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Protein Kinase C와 S6 Kinase에 關한 硏究
朴吉洪,黃祐翊 고려대학교 의과대학 1990 고려대 의대 잡지 Vol.27 No.1
To investigate the role of protein kinase C(PKC) and S6 kinase in the signal transduction of the factors which exhibit the growth promoting activity including serum growth factors, phorbol-12-myristate-13-acetate(PMA) and insulin. The enzymes were purified and characterized from human colon cancer cell, HRT-18. Meanwhile, biosynthetic labelling of phosphoproteins were performed with [³²P]H₃PO₄ in HRT-18 and mouse embryo fibroblast(MEF) under the influence of epidermal growth factor(EGF), fetal bovine serum(FBS), PMA and insulin, Furthermore, in vivo substrates of PKC and S6 kinase were observed in nucleus, mitochondria, cytosol and ribosome fractions of HRT-18, which were compared with phosphoproteins obtained by biosynthetic labelling. Accordingly, it was deduced that the enzymes are involved in phosphorylation of intracellular proteins, which lead to cell proliferation somehow and represent a component of the signal transduction pathway of growth promoting signals, and in addition, two enzymes as a signal transducer are surprisingly overlapping in physiologic activities and most probably closely related in sequence in the pathway. The data obtained are as follows. 1. Each of S6 kinase and PKC was purified 63 and 120 fold with a specific activity of 1790 and 4850 pmol of γ-phosphate of ATP transferred per min per mg of protein, respectively. 2. Both of S6 kinase and PKC were eluted at 0.07M NaCl concentration in 20mM Tris buffer pH7.5 on DEAE-Sephacel chromatography. 3. Molecular weights of PKC and S6 kinase were turned out to be 55,000 both. 4. As for the biosynthetic labelling of HRT-18 with [³²P]H₃PO₄, whole cell homogenate unravel 43KD, 36KD, 30KD and 20KD phosphoproteins, the activity of phosphorylation being highest in EGF stimulated cells. In the meantime, 40KD, 36KD and 22KD phosphorylation products were synthesized in mouse enibryo fibroblast, the generation of which was highest in the presence of PMA. 5. In the nucleus of HRT-18, elevated were the formation of 43KD, 36KD and 30KD phosphoproteins after biosynthetic labelling of HRT-18 with [³²P]H₃PO₄, wherein influences of growth factor, PMA or insulin conferred no influence on the profile. 6. Observation of in vivo substrates of S6 kinase and PKC are outlined as below. Two enzymes possess in common as their in vivo substates 43KD, 36KD, 20KD and 17KD proteins in the nucleus, 36KD, 20KD and 17KD proteins in the mitochondria, 67KD protein in the cytosol and 67KD, 36KD, 30KD, 30KD and 17KD proteins in the ribosome. Interestingly, 20KD and 17KD proteins existed in nucleus, mitochondria and ribosome in contrast to their absence in cytosol, which implies their possibility to be ribosomal proteins, where S6 kinase phosphorylated 17KD protein more strongly than PKC. On the other hand, phosphorylation of 30KD protein was discriminated only in ribosomal fractions, being more prominent also by S6 kinase than PKC. 7. The relatively consistent ratio of PKC to S6 kinase activity at each step of purification and the same NaCl concentration a t which two enzymes coeluted on DEAE-Sephacel chromatography and the similarities in physiologic substrates of two enzymes in subcellular fractions including nucleus, mitochondria, cytosol and ribosome, strongly suggest the intimate correlation of two enzymes in the signal transduction pathway of growth promoting signals, which can even lead to the assumption of their physically associated entity. 8. Discrepancy between the phosphoprotein profile generated by biosynthetic labelling with 32Pi and enzyme reaction with [γ-³²P]ATP reflect that the in vivo phosphate donor of S6 kinase and PKC may be a compartment separate from of the total cellular ATP.
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박길홍,JaegolChoe,Hyo-JungChoo,박윤규,손정원,Meyoung-konKim 생화학분자생물학회 2002 Experimental and molecular medicine Vol.34 No.3
Previous reports raised question as to whether 8-chloro-cyclic adenosine 3,5-monophosphate (8-Cl-cAMP) is a prodrug for its metabolite, 8-Cl-adenos-ine which exerts growth inhibition in a broad spec-trum of cancer cells. The present study was cariedout to clarify overall cellular afects of 8-Cl-cAMPand 8-Cl-adenosine on SK-N-DZ human neuroblas-toma cells by systematically characterizing geneexpression using radioactive human cDNA microaray.Microarray was prepared with PCR-amplified cDNAof 2,304 known genes spotted on nylon membranes,employing 33P-labeled cDNAs of SK-N-DZ cells as aprobe. The expression levels of approximately 100cDNAs, representing about 8% of the total DNA ele-ments on the array, were altered in 8-Cl-adenosine- or8-Cl-cAMP-treated cells, respectively. The genome-wide expression of the two samples exhibited partialoverlaps; different sets of up-regulated genes butthe same set of down-regulated genes. 8-Cl-adenos-ine treatment up-regulated genes involved in difer-entiation and development (LIM protein, connexin26, neogenin, neurofilament triplet L protein andp21WAF1/CIP1) and immune response such as naturalkiller cells protein 4, and down-regulated onesinvolved in proliferation and transformation (trans-forming growth factor-b, DYRK2, urokinase-typeplasminogen activator and proteins involved in tran-scription and translation) which were in close paral-lel with those by 8-Cl-cAMP. Our results indicatedthat the two drugs shared common genomic path-ways for the down-regulation of certain genes, butused distinct pathways for the up-regulation of dif-ferent gene clusters. Based on the findings, we sug-gest that the anti-cancer activity of 8-Cl-cAMPresults at least in part through 8-Cl-adenosine. Thus,the systematic use of DNA arrays can provideinsight into the dynamic cellular pathways involvedin anticancer activities of chemotherapeutics.
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홍성철,김덕래,김태연,정전은,박길홍 大韓成形外科學會 1991 Archives of Plastic Surgery Vol.18 No.1
The mechanism of tendon healing has been a controversial issue. Although there seems to be a dipolarizing opinion regarding the tendon healing, it is well known to us that the tendon has both intrinsic and extrinsic healing capacity which has proved in vivo and in vitro studies. Tendon adhesion is a common sequella after tendon repair, particularly after the ling period of immobilization. This can be explained by one wound concept or extrinsic tendon healing. If we can modify the natural healing process of the tendon by augmenting the intrinsic capacity, we can reduce the period of immobilization to allow early motion for better gliding. in this study, we used the electrical stimulation technique, which has been known to have good growth stimulatory effect on the living cells, to investigate its effect on tendon healing in organic culture for 6 weeks. Total 64 flexor tendons were harvested from 8 rabbit paws and they were cultured in separate petri-dishes.: 32 tendons were used for the control group and the rest 32 were used for the electric stimulation group. Tendons through which a continuous 7 μAmp current was passed at the repair site were compared with the non stimulated controls. In reference to the histologic study with Hematoxylin ?? Eosin stain and van Gieson stain, the following results were obtained. 1. There was found no necrosis in the specimens of the control as well as the elctrical stimulation group during 6 weeks' culture period. 2. Starting from 1 week, there was prominent increase of fibroblastic proliferation and collagen production in electrical stimulation group and these active repair processes persisted up to 6 weeks. Accordingly, I conclude that the intrinsic tendon healing capacity can be enhanced by the electrical stimulation in vitro.
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Restriction Fragment Length Polymorpohisms 분석을 이용한 진행성 근이양증의 보인자 검색
박길홍(PARK Gil Hong),박선화(PARK Sun Hwa) 대한체질인류학회 1991 해부·생물인류학 (Anat Biol Anthropol) Vol.4 No.1
진행성 근이양증을 가진 두 가족을 대상으로 혈청 creatine kinase치 측정과 더붙어 Restrietion Fragment Length Polvmorphi는(RFLPs) linkage 분석을 통해 보인자 검출을 시행하였다. probe는 진행성 근이양증 유전자내 위치한 것으로서 pERT 87 1 (Xmn 1), pFRT 87 8(Taq 1. Bst XI), pERT 87.15(Xmn 1, Bam HI), 근접한 prode로서 D2(Pvu Ⅱ) 및 pXUT23(B히 Ⅱ)dmf 사용하였다. 진단 대상 6명중 2명은 각각 97.1%, 99.9% 확률로 보인자였고, 4명은 99.9% 확률로 정상임을 진단하였다. 이상의 결과로 미루어 RFLPs linkage 분석법은 진행성 근이양증 뿐아니라 다른 X-염색체 열성 유전병에도 적용되어 대아 진단과 보인자 검색에 보다 정확한 진단법이 될 것으로 사료된다.
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항공사진을 이용한 우리나라 활엽수림의 임분구조에 (林分構造) 관한 연구
박길홍 ( Kil Hong Park ) 한국산림과학회 1986 한국산림과학회지 Vol.74 No.1
To investigate the stand structure of the stocked broad-leaved forests in Korea, 1,000 plots, allocated by systematic sampling method, were interpreted on the aerial photographs accompanied with ground survey. Total area of the stocked broad-leaved forests except Jeju island was 818,286㏊ and the percentage to total forest area was 12.7%. Total stock volume of the stocked broad-leaved forests was 38,890,779㎥ and the percentage to total stocked forest volume was 27.4%. Mean number of trees per ㏊ was 947 trees/㏊, basal area was 11.17㎥/㏊, DBH was 11.30㎝, tree height was 7.65m, stock volume was 44.96㎥/㏊, and current annual volume increment was 3.64㎥/㏊ in total land. The 64.7, 79.8 and 52.7 percent of the stocked broad-leaved forest area were distributed at elevations of 300-900m, in slope degree of above 25, and in northern aspect, respectively. Standfactors were apt to get better with the increase of distance from the car road way and the village, and with the increase of elevation belt.
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