• Proceedings of the Korea Society of Environmental Biology Conference
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• 2004.05a
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• pp.1-15
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• 2004

Higher plants can be valuable genetic assay systems for monitoring environmental pollutants and evaluating their biological toxicity. Two assays are considered ideal for in situ monitoring and testing of soil, airborne and aqueous mutagenic agents; the Tradescantia stamen hair assay for somatic cell mutations and the Tradescantia micronucleus assay for chromosome aberrations. Both assays can be used for in vivo and in vitro testing of mutagens. Since higher plant systems are now recognized as excellent indicators and have unique advantages over in situ monitoring and screening, higher plant systems could be accepted by regulatory authorities as an alternative first-tier assay system for the detection of possible genetic damages resulting from the pollutants or chemicals used and produced by industrial sectors. It has been concluded that potential mutagen and carcinogen such as the heavy metals among indoor air particulates, volatile compounds in the working places, soil, and water pollutants contribute to the overall health risk. This contribution can be considerable under certain circumstances. It is therefore important to identify the level of genotoxic activity in the environment and to relate it to the biomarkers of a health risk in humans. The results from the higher plant bioassays could make a significant contribution to assessing the risks of pollutants and protecting the public firom agents that can cause mutation anuor cancer. The plant bioassays, which are relatively inexpensive and easy to handle, are recommended for the scientists who are interested in monitoring pollutants and evaluating their environmental toxicity to living organisms.

• Journal of Plant Biotechnology
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• v.45 no.4
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• pp.315-321
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• 2018

Molecular and functional characterization of proteins and their levels is of great interest in understanding the mechanism of diverse cellular processes. In this study, we report on the convenient Escherichia coli-based protein expression system that allows recombinant of soluble proteins expression and cytosolic domain of membrane-localised kinases, followed by the detection of autophosphorylation activity in protein kinases. This approach is applied to regulatory proteins of Arabidopsis thaliana, including 14-3-3, calmodulin, calcium-dependent protein kinase, TERMINAL FLOWER 1(TFL1), FLOWERING LOCUS T (FT), receptor-like cytoplasmic kinase and cytoplasmic domain of leucine-rich repeat-receptor like kinase proteins. Our Western blot analysis which uses phospho-specific antibodies showed that five putative LRR-RLKs and two putative RLCKs have autophosphorylation activity in vitro on threonine and/or tyrosine residue(s), suggesting their potential role in signal transduction pathways. Our findings were also discussed in the broader context of recombinant expression and biochemical analysis of soluble and membrane-localised receptor kinases in microbial systems.

• Research in Plant Disease
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• v.19 no.4
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• pp.288-293
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• 2013

In worldwide, viral diseases of apple plants has caused the serious problems like reduced production and malformation of fruits. Also, the damages of apple plants by virus and/or viroid infection (Apple chlorotic leaf spot virus, Apple stem grooving virus, Apple mosaic virus, and Apple scar skin viroid) were reported in Korea. However there is few report about the protection approach against the infection by apple viruses. Therefore, this paper introduced the experimental protocol for the development of virus-free apple cultivars (Danhong, Hongan, Saenara, Summerdream). Apple plants were treated at $37^{\circ}C$ for 4 weeks and shoot tips were cultured in vitro. After heat treatment, the detection of apple viruses was performed by RT-PCR using virusspecific detection primers in new apple cultivars. With the heat treatments followed by in vitro shoot tip culture, the proportion of virus-free stocks of 'Danhong', 'Hongan', 'Saenara', and 'Summerdream' was 28%, 16%, 12%, and 12%, respectively. Taken together, this approach can be a good tool for production of virus-free apple stocks.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.3
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• pp.419-432
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• 1999

Objective: To establish the evaluation system of the quality of oocytes on the basis of the incidence of cumulus cells apoptosis, to investigate the relationships beween the incidence of cumulus cells and the outcomes of IVF-ET. Method: Thirth-four cycles undergoing controlled ovarian hyperstimulation for IVF-ET with tubal infertility (23 cycles) or unexplained infertility (11 cycles) were included in this study. Cumulus cell masses surrounding mature oocyte and co-culture of embryos with autologous cumulus cells during IVF-ET process. The incidence of apoptosis in cumulus cells was assessed by apoptosis detection kit fluorescein. The effect of co-culture using cumulus cells and the incidence of cumulus cells apoptosis. Results: The results were as follows: 1. The incidence of apoptosis in cumulus cells markedly increased in patients aged 40 or over, while the fertilization rate was greatly decreased in those age group. 2. Apoptosis in cumulus cells was found in both the fertilized oocytes and unfertilized oocytes, but the incidence of apoptosis was higher in unfertilized oocytes. 3. There is no clear correlation between apoptosis in cumulus cells and the number of oocytes retrieved. However, the incidence of apoptosis was increased when the number of oocytes retrieved was 5 and fewer in comparison with $6{\sim}10$. 4. Embryo grade was significantly affected by the incidence of apoptosis in cumulus cells. 5. Pregnancy rate of IVF-ET per cycle was 29.4%, and the pregnant group had the higher fertilization rate and a significantly lower incidence of apoptosis in cumulus cells compared with the nonpregnant group. 6. When cumulus cells were used as helper cells in the co-culture of the embryo, in vitro activity of cumulus cells based on morphological change and proliferation did not influence the quality of embryo, but was closely associated with the implantation rate and pregnancy rate, which was enhanced when morphological changes and proliferation of cumulus cells was more active. 7. This difference in the outcome of IVF-ET according to in vitro activity of cumulus cells used for co-cultue was not associated with the incidence of apoptosis in cumulus cells; but rather had likely relations with the different secretion pattern of protein, which may be an embryo trophic factor by cumulus cells. Conclusion: These results suggest that the incidence of apoptosis in cumulus cells can be used in predicting oocyte qualities and the outcomes of IVF-ET. And the effect of co-culture largely depends on the in vitro activity of cumulus cells as well.

• Journal of the Korean Society of Visualization
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• v.8 no.1
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• pp.46-52
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• 2010

In the present study, we employed the confocal laser scanning microscopy (CLSM) system to visualize the blood flow field with $1{\times}1{\mu}m^2$ spatial resolution. Based on the confocal microscopic image of red blood cells (RBCs), we performed the velocity vector field measurement and evaluated characteristics of cell migration from the cell depleted layer thickness calculation. The rat and mouse's blood were supplied into a micro glass tubes in vitro. The line scanning rate of confocal microscopy was 15 kHz for a $500{\times}500$ pixels image. As a result, the red blood cell itself can be used as a tracer directly without any kind of invasive tracer particle to get the velocity vector field of blood flow by performing particle image velocimetry (PIV) technique.

• Journal of Plant Biotechnology
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• v.39 no.3
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• pp.196-204
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• 2012

This study was carried out to develop reliable systems for somatic embryogenesis in oil palm tree (Elaeis guineensis Jacq.), and to verify the somaclonal variants by RAPD analysis. Embryogenic callus was induced successfully on modified half-strength MS medium containing $NaH_2PO_4{\cdot}2H_2O$ and casein. Embryogenic callus was further developed to somatic embryo mass (SEM), which is very hard and bonded tightly each other. Plantlets were proliferated when SEM was cultured on modified MS medium containing half strength $NH_4NO_3$, casein and L-ascorbic acid. Plantlets were transplanted into pots containing artificial soils. When RAPD analysis was conducted using randomly selected 95 in vitro plantlets and 19 random primers, somaclonal variation was detected using BNR35 primer. There was missing band around 1 kb in #22, #28, #35, and #77 plantlets. In addition, bands obtained from #28, #35, and #77 was much stronger than other normal bands. The blast results at NCBI revealed that somaclonal variation observed in this study was related to chloroplast genome of oil palm. The results also revealed that oil palm reproduction system through somatic embryogenesis is quite reliable and early detection of somaclonal variants seem to be possible at in vitro stage by RAPD analysis.

• Toxicological Research
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• v.18 no.1
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• pp.59-64
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• 2002

Substantial evidences have been accumulated about the hormone-like effects of exogenous substances such as pesticides and industrial chemicals during past years. The effects of these substances on the endocrine system are believed to be either enhancing or reducing of various endocrine action. It is necessary to identify putative causal agents by the batter system and to assess their ability to disrupt the endocrine system. A variety of in vitro and In vivo approaches have been used to determine the androgenic effects of environmental chemicals. To establish the method for assessment of the putative endocrine disruptors with androgenic activity, we carried out the cell proliferation assay by MTS method after treatment with the various concentration of testosterone in LNCaP cells (human prostatic cancer cell line) and also observed the expression of androgen-related genes by quantitative RT-PCR. In the cell proliferation assay, the results showed that the grouth of LNCaP cells increased within level of at least 10pM testosterone. We measured by quantitative RT-PCR method on the effects of testosterone on mRNA expression of androgen receptor (AR), prostate-specific antigen (PSA), bone morphogenetic protein (BMP) and BMP receptor (BMPR) In LNCaP cells. The results demonstrated that mRNA expression of PSA and BMPR-IB was observed differently within level of at least 0.01 pM testosterone compared with non-treated control. These observations suggest that the detection of PSA and BMPR-IB mRNA by the quantitative RT-PCR in LNCaP cells is very sensitive method to identify the endocrine disruptors to have the androgenic effects.

• Development and Reproduction
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• v.13 no.4
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• pp.217-226
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• 2009

Effects of sera from several fish species on monolayer formation, viability and functions of catfish hepatocytes were investigated to establish a primary hepatocyte culture system for screening endocrine disruptors. Hepatocytes of Korean catfish (Silurus asotus) were attached and formed monolayer using the media supplemented with their own serum or sera from eel and tilapia, but not with fetal bovine serum (FBS). The amount of fish sera (0.5~3%) for monolayer culture of the catfish hepatocytes was less than 1/10 of FBS (5~20%) that is commonly used for primary culture of hepatocytes of other species. The results indicate that FBS can be replaced with sera from some fish species and the fish sera are more effective than FBS in maintaining the shape and functions of the hepatocytes. The primary culture of catfish hepatocytes was maintained monolayer with fish sera for at least 10 days, which makes possible to be used for screening the activities of endocrine disruptors. In conclusion, the primary culture system of hepatocytes with fish sera in the present study could be a useful tool for screening and studying endocrine disruptors.

• The Korean Journal of Pharmacology
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• v.27 no.1
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• pp.7-12
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• 1991

We measured the developmental increase of tyrosine hydroxylase(TH) activity and dopamine content with high performance liquid chromatography with electrochemical detection(HPLC-EC) in dissociated cultures of fetal rat brainstem(E14). TH activity and dopamine content increased progressively upto 7 days in vitro, when the effects of various drugs on the dopamine contents were studied. ${\alpha}-Methyl-p-tyrosine$, a TH inhibitor and NSD-1015, an inhibitor of aromatic amiono acid decarboxylase effectively depleted dopamine contents. Dopamine contents were depleted by reserpine and increased by pargyline. When cultures grown for 1 week in control medium were then exposed to tetrodotoxin$(0.1\;{\mu}M$) for 7 days, exposure to tetrodotoxin markedly decreased TH activity. All the above results indicate that dopamine metabolism in the cultered cells reflect reliably the property of brain dopamine metabolism. We suggest that measuring TH activity and dopamine content in brainstem culture with HPLC-EC can be useful tool in the study of pharmacology as well as toxicology of the central dopaminergic system.

• Environmental Mutagens and Carcinogens
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• v.24 no.1
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• pp.33-39
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• 2004

To validate and to estimate the chemical hazard playa very important role to environment and human health. The detection of many synthetic chemicals including agrochemicals that may pose a genetic hazard in our environment is of great concern at present. Since these substances are not limited to the original products, and enter the environment, they have become widespread environmental pollutants, thus leading to a variety of chemicals that possibly threaten the public health. Pyrazosulfuron-ethyl [Ethyl-5-(4,6-dimethoxypyrimidin-2-ylcarbamoylsulfamoyl)-1-methylpyrazole-4-carboxylate, $C_{14}H_{18}N{6}O_{7}S,$ M.W. =414.39, CAS No. 93697-74-6], is one of well known rice herbicide belong in the sulfonyl urea group. To clarify the genotoxicity of this agrochemical, Ames bacterial reversion assay, in vitro chromosomal aberration assay with Chinese hamster lung (CHL) fibroblast and bone marrow micronucleus assay in mice were subjected. In Ames assay, although pyrazosulfuron-ethyl revealed cytotoxic at 5,000-140 $\mug/plate$ in Salmonella typhimurium TA100, no dose-dependent mutagenic potential in 4.4～70 $\mug/plate$ of S. typhimurium TA 98, TA 100, TA1535 and TA 1537 both in the absence and presence of S-9 metabolic activation system was observed. Using CHL fibroblasts, the 50% cell growth inhibition concentration $(IC_{50})$ of pyrazosulfuron-ethyl was determined as 1,243 $\mug/mL,$ and no chromosomal aberration was observed both in the absence and presence of S-9 mixture in the concentration range of 311-1,243 $\mug/mL.$ And also, in vivo micronucleus assay using mouse bone marrow, pyrazosulfuron-ethyl revealed no remarkable induction of MNPCE (micronucleated polychromatic erythrocytes/1000 polychromatic erythrocytes) in the dose range of 625-2,500 mg/kg body weight when administered orally. Consequently, Ames bacterial gene mutation with Salmonella typhimurium, in vitro chromosome aberration with mammalian cells and in vivo bone marrow micronucleus assay revealed no clastogenic potential of pyrazosulfuron-ethyl in this study.