• Journal of Microbiology and Biotechnology
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• v.18 no.12
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• pp.1938-1944
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• 2008

The procpb gene encoding human procarboxypeptidase B (proCPB, GeneBank access code AJ224866) was cloned and its Pichia expression plasmid, $pPIC9{\alpha}$/hproCPB (9.2 kb), was constructed, in which procpb was under the control of the AOXl promoter and connected to the downstream of the mating factor ${\alpha}$-1 ($MF{\alpha}1$) signal sequence. The plasmid was linearized by digestion with Sacl, and integrated into the genome of P. pastoris strain GS115. By culturing of Pichia transformant on methanol medium, the human proCPB was successfully expressed and secreted into the culture supernatant. Moreover, Western blot analysis of the extracellular proteins showed proCPB bands clearly at a molecular mass of 45 kDa, confirming the expression of proCPB with its right size. The CPB activity reached about 3.5 U/ml and 12.7 U/ml in the flask and fermentor batch cultures of Pichia transformant, respectively. No CPB enzyme activity was found in the intracellular fraction. When the fed-batch cultivation was performed with methanol and glycerol mixture as a feeding medium, the extracellular CPB activity was increased to 42.0 U/ml, which corresponds to a 3.3-fold higher level of CPB activity than that of batch culture. The $K_m$ and $k_{cat}$ values of recombinant human CPB enzyme for hippuryl-$_L$-Arg as a substrate were estimated to be 0.16 mM and $11.93\;sec^{-1}$, respectively.

• Archives of Pharmacal Research
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• v.26 no.10
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• pp.840-845
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• 2003

Temperature change is one of the major environmental factors that influence the human skin. However, the relationship between temperature and melanogenesis has received little attention. In the present study, we investigated the effects of temperature change on melanogenesis in a mouse melanocyte cell line (Mel-Ab), and primary cultured human melanocytes. We found that Mel-Ab cells cultured at low temperatures (31 and 34$^{\circ}C$) produce less melanin than cells at 37$^{\circ}C$. These results were confirmed by experiments upon human melanocytes, demonstrating that the hypopigmenting effect of low temperatures is not cell type dependent. The observed melanin production was found to be accompanied by tyrosinase activity at each temperature, indicating that tyrosinase activity is regulated by temperature. We further examined whether the incubation period at low temperatures plays an important role in the regulation of melanogenesis. Short exposures to 27$^{\circ}C$ for 1 h or 3 h did not affect tyrosinase activity or melanin synthesis, whereas long exposures to 31$^{\circ}C$ for 2 days or 6 days significantly reduced tyrosinase activity and melanin synthesis in a duration-dependent manner. Our results suggest that exposure to low temperature and the duration of this exposure are important regulators of melanogenesis.

• Archives of Pharmacal Research
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• v.20 no.6
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• pp.572-578
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• 1997

To gain further insight into how antiestrogens modulate cell function, the effects of antiestrogen on cell proliferation were studied in human breast cancer cells. We examined the effects of trans-tamoxifen on the proliferation of three human breast cancer cell lines that differed in their estrogen receptor contents. Trans-tamoxifen $(1{\mu}M)$ markedly inhibited the estrogen stimulated proliferation of MCF-7 human breast cancer cells that contained high levels of estrogen receptor $(1.15{\pm}0.03 pmole/mg protein)$ over that of control. In T47D cells that contained low levels of estrogen receptor $(0.23{\pm}0.05 pmole/mg protein)$, trans-tamoxifen $(1{\mu}M)$ showed minimal inhibition of estrogen stimulated cell proliferation over that of control. MDA-MB-231 cells, that contained no detectable levels of estrogen receptors, had their growth unaffected by trans-tamoxifen treatment. These results showed their sensitivity to growth inhibition by antiestrogen conrrelated well with their estrogen receptor content. Also we examined the effect of antiestrogen on cellular progestrone receptor level as well as plasminogen activator activity in MCF-7 cells. Trans-tamoxifen $(1{\mu}M)$ showed maximal inhibition of estrogen stimulated progestrone receptor level as well as plasminogen activator activity in MCF-7 cells that were stimulated by estrogen. It is not clear whether these inhibitions of progestrone receptor and plasminogen activator activity by estrogen are related to the antiestrogen inhibition of cell proliferation of MCF-7 cells. From the results of this study, it is clearly demonstrated that trans-tamoxifen is an antiestrogen in MCF-7 human breast cancer cells. Our data suggest that the biological effectiveness of trans-tamoxifen appear to result from its affinity of interaction with the estrogen receptor.

• The Korea Journal of Herbology
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• v.28 no.6
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• pp.155-160
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• 2013

Objectives : Gastric cancer is a leading cause of cancer-related deaths, worldwide. Houttuynia cordata Thunberg (H. cordata) has been used as a medicinal plants and it has an anti-cancer activity in human colorectal cancer and leukemic cancer. However, the potential anti-cancer activity and mechanisms of H. cordata for human gastric cancer cells have not been tested so far. Thus, this study examined the biological effects of H. cordata on the human gastric cancer cell line SNU-1 and AGS. Methods : Inhibition of cell proliferation and cell cycle by H. cordata was carried out by MTT assay and Muse cell cycle analysis and the expressions of protein associated with apoptosis and cell cycle regulation were investigated with Western blot analysis. Results : In MTT assay, the proliferation of SNU-1 and AGS cells was significantly inhibited by H. cordata in a time and dose dependent manner, Inhibition of cell proliferation by H. cordata was in part associated with apoptotic cell death, as shown by changes in the expression ratio of Bax to Bcl-2 by H. cordata. Also, H. cordata regulated the expression of cell cycle regulatory proteins such as pRb, cyclin D1, cyclin E, CDK4, CDK2, p21 and p15. Conclusion : The antiproliferative effect of H. cordata on SNU-1 and AGS gastric cancer cells revealed in this study suggests that H. cordata has intriguing potential as a chemopreventive or chemotherapeutic agent.

• Journal of Society of Preventive Korean Medicine
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• v.5 no.2
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• pp.106-113
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• 2001

The Cytotoxic activity of the ethyl acetate soluble fraction of Perilla frutescens on human oral epithelioid carcinoma cell lines was evaluated by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl-2H-tetrazalium bromide (MTT) colorimetric method. The light microscopic study was carried out to observe the morphological changes of cultured human oral epitheloid carcinoma cell lines (KB) These results were obtained as follows; 1. subfraction 1 of the ethyl acetate soluble fraction of perilla frutescens was shown significant cytotoxic activity (p<0.001) at 10-2 mg/ml concentration, this concentration was decreased the cytotoxic activity of 70.3% . 2. The comparison of IC50 values of various subfractions in human oral epithelioid carcinoma cell lines was exhibited that their susceptibility to these subfractions decreased in the following order ; subfraction 5 > subfraction 4 > subfraction 3 > subfraction 2 > subfraction 1 by MTT assay. In light microscopy, the subfraction 1 of the ethyl acetate soluble fraction of Perilla frutescens showed the highest cytotoxic activity. These findings suggest that subfraction 1 possessed the most cytotoxic constituents.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.207-212
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• 2012

The shortage of human organs for transplantation has induced the research on the possibility of using animal as porcine. However, pig to human transplantation as known as xeno-transplantation has major problem as immunorejection. Recently, the solutions of pig to human xenotransplantation are commonly mentioned as having a genetically modification which include alpha 1, 3 galatosyl transferase knockout (GTKO) and immune-suppressing gene transgenic model. Unfortunately, the expression level of transgenic gene is very low activity. Therefore, development of gene overexpression system is the most urgent issue. Also, the tissue specific overexpression system is very important. Because most blood vessels are endothelial cells, establishment of the endothelial-specific promoter is attractive candidates for the introduction of suppressing immunorejection. In this study, we focus the ICAM2 promoter which has endothelial-specific regulatory region. To detect the regulatory region of ICAM2 promoter, we cloned 3.7 kb size mini-pig ICAM2 promoter. We conduct serial deletion of 5' flanking region of mini-pig ICAM2 promoter then selected promoter size as 1 kb, 1.5 kb, 2 kb, 2.5 kb, and 3 kb. To analyze promoter activity, luciferase assay system was conducted among these vectors and compare endothelial activity with epithelial cells. The reporter gene assay revealed that ICAM2 promoter has critical activity in endothelial cells (CPAE) and 1 kb size of ICAM2 promoter activity was significantly increased. Taken together, our studies suggest that mini-pig ICMA2 promoter is endothelial cell specific overexpression promoter and among above all size of promoters, 1 kb size promoter is optimal candidate to overcome the vascular immunorejection in pig to human xenotransplantation.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.12
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• pp.4919-4923
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• 2014

Background: To investigate the effect of celecoxib on telomerase activity and apoptosis in a human laryngeal squamous carcinoma cell line (Hep-2 cells). Materials and Methods: The growth inhibition rate of Hep-2 cells in vitro was measured by MTT assay, and apoptosis by TUNEL assay and flow cytometry (FCM). The TRAP-ELISA method was used to determine telomerase activity in Hep-2 cells. The mRNA expression of human telomerase RNA component(hTR), human telomerase reverse transcriptase (hTERT) and human telomerase-associated protein(hTEP1) was determined by RT-PCR assay. Expression of Bax and Bcl-2 proteins was assessed by Western blotting. Results: Celecoxib can inhibit proliferation and induce apoptosis in a dose- and time-dependent manner, repress telomerase activity, decrease hTERT mRNA and Bcl-2 protein expression and increase Bax protein expression, PGE2 had no effect on telomerase. Conclusions: Celecoxib had the antiproliferative and pro-apoptotic effect in Hep-2 cells. Apoptosis was accompanied by a decrease in telomerase activity which was directly correlated with hTERT mRNA and up-regulation of Bax/Bcl-2. Bcl-2 may thus play an important role in telomerase activity as well as apoptosis.

• Journal of Periodontal and Implant Science
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• v.33 no.1
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• pp.49-60
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• 2003

Several growth factors and polypeptides are not commonly yet used for regenerators of bone tissue or alveolar bone because of the insufficiency of studies on their side effects, genetic engineering for mass production and stability for clinical application. Recently, many herbal medicines, which have advantage of less side effects and possibility of long-term use, have been studied for their capacity and effects of anti-bacterial, antiinflammatory and regenerative potential of periodontal tissues. Morindae Radix, Cibotium Barometz (L.), Albizziae Cortex, Cistandhis Herba have been traditionally used as medicines for treatment of bone disease in Eastern medicine. The objective of the present study is to examine the ability of alkaline phosphatase (ALP) activity of human fetal osteoblast (hFOB1) when several natural medicines were supplemented. hFOB1 were cultured with Dulbecuo's Modified Eagle's Medium Nutrient Mixture F-12 HAM ( DMEM/F-12 1:1 Mixture, Sigma, USA) and negative control, dexamethasone (positive control), and each natural medicines for 3 days. And then ALP activity was measured by spectrophotometer for enzyme activity and Alizarin red S staining for morphometry. Among the natural medicines of this study, Morindae Radix, Cibotium Barometz (L.) and Cistanchis Herba induced higher activity of ALP synthesis than negative controls in all experimental group. Albizziae Cortex showed mild increases than negative control group. According to measurement of positively stained area, all of the natural medicines of this study increased compared to negative control. Especially, Cibotium Barometz (L.) and Cistanchis Herba showed statistical significance compared to negative control (p<0.05). These results indicate that Morindae Radix, Cibotium Barometz (L.), Albizziae Cortex, Cistandhis Herba have an inducing ability of ALP synthesis on osteoblast.

• The Journal of Korean Medicine
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• v.28 no.4
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• pp.158-167
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• 2007

Background & Objective : Uncaria rhynchophylla is traditional medicine herb used for enhancing body resistance against various diseases. The aim of this study was to identify if Uncaria rhynchophylla extracts induce osteogenic activity in human osteoblast-like SaOS-2 cells. Methods : The osteogenic activity of Uncaria rhynchophylla was evaluated on cell proliferation assay by WST-8, and osteoblast-specific genes, such as VEGF, type I collagen (Col I), osteocalcin (OCN), and osteopontin (OPN) by RT-PCR analysis and ELISA assay in osteoblasts-like SaOS-2 cells. Bone mineralization was stained with Alizalin red method. Results : Uncaria rhynchophylla had significantly increased cell proliferation at a dose dependent manner in human osteoblast-like SaOS-2 cells. Uncaria rhynchophylla markedly increased alkaline phosphatase (ALP), vascular endothelial growth factor (VEGF) mRNA expression at 7 days and dose dependently increased ALP activity and VEGF secretion in human osteoblast-like SaOS-2 cells. Also, Uncaria rhynchophylla time-dependently increased type I collagen (Col I), osteopontin (OPN), and osteocalcin (OCN) mRNA in SaOS-2 cells. Extracellular accumulation of proteins such as Col I and OCN was maximal increased by Uncaria rhynchophylla at 10 ${\mu}g/ml$. Also, Uncaria rhynchophylla significantly induced mineralization in the culture of SaOS-2 cells. Conclusion : This study showed that Uncaria rhynchophylla had enhanced proliferation, ALP activity, VEGF, bone matrix proteins such as OCN, OPN, and Col I, and mineralization in SaOS-2 cells. These results propose that Uncaria rhynchophylla can play an important role in osteoblastic bone formation, osteogenesis, and may possibly lead to the development of bone-forming drugs.

• Journal of Food Hygiene and Safety
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• v.24 no.2
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• pp.124-127
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• 2009

The aim of this study is to evaluate the estrogenic activity of Cucurbita pepo seed extract which includes ${\beta}-sitosterol$ and other phytosterols. Sample was extracted from Cucurbita pepo seed by supercritical carbondioxide method and resuspended with ethanol. Estrogenic activity was measured by recombinant yeast assay which detects estrogenic activity using recombinant yeast with high level of estrogenic receptor. However, estrogenic activity of pumpkin seed extract was not found in this study. Based on this data, pumpkin seed extract will not cause estrogenic disturbance.