종양조직의 Transfer Ribonucleic Acid의 Methyl화에 관한 연구

이근배,이희성,이강석,김기수,Lee, Keun-Bai,Lee, Hi-Sung,Lee, Kang-Suk,Kim, Ki-Sao 생화학분자생물학회 1971 한국생화학회지 Vol.4 No.1

정상조직으로는 동물의 간, 종양조직으로는 Sarcoma 180 ascites tumor cells 및 Walker 256 carcinosarcoma 등을 시료로 사용하여 tRNA를 정제 하였고 또 같은 조직에서 methylase를 추출하여 in vitro에서 tRNA를 homologous 및 heterologous하게 methyl화를 행하여 다음과 같은 몇가지의 결과를 얻었다. 1. Homologous하게 methylate 시켰을 때 종양조직이 정상조직 보다 3 배의 methyl화 능을 나타냈다. 2. 종양조직의 methylase로 정상조직의 tRNA를 기질로 하여 heterologous 하게 methyl화 시켰을 때 정상조직의 homologous methylase에 비하여 4-5배의 methyl 화능을 나타냈다. 특히 Walker 256 carcinosarcoma의 methylase의 활성도가 가장 높았다. 3. 종양조직에 존재하는 tRNA methylase는 guanine 유도체 보다 adenine 유도체를 더 많이 methylate 시켰다. 4. Paper chromatography로 분리 한 methylated bases는 14종이며 그 중 13종의 화학구조가 확인 되었다. 5. 정상조직 및 종양조직에서 문헌에 보고되어 있지 않은 N,N'-dimethylcytosine을 종양조직인 Walker 256 carcinosarcoma에서 분리 확인 하였다. 이 물질은 Walker 256 carcinosarcoma의 methylase로서 다른 종의 tRNA를 methyl화 시켰을 때에도 분리 확인 할 수 있었다. 6. Adenine 유도체로 추정되는 1종류의 methylated bases를 종양조직인 Walker 256 carcinosarcoma 및 Sarcoma 180 ascites tumor cells에서 각각 분리 하였다. Studies on transfer ribonucleic acid (tRNA) methylases of normal tissues, Walker 256 carcinosarcoma and Sarcoma 180 ascites tumor cells have been carried out with respect to: 1) rate of methylation of tRNA from rabbit liver, Walker 256 carcinosarcoma, Sarcoma 180 ascites tumor cells and E. coli Band 2) an attempt was also made to search out the pattern of methylation, i.e., the specific bases methylated by the extracts of tumor tissues. The homogenate was centrifuged at 100,000 xg for 10 min and the supernatant fluid was used as the enzyme extracts. In vitro methylation of tRNA was carried out by incubating enzyme extract with substrate (tRNA) and ATP generating system. $^{14}C$-methyl-L-methionine was used as methyl donor. Resulting methylated tRNA was hydrolyzed and chromatographed on Whatman paper. Spots on the chromatograms were identified by measuring the Rf and UV absorption spectra and cut into strips and counted directly in a Beckman LS-100 liquid scintillation counter. The results obtained were as follows: 1. The tRNA methylases of tumor tissues exhibited the elevaton of methylating potencies about three-fold increase when methylated homologously. 2. The methylase activity of tumor tissues was increased 4-5 times over that of normal controls. The enzyme extracted from Walker 256 carcinosarcoma served as the most appropriate source. 3. Several lines of evidence for the binary nature of the tRNA methylase from tumor tissues were demonstrated. Results of heterologous methylations with a variety of substrate reveal that extracts of tumor tissues introduce more methyl group into adenine residue than into guanine residue of tRNA. 4. Exposure of tRNA to normal methylases followed by methylases from tumor tissues has indicated that the tumor tRNA methylases introduced methyl groups into different positions on the tRNA molecule than do normal enzymes. Among hydrolysate of methylated tRNA, a novel methylated pyrimidine, i.e., N,N'-dimethylcytosine and an unidentified base were detectable. The only extract from Walker 256 carcinosarcoma was found to possess methylase activity for the synthesis of N,N'-dimethylcytosine.

송아지 치수의 Histone

이근배,이희성,이강석 ( Keun Bai Lee,Hi Sung Lee,Kang Suk Lee ) 생화학분자생물학회 1973 BMB Reports Vol.6 No.2

Whole histone and histone fractions from calf dental pulp have been studied. Histones were extracted and fractionated according to the method of Johns and Phillips. The histone fractions were further purified by a polyacrylamide gel electrophoresis. The yield of whole histone recovered was 668 ㎎ per 100 g of dental pulp. This is very large amount as compared to the calf thymus and liver. However, nearly similar amount was obtained from transplantable ascites carcinoma cells. Quantitative differences were found between a number of corresponding histone fractions of calf dental pulp and thymus. Striking low contents of fraction f2a1 (arginine-rich histone) were observed in dental pulp, being about one third of those of calf thymus. Fraction f2a2 was also significantly decreased. However, slightly higher contents of histones were found in fractions f1 and f3 plus f2b. The electrophoretic behaviors were compared with those of histones from calf thymus and rat liver. Amino acid analysis of the individual histone fractions showed that the over-all compositions were remarkably similar to those of corresponding fractions from calf thymus. The data of comparable histone fractions of calf and rat isolated by other workers have been included for comparison. The evidence for the tissue-specific differences in the distribution of the histone fractions among different tissues of the single animal was presented.

송아지 치수의 Histone

이근배,이희성,이강석,Lee, Keun-Bai,Lee, Hi-Sung,Lee, Kang-Suk 생화학분자생물학회 1973 한국생화학회지 Vol.6 No.2

저자들은 송아지 치수에서 histone을 추출 분리하였다. Histone의 추출 및 분리는 Johns 및 Johns and Phillips 등의 방법으로 분리 하였으며, 각 histone 분획들은 15% polyacrylamide disc gel 전기영동에 의하여 순도 및 mobility를 다른 조직들의 그것과 비교 검토하였다. 즉 흰쥐 및 마우스의 간, 송아지의 흉선 및 Sarcoma 180 ascites tumor cells의 histone과 비교 및 약간의 고찰을 시도하였으며, 다른 사람의 업적을 인용하여 비교하였다. 1. 송아지 치수에서 histone 주분획인 f1, f1a1, f2a2, f2b 및 f3 분획을 분리하였다. 2. Histone의 수율은 조직 1g 당 6.68mg 이다. 3. 각 분획의 polyacrylamide disc gel 전기영동으로 mobility는 다른 조직의 histone과 거의 같았다. 4. 송아지 치수의 histone 분포는 송아지 흉선의 그것에 비하여 많은 차이가 있다. 즉 f3 분획이 전체 histone의 39%로 가장 많았고, f2a1 및 f2a2 분획은 각각 6% 및 10.8%로 가장 적었으며, 각 분획의 함량은 다른 조직의 그것에 비하여 많은 차이가 있다. 5. 각 histone 분획의 amino 산 조성은 다른 조직의 그것과 별차이가 없다. Whole histone and histone fractions from calf dental pulp have been studied. Histones were extracted and fractionated according to the method of Johns and Phillips. The histone fractions were further purified by a polyacrylamide gel electrophoresis. The yield of whole histone recovered was 668 mg per 100 g of dental pulp. This is very large amount as compared to the calf thymus and liver. However, nearly similar amount was obtained from transplantable ascites carcinoma cells. Quantitative differences were found between a number of corresponding histone fractions of calf dental pulp and thymus. Striking low contents of fraction f2a1 (arginine-rich histone) were observed in dental pulp, being about one third of those of calf thymus. Fraction f2a2 was also significantly decreased. However, slightly higher contents of histones were found in fractions f1 and f3 plus f2b. The electrophoretic behaviors were compared with those of histones from calf thymus and rat liver. Amino acid analysis of the individual histone fractions showed that the over-all compositions were remarkably similar to those of corresponding fractions from calf thymus. The data of comparable histone fractions of calf and rat isolated by other workers have been included for comparison. The evidence for the tissue-specific differences in the distribution of the histone fractions among different tissues of the single animal was presented.

Purification and Properties of Two Glyoxylate Reductases from Germinating Soybean Seed Embryos

이근배,이희성,이강석,Lee, Keun-Bai,Lee, Hi-Sung,Lee, Kang-Suk 생화학분자생물학회 1970 한국생화학회지 Vol.3 No.2

발아중인 대두의 배에서 두 종류의 glyoxylate reductase(glycoIIate-NAD oxidoreductase, EC. 1. 1. 1. 26)를 추출하고 ammonium sulfate에 의한 침전, Sephadex G-200 gel filtration, DEAE-cellulose 및 Sephadex G-100 column chromatography에 의하여 정제하였다. 두 종류의 효소는 각각 NADH 및 NADPH를 cofactor로 요구하는 바 각각 745배 및 789배 정제하였다. 최종적으로 분리 정제된 상기 효소는 sucrose density gradient centrifugation, starch 및 acrylamide gel electrophoresis를 행하여 단일 효소 단백임을 확인하였다. NADH-linked glyoxylate reductase의 Michaelis constant, Km은 $9.0{\times}10^{-3}M$ 이다. 또한 NADH-Iinked enzyme 및 NADPH-linked enzyme의 분자량은 Sephadex G-100 gel filtration으로 측정하여 각각 142,000 및 192,000 이었다. 이 효소의 최적 pH, 최적온도, thiol group 저해제 및 몇 가지의 금속 ion이 효소활성에 미치는 영향을 검토하였다. Two protein fractions which catalyse the reduction of glyoxylate to glycollate were isolated from germinating soybean seed embryos by ammonium sulfate precipitation, Sephadex G-200 gel filtration. DEAE-cellulose column chromatography and rechromatography on Sephadex G-100. One protein fraction required NADH and another protein fraction required NADPH as cofactor. These two enzymes were purified 745-fold and 789-fold, respectively. The final preparations were homogeneous by sucrose density gradient centrifugation, starch and acrylamide gel electrophoresis. The apparent Michaelis constant. Km, of NADH-linked glyoxylate reductase was estimated as approximately $9.0{\times}10^{-3}M$. Molecular weights of NADH-Iinked and NADPH-linked enzymes were estimated to be about 142,000 and 192,000, respectively, by Sephadex G-100 gel filtration. Effects of pH, temperature, thiol group inhibitors and various metal ions on the enzyme were also described.

종양조직의 Transfer Ribonucleic Acid 의 Methyl 화에 관한 연구

이근배,이희성,이강석,김기수 ( Keun Bai Lee,Hi Sung Lee,Kang Suk Lee ) 생화학분자생물학회 1971 BMB Reports Vol.4 No.1

Studies on transfer ribonucleic acid (tRNA) methylases of normal tissues, Walker 256 carcinosarcoma and Sarcoma 180 ascites tumor cells have been carried out with respect to: 1) rate of methylation of tRNA from rabbit liver, Walker 256 carcinosarcoma, Sarcoma 180 ascites tumor cells and E. coli B and 2) an attempt was also made to search out the pattern of methylation, i.e., the specific bases methylated by the extracts of tumor tissues. The homogenate was centrifuged at 100,000 xg for 10 min and the supernatant fluid was used as the enzyme extracts. In vitro methylation of tRNA was carried out by incubating enzyme extract with substrate (tRNA) and ATP generating system. ^(14)C-methyl-L-methionine was used as methyl donor. Resulting methylated tRNA was hydrolyzed and chromatographed on Whatman paper. Spots on the chromatograms were identified by measuring the Rf and UV absorption spectra and cut into strips and counted directly in a Beckman LS-100 liquid scintillation counter. The results obtained were as follows 1. The tRNA methylases of tumor tissues exhibited the elevaton of methylating potencies about three-fold increase when methylated homologously. 2. The methylase activity of tumor tissues was increased 4-5 times over that of normal controls. The enzyme extracted from Walker 256 carcinosarcoma served as the most appropriate source. 3. Several lines of evidence for the binary nature of the tRNA methylase from tumor tissues were demonstrated. Results of heterologous methylations with a variety of substrate reveal that extracts of tumor tissues introduce more methyl group into adenine residue than into guanine residue of tRNA. 4. Exposure of tRNA to normal methylases followed by methylases from tumor tissues has indicated that the tumor tRNA methylases introduced methyl groups into different positions on the tRNA molecule than do normal enzymes. Among hydrolysate of methylated tRNA, a novel methylated pyrimidine, i.e., N,N`-dimethylcytosine and an unidentified base were detectable. The only extract from Walker 256 carcinosarcoma was found to possess methylase activity for the synthesis of N,N`-dimethylcytosine.

경피적 자가 골수 및 이종골 이식을 이용한 고립성 골낭종의 치료

이근배,노성만,윤택림,손일진,정성택,Lee, Keun-Bae,Rowe, Sung-Man,Yoon, Taek-Rim,Son, Il-Jin,Jung, Sung-Taek 대한근골격종양학회 2003 대한골관절종양학회지 Vol.9 No.1

목적: 소아에 발생한 고립성 골낭종에 대해 경피적 자가 골수 및 이종골의 혼합 이식을 이용한 치료의 결과를 알아 보고자 하였다. 연구대상 및 방법: 1996년 1월부터 1999년 2월까지 경피적으로 자가 골수 및 이종골의 혼합 이식을 이용하여 치료한 고립성 골낭종 7예를 대상으로 하였다. 성별 분포는 남자가 4예, 여자가 3예였으며, 평균 연령은 10세(6~15), 평균 추시 기간은 35.6개월(20~52)이었다. 발생 부위별로 상완골 근위부 및 간부가 3예, 대퇴골 근위부가 3예, 장골(Ilium)이 1예였으며, 병소의 면적은 평균 14.7 $cm^2$(10~23)였다. 활성도는 활성형이 6예, 비활성형이 1예였으며, 과거력상 스테로이드 주입술을 시행받은 경우는 5명으로 평균 3.2회였다. 치료에 사용된 골수의 양은 평균 14.3 ml(10~20)였으며, 이종골로는 $Lubboc^{(R)}$(Transphyto S.A. Clermont Ferrand, France)을 사용하였으며 사용 갯수는 평균 6.4개(5~10)였다. 결과는 Neer의 분류법에 따라 판정하였다. 결과: 낭종의 전체가 신생골로 대체되어 완전한 치유를 보인 경우가 5예, 전체적으로 골 경화가 보이지만 부분적인 낭종이 관찰되는 경우가 2예로, 전예에서 만족스런 결과를 보였으며 수술중이나 술후에 합병증의 발생은 없었다. 결론: 고립성 골낭종의 치료를 위한 경피적 자가 골수 및 이종골의 혼합 이식술은 비교적 쉽고, 수술로 인한 합병증이 적으며, 치유율이 매우 높을 뿐만 아니라, 자가골 이식으로 인한 공여부의 문제점을 피할 수 있어 권장할 만한 좋은 치료법이라 생각된다. Purpose: To clarify the results of simple bone cyst (SBC) treatment in children by percutaneous autologous bone marrow grafting and xenografting. Materials and Methods: We studied seven cases (4 males, 3 females) of SBC, which were treated by percutaneous autologous marrow and heterograft bone grafting from January 1996 to February 1999. Their mean age at surgery was 10 years (6 to 15), and the mean follow-up period was 35.6 months (20 to 52). Three cases were located in the proximal and middle humerus; three cases were in the proximal femur; and one case occurred in the ilium. Mean volume was 14.7 $cm^2$ (10 to 23). Six cases were active, and one was inactive. Five patients had a history of receiving a mean of 3.2 steroid injections. The mean quantity of bone marrow used in treatment was 14.3 ml (10 to 20), and the mean amount of $Lubboc^{(R)}$ heterograft bone (Transphyto S.A. Clermont Ferrand, France) used was 6.4 blocks (5 to 10). Results were analyzed using the modified Neer classification. Results: Five cases completely healed with obliteration of the cyst cavity (Grade IV). Two cases demonstrated sclerosis around a partially visible cyst (Grade III). All treatment results were satisfactory and without intraoperative or postoperative complications. Conclusione: Percutaneous autologous marrow and heterograft bone grafting is recommended as an effective treatment method for simple bone cyst. It offers ease of operative technique, a high rate of healing, a low recurrence rate, low morbidity, a low incidence of postoperative complications, and free from bone graft donor site problems.

방사선 조사가 Sarcoma 180 Ascites Tumor Cells 의 Histone 생합성에 미치는 영향

이근배,이희성 ( Keun Bai Lee,Hi Sung Lee ) 생화학분자생물학회 1972 BMB Reports Vol.5 No.2

In the earlier works from this laboratory, biosynthesis, acetylation, methylation and phosphorylation of histone of transplantable ascites tumor cells have been studied. The present paper deals with the effects of X-ray irradiation on histone biosynthesis of Sarcoma 180 ascites tumor cells. The tumor-bearing mice were irradiated with a dose of 1250 roentgen prior to 24 hours double-labeling with 10 μCi of glycine-l-^(14)C and 20 μCi of ^(32)P-orthophosphate. Sham irradiated mice were used as control. Histones were prepared by Johns procedure and fractionated on polyacrylamide disc gel electrophoresis. The histones were hydrolyzed in acid and the digests were chromatographed on an amino acid analyzer. DNA was extracted according to the method of Ogur and Rosen. Radioactivity was counted in a liquid scintillation spectrometer. The time course changes in the incorporations of labeled compounds into histone and DNA have been followed. Results obtained were as follows ; 1. Contents of histone fractions from Sarcoma 180 ascites tumor cells have been shown to markedly differ from normal tissues, i.e. mouse liver, rat and calf thymus. 2. X-ray irradiation hindered ^(14)C-glycine incorporation into histone by 14 per cent. Uptake of ^(14)C was highest into f3 and was lowest into f2b among histone fractiones both in normal and irradiated mice. The incorporation of ^(14)C-glycine into histone reached a maximum 4 hours after glycine administration. 3. Radioactivity of ^(14)C-glycine was less distributed about 12 per cent in glycine fraction of histone hydrolysates from X-ray irradiated animal. 4. A decreased ^(32)P-incorporation (about 14%) into DNA was observed in irradiated mice. Relationships between histone biosynthesis and DNA metabolism were also discussed.

Histone 에 관한 연구 ( Ⅸ ) Walker 256 carcinosarcoma 의 Histone 에 대하여

이근배,이희성,라석찬 ( Keun Bai Lee,Hi Sung Lee,Suck Chan Rha ) 생화학분자생물학회 1977 BMB Reports Vol.10 No.3

In attempt to investigate histone fractions of tumor cells, nuclei was isolated from Walker 256 carcinosarcoma by the procedure of Pogo et al., Histone fractions Hl, H2a, H2b, H3 and H4 were prepared from isolated nuclei by the procedure of Johns. The five histone fractions found in most tissues were also present in the Walker 256 carcinosarc:oma histones. These histone fractions were characterized by amino acid analysis and by polyacrylamide disc gel electrophoresis. The results obtained were as follows: 1. The yield of whole histone was 3.75me per g of Walker 256 carcinosarcoma. 2. The yield of DNA was 3.35mg per g of Walker 256 carcinosarcorna. Consequently the DNA to histone ratio was 1 : 1.12. 3. The relative amounts of five fractions, i.e., Hl, H2a, H2b, H3 and H4 were 20.00%, 9.33 %, 26.67%, 33.33 % and 10.67%, respectively. 4. The electrophoretic mobility of individual histone fractions gave almost similar patterns to those of corresponding fractions of calf thymus. 5. Amino acid analysis of the individual histone fractions showed that the over-all com positions were similar but not identical to those of the corresponding fraction from calf thymus. 6. We found that histone H2b fraction of Walker 256 carcinosarcoma contained detectable amounts of ε-N-monomethyllysine and ε-N-dimethyllysine. No evidence for the presence of methylated lysine or other side-chain derivatives was reported on this histone fraction. We also found that histcne H1 fraction contained detectable amounts of 3-methylhistidine.

1. 건설재해 예방활동의 방향제고

이근배,Lee, Geun-Bae 한국건설안전협회 1997 建設 安全技術 Vol.24 No.-

마우스 간 핵소체의 In Vitro 단백질 생합성

이근배,이희성,Lee, Keun-Bai,Lee, Hi-Sung 생화학분자생물학회 1970 한국생화학회지 Vol.3 No.1

마우스 간 핵소체를 Busch 등의 방법에 의하여 분리하여 [$^{14}C$] amino acid의 핵소체 단백질에의 incorporation을 관찰하였다. 이 때 반응액의 조건 및 주요한 조성 성분, 즉 pH, 금속 이온, incubation의 기간, 단백질 생합성에 소요되는 효소 및 기질을 조절하면서 단백질 합성에 미치는 영향을 관찰하였다. 몇몇 hormone의 영향도 아울러 관찰하였다. 핵소체는 완전한 protein-synthesizing system을 구비하고 있는 것으로 생각된다. 즉 ATP-generating system 중의 구성성분을 하나씩 생략한 조건하에서도 [$^{14}C$] amino acid가 단백질에 incorporate하는 량에 거의 영향을 미치지 않는다. 이 때 creatine phosphate는 예외이며 이 물질을 첨가하지 않으면 단백질 생합성이 약 41% 저해된다. Chloramphenicol 및 cycloheximide는 단백질 생합성을 저해하며 저해율은 대체로 이 물질들의 첨가 농도에 비례한다. Thyroxine은 [$^{14}C$] amino acid의 단백질에의 incorporation을 증가시킨다. Nucleoli were isolated according to the method of Busch et at., and the acceptability of incorporating labelled amino acid into protein was studied with systematic changes in certain important variables of medium composition, i. e., pH, concentration of metal ions, incubation periods, protein-synthesizing system and the supply of amino acids. The effects of some hormones on the protein synthesis were also studied. The nucleoli appear to be complete protein-synthesizing system within themselves. The activity of the [$^{14}C$] amino acid incorporation into protein was very little affected by the omission of one of the components of ATP-generating system, except creatine phosphate. The inhibition of protein synthesis was found to be proportional to the concentration of chloramphenicol and cycloheximide used. The thyroxine enhanced the incorporation of labelled amino acid into protein.