1.Electron Microscopic Analysis of Apoptosis of HGPRT- Mouse Myeloma Cell Induced by Aminopterin, a de novo Pathway Blocking Agent.
Yong CHOI ; Yong Hoon CHUNG ; Yang Ja CHO ; Yong Keel CHOI
Korean Journal of Immunology 1998;20(4):389-396
Programrned cell death (PCD), or apoptosis, is a process by which cells die in response to specific physiological and toxicological signals. This genetically programmed form of cellular suicide is intirnately involved in many biological processes including growth, metamorphosis, embryogenesis, and oncogenesis. Cells undergoing PCD in normal and neoplasmic tissues display the following biochemical and morphological features: internucleosomal DNA fragmentation, reduced cell volume, condensed chromatin in nucleus, zeiosis and generation of apoptotic bodies containing intact organelles and plasma rnembrane. Hybridoma cell production, resulting from the fusion of myeloma cells with antibody producing spleen cells, is widely used in various fields of life science. This technique requires hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficient mutant myeloma cell line as a fusion partner. When these mutants cell is treated with aminopterin plus hypoxanthine-thymidine (HAT) after the cell fusion they are selectively and efficiently eliminated remaining fused hybridoma celis. But there hasn't been any report regarding the selective elimination mechanism of this HGPRT mutant myeloma cell. By using HGPRT myeloma P3-X 63-Ag8.653 (V653) as a model system, this study demonstrated that PCD was induced by aminopterin treatment of this V653 cell line. And the sequential ultrastructural changes during this death process were observed by using electron microscope. When V653 cells were incubated with 0.4 uM aminopterin, DNA fragmentation was detectable after 3 hours and peaked between 12 and 18 hours of aminopterin treatment and the cell viability decreased in a time dependant manner. V653 cells incubated with amiopterin showed following ultrastructural changes during the death process. Dilatation of rough endoplasmic reticulum (RER) and detachment of ribosomes were the earliest ultrastructural changes and first seen after 30 minute incubation. Dilatation of perinuclear cisternae began to appear after 1 hour and deformation of nucleoplasm such as decreased electron density of perinuclear heterochromatin and increased electron density of euchromatin were seen after 3 hours. Increased electron density of cytoplasm, decreased cell volume, condensation of chromatin and apoptotic bodies were observed in many cells after 9 hours but vacuolation by severe dilatation of RER was seen in some cells. 24 hours after incubation with aminopterin, many cells showed typical form of apoptosis characterized by cell shrinkage, increased electron density of cytoplasm and apoptotic bodies. On the contrary, some cells showed different type of cell death characterized by increased cell volume, decreased electron density of cytoplasm, severely dilated RER and apoptotic bodies. In both types of cells, mitochondrial cristae and limiting membrane of mitochondria are comparatively well preserved. In other cells, nuclei did not show significant changes but there were deformations of mitochondria such as markedly increased electron density and formation of lamella bodies. The death process of V653 cell was not synchronized among cells. The results of this study proved that aminopterin-induced selective elimination of fusion partner V653 myeloma cell is due to PCD. The earliest ultrastructural changes observed in this process were dilatation of RER and detachment of ribosomes. And there were two distinct morphological types in the PCD.
Aminopterin*
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Animals
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Apoptosis*
;
Biological Processes
;
Biological Science Disciplines
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Carcinogenesis
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Cell Death
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Cell Fusion
;
Cell Line
;
Cell Size
;
Cell Survival
;
Chromatin
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Cytoplasm
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Dilatation
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DNA Fragmentation
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Embryonic Development
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Endoplasmic Reticulum, Rough
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Euchromatin
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Female
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Guanine
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Heterochromatin
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Hybridomas
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Hypoxanthine
;
Hypoxanthine Phosphoribosyltransferase
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Membranes
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Mice*
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Mitochondria
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Organelles
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Plasma
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Pregnancy
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Ribosomes
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Spleen
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Suicide
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Transferases
2.Purification of the Protective Antigen from Bacillus anthracis.
Jeung Moon PARK ; Yong Keel CHOI ; Seong Kun CHO ; Young Gyu CHAI ; Seong Joo KIM
Journal of the Korean Society for Microbiology 1998;33(6):589-594
Anthrax toxin consists of three separate proteins, protective antigen (PA), edema factor (EF), and lethal factor (LF). PA binds to the receptor on mammalian cells and facilitates translocation of EF or LF into its cytosol. PA is the primary component of anthrax vaccines. In this study we purified PA from culture filtrates of Bacillus anthracis. The purification involved sequential chromatography through hydroxylapatite, DEAE-Sepharose CL-4B, followed by Mono-Q. The purified PA was judged to be homogeneous on SDS-PAGE, and consisted of a single polypeptide chain with a relative molecular weight of 85,000.
Anthrax
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Anthrax Vaccines
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Bacillus anthracis*
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Bacillus*
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Chromatography
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Cytosol
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Durapatite
;
Edema
;
Electrophoresis, Polyacrylamide Gel
;
Molecular Weight
3.Effects of interferon-alpha and -gamma on lymphokine-activated killer cell activity induced by interleukin-2.
Yong CHOI ; Yong Keel CHOI ; Jung Mogg KIM ; Yong Hoon CHUNG ; Kyung Hee KIM ; Wang Soo HAN
Journal of the Korean Society for Microbiology 1992;27(4):371-380
No abstract available.
Interferon-alpha*
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Interleukin-2*
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Killer Cells, Lymphokine-Activated*
4.Detection of Bacillus anthracis using a nested PCR Method.
Yong Keel CHOI ; Seong Kun CHO ; Myung Hee KIM ; Seung Yun BAIK ; Gyeong Hyun PARK ; Young Gyu CHAI
Journal of the Korean Society for Microbiology 1998;33(6):583-588
Bacillus anthracis is a soil pathogen capable of causing anthrax in animals and humans. To establish a method for specifically detecting B. anthracis, we used nested polymerase chain reaction. Outer and inner sets of oligonucleotide primers were designed from the protective antigen (pag) gene and from the cya gene of the plasmid pXO1. Ainplification of 482 bp or 208 bp DNA fragment obtained from a nested PCR method provided the basis for rapid and reliable assay for the detection and identification of B. anthracis.
Animals
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Anthrax
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Bacillus anthracis*
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Bacillus*
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DNA
;
DNA Primers
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Humans
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Plasmids
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Polymerase Chain Reaction*
;
Soil
5.Molecular Characterization of Fusarium solani and its Formae Speciales Based on Sequences Analysis of the Internal Transcribed Spacer (ITS) Region of Ribosomal DNA.
Young Mi LEE ; Yong Keel CHOI ; Byung Re MIN
Mycobiology 2000;28(2):82-88
The sequences of the internal transcribed spacer (ITS) and 5.8s ribosomal RNA gene (rDNA) from Fusarium solani and its four formae speciales belonging to section Martiella was determined to investigate intraspecific divergence of the ITS regions. The length of the 5.8S, a coding region, was equally 158 bp at all isolates, whereas the variable range of ITS region was shown at 147~152 bp (ITS1) and 148~174 bp (ITS2). According to the maximum-matching method, the matching percentage was 94~100 at 5.8s rDNA, 77~97 at ITS1, and 67~97 at ITS2, respectively. In dendrogram based on the alignment of the ITS sequence data, F.solani f. sp. piperis was distinguished from other isolates belonging to the same species and nucleotide identity was considerably low (41.5%).
Clinical Coding
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DNA, Ribosomal*
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Fusarium*
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RNA, Ribosomal, 5.8S
6.Gliotoxin induces the Apoptosis in HL-60 Cells.
Hun Taeg CHUNG ; Rae Kil PARK ; Yong Keel CHOI ; Sang Rock LEE ; Young Hee KIM ; Kwang Ho CHO ; Young Woo JANG
Korean Journal of Immunology 1998;20(4):397-403
Many fungi including Penicillium, Aspergillus, Gliocladium, and Thermoascus produce an epipolythiodioxopiperazine class of fungal metabolite, gliotoxin, which contirbutes the pathogenesis of fungal infection as an immunomodulator and cytotoxic agent. This study is designed to define the mechanism by which gliotoxin exerts the cytotoxic effect of gliotoxin on human promyelocytic leukemic cells, HL-60. Gliotoxin induces the apoptosis of HL-60 cells which is characterized by the ladder pattern fragmentation of DNA. Gliotoxin induces the activation of DEVD-specific cysteine protease in a time- and dose-dependent rnanner. It also increases the phosphotransferase activities of c-Jun N-terminal kinase1 (JNK1) and p38 in gliotoxin-treated HL-60 cells. Furthermore, gliotoxin decreases the activation of transcriptional activator, actiating protein (AP-1) and NF-kB. These results suggest that gliotoxin induces the apoptotic death of HL-60 cells via activation of DEVD- specific caspase as well as mitogen activated protein kinases (MAP kinases) including JNK1 and p38, and inhibition of transcriptional activators, AP-1 and NF-kB.
Apoptosis*
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Aspergillus
;
Caspase 3
;
Cysteine Proteases
;
DNA
;
Fungi
;
Gliocladium
;
Gliotoxin*
;
HL-60 Cells*
;
Humans
;
Mitogen-Activated Protein Kinases
;
NF-kappa B
;
Penicillium
;
Thermoascus
;
Transcription Factor AP-1
;
Transcription Factors
7.Induction of Apoptosis in Synovial Cells from Patients with Rheumatoid Arthritis.
Sung Yeoul CHANG ; Seong Yoon KIM ; Myung Hee KIM ; Young Gyu CHAI ; Jae Chan PARK ; Yong Keel CHOI ; Jae Bum JUN ; Sung Soo JUNG
Korean Journal of Immunology 1997;19(4):609-616
Homeostasis of multicellular organism is controlled by proliferation and differentiation of cells as well as by cell death. The defects in programmed cell death contribute to the pathogenesis of rheumatoid arthritis (RA) and systemic lupus erythematous. RA is considered to be a proliferating disorder of synovial tissue which is accompanied by inflammatory cell infiltration and bone erosion. The aim of the study was to find whether potent inducers of apoptosis could be induced apoptosis in RA synovial cells. We examined the effects of drugs, such as dexamethasone, methotrexate, hydrogen peroxide, and ceramide on induction of apoptosis in cultured RA synovial cells. Used drugs did not induced apoptosis in RA synovial cells. Finally Fas antigen-mediated apoptosis of RA synovial cells was investigated by the addition of anti-Fas antibody. To examine the ICE (interleukin-1p-convertase; caspase-1) expression in synovial cells, RT-PCR of caspase-1 gene was performed. In synovial cells of RA, Fas induces that caspase-1 activation cause apoptosis.
Apoptosis*
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Arthritis, Rheumatoid*
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Cell Death
;
Dexamethasone
;
Homeostasis
;
Humans
;
Hydrogen Peroxide
;
Ice
;
Methotrexate
8.Telomerase Activity in Rheumatoid Arthritis.
Hee Kwan KOH ; Mung Ju AHN ; Tae Hwan KIM ; Eun Young LEE ; Tae Seok YOO ; Je Kyung LEE ; Dae Kook CHANG ; Seung Cheol SHIM ; Jae Bum JUN ; Sung Soo JUNG ; In Hong LEE ; Sang Cheol BAE ; Dae Hyun YOO ; Yong Keel CHOI ; Seong Yoon KIM
The Journal of the Korean Rheumatism Association 1999;6(1):6-13
No abstract available.
Arthritis, Rheumatoid*
;
Osteoarthritis
;
Telomerase*