HPLC 에 의한 암의 지표로서의 뇨중 Ribonucleoside 의 분리 및 정량에 대한 연구

박경남(Kyung Nam Park),한중수(Joong Soo Han),고재경(Jai Kyung Koh) 대한내과학회 1987 대한내과학회지 Vol.33 No.2

N/A Urine contains certain metabolic end products of nucleic acid metabolism including sma1l amounts of modified nucleosides. They are methylated or otherwise structurally transformed ribosyl purines and pyrimidines and are not recycled through the salvage pathway but are excreted in urine. Their measurement in urine provides an accurate indicatar of the metabolism of RNA, especially transfer RNA. Altered patterns of excretion of these compounds might offer a sensitive biomarker for diagnosis and treatment of cancer. In order to find out whether the urinary ribonucleosides could be used as tumor markers for the hepatocellular carcinoma or stomach cancer, analysis of urine samples from normal controls and patients with hepatocellular carcinoma and stomach cancer was performed using reversed-phase HPLC. For this study, a rapid and precise chromatographic method for the determination of ribonucleosides in urine by high-performance liquid chromatography has been developed, The ribonucleosides were first isolated with an affinity gel containing immobilized phenylboronic acid. Contents of pseudouridine, 5-methylcytidine, 1-methyladenosine, 7-methylguanosine, guanosine, 1-methylinosine and 1-methylguanosine were significantly increased in urine of patients with hepatocellular carcinoma. Among these elevated ribonucleosides, excretions of pseudouridine, 5-methylcytidine, 1-methylinosine and 1-methylguanosine were higher than the upper limits (mean+2SD) in control group providing positive rates above 78% (positive rates for the excretions of 5-methylcytidine and 1-methylinosine were 100 %). In patients with stomach cancer, urinary excretions of pseudauridine, cytidine, 5-methylcytidine, 7-methylguanosine and adenosine were significantly in- creased. Positive rates for those of pseudouridine and 5-methylcytidine were above 50o. The results suggest that urinary levels of pseudouridine, 5-methylcytidine, 1-methylinosine and 1-methylguaaosine for the hepatocellular carcinoma and those of pseudouridine and 5-methylcytidine for the stomach cancer are useful as diagnostic tumor markers.

구연발표 : 간세포암종과 위암환자의 암조직 , 혈청 및 복수에서 핵산분해 효소활성과 뇨중 핵산분해산물의 배설량 변동에 관한 연구

박경남(Kyung Nam Park),한동수(Dong Soo Han),함준수(Jun Soo Ham),이민호(Min Ho Lee),기춘석(Choon Suck Kee),고재경(Jai Kyung Koh) 대한소화기학회 1987 대한소화기학회 추계학술대회 Vol.1987 No.-

위궤양의 발생기전에 대한 시험적 및 임상적연구 - Prednisolone 투여 백서의 위 Pepsin 의 성상에 관한 연구 -

박경남,기춘석,장호연,고재경 ( Kyung Nam Park,Choon Suhk Kee,Ho Yun Chang,Jai Kyung Koh ) 대한소화기학회 1980 대한소화기학회지 Vol.12 No.1

Because of its potent proteolytic nature, pepsin has been known to play an important role 'in ulcerogenesis of the stomach '>. The rate of pepsin secretion by stomach was reported to be stimulated by histamine'>, gastrin', pentagastrin >, caerulein >, secretin >, cholecystokinin> and glucocorticoids >, and inhibited by vagotomy>, histamin antag- .onists> and prostaglandins . It has been suggested that compounds which can timulate pepsin output may cause gastric ulcer and that drugs which exhibit antipeptic activity could be used as therapeutic agents for gastric ulcer. The introduction of glucocorticoids into medical practice with concomitant increase in the incidence of gastric ulcer made it clear that the adrenal glucocorticoids had a good deal to do with pepsin output and its activity-. However, no definite evidence for that tle pepsin secretion and its activity are affected by acute or chronic administration of glucocorticoids has been obtained in both experimental and clinical studies. In order to investigate the effect of glucocorticoids on pepsin activity in gastric juice, changes in the activity of pepsin vere observed in both basal a.nd gastopsin stimulated gastric juice collected from rats treated with prednisolone for fifteen days. Also studied were properties of pepsin in gastric juice to screen the compounds which specifically activate or inhibit the enzyme through the in vitro experiment .

肝細胞癌腫患者의 腹水 Ribonuclease의 活性度와 分離에 關한 硏究

孫樂志,朴炅南,高在炅 한양대학교 의과대학 1986 한양의대 학술지 Vol.6 No.1

In order to investigated whether some of the enzyme activities were changed in ascitic fluid from patients with hepatocellular carcinoma, activities of ribonuclease(RNase) and amylase were determined in ascitic fluid from patients with liver cirrhosis and hepatocellular carcinoma, and the results were compared. Neutral RNase in ascitic fluid from patient with hepatocellular carcinoma were separated and fractionated by both DEAE-cellulose column chromatography and polyacrylamid gel electrophoresis to find out whether the RNase specific to hepatocellular carcinoma was present in the ascitic fluid of hepatocellular carcinoma. 1) Concentrations of DNA, RNA and proteins in ascitic fluid higher in hepatocellular carcinoma than in liver cirrhosis, and the positive rates of DNA, RNA and protein contents in ascitic fluid as a marker for hepatocellular carcinoma against liver cirrhosis were observed to be 67%, 42% and 67% respectively. 2) Neutral RNase activities in ascitic fluid from patients with hepatocellular carcinoma was shown to be higher by 62% than those from patient with liver cirrhosis. As a marker for hepatocellular carcinoma, the positive rate of neutral RNase in ascitic fluid was shown to be high value of 67% against liver cirrhosis. 3) Amylase activities in ascitic fluid from patients with hepatocellular carcinoma was unchanged as compared with those from patients with liver cirrhosis. 4) DEAE-cellulose column chromatographical analyses revealed that neutral RNase in ascitic fluid were separated into 3 peaks in liver cirrhosis and into 4 peaks in hepatocellular carcinoma. The peak Ⅱ RNase absent in liver cirrhosis was observed in hepatocellular carcinoma. 5) Native polyacrylamide gel electrophoretic study on ascitic proteins revealed that electrophoretic protein patterns in liver cirrhosis are similar in the peak Ⅰ to, but different in the peaks Ⅲ and Ⅳ from those in hepatocellular carcinoma. These results indicated that proteins specific for hepatocellular carcinoma might be present not only in the peak Ⅰ, but also in the peaks Ⅲ and Ⅳ fractions.

위암 환자의 위암조직과 위액에서 위암에 특이한 단백에 관한 연구

박경남,신창록,고재경 한양대학교 의과대학 1994 한양의대 학술지 Vol.14 No.1

In order to investigate cancer specific proteins associated with stomach cancer, proteins of stomach cancer tissue and gastric juice from patients with stomach cancer were separated by a DEAE-cellulose column chromatography, high performance liquid chromatography(HPLC) and polyacrylamide gel electrophoresis, and the results were compared with those from control tissue and gastric juice. Protein concentration of cancer tissue and gastric juice of patients with stomach cancer was significantly increased and positive rate of protein contents in stomach cancer tissue and gastric juice as a marker for stomach cancer was high, indicating the possible use of of the protein contents as a biochemical marker for the stomach cancer. Proteins in the stomach cancer tissue were separated by a DEAE cellulose column chromatography into 6 peak proteins, of which a single peak protein (peak Ⅲb) was found to be specific to the cancer and proteins in the gastric juice from patient with stomach cancer were separated into 8 peak proteins, of which three protein peaks(peak Ⅱ, Ⅲ and Ⅳb) were specific to the cancer. Peak Ⅰ and Ⅴ proteins isolated from both cancer tissue and gastric juice from patient with stomach cancer were observed to be activated, suggesting a possible role of these proteins in carcinogenesis and suppression of the stomach cancer. Isolation patterns for proteins of the stomach cancer tissue appeared to be different form those of the control tissue, showing presence of more than 8 protein bands specific to the cancer and disappearance of more than 2 protein bands specific to the cancer and disappearance of more than 2 protein bands from the stomach cancer tissue. The results indicated that changes in proteins of cancer tissue and gastric juice from patient with stomach cancer did not take place in a single protein, but did occur in multiple proteins. Qualitative changes in the proteins were variable in nature, such as presence of cancer specific proteins, disappearance of proteins from the cancer tissue and gastric juice and activiation of the proteins.

胃癌組織內의 Acid Deoxyribonuclease 活性 및 性狀에 關한 硏究

咸駿洙,朴炅南,高在炅 한양대학교 의과대학 1985 한양의대 학술지 Vol.5 No.1

In the present study, activities of enzymes involved in deoxyribonucleic acid (DNA) degradation, deoxyribonuclease (DNase) Ⅰ, Ⅱ, Ⅲ and Ⅳ, were measured in extracts of stomach cancer tissues (20 cases) and were compared with those in extracts of normal control tissues. In order to clarify a possible association of acid DNase with carcinogenesis of stomach cancer, characteristic properties of acid DNase and mechanism of hydrolytic cleavage of DNA by acid DNase in stomach cancer tissues were studied. 1. Contents of DNA and protein were significantly higher in stomach cancer tissues than in normal control tissues, but that of RNA was not changed. 2. Of 4 types of DNase studied, the highest activity was observed with acid DNase (DNase Ⅱ) in both normal control and stomach cancer tissues, and the activity of acid DNase was significantly higher in stomach cancer tissues than in normal control tissues. 3. Acid DNase in stomach cancer tissues hydrolyzed double stranded DNA more rapidly than did single stranded DNA, but did not exhibit absolute specificity toward double stranded DNA. 4. Most of the products of enzymatic hydrolysis of DNA by acid DNsase in stomach cancer tissues were found to be polydeoxyribonucleotides with nucleotide length more than 20. Some of the hydrolytic products were, however, observed to be monodeoxyribonucleotides. Observations that the activity of acid DNase in stomach cancer tissues 1) was significantly increased, 2) was highly active against double stranded DNA and 3) cleaved DNA endonucleolytically support the assumption that the DNase might play a role in transforming normal cells into cancer cells by modifying a part of host genome.

간세포암종 환자의 혈청,복수 및 간조직의 Nuclease활성과 단백분획에 대한 연구

정선근,박경남,고재경 한양대학교 의과대학 1987 한양의대 학술지 Vol.7 No.1

Activities of nucleases were determined in liver tissues, serum, and ascitic fluid of patients with liver cirrhosis and hepatocellular carcinoma and were compared with those of normal control. Ribonucleases (RNases) and proteins in these liver tissues and body fluid were isolated and fractionated by a polyacrylamide gel electrophoresis (PAGE) and a DEAE-cellulose column chromatography to investigate the prescence of the enzyme and proteins specific to hepatocellular carcinoma. 1. In hepatocellular carcinoma tissues, acid deoxyribonuclease (DNase) activity was greatly increased and neutral RNase activity was significantly decreased as compared with those of normal control liver tissues. Activities of acid DNase and neutral RNase were, however, unchanged in liver cirrhosis tissues. 2. Activities of neutral RNase in both serum and ascitic fluid of patients with hepatocellular carcinoma were significantly increased, and the positive rate of the serum and ascitic fluid enzymes as a marker for hepatocellular carcinoma were over 50%, indicating that activities of neutral RNase in serum and ascitic fluid might be useful for the diagnosis of hepatocellular carcinoma. 3. PAGE patterns for proteins in liver tissues, serum, and ascitic fluid of patients with hepatocellular carcinoma appeared to be different slightly from those of normal control and of liver cirrhosis. No distinct new protein bands were, however, found in the PAGE protein patterns of hepatocellular carcinoma. 4. DEAE-cellulose column chromatography for RNases and proteins in ascitic fluid of patients with hepatocellular carainoma revealed that five RNase and protein peaks were isolated, of which two peaks (peak Ⅱand Ⅲ) were apparently specific to hepatocellular carcinoma.

위암조직에서 Ribonuclease와 Ribonuclease Inhibitor의 상호작용에 관한 연구

박경남,안광무,고재경 한양대학교 의과대학 1994 한양의대 학술지 Vol.14 No.1

In order to understand the processes involved in carcinogenesis and suppression of stomach cancer, activities of ribonuclease(RNase) and RNase inhibitor were measured in the stomach cancer tissue and were compared with those in the control tissue of the stomach. Also separated were RNases and RNase inhibitors from stomach cancer tissue by a DEAE cellulose colimn chromatography to find out the RNase and RNase inhibitor specific to the stomach cancer. Activities of acid and neutral RNase were signigicantly decreased in stomach cancer tissue and positive rates of the enzymes as markers for the stomach cancer were high suggesting the use of RNases as a biochemical marker for the stomach cancer. Activity of RNase inhibitor expressed as latent RNase activity was unchanged, although it tended to be decreased. RNases in the stomach cancer tissue was separated by a DEAE-cellulose column chromatography into six isozymes, of which RNase isozyme Ⅱ was found to be specific to the cancer and RNase isozyme V was activated. The RNase isozyme Ⅲb isolated in the control tissue disappeared from the cancer tissue. Of the RNase isozymes isolated from the stomach cancer tissue, RNase Ⅰ,Ⅱ and Ⅲ isozymes were nonseretory type of RNase active against RNA as substrate, RNase Ⅳ and Ⅴ isozymes secretory type of RNase active against poly C and RNase Ⅳ mixed type of RNase. On the other hand, RNase isozymes from the control tissue of the stomach were all secretory type of RNase except for RNase isozyme I. This indicated that RNase isozymes Ⅲ and Ⅵ isolated from the cancer tissue appeared to be different in nature from those from the control tissue. Activity of RNase inhigitor measured with poly C as a substrate was increased in RNase isozymes Ⅳ,Ⅴ and Ⅵ of the stomach cancer tissue, being increased greatly in the RNase isozyme Ⅴ. Observations that the RNase isozyme V isolated from the stomach cancer tissue was activated and exhibited higher acctivity of RNase inhibitor suggested that interaction between RNase isozyme V and RNase ingibitor play an important role in carcinogenesis and suppression of stomach cancer.

방광암조직 acid deoxyribonuclease의 작용기전에 관한 연구

김용석,박무남,고재경 한양대학교 의과대학 1993 한양의대 학술지 Vol.13 No.1

Deoxyribonuclease (DNase) activity was determined in the bladder cancer tissue and was compared with that of the control tissue. The acid DNase known to be associated with carcinogenesis was isolated and purified from the bladder cancer tissue, and the substrate specificity and mechanism of action of the enzyme were studied to investigate the role of the acid DNase in the process involved in carcinogenesis of the bladder cancer. Activities of DNase Ⅰ,Ⅲ and Ⅳ were unchanged, but activity of DNase Ⅱ (acid DNase) was greatly increased in the bladder cancer tissue by a DEAE-cellulose column chromatography and the peak from the enzyme was greater in the cancer than in the control. The partially purified acid DNase from the bladder cancer tissue was highly active toward ds DNA, but still active toward ss DNA (18% of activity with ds DNA). The enzyme did not exhibit species specificity toward the substrates studied. Analyses for the ds DNA digest by the acid DNase from the bladder cancer tissue showed that majority of the products was oligodeoxyribonucleotides with chain length of 5-25. This indicated that the acid DNase was an endonuclease in nature. The acid DNase purified from the bladder cancer tissue was inhibited by RNA and polyribonucleotides, the degree of inhibition being changed with base sequence of the polyribonucleotides studied. The present study indicated that 1) the acid DNase in the bladder cancer tissue was greatly increased, 2) the enzyme was endonuclease in nature and 3) the enzyme activity was inhibited by RNA and polyribonucleotides, suggesting that the acid DNase in the bladder cancer tissue might play an important role in the process involved in carcinogenesis of the bladder cancer and that the process could be modified by RNA or polyribonucleotides.

암의 지표로서의 위암조직내 ribonucleoside와 tRNA의 조성에 관한 연구

박경남,한중수,고재경,윤영준 한양대학교 의과대학 1989 한양의대 학술지 Vol.9 No.1

The level of ribonucleosides in perchloric acid extracts of the stomach cancer tissues was determined by reversed-phase high-performance liquid chromatography (HPLC). By comparing the levels of these compounds in the normal control portions with the neoplastic portions of tissues resected from malignant cancer patients, it was found that there was elevation of the pseudouridine level in all of the cancer tissue studied(35% increase over that in the normal control tissues). The concentrations of adenosine and cytidine in the stomach cancer tissue samples were decreased by 50% and 15% as compared to normal control levels, respectively. Determination of the modifications present in a transfer RNA (tRNA) and the extent to which they are modified is essential to the study of cellular development and differentiation. A HPLC method has been developed to quantify the major and modified nucleoside composition of hydrolyzed unfractionated tRNA. The method is rapid and sensitive and offers a high degree of resolution for analyzing both stable and unstable modified nucleosides. The method was applied for the analysis of 17 ribonucleosides in tRNA extracted from normal and neoplastic stomach tissues. For the major nucleosides, differences between cancer and normal control tissues were small. Contents of 7-methylinosine and 1-methylguanosine in the cancer tissue were almost equal to those in the normal control, but the other eleven modified nucleoside contents were increased in tRNA extracted from the cancer tissues. Among these elevated nucleosides, contents of pseudouridine, 4-thiouridine and N??-methyladenosine were significantly increased. When compared to normal control values, total modified nucleoside contents in tRNA extracted from the cancer tissues were increased to as high as 43%. The results indicated that intracellular levels of pseudouridine, adenosine and cytidine might be useful for markers for the stomach cancer and tRNA in the cancer tissues was observed to be modified more intensely than that in the normal control tissues with methylation, paseudouridylation and thiolation.