- Homogeneity of Phospholipase C of Bovine Uterus and Seminal Vesicle Compared with Brain Isozymes.
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Jung Hye Kim, Ki Yung Lee, Sue Goo Rhee
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Yeungnam Univ J Med. 1988;5(2):37-45. Published online December 31, 1988
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DOI: https://doi.org/10.12701/yujm.1988.5.2.37
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- Phosphoinositide-specific Phospholipase C (PI-PLC) is a second messenger of signal transducer on cell membrane. In the previous study, PLC of bovine brain has been purified three isozymes. In this paper, uterus and seminal vesicle have been purified. Two peaks of PI-PLC activity were resolved when bovine uterus and seminal vesicle proteins were chromatographed on a DEAE and phenyl TSK 5PW HPLC column. Each two peak was compared with PI-PLC I, II and III from bovine brain and we got the retention time on HPLC. The peak fractions with PLC activity were tested homogeneity with brain PLC monoclonal antibodies (Mab). Mab-labeled affigels were bounded in the range of 73.8%~97.5% with PLC I, II and III. Homogeneity of fractions were revealed that DEAE F-1 and phenyl F-1-I were highest level of PLC III in uterus and seminal vesicle and DEAE F-2 and phenyl F-2-I were mixed PLC I and II.
- Production of Monoclonal Antibody to Polychlorinated Biphenyl Induced Cytochrome P-450 LMII in Rat Liver.
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Jung Hye Kim, Jae Ryong Kim, Ki Yung Lee
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Yeungnam Univ J Med. 1986;3(1):103-110. Published online December 31, 1986
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DOI: https://doi.org/10.12701/yujm.1986.3.1.103
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- Cytochrome P-450 (CP-450) is one of the three components of the liver microsomal enzyme system which hydroxylates fatty acids, hydrocarbons and a variety of drugs and other foreign compounds. Female Balb/c mice were immunized with purified polychlorinated biphenyl (PCB) – induced CP-450 LMII. The spleen cells derived from immunized mice were fused with SP2 myeloma cells using polyethylene glycol (PEG 3500). The hybrid cells were selected by hypoxanthine-aminopterin-thymidine (HAT) medium and the culture fluid were screened by enzyme-linked immunosorbent assay to CP450 LMII. The hybrid cells(×107) were inoculated into intraperitoneal cavity of Balb/c mice for the purpose of production of ascetic fluids. Monoclonal antibody (Mab) was purified from ascitic fluid by DEAE cellulose ion exchange chromatography and I¹²⁵ labeled Mab was also confirmed by autoradiography and SDS-polyacrylamide gel electrophoresis (MW:55,000 and 110,000)
- Purification of Band 3 from the Human Erythrocyte Membrane and its Incorporation into Liposome.
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Jae Ryong Kim, Jung Hye Kim, Ki Yung Lee
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Yeungnam Univ J Med. 1986;3(1):41-48. Published online December 31, 1986
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DOI: https://doi.org/10.12701/yujm.1986.3.1.41
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- Band 3, the predominant 95,000 dalton anion transport protein, is the major intrinsic glycoprotein of the human erythrocyte membrane. This anion carrier exists as a dimer and binds the cytoskeletons such as spectrin, ankyrin and actin. And the liposomes are vesicular structures which form spontaneously upon hydration of phospholipids. These artificial lipid vesicles have been investigated as model of the biological membranes and as a mean of improving the delivery of nucleic acids, drugs, proteins and biological substances to specific target tissues and cells. In this study, we were purified Band 3 from the human erythrocyte membrane (ghost) was prepared by hemolysis of intact human erythrocyte with weak alkali-hypotonic solution. Band 6 was removed from ghost by extracting with solution of an ionic strength of 0.15. Band 3 and Band 4 were solubilized selectively by extracting Band 6-depleted ghosts with Triton X-100 under nondenaturing conditions. Band 3 was then purified from Triton X-100 extract treated with p-chloromercuribenzoate by sucrose density gradient ultracentrifugation. This purified Band 3 was incorporated into liposomes prepared by reverse-phase evaporation. Phosphatidyl L-serine and cholesterol (1:1 molar ratio) were dissolved in chloroform and the chloroform was removed by rotator evaporation under reduced pressure. Band 3 solution without Triton X-100 was introduced into a mixture of lipids and diethylether. Diethylether was subsequently removed by evaporation. This purified Band 3 and its incorporation into liposomes were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
- Cytological Study of the Introduction of Agrobacterium tumefaciens Spheroplasts into Nicotiana tabacum Protoplasts.
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Jung Hye Kim, Yong Bum Koo, Ki Yung Lee
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Yeungnam Univ J Med. 1985;2(1):175-181. Published online December 31, 1985
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DOI: https://doi.org/10.12701/yujm.1985.2.1.175
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- Agrobacterium tumefaciens induces cancerous growths called crown galls at wound sites on dicotyledonous plants. A large plasmid called T1 plasmid is responsible for virulence. Upon tumor induction, part of the plasmid, termed T-DNA, becomes integrated into plant genome and its genetic sequences are expressed. These properties allow T1 plasmids to be used as gene vectors in plants. Several in vitro methods for the transfer of T1 plasmid into plant cell have been developed. One of them is the treatment of bacterial spheroplasts and plant protoplasts mixture with polyethylene glycol that is generally used as fusogen in cell-to-cell fusion. Several workers investigated the interaction of bacterial spheroplasts with plant protoplasts in the presence of polyethylene glycol and suggested that the interaction is not fusion but endocytosis. In this report we observed the interaction of Agrobacterium tumefaciens spheroplasts with Nicotiana tabacum protoplasts by electron microscope. Agrobacterium tumefaciens spheroplasts with Nicotiana tabacum protoplasts were prepared and mixed in the presence of polyethylene glycol and high pH-high Ca²⁺ buffer. Then the interaction of the spheroplasts with the protoplasts was examined by transmission electron microscope. After the treatment of polyethylene glycol the spheroplasts adhered to the surface of the protoplasts and then they were engulfed by the protoplasts. After the high pH-high Ca²⁺ buffer treatment the engulfed spheroplasts lost their cell integrity. No fusion process was observed. Thus all these observation suggest that the introduction process of Agrobacterium tumefaciens spheroplasts into Nicotiana tabacum protoplasts with the aid of polyethylene glycol is endocytosis.
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