Gene Replacement Therapy

고재경 생화학분자생물학회 1974 BMB Reports Vol.4 No.1

活性슬럿지法에 의한 炭化水素 함유 廢水의 淨化

고정삼,김재하,강경수,고영환 濟州大學校工科大學附屬産業技術硏究所 1991 尖端技術硏究所論文集 Vol.2 No.-

Activated sludge process which has been widely applied to the treatment of waste-water was slightly modified to remove hydrocarbons from wastewater. The process of wastewater treatment consisted of two consecutive reactors. Cells of Acinetobacter calcoaceticus were first cultivated in synthetic wastewater containing 3%(W/V) of hydrocarbons. The resulting culture was then exposed to acclimatized active sludge. Hydrocarbon concentrations of the effluent from the process were 0.19-0.21%(W/V). The contents of suspended solid were reduced to 17-53 ㎎/l. The data imply that A.calcoaceticus can be used for the treatment of wastewater containing hydrocarbons.

폐선암조직에서 Neutral Ribonuclease의 분리와 성상에 관한 연구

김응수,고재경,지행옥 한양대학교 의과대학 1993 한양의대 학술지 Vol.13 No.1

Concentrations of nucleic acids and proteins were determined in adenocarcinoma tissue of the lung and were compared with those in the control lung tissue. Also studied were properties of the neutral RNase specific to the lung cancer to investigate the possible role of the RNase in process involved in carcinogenesis of adenocarcinoma of the lung. DNA and protein centents were unchanged, but RNA content was increased in adenocarcinoma tissue of the lung. Neutral RNase activity was unchanged in the cancer tissue, indicating that the RNase could not be used as a marker for the lung cancer. Proteins and neutral RNase in adenocarcinoma tissue of the lung were separated by a DEAE-cellulose column chromatography into 7peaks each, of which the peak I neutral RNase isozyme was not found in the control lung tissue. This indicated that the peak I neutral RNase was specific to the adenocarcinoma of the lung. The peak I neutral RNase isolated from the adenocarcinoma tissue of the lung did not hydrolyze single stranded (ss) polydeoxyribonucleotides and double stranded (ds) polynucleotides, but hydrolyzed ss polyribonucleotides. The enzyme was observed to be highly active toward poly C, poly U and RNA, indicating that the RNase appeared to be mixed type of secretory and nonseretory RNase. The peak I RNase was not active toward A-A and G-G linkages, but unusually highly active toward A-C and A-U linkages (4 to 6 fold as active as C-C linkage). These results indicated that the peak I neutral RNase isolated from the adenocarcinoma of the lung was (1)specific to the lung cancer. (2) mixed type of seretory and nonsecretory enzymes, (3) unusually highly active toward A-C and A-U linkages of ss polyribonucleotides and RNA, suggesting that the RNase might play roles in processes involved in carcinogenesis and suppression of cancer.

Human Serum Albumin에 대한 단세포군 항체의 생성, 특성분석 및 microalbuminuria 검색을 위한 sandwich ELISA의 개발에 관한 연구

구자욱,고광욱,고재경 한양대학교 의과대학 1992 한양의대 학술지 Vol.12 No.1

Alumin of a variety of species has been the subject of extensive studies by protein chemists and immunologists for over 3 deades. Human serum albumin (HSA) is the most abundant protein in plasma. It is a nonglycosylated protein consisting of a single polypeptide chain of 585 amino acids with 34 cystive residues. The molecule contains 9 serial double loops formed by 17 disulfide bridges arranged as 3 repeat units or domains. Since polyclonal antibodies bind to almost all antigenic determinants of antigen, the experiments base4d on polyclonal antibodies yielded frequently low-titre, of broad specificity, and are not completely reproducible, although the heterogeneity of fine specificity could be minimized by affinity chromatography on fragments of the immunogens. Recently, the development of hybridoma techniques has made it possible to produce a large amount of homogenous and specific anti-protein antibody which reacts with a single epitope of antigen. Diabetic nephropathy is the major cause of the increased mortality in insulin-dependent diabetes mellitus (IDDM), affecting over 40% of these patients. Recent studies have shown that an increased urinary albumin excretion rate (microalbuminuria) in IDDM patients is a good predictor of the development of diabetic nephropathy. The possibility that therapeutic intervention instituted at this early stage of the disease might reverse, arrest or at least slow its progression to late and irreversible stages promotes the necessity to develop a screening test for microalbuminuria. Microalbuminuria can be measured by radioimmunoassay(RIA), nephelometric laser, immunoturbidimetry and enzyme linked immunosorbent assay(ELISA). However, RIA has some disadvanges such as isotope-related health hazards, and expense of equipment used to measure gamma-emitting isotope. The aims in this study were to produce monoclonal antibodies against HSA and to develop a simple, rapid, and sensitive sandwich ELISA using anti-human albumin monoclonal antibodies for detecting microalbuminuria. Four monoclonal antibodies(MHA-1, MHA-2, MHA-3, and MHA-4) which reacted selectively against HSA were produced by hybridoma technology. Isotype of each monoclonal antibody was found to be IgG₁. All monoclonal antibodies except MHA-1 were species-specific which were demonstrated on ELISA and immunoblotting. Since MHA-4 reacted with albumin in only nonreducting condition, it was suggested that this antibody recognized the conformational epitope of the albumin. Utilizing ingibition ELISA, it was speculated that these four antibodies recognized respectively 4 different epitopes of albumin and among those epitopes, epitopes of MHA-2 and MHA-3 same or located very approximately. Sandwich ELISA using two monoclonal antibodies(MHA-4 as capture antibody and MHA-2 as detector antibody), which reacted with different epitopes, were developed to detect microalbuminuria. Dynamic range of this assay was from 30 to 5000㎍/1 and its lowere limit of detection was 30㎍/1, corresponding to 1.5ng of albumin per well. The intra-assay and the inter-assay coefficient of variation were 3.3-4.0% and 6.8-8.0%, respectively. Analytical recovery ranged from 92 to 103%. Concentrations of 84 urine samples were measured by the sandwich ELISA and competitive RIA. Correlation between two assays was good(r=0.983, p<0.005) over a range of albumin concentration tested. Albumon excretions in 24 hour urine samples from non-insulin-dependent diabetes mellitus(NIDDM) patients (mean 16.7mg/day, n=39, 8yr duration) were significantly increased (t=2.53, p=0.015) as compared with those from healthy subjects(mean 5.6mg/day). However, 24 hour urine albumin excretions in IDDM patient(mean 11.1mg/day, n=25, 6.8yr duration) werer not significantly different from those in normal control(t=1.16, p=0.26). The 24 hour urine albumin(mg/day) correlated well with the urine albumin/creatinine ratio(r=0.816, p<0.005) on log data. This study indicates that this newly established sandwich ELISA is found to have good analytical characteristics, outstanding in its specificty, sensitivity, accuracy, and precision, and is suitable and easily accessable for detection of microalbuminuria in clinical medicine.

위암환자의 위액단백과 Amylase의 분리와 성상에 관한 연구

정부근,고재경,노연희 한양대학교 의과대학 1992 한양의대 학술지 Vol.12 No.1

Amylase activity was measure din gastric juice and cancer tissue of patients with stomach cancer to find out the possible use of the enzyme as a biochemical marker for stomach cancer. Amylase and proteins in gastric juice from patients with stomach cancer were separated by a DEAE-cellulose column chromatography to investigate the enzyme and proteins specific to the stomach cancer. (1)Protein content and amylase activity were not changed in gastric juice of patients with gastritis and duodenal ulcer, but significantly increased in gastric juice of patients with stomach cancer. The positive rate of protein content and the enzyme activity in the gastric juice as a marker for stomach cancer was relatively high. (2)Protein content in the stomach cancer tissue was significantly increased and amylase activity was greatly increased in 3 cases out of 12 cases of the stomach cancer tissues studied. (3)Substrate specificity and effect of halogen ions on amylase in gastric juice of patient with the stomach cancer was similar to those on the enzyme in the stomach cancer tissue. (4)DEAE-cellulose columkn chromatography revealed that proteins in gastric juice were separated into 8 peaks in the stomach cancer and into 6 peaks in the control. Amylase in the gastric juice was separated by the chromatography into 2 isozymes in the stomach cancer, but not in the control. Substrate specificity for the two gastric juice amylase isozymes was observed to be different. These results indicate that protein content and amylase activity in the gastric juice could be used as a biochemical marker for the stomach cancer and that amylases specific to the stomach cancer appear to be released from the stomach cancer tissue.

폐 상피양 세포암에 특이한 Ribonuclease의 작용기전에 관한 연구

이성윤,지행옥,고재경 한양대학교 의과대학 1993 한양의대 학술지 Vol.13 No.1

A neutral ribonuclease (RNase) specific to the epidermoid carcinoma of the lung was isolated from the lung cancer tissue to investigate processes involved in carcinogenesis and suppression of the lung cancer. Also studied were the substrate specificity and mechanism of action of the neutral RNase specific to the lung cancer. Neutral RNase activity in the lung tissue obtained by surgery was unchanged in four varieties of lung cancers (epidermoid carcinoma in 20 cases, 472±1859 umole/g/hr; adenocarcinoma in 5 cases, 5165±1575 umole/g/hr; karge cell carcinoma in 3 cases, 5870±2305 umole/g/hr; small cell carcinoma in 3 cases, 5405±2822 umole/g/hr; control in 31 cases, 4380±1520 umole/g/hr), indicating that RNase assay in the lung tissue could not be used as a biochemical marker for the lung cancer. Neutral RNases in the epidermoid carcinoma tissue of the lung were separated by a DEAE-cellulose column chromatography into 6 peaks, of which the peak Ⅰ neutral RNase isozyme was specific to the epidermoid cancer of the lung. High performance liquid chromatorgraphy (HPLC) and polyacrylamide gel electrophoresis (PAGE) patterns for peak Ⅰ protein from epidermoid carcinoma tissue of the lung appeared to be different from those of control lung tissue. The subpeak Ⅰ-5~8 (in HPLC pattern) that was supposed to be associated with RNase was greatly increased in the lung cancer, indicating that peak Ⅰ protein from epidermoid carcinoma tissue of the lung was specific to the lung cancer and that peak Ⅰ RNase specific to the cancer was located in HPLC subpeak Ⅰ-5~8. The peak Ⅰ neutral RNase was observed to be highly active toward poly C, poly AC and poly ACU and less active toward poly U and RNA, indicating that the peak Ⅰ neutral RNase was the secretory type of RNase. No activity was found toward polydezyribonucleotides and double stranded polyribonucleotides. Majority of products of poly C digest by the peak Ⅰ neutral RNase was analyzed to be oligoribonucleotides, indicating that the RNase was endonuclease in nature. Observations that the peak Ⅰ neutral RNase was specific to the epidermoid carcinoma of the lung and that the enzyme was endonuclease in nature suggested that the RNase might play an important role in process involved in the suppression of the lung cancer.

급성 백혈병환아의 혈청내 변형 Ribonucleoside의 변동에 관한 연구

한중수,이정국,고재경 한양대학교 의과대학 1993 한양의대 학술지 Vol.13 No.1

In order to find out whether the modified ribonucleosides in serum could be used as specific tumor markers for acute lymphocytic leukemia (ALL) and acute myelogenous leukemia (AML), analysis of serum samples from normal child hood controls and patients with the leukemia was performed using reversedphase HPLC. For this study, a rapid and precise chromatographic method for the determination of very small amounts of modified nucleosides in serum by HPLC has been developed. The ribonucleosides were prefractionated with boronate affinity gel column. 1. The concentration of total modified nucleosides in serum of patients with ALL was increased by 46% as compared to normal control level. Patients with ALL had significantly higher concentrations of pseudouridine (ø), 1-me-thyladenosine(m A), 4-thiouridine(sU), 1-methylinosine(mI), 1-methylguanosine(m G ), N,N-dimethylguanosine(m G ) and -methyl-adenosine( ) than normals. Among these nucleosides elevated, positive rates of ø, m A, m I, m G and N A as a marker for the leukemia were observed to be 79%,79%,64%,93% and 93%, respectively. 2. When compared to normal control value, total modified nucleoside content in serum of patients with AML was increased by 21%, Contents of , mA, sU, mI,mG and NA were significantly incresed in serum of patients with AML. Positive rate of mI, mG and NA were 83%, and that of mA was 67%, respectively in AML studied in this experiment. 3. In follow-up study for seven of fourteen patients with ALL, contents of , mA, mI and mG were decreased to normal value after effective chemotherapy. The results suggested that serum levels of ø, m A,m I, m G and N A for ALL and those of m A, m I, m G and N A for AML could be used as diagnostic or biochemical markers. These markers might be useful to monitor the effectiveness of chemotherapy for leukemia.

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.

골육종의 억제과정에 미치는 Ribonuclease 의 작용

김성준,이경태,고재경 한양대학교 의과대학 1993 한양의대 학술지 Vol.13 No.1

Neutral ribonuclease (RNase) highly active toward polycyidylate (poly C) was isolated from osteosarcoma tissue by DEAE-cellulose column chromatography and was found to be specific to the bone sarcoma by means of a high performance liquid chromatography (HPLC) and polyacrylamide gel electrophoresis (PAGE). Also studied were substrate specificity and the product analysis of the neutral RNase to understand the role of the enzyme specific to osteosarcoma in pathogenesis of the bone sarcoma. Neutral RNase activity was greatly increased in osteosarcoma tissue, suggesting that in could by used as a marker for the bone sarcoma. Neutral RNases in osteosarcoma tissue were separated by a DEAE-cellulose column chromatography into 5 peaks, of which peaks Ⅵ and Ⅶ RNase isozymes were not found in the control bone tissue. The activity of peak Ⅰ RNase isozyme was most active among the RNase isozymes separated. HPLC and PAGE patterns for the proteins associated with the peak Ⅰ neutral RNase of osteosarcoma appeared to be different those of the control, suggesting that the enzyme might be specific to the bone sarcoma. The peak Ⅰ neutral RNase isozyme specific to the osteosarcoma was found to be active toward ss polyribonucleotide (highly active toward C-C, C-U and A-U linkages). Majority of the poly C digest by the RNase specific to the osteosarcoma was observed to be oligoribonucleotides with chain length of 4-25, indicating that the enzyme was endonuclease in nature. The present study indicate that 1) neutral RNase activity was greatly increased in osteosarcoma tissue, 2) the enzyme could be used as a marker for osteosarcoma, 3) the peak Ⅰ neutral RNase isozyme isolated from osteosarcoma tissue might be specific to the bone sarcoma, 4) the RNase isozyme was active toward ss polyribonucleotide and 5) the RNase isozyme was endofibonuclease in nature, suggesting a possible role of the RNase isozyme in the suppression of the osteosarcoma.

난소암조직 Acid Deoxyribonuclease의 분리와 성상에 관한 연구

김두상,김영신,고재경,한중수 한양대학교 의과대학 1992 한양의대 학술지 Vol.12 No.1

In order to find out a possible role of acid deoxyribonuclease (DNase) in carcinogenesis of the ovary, activity of the enzyme was measured and the nature of the enzyme was studied in serous cystadenocarcinoma and endodermal sinus tumor of the ovary following the purification of the acid DNase in the tumor tissue of the ovary. (1)The acid DNase activity was greatly increased in the tumor tissues of the ovary, serous cystadenocarcinoma and endodermal sinus tumor tissues, while neutral and alkaline DNase activities were unchanged in the tumor tissues. This may indicate that the acid DNase can be used as a biochemical marker for the ovary tumors. (2)Proteins in the tumor tissues of the ovary were separated by a DEAE-cellulose column chromatography into 7 peak, respectively, of which one peak protein each appeared to be specific for serous cystadenocarcinoma and endodermal sinus tumor. (3)Acid DNases in the tumor tissues of the ovary were isolated into a single peak, respectively. The size of peak in the tumor tissues was greater than that in the control tissue of the ovary, indicating that the acid DNases in the tumor tissues were activated, but not specific for the tumors. (4)Acid DNases in the tumor tissues of the ovary were partially purified by centrifugation and a DEAE-cellulose column chromatography. The purified enzyme was highly active against double stranded DNA (ds-DNA), even though some activity was found against single stranded DNA (ss DNA). Observations that acid DNase from serous cystadenocarcinoma tissue was highly active and specific to ds DNA compared with ss DNA suggested that the enzyme might play a role in transforming normal ovarian cells into cancer cells.