• Toxicological Research
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• v.18 no.3
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• pp.293-300
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• 2002

Chemical carcinogen-induced alteration of aldehyde metabolic enzymes were examined in clone 9 cell. Diethylnitrosamine (DENA), N-nitrosoethylurea (NEU) and N-nitrosomorpholine (NNM) were wed as model carcinogens. Changes in enzyme activities by repetitive treatment of DENA, NEU or NNM were analyzed in terms of specific activities and activity stainings of the enzymes on the gel. Upon treatment of DENA, lipid peroxide level increased upto 10 fold, indicating strong oxidative stress state of the cell. Notable enhancement of ADH and ALDH activity occurred after DENA treatment, while glutathione-S-transferase activity was slightly increased. Furthermore, about 2.5 fold higher superoxide dismutase (SOD) activity was detected during deactivation of catalase (CAT) activity by repetitive treatment of DENA. However in NEU-treated cell, about 2.3 fold higher ALDH activity was found while ADH activity was slightly increased. Notable increase CAT and SOD could also be found. In contrast, maximum 3.5 fold higher CAT activity occurred during SOD deactivation in NNM-treated cell. These results suggest that there might be different enzymatic responses in relation to cell protection against DENA, NEU or NNM.

• Journal of Korean Ophthalmic Optics Society
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• v.12 no.3
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• pp.19-25
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• 2007

It was studied how using soft contact lens multi-purpose solution (MPS), often used for medical treatment, effects the inhibition on cell growth, and how using the MPS demages eye cells, on rabbit eye's corneal epithelium and endothelium tissue. $ReNu^{(R)}$ (Baush & Lomb, USA), Opti-free $express^{(R)}$ (Alcon, USA), Free-sol $plus^{(R)}$ (Hanamedicon, Korea) were used as MPS. After culturing Clone 1-5C-4 cell lines (Human conjunctival cell lines), cell growth inhibition rate was measured by MIT assay. By making Hematoxylin and Eosin stain specimen, the morphology was observed by optical microscope. In the In vivo experiment, 9 white rabbit eyes (18 eyes) were classified into 3 groups. The experiment group is left eyes (9 eyes) of rabbit, and MPS were dropped; however the control group, the right eyes (9 eyes), were only used a saline solution without a preservatives. After the dropping within the period, the cornea surface of rabbit endothelium tissue was observed using scanning electron microscopy (SEM).

• International Journal of Industrial Entomology and Biomaterials
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• v.9 no.2
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• pp.173-181
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• 2004

To delay the onset of apoptosis in the culture, transformed Tn 5B1-4 cells harboring anti-apoptotic genes, bcl-2 and baculovirus p35, have been established and analyzed for their anti-apoptotic ability in suspension culture using spinner flasks. In the suspension culture at agitation speeds of 100 rpm and 200 rpm, the cell growth of cell clone expressing Bcl-2 protein was much higher than other two clones and the maximum cell density of the clone was 6.0 ${\times}$ 10$^{6}$ cells/ml and 6.2 ${\times}$ 10$^{6}$ cells/ml at day three of the incubation. On the other hand, the cell growth of cell clone expressing baculovirus protein P35 was much higher than other two clones in suspension culture at agitation speed of 300 rpm and the maximum cell density of the clone was 6.1 ${\times}$ 10$^{6}$ cells/ml at day three of the incubation. Based on the pattern of genomic DNA laddering and the microscopic observation of apoptotic bodies, the more apoptotic bodies are induced in Tn 5B1-4 control cell clone at higher agitation speed. This result shows that the shear stress can be a main factor in inducing apoptosis in spinner flask culture. At low agitation speed, cell clone expressing Bcl-2 was more effective in delaying the onset of apoptosis than the cell clone expressing P35. On the other hand, at high agitation speed, cell clones expressing baculovirus P35 was more effective in delaying the onset of apoptosis than the cell clone expressing Bcl-2. Therefore, anti-apoptotic genes, bcl-2 and baculovirus p35, can playa distinct role depending on agitation speed in the suspension culture.

• Journal of Life Science
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• v.18 no.11
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• pp.1499-1506
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• 2008

To examine antitumor activity of the edible plant Zanthoxylum schinifolium, the cytotoxic effect of various organic solvent extracts of its stems on human acute leukemia Jurkat T cells was investigated. Among these extracts such as methanol extract (SS-7), methylene chloride extract (SS-8), ethyl acetate extract (SS-9), n-butanol extract (SS-10), and residual fraction (SL-11), SS-8 exhibited the most cytotoxic activity against Jurkat T cells. The methylene chloride extract (SS-8) possessed the apoptogenic activity capable of inducing sub-G1 peak along with apoptotic DNA fragmentation in Jurkat T cells. Western blot analysis revealed that SS-8 induced apoptosis via mitochondrial cytochrome c release into cytoplasm, subsequent activation of caspase-9 and caspase-3, and cleavage of PARP, which could be blocked by overexpression of Bcl-xL. Jurkat T cell clone I2.1 $FADD^{-/-}$) and Jurkat T cell clone I9.2 (caspase-$8^{-/-}$ were as sensitive as was the wild-type Jurkat T cell clone A3 to the cytotoxic effect of SS-8, suggesting no contribution of Fas/FasL system to the SS-8-mediated apoptosis. The GC-MS analysis of SS-8 showed that it was composed of 16 ingredients including 9,12-octadecanoic acid (18.62%), 2,4-dihydro-5-methyl-4- (1-methylethylidene)- 2-(4-nitrophenyl)-3H- pyrazol-3-one (14.97%), hexadecanoic acid (14.23%), (z,z)-6,9-pentadecadien- 1-ol (13.73%), 5,6-dimethoxy-2-methyl benzofuran (10.95%), and 4-methoxy-2-methylcinnamic acid (5.38%). These results demonstrate that the methylene chloride extract of the stems of Z. schinifolium can induce apoptotic cell death in Jurkat T cells via intrinsic mitochondria-dependent caspase cascade regulated by Bcl-xL without involvement of the Fas/FasL system.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.712-720
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• 2007

To investigate clonal variations of recombinant Chinese hamster ovary(rCHO) clones in response to culture pH and temperature, serum-free suspension cultures of two antibody-producing CHO clones(clones A and B), which were isolated from the same parental clone by the limiting dilution method, were performed in a bioreactor at pH values in the range of 6.8-7.6, and two different temperatures, $33^{\circ}C\;and\;37^{\circ}C$. In regard to cell growth, clone A and clone B displayed similar responses to temperature, although their degree of response differed. In contrast, clones A and B displayed different responses to temperature in regard to antibody production. In the case of clone A, no significant increase in maximum antibody concentration was achieved by lowering the culture temperature. The maximum antibody concentration obtained at $33^{\circ}C$(pH 7.4) and $37^{\circ}C$(pH 7.0) were $82.0{\pm}2.6$ and $73.2{\pm}4.1{\mu}g/ml$, respectively. On the other hand, in the case of clone B, an approximately 2.5-fold increase in maximum antibody concentration was achieved by lowering the culture temperature. The enhanced maximum antibody concentration of clone B at $33^{\circ}C$($132.6{\pm}14.9{\mu}g/ml$ at pH 7.2) was due to not only enhanced specific antibody productivity but also to prolonged culture longevity. At $33^{\circ}C$, the culture longevity of clone A also improved, but not as much as that of clone B. Taken together, CHO clones derived from the same parental clone displayed quite different responses to culture temperature and pH with regards antibody production, suggesting that environmental parameters such as temperature and pH should be optimized for each CHO clone.

• Biomedical Science Letters
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• v.15 no.1
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• pp.55-60
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• 2009

Insect cells such as Spodoptera frugiperda Clone 9 (Sf9) cells are widely chosen as the host for heterologous expression of a mammalian sugar transport protein using the baculovirus expression system. Characterization of the expressed protein is expected to include assay of its function, including its ability to transport sugars and to bind inhibitory ligands such as cytochalasin B. It is therefore very important first to establish the transport characteristics and other properties of the endogenous sugar transport proteins of the host insect cells. However, very little is known of the transport characteristics of Sf9 cells, although their ability to grow on TC-100 medium strongly suggested the presence of endogenous glucose transport system. In order to investigate the substrate and inhibitor recognition properties of the Sf9 cell transporter, the ability of pentoses to inhibit 2-deoxy-D-glucose (2dGlc) transport was investigated by measuring inhibition constants $(K_i)$. To determine the time period over which of sugar into the Sf cells was linear, the uptake of 2dGlc 0.1mM extracellular concentration was measured over periods ranging from 30 seconds to 30 minutes. The uptake was linear for at least 2 minutes at the concentration, implying that uptake made over a 1 minute time course would reflect initial rates of the sugar uptake. The data have also revealed the existence of a saturable transport system for pentose uptake by the insect cells. The transport was inhibited by D-xylose and D-ribose, although not as effective as hexoses. However, L-xylose had a little effect on 2dGlc transport in the Sf9 cells, indicating that the transport is stereoselective. Unlike the human erythrocyte-type glucose transport system, D-ribose had a somewhat greater apparent affinity for the Sf9 cell transporter than D-xylose. It is therefore concluded that Sf9 cells contain an endogenous sugar transport activity that in some aspects resembled the human erythrocyte-type counterpart, although the Sf9 and human transport systems do differ in their affinity for cytochalasin B.

• Journal of Microbiology and Biotechnology
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• v.9 no.4
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• pp.475-481
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• 1999

The study of lignin, a major component of secondary cell wall, has been partly focused on its removal from the woody part in the kraft pulping industry. Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.l95) catalyzes the synthesis of cinnamyl alcohols from corresponding cinnamaldehydes. A cDNA clone, MdCADl, encoding putative CAD from apples (Malus domestica Borkh. cv Fuji) was characterized in this study. The clone contains an open reading frame of 325 amino acid residues, which shows a greater than 80％ identity with Eucalyptus CADl. MdCADl mRNA was detectable in vegetative tissues and was strongly expressed in the fruit. The expression pattern of MdCADl mRNA in the fruit peel after light exposure was also examined. The mRNA was rapidly increased until 1 day after light exposure and remained stable thereafter, suggesting that MdCADl is light inducible. The inducibility of the MdCADl gene was examined using several environmental stresses. Mechanical wounding of leaves increased the MdCADl mRNA level and the induction was further increased by salicylic acid. Southern blot hybridization showed that there is either one or a few copies of CAD genes in apples. To our knowledge, it is believed that MdCADl is the first CAD clone expressed predominantly in fruit.

• Biomedical Science Letters
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• v.7 no.4
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• pp.161-166
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• 2001

Most mammalian cells take up glucose by passive transport proteins in the plasma membranes. The best known of these proteins is the human erythrocyte glucose transporter, GLUT1. High levels of heterologous expression far the transporter are necessary for the investigation of its three-dimensional structure by crystallization. To achieve this, the baculovirus expression system has become popular choice. However, Spodoptera frugiperda Clone 9 (Sf9) cells, which are commonly employed as the host permissive cell line to support baculovirus replication and protein synthesis, grow well on TC-100 medium that contains 0.1% D-glucose as the major carbon source, suggesting the presence of endogenous glucose transporters. Furthermore, very little is known of the endogenous transporters properties of Sf9 cells. Therefore, human GLUT1 antibodies would play an important role for characterization of the GLUT1 expressed in insect cell. However, the successful use of such antibodies for characterization of GLUT1 expression m insect cells relies upon their specificity for the human protein and lack of cross-reaction with endogenous transporters. It is therefore important to determine the potential cross-reactivity of the antibodies with the endogenous insect cell glucose transporters. In the present study, the potential cross-reactivity of the human GLUT1 antibodies with the endogenous insect cell glucose transporters was examined by Western blotting. Neither the antibodies against intact GLUT1 nor those against the C-terminus labelled any band migrating in the region expected fur a protein of M$_r$ comparable to GLUT1, whereas these antibodies specifically recognized the human GLUT1. Specificity of the human GLUT1 antibodies tested was also shown by cross-reaction with the GLUT1 expressed in insect cells. In addition, the insect cell glucose transporter was found to have very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.

• IMMUNE NETWORK
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• v.5 no.3
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• pp.163-171
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• 2005

Background: To perform the successful dendritic cell-based cancer immunotherapy one of the main issues to be solved is the source of antigen for DC pulsing. Limitations occur by using auto-tumor lysate due to the difficulties obtaining enough tumor tissue(s) quantitatively as well as qualitatively. In this study the possibility of allogeneic tumor cell lysate as a DC pulsing antigen has been tested in mouse melanoma pulmonary me tastasis model. Methods: B16F10 melanoma cells $(1{\timeS}10^5/mouse)$ were inoculated intra venously into the C57BL/6 mouse. Therapeutic DCs were cultured from the bone marrow myeloid lineage cells with GM-CSF and IL-4 (1,000 U/ml each) for 7 days and pulsed with lysate of either autologous B16F10 (B-DC), allogeneic K1735 (C3H/He origin; K-DC) or CloneM3 (DBA2 origin; C-DC) melanoma cells for 18 hrs. Pulsed-DCs $(1{\times}10^6/mouse)_{[CGP1]}$ were injected i.p. twice with one week interval starting from the day 1 after tumor cell inoculation. Results: Without observable toxicity, allogeneic tumor cell lysate pulsed-DC induced the significantly better anti-tumor response (tumor scale: $2.7{\pm}0.3,\;0.7{\pm}0.3\;and\;0.3{\pm}0.2$ for saline, B-DC and C-DC treated group, respectively). Along with increased tumor specific lymphocyte proliferations, induction of IFN-${\gamma}$ secretion against both auto- and allo-tumor cell lysates was observed from the DC treated mice. (w/B16F10-lysate: $44.97{\pm}10.31,\;1787.94{\pm}131.18,\;1257.15{\pm}48.27$, w/CloneM3 lysate: 0, $1591.13{\pm}1.83,\;1460.47{\pm}86.05pg/ml$ for saline, B-DC and C-DC treated group, respectively) Natural killer cell activity was also increased in the mice treated with tumor cell lysate pulsed-DC ($8.9{\pm}_{[CGP2]}0.1,\;11.6{\pm}0.8\;and\;12.6{\pm}0.7%$ specific NK activity for saline, B-DC and C-DC treated group, respectively). Conclusion: Conclusively, promising data were obtained that allogeneic-tumor cell lysate can be used as a tumor antigen for DC-based cancer immunotherapy.

• Korean Journal of Animal Reproduction
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• v.13 no.2
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• pp.93-97
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• 1989

These experiments were carried out to develop the technique of nuclear transplantation necessary for elevating utilization efficiency of high quality embryos and the production of clone animals. Embryos of pronucleus stages were obtained from ICR mice. Removal of pronuclei and their transfer to recipient embryos were carried out by micromanipulation and virus mediation. The results obtained in these experiments were summarized as follows ; 1. Total 337 pairs of pronuclear stage embryos were subjected to nuclear transplantatin and 247 pairs(73.3%) of them were successfully transplanted and the number of fused embryos between transplanted nucleus and cytoplasm was 188 pairs(55.8%). 2. Of the 188 fused embryos cultured in vitro, 174(92.4%), 131(69.7%) and 117(62.2%) embryos were developed to 2-cell, morula and blastocyst stages, respectively. 3. When total 104 nuclear transplanted embryos were transferred to uteri of recipient mice on day 2-3 of pseudopregnancy, 4 of 12 recipient mice were pregnant and the number of embryos developed to young was 28(26.9%).