• Molecular & Cellular Toxicology
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• 제4권3호
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• pp.211-217
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• 2008

To clarify the DNA damage from reactive oxygen species, we measured the DNA damage through Fpg/Endo III FLARE (Fragment Length Analysis with Repair Enzyme) assay and real time RT-PCR. The 80 SD rats assigned to 4 dose groups exposed to cumene vapor for 90 days. With Fpg/Endo III FLARE assay in hepatocytes, we found the OTM (Olive Tail Moment) and TL (Tail Length) significantly increased in no-enzyme treated and Fpg-treated control and 8 ppm groups with 28 days exposure. In Endo III-treated 8 ppm group, significantly increased the values with 90 days exposure. With lymphocytes, it was founded the values significantly increased in no-enzyme treated 800 ppm group in 28 and 90 days. It was significantly increased in Endo III-treated 80 ppm for 28 days and 800 ppm for 90 days. From the above findings, FLARE assay was suggested as being available as a biological marker for DNA damage induced by cumene exposure in SD rats. And we used real time RT-PCR for the OGG1 mRNA expression, it had dose-dependent biologic effects in 1 day exposure, but decrease the levels of rOGG1 mRNA. Our findings provide evidence that cumene exposure may cause suppression of rOGG1 in the rat hepatocytes or lymphocytes.

• Environmental Analysis Health and Toxicology
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• 제9권1_2호
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• pp.37-51
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• 1994

To evaluate the genotoxicities of workers exposed to glue and glue cleaning solution, ambient air monitoring of working place, animal study and human monitoring were carried out. By GC-MS analysis, air samples collected from shoesmaking plant were found to be toluene, xylene, cyclohexane, n-hexane, methyl ethyl ketone, trichloroethylene, butylacetate, isopropyl alcohol. Glue and glue cleaning solution from shoesmaking plant were applicated topically to the CD-1 mice. DNA was isolated from skin 24 hr following the application and analysed for DNA-adducts using the nuclease $P_1$version of $^{32}$P-postlabelling assay. RAL (Relative Adduct Labelling, adducts$10^8$ nucleotides) was significantly increased in a dose-dependent manner in the glue cleaning solution treated mice skin. Peripheral blood DNA-adducts of workers exposed to glue and glue cleaning solution were also analysed by the same method, but there were not significant differences in the peripheral blood DNA-adducts level between exposed and control workers. In addition, glue cleaning solution from shoes factory was evaluated for mutagenicity in the Salmonella plate incorporation assay using strains TA 100 and TA 1535 in the presence and absence of Arochlor 1254-induced rat liver S$_{9}$. There was evident mutagenicity for cleaning solution in TA 100 regardless of $S_9$, but TA 1535 showed positive only in the absence of $S_9$when predicted by Stead model of mutagenicity prediction (p=0.0000). The urine concentrates from workers and controls were also assayed for mutagenicity towards strain TA 100 of Salmonella typhimurium in the presence of $S_9$ using Kado's microsuspension assay, but their mutagenic activities were not found to be significant. These data suggest that shoesmaking workers are exposed to genotoxic compounds and need to be monitored by testing the mutagenicity of human urines. However, $^{32}$P-postlabelling application requires further validation for the routine monitoring of human exposure.osure.

• Journal of Microbiology and Biotechnology
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• 제16권6호
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• pp.952-960
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• 2006

As a unicellular green alga that possesses many of the metabolic pathways present in higher plants, Chlorelia offers many advantages for expression of heterologous proteins. Since strong and constitutive promoters are necessary for efficient expression in heterologous expression systems, the development of such promoters for use in the Chlorella system was the aim of this study. Proteins encoded by the early genes of algal viruses are expressed before viral replication, probably by the host transcriptional machinery, and the promoters of these genes might be useful for heterologous expression in Chlorella. In this study, putative promoter regions of DNA polymerase, ATP-dependent DNA ligase, and chitinase genes were amplified from eight Korean Chlorella virus isolates by using primer sets designed based on the sequence of the genome of PBCV-1, the prototype of the Phycodnaviridae. These putative promoter regions were found to contain several cis-acting elements for transcription factors, including the TATA, CAAT, NTBBF1, GATA, and CCAAT boxes. The amplified promoter regions were placed into Chlorella transformation vectors containing a green fluorescence protein (GFP) reporter gene and the Sh ble gene for phleomycin resistance. C. vulgaris protoplasts were transformed and then selected with phleomycin. The GFP fluorescence intensities of cells transformed with chitinase, DNA polymerase, and DNA ligase gene promoter-GFP fusion constructs were 101.5, 100.8, and 95.8%, respectively, of that of CaMV 35S-GFP-transformed Chlorella cells. These results demonstrate that these viral promoters are active in transformed Chlorella.

• BMB Reports
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• 제39권5호
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• pp.492-501
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• 2006

Since differentiation therapy is one of the promising strategies for treatment of leukemia, universal efforts have been focused on finding new differentiating agents. In that respect, we used guanosine 5'-triphosphate (GTP) to study its effects on K562 cell line. GTP, at concentrations between 25-200 ${\mu}M$, inhibited proliferation (3-90%) and induced 5-78% increase in benzidine-positive cells after 6-days of treatments of K562 cells. Flow cytometric analyses of glycophorine A (GPA) showed that GTP can induce expression of this marker in more mature erythroid cells in a time- and dose-dependent manner. These effects of GTP were also accompanied with inhibition of DNA synthesis (measured by [$^3H$]-thymidine incorporation) and early S-phase cell cycle arrest by 96 h of exposure. In contrast, no detectable effects were observed when GTP administered to unstimulated human peripheral blood lymphocytes (PBL). However, GTP induced an increase in proliferation, DNA synthesis and viability of mitogen-stimulated PBL cells. In addition, growth inhibition and differentiating effects of GTP were also induced by its corresponding nucleotides GDP, GMP and guanosine (Guo). In heat-inactivated medium, where rapid degradation of GTP via extracellular nucleotidases is slow, the anti-proliferative and differentiating effects of all type of guanine nucleotides (except Guo) were significantly decreased. Moreover, adenosine, as an inhibitor of Guo transporter system, markedly reduced the GTP effects in K562 cells, suggesting that the extracellulr degradation of GTP or its final conversion to Guo may account for the mechanism of GTP effects. This view is further supported by the fact that GTP and Guo are both capable of impeding the effects of mycophenolic acid. In conclusion, our data will hopefully have important impact on pharmaceutical evaluation of guanine nucleotides for leukemia treatments.

• Asian Pacific Journal of Cancer Prevention
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• 제14권11호
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• pp.6273-6280
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• 2013

Goniothalamin, a natural compound extracted from Goniothalamus sp. belonging to the Annonacae family, possesses anticancer properties towards several tumor cell lines. This study focused on apoptosis induction by goniothalamin (GTN) in the Hela cervical cancer cell line. Cell growth inhibition was measured by MTT assay and the $IC_{50}$ value of goniothalamin was $3.2{\pm}0.72{\mu}g/ml$. Morphological changes and biochemical processes associated with apoptosis were evident on phase contrast microscopy and fluorescence microscopy. DNA fragmentation, DNA damage, caspase-9 activation and a large increase in the sub-G1 and S cell cycle phases confirmed the occurrence of apoptosis in a time-dependent manner. It could be concluded that goniothalamin show a promising cytotoxicity effect against cervical cancer cells (Hela) and the cell death mode induced by goniothalamin was apoptosis.

• 한국동물학회지
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• 제23권3호
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• pp.115-123
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• 1980

DNA 회복합성과 염색체이상과의 연관성여부를 추구하기 위하여 CHO 세포를 재료로 MNNG의 농도와 처리후 시간경과에 따른 절제회복율을 조사하고 이들 염색체 이상율과 비교하여 다음과 같은 결과를 얻었따. 1. MNNG에 의한 절제회복율은 $0.5 \\times 10^-5M$에서 $0.5 \\times 10^-5M$까지 농도의 증가에 따른 절제회복율의 증가를 보인다. 또 $1 \\times 10^-5M$ 처리후 0시간째는 절제회복율의 최고치를 보이다가 그후 점자 감소하여 24시간에는 0시간의 66%정도 나타난다. 2. MNNG에 의한 염색체이상은 $1 \\times 10^-5M$ 처리후 6시간에 최대치를 보이며 이상형의 대부분은 염색분체 절단을 나타낸다. 그러나 시간이 경과함에 따라 염색체분체절단은 감소하고 염색분체교환은 증가하여 24시간에는 두종류의 이상율이 비슷하게 이른다. 3. MNNG 처리후 시간경과에 따른 절제회복율은 전체이상율과 대체로 일치한다. 그러나 염색분체교환 또는 염색분체절단과는 어떤 비례관계도 보이지 않는다. 따라서 이같은 결과들은 MNNG에 의한 DNA 상해 및 그 회복은 염색체의 회복 현상과는 연관성이 없음을 암시하는 것이다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권14호
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• pp.6047-6053
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• 2015

Background: Hepatocellular carcinoma is one of the most common cancers worldwide. Its prevalence is increasing in many countries. Plant products can be used to protect against cancer due to natural anticancer and chemopreventive constituents. Strobilanthes crispus is one of plants with potential chemopreventive ability. Objective: This study aimed to evaluate the anticancer effects of Strobilanthes crispus juice on hepatocellular carcinoma cells. Materials and Methods: MTT assays, flow cytometry, comet assays and the reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the effects of juice on DNA damage and cancer cell numbers. Results: This juice induced apoptosis after exposure of the HepG2 cell line for 72 h. High percentages of apoptotic cell death and DNA damage were seen at the juice concentrations above 0.1%. It was found that the juice was not toxic for normal cells. In addition, juice exposure increased the expression level of c-myc gene and reduced the expression level of c-fos and c-erbB2 genes in HepG2 cells. The cytotoxic effects of juice on abnormal cells were in dose dependent. Conclusions: It was concluded that the Strobilanthes crispus juice may have chemopreventive effects on hepatocellular carcinoma cells.

• BMB Reports
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• 제34권6호
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• pp.551-558
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• 2001

We extended the measurable time scale of DNA dynamics to submicrosecond using a long-lifetime metal-ligand complex, $[Ru(phen)_2(dppz)]^{2+}$ (phen=1,10-phenanthroline, dppz=dipyrido[3,2-a:2',3'-c]phenazine) (RuPD), which displays a mean lifetime near 350 ns. We partially characterized the fluorescence resonance energy transfer (FRET) in calf thymus DNA from RuPD to nile blue (NB) using frequency-domain fluorometry with a high-intensity, blue light-emitting diode (LED) as the modulated light source. There was a significant overlap of the emission spectrum of the donor RuPD with the absorption spectrum of the acceptor NB. The F$\ddot{o}$rster distance ($R_0$) that was calculated from the spectral overlap was $33.4\;{\AA}$. We observed dramatic decreases in the steady-state fluorescence intensities of RuPD when the NB concentration was increased. The intensity decays of RuPD were matched the closest by a triple exponential decay. The mean decay time of RuPD in the absence of the acceptor NB was 350.7 ns. In a concentration-dependent manner, RuPD showed rapid intensity decay times upon adding NB. The mean decay time decreased to 184.6 ns at $100\;{\mu}M$ NB. The FRET efficiency values that are calculated from the mean decay times increased from 0.107 at $20\;{\mu}M$ NB to 0.474 at $100\;{\mu}M$ NB concentration. The use of FRET with a long-lifetime metal-ligand complex donor is expected to offer the opportunity to increase the information about the structure and dynamics of nucleic acids.

• Preventive Nutrition and Food Science
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• 제9권2호
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• pp.150-155
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• 2004

The free radical scavenging activities and the protective effects of Rhus javanica extracts against oxidative damage induced by reactive oxygen species (ROS) were investigated. n-Hexane, ethyl acetate and water fractions were prepared from a methanol extract. DPPH radical, superoxide anion and hydroxyl radical scavenging activities were estimated. Intracellular ROS formation was quantified using fluorescent probes, 2', 7'-dichlorofluorescin diacetate (DCFH-DA) for hydroxyl radical and dihydroethidium (DHE) for superoxide anion. The oxidative DNA damage was investigated by the comet assay in HepG$_2$ cells exposed either to $H_2O$$_2$ or to menadione. The highest $IC_{50}$/ values for DPPH radical scavenging activity was found in the ethyl acetate fraction with a value of 5.38 $\mu\textrm{g}$/mL. Cells pretreated with $\geq$ 1 $\mu\textrm{g}$/mL of the ethyl acetate extract had significantly increased cell viability compared to control cells, which were not pretreated with the extract. Intracellular ROS formation and DNA damage in HepG$_2$ cells, which were pretreated with the various concentrations of Rhus javanica ethyl acetate extract and then incubated either with $H_2O$$_2$ or with menadione, reduced in a dose-dependent manner. These findings suggest that Rhus javanica might have biologically active components which have strong protective effects against ROS induced oxidative damages to the biomolecules, such as cell membranes and DNA.

• 한국가축번식학회지
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• 제26권4호
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• pp.361-368
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• 2002

The onset of pronucleus formation and DNA synthesis in porcine oocytes following the injection of porcine or murine sperm was determined in order to obtain insights into species-specific paternal factors that contribute to fertilization. After 44h in vitro maturation, spermatozoa was injected into the cytoplasm of oocytes. After injection, all oocytes were transferred to NCSU23 medium and cultured at 39'E under 5% CO2 in air. Similar frequencies of oocytes with female pronuclei were observed after injection with porcine sperm or with murine sperm. In contrast, male pronuclei formed 8 to 9 h following the injection of porcine sperm, and 6 to 8 h following the injection of murine sperm. After pronucleus formation maternally derived microtubules were assembled and appeared to move both male and female pronuclei to the oocyte center. A few porcine oocytes entered metaphase 22 h after the injection of murine sperm, but normal cell division was not observed. The mean time of onset of S-phase in male pronuclei was 9.7 h following porcine sperm injection and 7.4 h following mouse sperm injection. These results suggested that DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, and the sperm nuclear decondensing activity and microtubule nucleation abilities of the male centrosome are cell cycle dependent.