• Korean Journal of Medicinal Crop Science
• /
• v.25 no.1
• /
• pp.45-51
• /
• 2017

Background: This study was performed to evaluate the protective effect of Saururus chinensis ethanol extract (SCE) against styrene toxicity in mouse spermatocyte cells [GC-2spd (ts) cell line]. Methods and Results: Cytotoxicity in mouse spermatocyte cells was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Generation of reactive oxygen species (ROS) was determined using 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) assay. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blotting were performed to quantify the mRNA and protein expression levels, resepectiviely, of stress or apoptosis-related genes including p21, p53, heat shock protein 70 (Hsp70), heat shock protein 90 (Hsp90), Bax, Bcl-2, and caspase-3. The results of the MTT assay showed that $50 {\mu}g/m{\ell}$ SCE did not affect cell viability. ROS generation in mouse spermatocyte cells increased by treatment with $100{\mu}M$ styrene, and decreased by co-treatment with SCE. SCE repressed the mRNA expression of stress-related genes, which increased by styrene treatment. In addition, SCE inhibited the apoptosis of mouse spermatocyte cells by ameliorating mRNA and protein levels of apoptotic genes that were altered by styrene treatment. Conclusions: These results suggest that SCE may alleviate styrene toxicity in mouse spermatocyte cells by reducing ROS stress and regulating genes related to styrene toxicity.

• YAKHAK HOEJI
• /
• v.45 no.3
• /
• pp.293-301
• /
• 2001

Genetically modified organism (GMO) using recombinant DNA technique has been exponentially increased, however there are still arguments for the safety of GM foods. The objective of this research was to compare the allergens of GM soybean(Roundup Ready$^{TM}$) with conventional soybeans. Each soybean extracts were prepared as crude extracts, heated extracts, and as heated and simulated gastric quid (SGF)-digested samples to characterize the stability of allergens to physicochemical treatment. Positive sera from 20 soybean-sensitive patients and control sera from 5 normal subjects were used to identify the endogenous allergens in soybeans. Specific-IgE binding activities to each soybean preparations were evaluated by ELISA and immunoblot technique. In ELISA result, IgE binding activities of positive sera to soy crude extracts generally showed two fold higher mean value than those of control sera, how-ever there was no significant difference between GM soybean and natural soybean varieties. Extracted proteins form each of the soybean preparations were separated with SDS-PAGE. The band pattern of GM soybean was very similar to those of natural soybean varieties. Immunoblots for the different soybeans revealed no differences in IgE-binding protein patterns, moreover, disclosed five prominent IgE-binding bands (75, 70, 50, 44 and 34 kDa) in crude extracts, four (75, 70, 44 and 34 kDa) in heated preparations, one (50 kDa) in heated and SGF-digested preparations. These IgE binding bands were consistent with previously reported results on the soybean. These results indicate that GM soybean (Roundup Ready$^{TM}$) is no different from natural soybean in terms of its allergen.gen.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.23 no.2
• /
• pp.124-136
• /
• 2001

Heat shock protein (HSP) expression is unregulated in tumor cells and, HSP expression is likely marker of the malignant potential of oral epithelial lesion. Furthermore, the 70kDa HSP is implicated in the degree of tumor differentiation, the rate of tumor proliferation and the magnitude of the anti-tumor Immune response. Accordingly, the distribution and intensity of HSP70 and HSP47 expression was assessed in the DMBA induced oral carcinogenesis in hamster. Golden Syrian hamsters which were 3 months-age and $90{\sim}120g$ were collected. 9,10-dimethyl -1,2-benzanthracene (DMBA) in a 0.5% solution in mineral oil was painted on the buccal pouch mucosa 3 times per week in the study group. In each control and experimental groups of 6, 8, 10, 12, 14, 16, 18, 20 weeks, specimen were sectioned for immunohistochemical study with anti-HSP47 and anti-HSP70 antibody. The following results were obtained. 1. HSP47 positive cells were race or negative of normal oral mucosa, increased mildly in basal and suprabasal basal layer, and spinous cell layer after experimental 6 weeks (dysplastic or CIS stage). In CIS stage, HSP47 expression is prominent in dysplastic free or normal adjacent epithelium. 2. HSP47 positive cells in connective tissue were mainly inflammatory cells, which is gradually increased from control to precancerous and cancer stage. But HSP47 positive cells after 14 weeks were decreased, especially normal and cancer adjacent epithelium. 3. The positive staining cells of HSP70 in control, dysplastic, and CIS stage were not seen. But they were mild findings in basal layer and moderate findings in spinous layer after experimental 14 weeks (cancer stage). 4. HSP70 positive cells were increased in precancerous and cancer stage than control group in connective tissue. After experimental 16 weeks, we could not find the HSP expression in cancer cells according to cancer differentiation or cancer stage. It is concluded that HSP70 or HSP47 expression is not a definitive marker of oral malignancy or malignant potential. However, with further development, HSP immunoreactivity may be valuable as an adjunct to conventional histology for assessing the malignant potential of oral mucosal lesions.

• Microbiology and Biotechnology Letters
• /
• v.44 no.1
• /
• pp.1-8
• /
• 2016

The purpose of this study was to assess the whitening effects of an extract from Smilax china L., which is a vine shrub belonging to the lily family. With regard to the whitening effects, 70% ethanol and water extracts from Smilax china L. showed more than 77.6% and 40.2% tyrosinase inhibition at a concentration of $1,000{\mu}l$. Furthermore, the 70% ethanol extract showed cytotoxicity of 89% at a concentration of $100{\mu}g/ml$ in melanoma cells. Western blot showed that the inhibitory effect of the 70% ethanol extract on MITF, TRP-1, TRP-2, and tyrosinase protein expression decreased by 89.9%, 46.2%, 57.6%, and 55.8%, respectively, at a concentration of $50{\mu}g/ml$. Moreover, reverse transcription-PCR showed that the inhibitory effect of the 70% ethanol extract on MITF, TRP-1, TRP-2, and tyrosinase mRNA expression decreased by 78.5%, 58.0%, 78.8%, and 70.8%, respectively, at the same concentration of $50{\mu}g/ml$ concentration. Further, realtime PCR showed that the 70% ethanol extract-induced decrease in MITF, TRP-1, TRP-2, and tyrosinase quantitative mRNA expression rate was concentration-dependent. The findings suggest that the extract from Smilax china L. has great potential as a cosmetic ingredient with whitening effects.

• The Korean Journal of Physiology and Pharmacology
• /
• v.7 no.1
• /
• pp.9-14
• /
• 2003

Recent studies indicated that cancer cells become resistant to ionizing radiation (IR) and chemotherapy drugs by enhanced DNA repair of the lesions. Therefore, it is expected to increase the killing of cancer cells and reduce drug resistance by inhibiting DNA repair pathways that tumor cells rely on to escape chemotherapy. There are a number of key human DNA repair pathways which depend on multimeric polypeptide activities. For example, Ku heterodimer regulatory DNA binding subunits (Ku70/Ku80) on binding to double strand DNA breaks (DSBs) are able to interact with 470-kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and are essential for DNA-dependent protein kinase (DNA-PK) activity. It has been known that DNA-PK is an important factor for DNA repair and also is a sensor-transmitting damage signal to downstream targets, leading to cell cycles arrest. Our ultimate goal is to develop a treatment of breast tumors by targeting proteins involved in damage-signaling pathway and/or DNA repair. This would greatly facilitate tumor cell cytotoxic activity and programmed cell death through DNA damaging drug treatment. Therefore, we designed a domain of Ku80 mutants that binds to Ku70 but not DNA end binding activity and used the peptide in co-therapy strategy to see whether the targeted inhibition of DNA-PK activity sensitized breast cancer cells to irradiation or chemotherapy drug. We observed that the synthesized peptide (HNI-38) prevented DNA-PKcs from binding to Ku70/Ku80, thus resulting in inactivation of DNA-PK activity. Consequently, the peptide treated cells exhibited poor to no DNA repair, and became highly sensitive to IR or chemotherapy drugs, and the growth of breast cancer cells was inhibited. Additionally, the results obtained in the present study also support the physiological role of resistance of cancer cells to IR or chemotherapy.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.20 no.4
• /
• pp.362-372
• /
• 1998

Heat shock protein (HSP) expression is unregulated in tumor cells and, HSP expression is likely marker of the malignant potential of oral epithelial lesion. Furthermore, the 70kDa HSP is implicated in the degree of tumor differentiation, the rate of tumor proliferation and the magnitude of the anti-tumor immune response. Accordingly, the distribution and intensity of HSP 70 and HSP 47 expression was assessed in the DMBA induced oral carcinogenesis in hamster. Golden Syrian hamsters which were 3 months-age and 90-120g were collected. 9,10-dimethyl-1,2-benzanthracene (DMBA) in a 0.5% solution in mineral oil was painted on the buccal pouch mucosa 3 times per week in the study group. In each control and experimental groups of 6, 8, 10, 12, 14, 16, 18, 20 weeks, specimen were sectioned for immunohistochemical study with anti-HSP47 and anti-HSP70 antibody. The following results were obtained. 1. HSP47 positive cells were rare or negative of normal oral mucosa, increased mildly in basal and suprabasal basal layer, and spinous cell layer after experimental 6 weeks (dysplastic or CIS stage). In CIS stage, HSP47 expression is prominent in dysplastic free or normal adjacent epithelium. 2. HSP 47 positive cells in connective tissue were mainly inflammatory cells, which is gradually increased from control to precancerous and cancer stage. But HSP47 positive cells after 14 weeks were decreased, especially normal and cancer adjacent epithelium. 3. The positive staining cells of HSP70 in control, dysplastic, and CIS stage were not seen. But they were mild findings in basal layer and moderate findings in spinous layer after experimental 14 weeks (cancer stage). 4. HSP70 positive cells were increased in precancerous and cancer stage than control group in connective tissue. After experimental 16 weeks, we could not find the HSP expression in cancer cells according to cancer differentiation or cancer stage. It is concluded that HSP70 or HSP47 expression is not a definitive marker of oral malignancy or malignant potential. However, with further development, HSP immunoreactivity may be valuable as an adjunct to conventional histology for assessing the malignant potential of oral mucosal lesions.

• BMB Reports
• /
• v.39 no.1
• /
• pp.46-54
• /
• 2006

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of $11,180{\pm}50$ Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S], type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa $\alpha$ and 34 kDa $\beta$ subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at $55^{\circ}C$, pH 7.0. Maximum activity was observed at $70^{\circ}C$ and the optimum pH for enzymatic activity was 7.0 -8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with $K_m$ and $k_{cat}$ values of $163\;{\mu}M$ and $452\;min^{-1}$, respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.

• Journal of Life Science
• /
• v.14 no.2
• /
• pp.325-330
• /
• 2004

The RAD3 gene of Saccharomyces cerevisiae is required for excision repair and is essential for cell viability. RAD3 encoded protein possesses a single stranded DNA-dependent ATPase and DNA and DNA-RNA helicase activities. To examine the extent of conservation of structure and function of RAD3 during eukaryotic evolution, the RAD3 homolog gene was isolated by screening of genomic DNA library. The isolated gene was designated as HRD3 (Homologue of RAD3 gene). The over-expressed HRD3 protein was estimated to be a 75 kDa in size which is in good agreement with the estimated by the nucleotide sequence of the cloned gene. Two-dimensional gel electrophoresis showed that a number of other protein spots dramatically disappeared when the HRD3 protein was overexpressed. The overexpressed RAD3 protein showed a toxicity in E. coli host, suggesting that this protein may be involved in the inhibition of protein synthesis and/or degradation of host protein. To determine which part of HRD3 gene contributes to the toxicity in E. coli, various fusion plasmids containing a partial sequence of HRD3 and lac'Z gene were constructed. These results suggest that the C-terminal domain of HRD3 protein may be important for both toxic effect in E. coli and for its role in DNA repair in S. pombe.

• Korean Journal Plant Pathology
• /
• v.14 no.4
• /
• pp.339-344
• /
• 1998

Since the leaf inoculation procedures are time-consuming and require considerable growth chamber space, a rapid dioassay method for screening of pathogenicity of Didymella bryoniae, a casual agent of gummy stem blight in watermelon, was established in this paper. The method produced reliable results within 8 days ( 5 days for growing seedlings and 3 days for rapid disease response in the seedlings). After contaminants in the root of 4~5 day-old seedlings had been washed using sterilized water, 5 seedlings were dipped into a vial containing 12 ml of conidial suspension (106 cells/ml). After the vials were placed in a growth chamber (22$^{\circ}C$, RH 50%, 14hr light/10hr darkness) for 3 days, susceptibility and resistance of cultivars were determined by the degree of disease response on cotyledon. The result of obtained by the dip-inoculation method was well coincided with the results by the leaf inoculation procedures and the result that had been observed for several years in the field. Screening of collected watermelon cultivars by the dip-inoculation method revealed that all the 21 domestic cultivars collected were susceptible and only 3 foreign cultivars (PI 189225, PI 482322 and IT 188207) were resistant among 18 cultivars A cucumber cultivar (Marketer) and bitter cucumber were proven to be resistant against the D. bryoniae among 8 other different cucurbits tested. The SDS-PAGE patterns of total proteins from a susceptible (Keumcheon) and a resistant (PI 189225) watermelon cultivars were compared 0, 12, 24 and 36 hrs after inoculation. The amounts of two distinct protein bands (24 kDa and 70 kDa) were gradually increased after inoculation in both cultivars.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.46 no.4
• /
• pp.435-441
• /
• 2017

The present study was carried out to evaluate the applicability of Tenebrio molitor larvae (mealworm) as a health functional food material in order to contribute to the development of the domestic insect industry and health functional food industry. Protein hydrolysates were prepared from mealworm powder by enzymatic hydrolysis using five different proteases (alcalase, bromelain, flavourzyme, neutrase, and papain), and the hydrolysates were then tested for their antioxidant activities. Based on available amino group contents and sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses, mealworms treated with alcalase ($4,781.39{\mu}g/mL$), flavourzyme ($5,429.35{\mu}g/mL$), or neutrase ($3,155.55{\mu}g/mL$) for 24 h showed high degree of hydrolysis (HD) value, whereas HD values of bromelain ($1,800{\mu}g/mL$) and papain-treated ($1,782.61{\mu}g/mL$) mealworms were much lower. Protein hydrolysates showing high HD values were further separated into > 3 kDa and ${\leq}3kDa$ fractions by a centrifugal filter system and then lyophilized, and the production yields of the low molecular weight protein hydrolysates (${\leq}3kDa$) by alcalase, flavourzyme, and neutrase were 42.05%, 26.27%, and 30.01%, respectively. According to the RC_{50} values of the protein hydrolysates (${\leq}3kDa$) obtained from three different antioxidant analyses, all three hydrolysates showed similar antioxidant activities. Thus, alcalase hydrolysates showing the highest production yield of low molecular weight protein hydrolysates were further tested for their inhibitory effects on peroxidation of linoleic acid by measuring thiobarbituric acid values, and the results show that peroxidation of untreated linoleic acid increased dramatically during 6 days of incubation. However, pretreatment with the hydrolysates ($100{\sim}800{\mu}g/mL$) significantly inhibited linoleic acid peroxidation in a dose-dependent manner over 6 days.