肝癌細胞의 γ-Glutamyltranspeptidase에 對한 Monoclonal antibody에 關한 硏究

金明坤,柳總根 고려대학교 의과대학 1992 고려대 의대 잡지 Vol.29 No.2

This study was devised to produce monoclonal antibody against hepatocelullar carcinoma speclfic γ-glutamyltranspeptidase (HCC GGT), which proceeded by first purifying the HCC GGT using ion exchange chromatography and immunoaffinity chromatography. Subsequently, a mouse was immunized with the purified enzyme, and then spleen cells taken from the immunized mouse was fused with mouse myeloma cells (SP2-Ag 14) to form the hybridoma. The following results were obtained by characterization of the monoclonal antibody of the hybridoma using immunoglobulin isotyping and immunoblot. 1. The purification of HCC GGT by means of ion exchange chromatography and immunoaffinity chromatogiaphy produced a yield of 29.41%, a purification fold of 167.87, and final specific activity of 21units per mg of protein. 2. Direct enzyme linked immunoadsorbent assay (ELISA) method was used to measure the antibody level against HCC GGT. An antibody titration of the blood sample taken from the mouse immunized with the above enzyme showed value over 1:5, 120. 3. The cell fusion of the mouse myeloma cell and the spleen cell of the immunized mouse was generated 14 positive wells in 221 wells. (specific fusion efficacy of 6.33%) Only one IgG2a monoclonal antibody was determined by both repetitive cloning procedures and immunoglobulin subisotyplng. 4. A antigen-antibody cross matching after the western blot revealed a specific reaction between the HCC GGT antigen at the 40Kd band and the monoclonal antibody of the hybridoma. And the monoclonal antibody did not react with GGT derived from normal liver or kidney.

단일클론 항γ-Glutamyltransferase항체를 이용한 효소 및 방사 면역측정법 적용에 관한 연구

金明坤,柳總根 고려대학교 의과대학 1994 고려대 의대 잡지 Vol.31 No.1

γ-Glutamyltransferase (GGT: E. C. 2. 3. 2. 2.) is a glycoprotein enzyme which is involved in glutathione metabolism and amino acid transport through the plasma membrane. It is distributed widely in several organs including liver and kidney. Several isozymes of GGT have been reported and some of the isozymes may be associated with hepatocarcinogenesis. We have produced six monoclnal antibodies (mAbs) against GGT purified from the liver of 2-acetamidofluorene (AAF) treated rats. All of the six mAbs were obtained by immunizing mice with liver GGT. Liver GGT from AAF treated rats was biochemically purified to a specific activity of 125.6 units per mg of protein. The overall purification was 551 folds and the final yield was 20. 8. Six hybridomas which produced anti-GGT Abs were extensively subcloned and injected into the peritoneal cavity of BALB/c mice to obtain large quantities of Abs. These mAbs were purified from ascites by ammonium sulfate precipitation and protein A sepharose CL-4B column chromatography. Using these mAbs we performed enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry (IHC), and autoradiography (ARG) to study the distribution of GGT isozymes in tissue. The results indicate that GGT-mAb 1 is specific for the AAF treated liver GGT, GGT-mAb 5 for the normal liver GGT, and GGT-mAb 6 for the normal kidney GGT. These mAbs may be used to evaluate the distribution of GGT isozymes in different tissues.

단일클론 항 γ-Glutamyltrasferase(GCT) 항체를 이용한 혈뇌장벽 내 GGT의 분포에 관한 연구

이병규,김명곤,신규만,류총근 대한신경외과학회 1995 Journal of Korean neurosurgical society Vol.24 No.3

γ-Glutamyltransferase(GGT: E.C. 2.3.2.2.) is a glycoprotein enzyme which is involved in glutathione metabolism and amino acid transport through the plasma membrane. It is distributed widely in several organs including liver, kidney, pancrease and brain. GGTs derived from the brain of Wister rats and BALB/c mice were biochemically purified to a specific activity of 4246.2, 862.1 units per mg of protein, a purification folds 93.7, 43.8 and the final yield 65.8, 44.0% respectively. Electrophoretic pattern of purified GGTs from rats and mice brain shows very similar protein fraction each other. We have produced six monoclonal antibodies(GGT-MAb 1-6) against 2-acetamidofluorene treated rat liver GGT. Using these GGT-MAb 1-6 we performed immunohistochemistry(IHC) to study the distribution of GGY isozymes in normal tissues of rat brain and in ncoplastic tissues of human brain. The results indicated that human brain GGT was localized in pericytes of blood-brain barrier, especially in the blood-rich portion of the brain(e.g. cerebellum of rat, meningioma and craniopharyngioma of human). Therefore these MAbs may be used to evaluate the distribution of GGT isozymes in different tissues.

신경정신 의학분야의 방사성동위원소 표지 cDNA 마이크로어레이

최재걸,신경호,이민수,김명곤 대한핵의학회 2003 핵의학 분자영상 Vol.37 No.1

Microarray technology allows the simultaneous analysis of gene expression patterns of thousands of genes, in a systematic fashion, under a similar set of experimental conditions, thus making the data highly comparable. In some cases arrays are used simply as a primary screen leading to downstream molecular characterization of individual gene candidates. In other cases, the goal of expression profiling is to begin to identify complex regulatory networks underlying developmental processes and diseases states. Microarrays were originally used with cell lines or other simple model systems, More recently, microarrays have been used in the analysis of more complex biological tissues including neural systems and the brain. The application of cDNA arrays in neuropsychiatry has lagged behind other fields for a number of reasons. These include a requirement for a large amount of input probe RNA in fluorescent-glass based array systems and the cellular complexity introduced by multicellular brain and neural tissues. An additional factor that impacts the general use of microarrays in neuropsychiatry is the lack of availability of sequenced clone sets from model systems. While human cDNA clones have been widely available, high quality rat, mouse, and drosophilae, among others are just becoming widely available. A final factor in the application of cDNA microarrays in neuropsychiatry is cost of commercial arrays. As academic microarray facilitates become more commonplace custom made arrays will become more widely available at a lower cost allowing more widespread application. In summary, microarray technology is rapidly having an impact on many areas of biomedical research. Radiosotope-nylon based microarrays offer alternatives that may in some cases be more sensitive, flexible, inexpensive, and universal as compared to other array formats such as fluorescent-glass arrays. in some situations of limited RNA or exotic species, radioactive membrane microarrays may be the most practical experimental approach in studying psychiatric and neurodegenerative disorders, and other complex questions in the brain. (Korean J Nucl Med 2003;37:43-52)

Profiling of Gene Expression in Human Keratinocyte Cell Line Exposed to Quantum Dot Nanoparticles

Kim, In-Kyoung,Lee, Seung-Ho,Kim, Yu-Ri,Seo, Sang-Hui,Jeong, Sang-Hoon,Son, Sang-Wook,Kim, Meyoung-Kon The Korean Society of Toxicogenomics and Toxicopro 2009 Molecular & cellular toxicology Vol.5 No.1

Quantum Dot (QD) nanoparticles are used in various industrial applications, such as diagnostic, drug delivery, and imaging agents of biomedicine. Although QDs are extensively used in many medical science, several studies have been demonstrated the potential toxicity of nanoparticles. The first objective of this study was to investigate the nanotoxicity of QDs in the HaCaT human keratinocyte cell line by focusing on gene expression pattern. In order to evaluate the effect of QDs on gene expression profile in HaCaT cells, we analyzed the differential genes which related to oxidative stress and antioxidant defense mechanisms by using human cDNA microarray and PCR array. A human cDNA microarray was clone set, which was sorted for a list of genes correlated with cell mechanisms. We tried to confirm results of cDNA microarray by using PCR array, which is pathway-focused gene expression profiling technology using Real-Time PCR. Although we could not find the exactly same genes in both methods, we have screened the effects of QDs on global gene expression profiles in human skin cells. In addition, our results show that QD treatment somehow regulates cellular pathways of oxidative stress and antioxidant defense mechanisms. Therefore, we suggest that this study can enlarge our knowledge of the transcriptional profile and identify new candidate biomarker genes to evaluate the toxicity of nanotoxicology.

Anti-proliferation Effect of Damina 909 on Pancreatic Cancer Cells in Tumor-Xenografted Nude Mice Model

Kim, Yu-Ri,Lee, Seung-Min,Seo, Sang-Hui,Lee, Seung-Ho,Kim, In-Kyoung,Jun, Hwang-Jeok,Nam, Jong-Hyun,Kim, Meyoung-Kon The Korean Society of Toxicogenomics and Toxicopro 2009 Molecular & cellular toxicology Vol.5 No.1

In this study, we investigated the anti-proliferative effect of Damina 909 in human cancer cell lines and tumor-xenografted nude mice to elucidate its potential in treating many cancers. Damina 909 treatment resulted in inhibition of cell proliferation of human pancreatic cancer cells. Our in vivo study showed that the weight of pancreatic tumors in Damina 909-treated group were the lighter than control group. Consequently, the intake of food and water in Damina 909-treated group did not change, while those in control group were steadily decreased over a period of treatment. Moreover, Damina 909 treatment elevated the protein expression of p53 and p21 in pancreatic tumor of xenografted nude mice. In summary, compare to other human cancer cells such as prostate and hepatocyte, Damina 909 is most effectively inhibited proliferation of pancreatic cancer cells by increasing the expression of tumor suppressor genes. This led us to speculate that a candidate substance for effective cancer therapy of pancreatic cancer might be contained in Damina 909.

Effect of toluene on RANTES and eotaxin expression through the p38 and JNK pathways in human lung epithelial cells

Kim, Yu-Ri,Kim, In-Kyoung,Seo, Sang-Hui,Lee, Seung-Ho,Lee, Hee-Ra,Pie, Jae-Eun,Kim, Meyoung-Kon The Korean Society of Toxicogenomics and Toxicopro 2010 Molecular & cellular toxicology Vol.6 No.4

The increasing prevalence and severity of environmental diseases have been attributed the rise in environmental pollutants occurring as a consequence of industrial development. In this study, we examined that underlying mechanisms of toluene in human lung epithelial cells. We selected two genes known to be involved in environmental disease such as RANTES and eotaxin for the present study based on published reports and prior observation. We observed that toluene increased the mRNA and protein levels of RANTES and eotaxin in a dose-dependent manner, together with the activation of p38 MAPK and JNK. Moreover, the inhibition of p38 MAPK and JNK activation prevented the release of RANTES and eotaxin induced by toluene. The present study is the first to show that toluene exposure induces the expression of RANTES and eotaxin in human bronchial epithelial cells through two distinct MAPKs such as p38 and JNK. Our observations suggest that modulation of the expression of immunerelated chemokines and cytokines may be important factors in development of environmental diseases induced by air pollutants.

Similarity of Gene Expression Profiles in Primary Brain Tumors with the Toxic Mechanism by Environmental Contaminants

Kim, Yu-Ri,Kim, Ki-Nam,Park, Yoon-Hee,Ryu, Yeon-Mi,Sohn, Sung-Hwa,Seo, Sang-Hui,Lee, Seung-Ho,Kim, Hye-Won,Lee, Kweon-Haeng,Kim, Meyoung-Kon The Korean Society of Toxicogenomics and Toxicopro 2005 Molecular & cellular toxicology Vol.1 No.3

Recently, a large number of clinical experiments have shown that exposure of organic pollutants lead to various cancers through the abnormal cell growth. Environmental pollutants, such as 2, 3, 7, 8-Tetrachloro dibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons (PAHs), are carcinogen and are known to cause the cognitive disability and motor dysfunction in the developing of brain. The effects of these pollutants on neurodevelopmental disorder is well established, but the underlying mechanism(s) and similarity of gene expression profiles in human brain tumors with organic pollutants still remain unclear. In this study, we first examined the gene expression profiles in glioblastomas compared with meningioma that are kinds of primary human brain tumor by using human cDNA microarray. The results of cDNA microarray analysis revealed that 26 genes were upregulated (Z-ratio>2.0) and 14 genes were downregulated (Z-ratio<-2.0) in glioblastoma compared with meningioma. From the altered gene patterns, mitogen-activated protein kinase (MAPK) signaling related genes, such as MAP2K3, MAP3K11 and jun activated domain binding protein, and transcription factors, such as UTF2 and TF12, were upregulated in glioblastoma. Also, we tried to investigate the relation between important genes up- and down-regulated in giloblastoma and various organic pollutants. Therefore, the identification of changes in the patterns of gene expression may provide a better understanding of the molecular mechanisms involved in human primary brain tumors and of the relation between gene expression profiles and organic pollutants in brain tissue.

Gene Expression Profile in Carpal Tunnel Syndrome Patients

Kim, Hye-Won,Kim, Ki-Nam,Seo, Sang-Hui,Lee, Seung-Ho,Sohn, Sung-Hwa,Kim, Yu-Ri,HaLee, Young-Mie,Shim, Jae-Sun,Ahn, Duck-Sun,Kim, Meyoung-Kon The Korean Society of Toxicogenomics and Toxicopro 2006 Molecular & cellular toxicology Vol.2 No.4

Carpal tunnel syndrome (CTS) is one of the most common disorders by under pressure of the median nerve at the wrist in these days. However, pathological mechanism of CTS is unknown. We carried out this study to identify the changes of gene expression and to evaluate possible mechanism in CTS. 120 CTS patients and 30 control patients were included in this study. Patients with a history of diabetes, hypertension, thyroid diseases, and arthritis were excluded. CTS patients were divided to three experimental groups-Mild, Moderate, and Severe group-according to elecrodiagnosis. Radioactive cDNA microarrays (Nylon membrane including 1,152 genes) were used to examine the difference of gene expression profile in CTS. We identified up-regulated genes by more than 2.0 value of z-ratio, and down-regulated genes by less than-2.0 value of z-ratio. 20 genes such as the ITGAL, ITGAM, PECAM1, VIL2, TGFBR2, RAB7, RNF5 and NFKB1 were up-regulated, and 28 genes such as PRG5, CASP8, CDH1, IGFBP5, CBX3, HREV107, PIN, and WINT2 were down-regulated. These genes were related with TGF beta signaling pathway, NF-Kb signaling pathway, antiapoptotic pathway and T cell receptor signaling pathway. However, there were no differences in gene expression profiles according to severities of symptoms. We suggest that CTS could be related with proinflammatory mechanism and antiapoptotic mechanism.

The Efficiency of Zinc-Aspartate Complex on Zinc Uptake in Plasma and Different Organs in Normal SD Rats

Kim, Yu-Ri,Kim, Ki-Nam,Shim, Boo-Im,Lee, Seung-Min,Kim, In-Kyoung,Sohn, Sung-Hwa,Park, Myung-Gyu,Park, Hong-Suk,Kim, Meyoung-Kon The Korean Society of Toxicogenomics and Toxicopro 2007 Molecular & cellular toxicology Vol.3 No.2

Zinc is essential metal and plays a role in a wide variety of physiological and biochemical processes. Prostate gland contains high level of zinc, generally 3-10 folds higher than other organs. Prostatic zinc uptake is resulted from the existence of zinc transporter (ZnT) protein families in membrane. In this study, we investigated the difference of zinc uptake efficiency of zinc-aspartate complex (Zn-Asp) into various organs compared with $ZnSO_4$. We observed that Plasma zinc concentration in both $ZnSO_4$ and Zn-Asp administrated group was increased progressively following administration, and reached a peak level at 2 hr. The increasing pattern of zinc concentration was similar to each groups, however the zinc concentration of Zn-Asp administrated group was higher than that of $ZnSO_4$ administrated group. We found that prostatic zinc level of Zn-Asp administrated group was higher than $ZnSO_4$ administrated group, and was increased approximately $\sim$2.7 fold and $\sim$4.2 fold at 4 and 8 hr after administration. From these observations, we suggest than Zn-Asp has high uptake efficiency of zinc into the prostate gland. Therefore, Zn-Asp is potentially useful treatment of many prostatic diseases.