• Proceedings of the Korea Environmental Mutagen Society Conference
• /
• 2002.11a
• /
• pp.80-88
• /
• 2002

Recent studies demonstrate that collagen IV selectively pro-motes the repair of physiological processes in sublethally injured renal proximal tubular ceils (RPTC). We sought to further define the mechanisms of cell repair by measuring the effects of toxicant injury and stimulation of repair by L-ascorbic acid-2-phosphate (AscP), exogenous collagen IV, or function-stimulating integrin antibodies on the expression and subcellular localization of collagen-binding integrins (CBI) in RPTC. Expression of CBI subunits ${\alpha}_1$, ${\alpha}_2$, and ${\beta}_1$ in RPTC was not altered on day 1 after sublethal injury by S-(1,2-dichlorovinyl)-L-cysteine (DCVC). On day 6, expression of ${\alpha}_1$ and ${\beta}_1$ subunits remained unchanged, whereas a 2.2-fold increase in ${\alpha}_2$ expression was evident in injured RPTC. CBI localization in control RPTC was limited exclusively to the basal membrane. On day 1 after injury, RPTC exhibited a marked inhibition of active $Na^+$ transport and a loss of cell polarity characterized by a decrease in basal CBI localization and the appearance of CBI on the apical membrane. On day 6 after injury, RPTC still exhibited marked inhibition of active $Na^+$ transport and localization of CBI to the apical membrane. However, DCVC-injured RPTC cultured in pharmacological concentrations of AscP (500 ${\mu}$M)or exogenous collagen IV (50 ${\mu}$g/ml) exhibited an increase inactive $Na^+$ transport, relocalization of CBI to the basal membrane, and the disappearance of CBI from the apical membrane on day 6. Function-stimulating antibodies to CBI ${\beta}_1$ did not promote basal relocalization of CBI despite stimulating the repair of $Na^+$/$K^+$-ATPase activity on day 6 after injury. These data demonstrate that DCVC disrupts integrin localization and that physiological repair stimulated by AscP or collagen IV is associated with the basal relocalization of CBI in DCVC-injured RPTC. These data also suggest that CBI-mediated repair of physiological functions may occur independently of integrin relocalization.

• Biomedical Science Letters
• /
• v.2 no.1
• /
• pp.121-126
• /
• 1996

Specific monoclonal antibodies(mAbs) against bis-indole alkaloids related to vinblastine were established to develop a simple and specific immunoassay system for the quantitation of Vinca alkaloids. Vinca alkaloids were extracted from tissue cultured cells of periwinkle plant (Vinca rosea L.). Spleen cells from Balb/c mice immunized with vinblastine-bovine serum albumin(VBL-BSA) conjugate as immunogen were fused with myeloma cells(Sp2/0-Ag.14) in the presence of polyethylene glycol. In the preliminary experiments, 32 clones which highly reacted with VBL-BSA conjugate were selected by ELISA(Enzyme-linked immunosorbent assay).These clones were further analyzed by inhibition assay of ELISA. The results obtained with two typical monoclonal antibodies, KN-1 and KN-2, were described. KN-1 exhibited considerable reactivities with soluble dimeric bis-indole alkaloids, whereas no cross reacted with monomeric bis-indole alkaloids. However KN-2 showed cross reactivity with mono- and di-meric bis-indole alkaloids. Furthermore, KN-1 was applied to the immunoassay system for determining the VBL amounts of in vitro cultured cell extracts. This assay system could detect dimeric vinca alkaloid as low as 0.05 nM.

• Korean Journal of Food Science and Technology
• /
• v.36 no.1
• /
• pp.152-157
• /
• 2004

Gluten was extracted from domestic wheat flour using UTH buffer (4 M urea in 0.1 M Tris-HCl, pH 8.6) and validated by SDS-PAGE analysis for production of wheat flour products with reduced gluten content.. Anti-gluten polyclonal antibody was made by administering extracted gluten fraction on animal model. Anti-gluten serum titer of extracted gluten fraction was evaluated by ELISA, and that of antibody titer according to administration period. Anti-gluten sera were used for ELISA and immunoblot analysis before and after hydrolysis of gluten fraction at optimal pH and temperature condition for each protease. Gluten fraction separated by SDS-PAGE showed several bands covering 75 to 10 kDa, in which anti-gluten sera were 25, 34, and 45 kDa. Enzyme hydrolysis of gluten fraction revealed protein band sizes to be lower than 15 kDa. Content of pretense from bovine pancreas (b.p. protease) for gluten hydrolysis was estimated as 1 mg in 10 mL gluten fraction extracted for 4 hr.

• Korean Journal of Food Science and Technology
• /
• v.22 no.2
• /
• pp.177-182
• /
• 1990

The changes in the partition coefficient of model proteins (lysozyme, myoglobin, conalbumin, bovine serum albumin) in an aqueous two·phase system formed by polyethylene glycol and dextran were examined in order to improve the capacity of counter current distribution (CCD) for the protein fractionation and concentration . The protein distribution pattern in CCD with 30 tubes varied with the pH (4.5, 5.5, 6.5, 9.0, 12.0) and KCl concentration (0mM, 50mM, 250mM, 500mM) of the system. From the mixture of model proteins, pure myoglobin was appeared at the upperphase of 14th tube having 50mM of KCl at pH 5.5 and the upper-phase of 13th tube having 250mM of KCl at pH 6.5. Similarly pure BSA was obtained at the 14th tube having KCl 250mM with pH 4.5, pure lysozyme at the 19th tube having 500mM of KCl at pH 4.5 and the upper-phase of 16th tube 50mM of KCl at pH 5.5.

• Analytical Science and Technology
• /
• v.28 no.5
• /
• pp.309-316
• /
• 2015

The analytical method of lead in plasma by ICP-MS was validated after securing environment within class 1,000 classification. We tested specificity and accuracy of within-run and between-run. According to measurement of the amount of suspended particulates in a clean room, 0.3~62 particles were detected in 0.3 µm size while 0.0~28.3 particles were observed in 0.5 µm size. Total suspended particulates met required environment with up to 90.3 particles. The MDL (Method detection limit) of the sample which has been fabricated using fetal bovine serum (FBS) blank was 1.77 ng/L, and LOQ (Limit of quantification) was 5.55 ng/L. The slope, intercept and correlation coefficient of the calibration curve were y=1.09×10⁻³x+4.88×10⁻² and r=0.9999, which showed good correlation. The specificity, within-run and between-run accuracy satisfied the standard at more than 50 ng/L. The average lead concentration in plasma of the general people, current workers and retired workers was 55.4 ng/L, 440 ng/L, and 132 ng/L.

• Clinical and Experimental Reproductive Medicine
• /
• v.28 no.2
• /
• pp.121-129
• /
• 2001

Objective: The aim of this study is to compare the efficiency of a method for the cryopreservation of mouse blastocyst.. Methods: Mouse embryos were obtained at 2-cell stage and cultured to blastocyst stage in T6 medium supplemented with 10% fetal bovine serum. Morphologically normal blastocysts were collected and randomly divided to one control and four experimental groups. In control group, blastocysts were cultured in vitro continuously for additional two days. In group 2, blastocysts were exposed to vitrification solution (ethylene glycol) only without cryopreservation (exposure only group). In group 3, 4 and 5, blastocysts were cryopreserved by slow-freezing procedure with glycerol (slow-fteezing group) or by vitrification procedure using EM grids (EM grids group) and cryoloop (cryoloop group), respectively. Frozen blastocysts were thawed and cultured for additional two days. Twenty four hours after thawing, some blastocysts were fixed and stained with Hoechst 33342 (bisbenzimide) and the number of nuclei in each blastocysts were counted to confirm the survival of bias to cysts in experimental groups. Results: Survival rate and hatching rate of the blastocysts in slow-freezing group (24 h: 72.4% and 66.0%, 48 h: 63.2% and 64.6%) and EM grids group (24 h: survival rate 77.3%, 48 h: 70.1% and 71.4%) were significantly lower ($X^2$-test p<0.05) than those of control group (24 h: 93.4% and 86.0%, 48 h: 88.5% and 90.7%). In contrast, the survival rate and hatching rate of the blastocysts in cryoloop group (24 h: 84.1% and 84.1%,48 h 79.3% and 87.7%) is well compared with those in the control group. The mean (${\pm}SD$) cell number of blastocyst in the exposure only ($89.2{\pm}11.5$), EM grids ($85.0{\pm}10.3$) and cryoloop ($89.0{\pm}11.0$) groups, except slow-freezing group ($79.0{\pm}10.0$), were not significantly different from that of control group ($93.1{\pm}13.9$) 24 h after thawing (Student's t-test). Conclusion: This study demonstrates that higher survival rate of vitrified-thawed mouse blastocyst can be obtained using cryoloop as the embryo container at freezing rather than slow-freezing or vitrification using EM grids. The results of this study suggest that vitrification using cryoloop (with ethylene glycol) may be a preferable procedure for mouse blastocyst cryopreservation and could be applied to the human blastocyst cryopreservation.

• Asian-Australasian Journal of Animal Sciences
• /
• v.22 no.3
• /
• pp.328-335
• /
• 2009

This study was undertaken to investigate the influence of exposure and removal of four different cryoprotectants (CPAs) on the ATP content of cumulus cell-enclosed (COs) and cumulus cell-denuded (DOs) immature porcine oocytes. The in vitro nuclear maturation of the COs, exposed to and removed from the CPAs was also assessed. Both COs and DOs were exposed to 1.5 M concentrations of each CPA (ethylene glycol (EG); propylene glycol (PG); dimethyl-sulfoxide (DMSO); and glycerol (G)) for durations of 5, 15, and 30 minutes at room temperature ($23.5{\pm}1.5^{\circ}C$), and immersed in physiological saline supplemented with 20% (v/v) fetal bovine serum for 5 minutes ($39^{\circ}C$) to remove each CPA. Before, during and after exposure to each CPA, the ATP content of both the COs and the DOs was measured. After removal from each CPA an aliquot of the COs was matured for 44${\pm}$2 h, and their nuclear maturation rates were measured up to the metaphase stage of the second meiotic division (the M-II stage). The maturation rates up to the M-II stage were not significantly different between all the groups that were exposed to each CPA for 5 minutes. For 15 and 30 minute exposures, the maturation rates of the COs exposed to PG, DMSO and EG were almost the same as those of the control groups; however, the rates of G group exposed for 15 and 30 minutes were significantly lower (p<0.05) than the control group. These groups were also found to have a decrease in the ATP content of COs and DOs during and after exposure for the same periods (p<0.05). In addition, although the ATP contents of the COs after exposure to EG for any period were the same as the controls, the ATP content of the DOs exposed to EG for any period were significantly lower (p<0.05) than those of the controls. When the ATP content of the COs and DOs of each CPA were compared, the DOs exposed to PG were found to have a significantly greater level (p<0.05) than DOs exposed to G for any duration. In addition, the ATP content of DOs exposed to PG for 30 min and removal was also higher (p<0.05) than when exposed to DMSO for the same period. These findings indicate that PG may be a useful CPA for the cryopreservation of immature porcine oocytes.

• THE JOURNAL OF KOREAN ORIENTAL ONCOLOGY
• /
• v.5 no.1
• /
• pp.47-60
• /
• 1999

Traditional oriental medicines have been used for treatment of various kinds of human cancers for long times and some of them proven to be effective clinically. However, the pharmacological actions and mechanisms related to cancer treatment are generally unknown. In an effort to clarify the action mechanisms of several oriental medicines used for cancer treatments, we planned this experimental procedures. We selected Cordyceps sinensis (冬蟲下草), Punellae Herba (夏枯草), Rehmanniae Radix (熟地黃), Paeoniae Radix (白芍藥), Oldenlandiae Herba (白花蛇舌草), Partulaceae Herba (馬齒? ), Scdopendra subspinipes mutilans (蜈蚣), Mylabris Phalerara (班蟄), Phellinus igniarius(桑黃), Ganodermae Lignum(靈芝) for evaluation, which have been used for patients of gastric cancers. The twenty grams of medicines were boiled in 100ml of water for 1 hour and filtered with $0.2\;{\mu}m$ pore-sized filter unit to remove insoluble particles. Initially we evaluated the effects oriental medicines on growth inhibition in stomach cancer cells. The gastric cancer cell line, AGS, was cultured in RPMI 1640 supplemented with l0% heat-inactivated fetal bovine serum and treated with $10{\mu}l$ oriental medicines per 1ml of medium up to 48 hours. The specimens were subjected to MTT assay for evaluation of growth inhibition. We found mat Mylabris phalerata (班蟄) markedly suppressed the growth of cancer cells as shown in results. Next, we checked the effects of oriental medicines on cancer on cell cycles and apptosis. Mylabrls phalerata (班蟄) inhibited cell cycle progression of cancer cells a compared with control cells and cells treated with other medicines. In addition, Mylabri phalerata (班蟄) induced apoptosis in 30-40% of stomch cancer cells within 24 hours. Other oriental medicines used for this experiments did not show apoptosis-inducing effects on cancer cells. Finally, we determined the expression levels of genes associated with cell cycle and apoptosis. The expressions of Bcl-2 and bcl-XL were downregulated by the treatment of Mylabris phalerata (班蟄). However, the expression levels of genes related to cell cycles were not altered significantly. In conclusion, we found that Mylabris phalerata (班蟄) has in vivo gowth-inhibiting and apptosis-inducing effects on stomach cancer cells. However, we think that at least animal experiments are necessay for evduations.

• Journal of Acupuncture Research
• /
• v.25 no.1
• /
• pp.25-55
• /
• 2008

Objectives : The aim of the present study was to observe the effect of Astragali Radix Herbal-Acupuncture Solution(AR-HAS) at $ST_{36}$(jok-samni, $Z\acute{u}s\bar{a}n$ Li) on collagen- II -induced arthritis(CIA) in mice. Methods : DBA/1J mice were immunized with bovine type II collagen(CII) on days 0 and 21 to induce arthritis. The mice were divided into 5 groups : normal group(no CIA), control group(CIA+no treatment), needle prick group(CIA+single prick with an injection needle), saline group(CIA+saline injection) and ARHA group(CIA+ R-HA treatment). The needle prick, saline injection, and AR-HA groups were injected on the right $ST_{36}$(jok-samni) of mice for 9 weeks, 3 times a week, beginning 4 weeks after the booster immunization. Results : 1. The incidence of arthritis, AI(arthritis index), and joint edema decreased in the AR-HA group. 2. Weight gain, hypertrophy of the spleen, adhesion of the tissues, and transformation of the joint were restrained in the AR-HA group. 3. The concentrations of $IL-1{\beta}$, IL-6, $TNF-{\alpha}$, $IFN-{\gamma}$ in CIA mouse serum and $IFN-{\gamma}$, IL-10 in the CIA mouse spleen cell culture decreased significantly at $ST_{36}$ in the AR-HA group. 4. Total cell counts increased significantly, and the ratio of $CD3e^+$ to $CD45R^+$, $CD4^+$ to $CD8^+$, and $CD4^+$ to $CD25^+$ cells decreased significantly at $ST_{36}$ in the CIA mouse spleen cell culture of the AR-HA group. 5. Total cell counts decreased significantly, and $CD4^+/CD25^+$ and $CD45R^+/CD69^+$ decreased significantly at $ST_{36}$ in the CIA mouse lymph nodes of the AR-HA group. 6. $CD3e^+/CD69^+$ and $CD11b^+/Gr-1^+$ cells decreased significantly at $ST_{36}$ in the CIA mouse joints of the AR-HA group. 7. The histological examination showed that cartilage destruction and synoviocyte proliferation decreased in the CIA mouse joints of the AR-HA group, and collagen fiber was expressed similar to that seen in the normal group. Conclusions : Our experiments show that at $ST_{36}$, an anti-inflammatory mechanism of AR-HA controls synovial cell proliferation and protects against cartilage destruction in rheumatoid arthritis.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.38 no.6
• /
• pp.343-353
• /
• 2012

Objectives: This aim of this study was to effectively isolate mesenchymal stem cells (hSMSCs) from human submandibular skin tissues (termed hSMSCs) and evaluate their characteristics. These hSMSCs were then chemically induced to the neuronal lineage and analyzed for their neurogenic characteristics in vitro. Materials and Methods: Submandibular skin tissues were harvested from four adult patients and cultured in stem cell media. Isolated hSMSCs were evaluated for their multipotency and other stem cell characteristics. These cells were differentiated into neuronal cells with a chemical induction protocol. During the neuronal induction of hSMSCs, morphological changes and the expression of neuron-specific proteins (by fluorescence-activated cell sorting [FACS]) were evaluated. Results: The hSMSCs showed plate-adherence, fibroblast-like growth, expression of the stem-cell transcription factors Oct 4 and Nanog, and positive staining for mesenchymal stem cell (MSC) marker proteins (CD29, CD44, CD90, CD105, and vimentin) and a neural precursor marker (nestin). Moreover, the hSMSCs in this study were successfully differentiated into multiple mesenchymal lineages, including osteocytes, adipocytes, and chondrocytes. Neuron-like cell morphology and various neural markers were highly visible six hours after the neuronal induction of hSMSCs, but their neuron-like characteristics disappeared over time (24-48 hrs). Interestingly, when the chemical induction medium was changed to Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS), the differentiated cells returned to their hSMSC morphology, and their cell number increased. These results indicate that chemically induced neuron-like cells should not be considered true nerve cells. Conclusion: Isolated hSMSCs have MSC characteristics and express a neural precursor marker, suggesting that human skin is a source of stem cells. However, the in vitro chemical neuronal induction of hSMSC does not produce long-lasting nerve cells and more studies are required before their use in nerve-tissue transplants.