domestic variety, and about 260 to 280 hours were required for "Jiwhang 1", but their yields were ranged about 71.42∼71.6%. Fructose and glucose were major free sugar of R. Radix preparata, and their composition rates were 63∼65% and 30%, respectively, but sucrose and maltose were traced only small amount. The Catalpol content was decreased as increasing the frequencies of steam heating repetion. But the catalpol was not completely removed. After processing, R. Radix preparata packed with paper-bag or PE-bag, and storaged at common temperature was keeping a best quality.

F9 기형암종 세포의 분화에 따른 small GTP-binding protein변화

박혜성,이준승,김규원,허규정 이화여자대학교 생명과학연구소 1994 생명과학연구논문집 Vol.5 No.-

세포분화에 따른 Small GTP-binding protein의 역할을 밝히기 위하여 Retinoic acid(RA)와 dibutyryl cyclic AMP(dbcAMP)로 분화를 유도한 F9 기형암종세포의 형태적인 변화와 함께 Small GTP-binding protein의 분포를 조사하였다. RA와 dbcAMP를 처리한 세포는 분화유도 5일경(초기 분화 단계)에 분명한 세포의 경계를 보이기 시작하여 7일경(분화 후기 단계)에는 거의 모든 세포가 둥근 분화된 형태로 전환되었다. 이 분화과정 동안 세포막에는 많은 microvilli와 lamellopodia 같은 구조물이 나타났다. 아울러 초기 분화 단계에 많은 량의 laminin이 발현되었으며 분화 후기에 microtubule의 재분포가 관찰되었다. 세종류의 Small GTP-binding protein(25, 23, 21 KD)이 F9 세포의 막성분과 세포질에서 관찰되었으며 분화가 진행됨에 따라서 세단백질 모두 증가되는 양상을 보였다. 이러한 결과는 Small GTP-binding protein이 F9 세포의 분화에 특별한 기능을 가지고 있음을 시사해 주고 있다. To address possible roles of the small GTP-binding protein in differentiation of F9 teratocarcinoma stem cells, the distribution and amount of these proteins together with morphological changes of the cells were sstudied during the differentiation of F9 cells induced by retinoic acid and dibutyryl cyclic AMP (dbcAMP). When monolayer cultures of F9 cells were induced to differentiate with retinoic acid and dbcAMP, the cells started to show distinctive cell boundaries in about 5 days (early differentiation stage) and reached fully differentiated shapes in about 7 days (late differentiation stage). During this differentiation process, membranes of the cells developed a large number of microville and lamellopodia. In addition, expression of laminin increased maximally in early stage, then microtubule rearrangement followed in late differentiation stage. Three classes of small GTP-binding proteins with sizes of 25, 23 and 21 kD were found in both microsomes and cytosols of F9 cells. These proteins increased along with progress of the differentiation. These results suggest that these small GTP-binding proteins likely have specific roles in the differentiation of F9 cells.

F9 기형암종 세포의 분화에 따른 small GTP-binding protein변화

박혜성,이준승,김규원,허규정 부산대학교 유전공학연구소 1994 분자생물학 연구보 Vol.10 No.-

세포분화에 따른 Small GTP-binding protein의 역할을 밝히기 위하여 Retinoic acid(RA)와 dibutyryl cyclic AMP(dbc AMP) 로 분화를 유도한 F9 기형암종세포의 형태적인 변화와 함께Small GTP-binding protein의 분포를 조사하였다. RA와 dbc AMP를 처리한 세포는 분화유도 5일경(초기 분화 단계)에 분명한 세포의 경계를 보이기 시작하여 7일경(분화 후기 단계)에는 거의 모든 세포가 둥근 분화된 형태로 전환되었다. 이 분화과정 동안 세포막에는 많은 microville와 lamellopodia 같은 구조물이 나타났다. 아울러 초기 분화 단계에 많은 량의 laminin이 발현되었으며 분화 후기에 microtuble 의 재분포가 관찰되었다. 세종류의Small GTP-binding protein(25,23,21 KD )이 F9 세포의 막성분과 세포질에서 관찰되었으며 분화가 진행됨에 따라서 세단백질 모두 중가되는 양상을 보였다. 이러한 결과는Small GTP-binding protein이 F9세포의 분화에 특별한 기능을 가지고 있음을 시사해주고 있다. To adress possible roles of the small GTP-binding proteins in differentation of F9 teratocarcinoma stem cells, the distribution anf amount fo these proteins together with morphological changes of the cells were studied during the differentiation of F9 cells induced by retionic acid and dibutyryl cyclic AMP(dbc AMP). When monolayer cultures of F9 cells were induced to differentiate with retionic acid dbc AMP, the cells started to show distinctive cell boundaries in about 5 days(early differentiation stge) and reached fully differentiated shaper in about 7 days(late differentiation stage). During this differentiation process, membranes of the cells developed a large number of microville and lamellopodia. In addition, expression of laminin increased maximally in early stage, then microtuble rearrangement followed in late differentiation stage. Three classes of small GTP-binding proteins with sizes of 25,23 and 21 kD were fiund in both microsomes and cytosols of F9 cells. These proteins increased along with progress of the differentiation. These results suggest that these small GTPbinding proteins likely have specific roles in the differentiation of F9 cells.

Protein Kinase A Functions as a Negative Regulator of c-Jun N-terminal Kinase but not of p38 Mitogen-activated Protein Kinase in PC12 Cells

Hur, Kyu-Chung The Korean Society for Integrative Biology 2005 Integrative biosciences Vol.9 No.3

Cyclic-AMP-dependent protein kinase (PKA) seems to function as a negative regulator of the c-Jun $NH_2-terminal$ kinase (JNK) signaling pathway. We demonstrate here that the activity of the PKA catalytic subunit (PKAc) is reduced in apoptotic PC12 pheochromocytoma cells. Apoptotic progress was inhibited by dibutyryl cyclic AMP (dbcAMP), an analog of cAMP. The rescue by dbcAMP was attributable to inhibition of the JNK but not of the p38 signaling pathway, due to the induction of PKA activity. JNK was present in immunocomplexes of PKAc, and PKAc phosphorylated JNK in vitro. Presence of p38 kinase, however, was not prominent in immunocomplexes of PKAc. Our data suggest that JNK is a target point of negative regulation by PKAc in the JNK signaling pathway.

Insulin mediated H₂O₂ generation is necessary for the inhibition of cell death mediated by serum deprivation

Hur, Kyu Chung 이화여자대학교 세포신호전달연구센터 1999 고사리 세포신호전달 심포지움 Vol. No.1

Effect of insulin on cell death induced by serum deprivation was examined in SK-N BE(2) and SK-N-SH human neuroblastoma cell lines. Insulin suppressed cell death by serum deprivation in SH cell line, however, accelerated cell death in BE(2) cell line. To address why insulin stimulates cell death by serum deprivation in BE(2) cells, we examined insulin mediated signaling in two cell lines. Phosphatidylinositol 3-kinase(PI3-kinase) was normally activated by insulin in both cell lines. H₂O₂ generation increased in response to insulin in SH cells, but not in BE(2) cell. When BE(2) cells were cultured in serum-free media in the presence of exogenous H₂O₂, cell death was suppressed by insulin. The generation of H₂O₂ by insulin in BE(2) cells was restored with transfection of wild type Rac1 or constitutively active Rac 1(V12) but not in cells transfected with dominant-negative Rac(N17). Moreover, insulin suppressed cell death In cells overexpressing wild type Rac 1. These result indicate that the failure of H₂O₂ generation in BE(2) cells in response to insulin could be a reason for the acceleration of the cell death induced by serum deprivation.

Protein kinase A functions as a negative regulator of c-Jun N-terminal kinase but not of p38 mitogen-activated protein kinase in PC12 cells

Kyu Chung Hur 한국통합생물학회 2005 Animal cells and systems Vol.9 No.3

Cyclic-AMP-dependent protein kinase (PKA) seems to function as a negative regulator of the c-Jun NH2-terminal kinase (JNK) signaling pathway. We demonstrate here that the activity of the PKA catalytic subunit (PKAc) is reduced in apoptotic PC12 pheochromocytoma cells. Apoptotic progress was inhibited by dibutyryl cyclic AMP (dbcAMP), an analog of cAMP. The rescue by dbcAMP was attributable to inhibition of the JNK but not of the p38 signaling pathway, due to the induction of PKA activity. JNK was present in immunocomplexes of PKAc, and PKAc phosphorylated JNK in vitro. Presence of p38 kinase, however, was not prominent in immunocomplexes of PKAc. Our data suggest that JNK is a target point of negative regulation by PKAc in the JNK signaling pathway.

Reactive oxygen species (ROS) is needed for insulin-mediated signaling in SK-N-BE(2) human nueroblastoma cell line

Hur, Kyu Chung 이화여자대학교 세포신호전달연구센터 2000 고사리 세포신호전달 심포지움 Vol. No.2

In multicellular organisms, the behavior of individual cells such as their growth, differentiation, and migration is tightly controlled by their environment. Two major classes of controlling components are diffusible peptide growth factors and insoluble extracellular matrix(ECM) proteins. These molecules act independently and cooperatively on the cells. This study focused on the signal transduction pathway activated by peptide growth factors in the human neuroblastoma cell line, SK-N-BE(2) [BE(2)]. A previous report in my laboratory demonstrated that BE(2) cells did not respond normally to the peptide growth factor such as insulin and NGF. To figure out why these cells did not respond to these growth factors, I examined insulin signaling in these cells. Tyrosine phosphorylation induced by insulin in BE(2) cells was much less than in PC12 cells although the tyrosine phosphorylation of insulin receptor was increased. The phosphorylation of IRS-1 in BE(2) cells was also increased less than in PC12 cells, and neither Grb2 nor GAP that regulates Ras activity interacted with the phosphorylated IRS-1. Moreover, MAP kinase, the downstream of Ras, was rarely activated by insulin in BE(2) cells. PI3-kinase bound to IRS-1 and the activity of PI3-kinase was increased by insulin in BE(2) cells. These results indicate that these cells may have some defects in the insulin-mediated Ras-MAP kinase activation. To find out whether Ras-MAP kinase pathway in BE(2) cells has defects, BE(2) cells were treated with pervanadate, a PTPase inhibitor. The tyrosine phosphorylation of many proteins, interaction of IRS-1 with Grb2, and MAP kinase activity were observed. These results suggest that BE(2) cells have defects not in Ras-MAP kinase cascade but in upstream of Ras-MAP kinase pathway activated by insulin. Recently it has been demonstrated that reactive oxygen species(ROS) including H₂O₂ and superoxide anion(O₂^(-)) is produced by cytokines and growth factors and may have a general role in mediating tyrosine phosphorylation through the inhibition of protein tyrosine phosphatases(PTPases). Insulin decreased ROS in BE(2) cells. To examine the possibility that the decrease of ROS by insulin could inactivate Ras-MAP kinase pathway, I applied H₂O₂ to BE(2) cells by adding glucose oxidase to the culeutr medium then treated the cells with insulin. Insulin enhanced the tyrosine phosphorylation of many proteins, interaction of IRS-1 with Grb2, and MAP kinase activity. To confirm the role of ROS in Ras-MAP kinase pathway, PC12 cells which respond normally to insulin were treated with N-acetylcysteine(NAC), a ROS scavenger, and the treatment of insulin was followed. Those results represented the opposite effects: NAC reduced the total tyrosine phosphorylation, interaction of IRS-1 with Grb2 and MAP kinase activity by insulin. These data represent that insulin-induced Ras-MAP kinase pathway requires an increase in intracellular H₂O₂. To identify a signaling molecule responsible for the defect of BE(2) cells in ROS generation, Rac1 gene from BE(2) cells was sequenced because Rac1 plays an important role in generating ROS in both phagocytic and nonphagocytic cells. The sequence of Rac1 in BE(2) cells has three substitutions in nucleic acids resulting amino acid changes. From these results, I concluded that the unresponsiveness of BE(2) cells to insulin is likely due to the abnormality of a ROS generation system that regulates Ras-MAP kinase pathway, and that abnormal Rac1 protein is the cause of this defect.

Effect of Calmodulin on the Phosphorylation of the Lens Membrane Sunstrates for Protein Kinase A

Hur, Kyu Chung,Kim, Kyu-Won Korean Society of Molecular Biology 1993 Molecules and cells Vol.3 No.4

전력계통의 안정도 향상을 위한 유전알고리즘을 이용한 TCSC 안정화장치 설계

정문규(Mun-Kyu Chung),정형환(Hyeong-Hwan Chung),허동렬(Dong-Ryol Hur),주석민(Seok-Min Joo),정동일(Dong-Il Chung) 대한전기학회 2003 대한전기학회 학술대회 논문집 Vol.2003 No.7