• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.1
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• pp.163-170
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• 2006

Nardostachyos Rhizoma (N. Rhizoma) belonging to the family Valerianaceae has been anti-arrhythmic effect, and sedation to the central nerve and a smooth muscle. We reported that the water extract of N. Rhizoma induced apoptotic cell death and differentiation in human promyelocytic leukemia (HL-60) cells. Cytotoxicity of N. Rhizoma was detected only in HL-60 cells (IC50 is about 200 ${\mu}g/ml$). The cytotoxic activity of N. Rhizoma in HL-60 cells was increased in a dose-dependent manner. We used several measures of apoptosis to determine whether these processes were involved in N. Rhizoma-induced apoptotic cell death. The high-dose (200 ${\mu}g/ml$) treatment of N. Rhizoma to HL-60 cells showed cell shrinkage, cell membrane blobbing, apoptotic bodies, and the fragmentation of DNA, suggesting that these cells underwent apoptosis. Treatment of HL-60 cells with N. Rhizoma time-dependently induced activation of caspase-3, caspase-8, and caspase-9 and proteolytic cleavage of poly(ADP-ribose) polymerase. Also, we investigated the effect of N. Rhizoma on cellular differentiation and proliferation in HL-60 cells. Differentiation and proliferation of HL-60 cells was determined through expression of CD11b and CD14 surface antigens using flow cytometry and nitroblue tetrazolium (NBT) assay, and through analysis of cell cycle using propidium iodide assay, respectively. N. Rhizoma induced the differentiation of HL-60 at the low-dose (100 ${\mu}g/ml$) treatment, as shown by increased expression of differentiation surface antigen CD11b, but not CDl4 and increased reducing activity of NBT. When HL-60 cells were treated with N. Rhizoma at concentration of $50{\mu}g/ml\;and\;100{\mu}g/ml$, NBT-reducing activities induced approximately 1.5-fold and 20.0-fold as compared with the control. In contrast, HL-60 cells treated with the N. Rhizoma-ATRA combination showed markedly elevated levels of 26.3-fold at $50{\mu}g/ml$ N. Rhizoma-0.1 ${\mu}M$ ATRA combination and 27.5-fold at 50 ${\mu}g/ml$ N. Rhizoma-0.2 ${\mu}M$ ATRA combination than when treated with N. Rhizoma alone or ATRA alone. It may be that N. Rhizoma plays important roles in synergy with ATRA during differentiation of HL-60 cells. DNA flow-cytometry indicated that N. Rhizoma markedly induced a G1 phase arrest of HL-60 cells. N. Rhizoma-treated HL-60 cells increased the cell population in G1 phase from 32.71% to 42.26%, whereas cell population in G2/M and S phases decreased from 23.61% to 10.33% and from 37.78% to 33.98%, respectively. We examined the change in the $p21^{WAF1/Cip1}\;and\;p27^{Kip1}$ proteins, which are the CKIs related with the G1 phase arrest. The expression of the CDK inhibitor $p27^{Kip1},\;but\;not\;p21^{WAF1/Cip1}$ were markedly increased by N. Rhizoma. Taken together, these results demonstrated that N. Rhizoma induces apoptotic cell death through activation of caspase-3, and potently inhibits the proliferation of HL-60 cells via the G1 phase cell cycle arrest in association with $p27^{Kip1}$ and granulocytic differentiation induction .

• Journal of Life Science
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• v.29 no.4
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• pp.410-420
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• 2019

Citrus unshiu peel extracts possess a variety of beneficial effects, and studies on their anticancer activity have been reported. However, the exact mechanisms underlying this activity remain unclear. In the current study, the apoptotic effect of ethanol extract of C. unshiu peel (EECU) on human breast adenocarcinoma MDA-MB-231 cells and related mechanisms were investigated. The results showed that the survival rate of MDA-MB-231 cells treated with EECU was significantly inhibited in a concentration-dependent manner, which was associated with the induction of apoptosis. EECU-induced apoptosis was associated with the activation of caspase-8 and caspase-9, which initiate extrinsic and intrinsic apoptosis pathways, respectively, and caspase-3, a representative effect caspase. EECU suppressed the expression of the inhibitor of apoptosis family of proteins, leading to an increased Bax/Bcl-2 ratio and proteolytic degradation of poly (ADP-ribose) polymerase. EECU also enhanced the loss of the mitochondrial membrane potential and cytochrome c release from the mitochondria to the cytosol, along with truncation of Bid. In addition, EECU activated AMP-activated protein kinase (AMPK), and compound C, an AMPK inhibitor, significantly weakened EECU-induced apoptosis and cell viability reduction. Furthermore, EECU promoted the generation of reactive oxygen species (ROS), which acted as upstream signals for AMPK activation as pretreatment of cells, with the antioxidant N-acetyl cysteine reversing both EECU-induced AMPK activation and apoptosis. Collectively, these findings suggest that EECU inhibits MDA-MB-231 adenocarcinoma cell proliferation by activating intrinsic and extrinsic apoptotic pathways, which was mediated through ROS/AMPK-dependent pathways.

• Korean Journal of Agricultural Science
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• v.25 no.2
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• pp.181-198
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• 1998

In order to determine the optimum pasteurization conditions by microwave heating(MWH) at $50^{\circ}C{\sim}70^{\circ}C$ for 30 minute compared with water bath heating(WBH) at $65^{\circ}C$ for 30minute during storage at $5^{\circ}C$, the chemical composition, microbiological changes and keeping quality were examined and the results were as follows: 1. The fat protein lactose, total solid contents of raw milk, at $50{\sim}70^{\circ}C$ for 30 min. in MWH and at 65 for $30^{\circ}C$ min. in WBH did not changed significantly during the storage at $5^{\circ}C$. 2. The pH and acidity for the raw milk untreated were 6.75 and 0.16%, and those of MWH heated and WBH milk wee 6.75~6.50 and 0.16%~0.19%, phosphatase test were negative at $61^{\circ}C$ for 20 min. at $62^{\circ}C$ for 15 min. at $63^{\circ}C$ for 10 min. at $64^{\circ}C$ for 5 min. at $65^{\circ}C$ for 5 min. in MWH and at $65^{\circ}C$ for 30 min. in WBH. 3. Whey protein content was $18.53mg/m{\ell}$ in raw milk untreated, however, those were decreased as the heating temperature increased. The proteolytic activity of treated milk by WBH(44%) was lower than that by MWH(94%). 4. Total bacteria counts were $2.8{\times}10^5CFU/m{\ell}$ in raw milk untreated, $2.8{\times}10^3CFU/m{\ell}$ at $65^{\circ}C$ for 30 min. $2.4{\times}10^3CFU/m{\ell}$ at $70^{\circ}C$ for 30 min. in MWH and $3.0{\times}10^3CFU/m{\ell}$ at $65^{\circ}C$ for 30 min. in WBH. Because total bacteria count did not increased in MWH at $65^{\circ}C$, $70^{\circ}C$ for 30 min. and $65^{\circ}C$ for 30 min. in WBH during the 10 days storaging, Also, total bacteria counts for treated milk were a most drastic decrease after $61^{\circ}C$, $62^{\circ}C$, $63^{\circ}C$, $64^{\circ}C$, $65^{\circ}C$ for 5 min. in MWH. 5. Coliform bacteria counts were $2.6{\times}10^3CFU/m{\ell}$ in raw milk untreated. There were not detected at $55^{\circ}C{\sim}70^{\circ}C$ for 30 min. in MWH and at $65^{\circ}C$ for 30 min. in WBH. Coliform bacteria counts were not detected after $61^{\circ}C$, $62^{\circ}C$, $63^{\circ}C$, $64^{\circ}C$, $65^{\circ}C$ for 5 min. in MWH. 6. Thermoduric bacteria counts were $5.2{\times}10^4CFU/m{\ell}$ in raw milk untreated, $2.0{\times}10^3CFU/m{\ell}$ at $65^{\circ}C$ for 30 min. $1.9{\times}10^3CFU/m{\ell}$ at $70^{\circ}C$ for 30min. in MWH and $2.2{\times}10^3CFU/m{\ell}$ at $65^{\circ}C$ for 30 min. in WBH. Because thermoduric bacteria counts did not increased in MWH at $65^{\circ}C$, $70^{\circ}C$ for 30 min. and $65^{\circ}C$ for 30 min. in WBH during the 10days storaging. Also, thermoduric bacteria counts were a most drastic decrease after $61^{\circ}C$, $62^{\circ}C$, $63^{\circ}C$, $64^{\circ}C$, $65^{\circ}C$ for 5 min. in MWH. 7. Psychrotrophic bacteria counts were $2.8{\times}10^5CFU/m{\ell}$ in raw milk untreated, $2.0{\times}10^1CFU/m{\ell}$ at $65^{\circ}C$ for 30 min. $2.0{\times}10^1CFU/m{\ell}$ at $70^{\circ}C$ for 30 min. in MWH and $3.0{\times}10^1CFU/m{\ell}$ at $65^{\circ}C$for 30 min. in WBH. Because psychrotrophic bacteria counts did not increased in MWH at $65^{\circ}C$, $70^{\circ}C$ for 30min. and $65^{\circ}C$ for 30 min. in WBH during the 10 days storaging. Also, psychrotrophic bacteria counts were a most drastic decrease after $61^{\circ}C$, $62^{\circ}C$, $63^{\circ}C$, $64^{\circ}C$, $65^{\circ}C$ for 5 min. in MWH.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.27 no.3
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• pp.223-231
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• 1994

In order to develop the squid(Todarodes pacificus) sik-hae, the changes of TBA, fatty acids, free amino acids, and the number of microflora fermented at different fermentation temperatures and periods were determined. In addition, pretense from squid sik-hae was partially purified. The number of TBA was the highest after 5-day storage and decreased after that, and lipid oxidation was the highest at $10^{\circ}C$. The amounts of linoleic aid(18:2) and oleic acid (18:1) were about $60\%$ of fatty acid composition of squid sik-hae, and linolenic acid(18:3) and EPA(20:5) significantly decomposed with increasing fermentation periods and temperatures. Pro, His, Aeg, Leu, and Glu were composed mainly of amino acid and the composition ratios of Ser, His, and Arg decreased with increasing fermentation periods whereas, those of Glu, Ala, Val, and Tyr increased. The composition ratios of Glu, Val, and Met increased with increasing fermentation temperatures whereas, those of Ala, Cys, Thr, and Gly decreased. The number of microflora generally increased up to 15-days of storage and decreased after that. The rates of increass and decreass of the microbial number increased in proportion to fermentation temperatures. In addition, the bacteria producing proteases were identified as Bacillus spp. Proteases from $60{\sim}80\%$ ammonium sulfate concentration showed the highest activity and had about 15 binds with molecular weights between 20,000 and 40,000 Dalton by the SDS-PAGE.

• Microbiology and Biotechnology Letters
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• v.44 no.4
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• pp.432-441
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• 2016

Machaerium cuspidatum, a canopy liana, is a species of genus legume in the Fabaceae family and contributes to the total species richness in the tropical rain forests. In the present study, we investigated the antioxidative and anti-cancer effects of M. cuspidatum and its mode of action. The methanol extract of M. cuspidatum (MEMC) exhibited anti-oxidative activity with an $IC_{50}$ value of $1.66{\mu}g/ml$, and this was attributable to its 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity. MEMC also exhibited a cytotoxic effect and induced morphological changes in a dose-dependent manner in several cancer cell lines including human lung adenocarcinoma A549 cells, human hepatocellular carcinoma HepG2 cells, and human colon carcinoma HT29 cells. Moreover, MEMC treatment induced the accumulation of subG1 population, which is indicative of apoptosis in A549 and HepG2 cells. MEMC-induced apoptosis was confirmed by the increase in Annexin V-positive apoptotic cells and apoptotic bodies using Annexin-V staining and DAPI staining, respectively. Further investigation showed that MEMC-induced apoptosis was associated with the increase in p53 and Bax expression, and the decrease in Bcl-2 expression. In addition, MEMC treatment led to proteolytic activation of caspase-3, 8, and 9 and degradation of poly-ADP ribose polymerase (PARP). Taken together, these results suggest that MEMC may exert a beneficial anti-cancer effect by inducing apoptosis via both the extrinsic and intrinsic pathways in A549 and HepG2 cells.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.4
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• pp.229-236
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• 2006

Objective: We investigated the expression of uPA and uPAR in eutopic endometrium of advanced stage endometriosis and control patients. Methods: The 33 endometriosis patients and 32 controls were enrolled. Endometrial samples were obtained from 65 premenopausal women aged 29-44 years, undergoing laparoscopic surgery or hysterectomy for non-malignant lesions. Sufficient samples were collected from 33 patients with endometriosis stage Ill and IV and 32 controls without endometriosis confirmed by laparoscopic surgery. The mRNA expression of uPA and uPAR from eutopic endometrium were analyzed by RT-QC PCR. Results: The mRNAs of uPA and uPAR were expressed in eutopic endometrium from endometriosis and normal controls throughout the menstrual cycle. Uterine endometrium from women with endometriosis expresses significantly (p<0.05) higher levels of u-PA mRNA than endometrium from normal women without endometriosis in the proliferative phase. There were no significant differences in expression of uPAR in eutopic endometrium between controls and endometriosis patients. Conclusion: These results suggest that eutopic endometrium from endometriosis patients may be more invasive and prone to peritoneal implantation because of greater u-PA mRNA expression than endometrium from women without endometriosis. Thus, increased proteolytic activity may be one etiology for the invasive properties of the endometrium resulting in the development of endometriosis.

• Clinical and Experimental Pediatrics
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• v.46 no.6
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• pp.548-553
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• 2003

Purpose : Matrix metalloproteinase(MMP)-9 is known to breakdown the blood-brain barrier by degrading the extracellular matrix of the subendothelial basement membrane in meningitis. Tissue inhibitor of metalloproteinase(TIMP)-1, a known inhibitor of MMP-9, has been postulated to inhibit the proteolytic activity of MMP-9 by bindng to MMP-9, but their interaction has not been fully understood yet. So far, there have been some reports on the relationship of MMP-9 and TIMP-1 in bacterial meningitis, but few reports in viral meningitis. Furthermore, there has been no report on this in Korea. We investigated the concentrations of MMP-9 and TIMP-1 in cerebrospinal fluid (CSF) and serum of patients with viral meningitis and control subjects, and evaluated their relationship with other clinical parameters of meningitis. Methods : CSF and blood were obtained from 25 subjects with viral meningitis and 14 control subjects. After centrifugation, supernatants were stored at $-20^{\circ}C$ and we assayed concentrations of MMP-9 and TIMP-1 by the sandwich ELISA method. Results : Concentrations of CSF MMP-9 and TIMP-1 were significantly elevated in patients with viral meningitis, when compared with those in control subjects. Their serum levels showed no differences between the two groups. MMP-9 levels were closely correlated with TIMP-1 levels in the CSF($r_s=0.42$, P<0.05). CSF MMP-9/TIMP-1 ratios were significantly higher in patients with viral meningitis than those in the control subjects(P<0.05). Both CSF MMP-9 and TIMP-1 levels positively correlated with CSF total leukocyte counts($r_s=0.43$, P<0.05, $r_s=0.48$, P<0.05). TIMP-1 levels positively correlated with total protein concentrations in the CSF($r_s=0.43$, P<0.05). Conclusion : MMP-9 and TIMP-1 may play an important role in the breakdown and maintenance of BBB in viral meningitis, respectively.

• Tuberculosis and Respiratory Diseases
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• v.49 no.1
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• pp.60-71
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• 2000

Backgrounds : Cathepsin D, an aspartic lysosomal proteinase, is believed to be involved in local invasion and metastasis of tumor cells by its proteolytic activity and has been described to be associated with tumor progression and prognosis in some human malignancies including breast cancer. But, its prognostic value for human lung cancer remains to be determined. The purpose of this study is to determine clinicopathological and prognostic significance of cathepsin D expression in non-small cell lung cancer. Method : Using a polyclonal antibody, immunohistochemical analysis of cathepsin D was performed on paraffin embedded sections of tumors obtained surgically from 54 patients with non-small cell lung cancer (37 squamous cell carcinoma, 14 adenocarcinoma, 2 large cell carcinoma, and 1 undifferentiated carcinoma). Results : Eighteen patients (33.3%) showed positive immunoreactivities of cathepsin D in tumor cells. No significant correlation of cathepsin D expression in tumor cells was found in p-stage (surgical-pathologic stage), tumor size, tumor factor, nodal involvement, and differentiation. Of 54 patients, 29 (53.7%) patients showed moderate to massive cathepsin D-positive stromal cells within the tumor tissues, while the rest (46.3%) showed few cathepsin D-positive stromal cells within the tumor tissues. Cathepsin D expression in stromal cells was significantly associated with p-stage in non-small cell lung canær (p=0.031). No significant correlation of the degree of cathepsin D-positive stromal cells was found in tumor size, T -factor, nodal involvement, differentiation Cathepsin D expression status in tumor cells and stromal cells was not significantly associated with prognosis expressed by survival rate. The results of multivariate analyses of variables possibly associated with prognosis showed that nodal involvement was the only independent prognostic factor in all patients. Conclusion : Cathepsin D expression in stromal cells was significantly associated with p-stage in non-small cell lung cancer. However, it was not related to other clinicopathologic features and prognosis, and Cathepsin D expression in tumor was not related to p-stage and prognosis.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.409-415
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• 1985

This study has been carried out in order to investigate the bacterial quality of fish meat paste products and the characteristics of isolated thermodurics from the products. Twenty samples of crab-flavored fish stick (Kematsal), 23 samples of plate fish meat paste (Panomuk, Kamaboko), 5 samples of fried fish meat paste (Tigimomuk), 2 samples of roasted fish meat paste (Puduromuk, Chikuwa), 20 samples of fish sausage were collected from processing plants and supermarkets in Pusan, Korea during the period from May to October in 1984. The results obtained are as follows. Amont the samples collected from supermarkets, roasted fish meat paste and fried fish meat paste marked hish counts in coliforms and fungi while very low in the samples of crab-flavored fish stick and plate fish meat paste. Salmonella was not detected in all the samples examined and Staphylococcus aureus was detected only in fried fish meat paste, Thermoduric bacteria were detected less than 10$^2$/g in the samples of crab-flavored fish stick and plate fish meat paste, which might come from subsidiary materials such as starch and seasonings. Among the isolated bacteria, distribution of the proteolytics were more than 87％ and the lipolytics were less than 20％. Gram positive bacteria was more than 70％ in crab-flavored fish stick and plate fish meat paste, 47.3％ in fried fish meat paste. And rod in shape was almost more than 90％ in all the samples. The most heat resistant bacterium isolated from the samples was identified as a Bacillus licheniformis(named B. licheniformis CR-11). The strain showed strong proteolytic activity and also grew well at above 2$0^{\circ}C$. The growth rate and generation time of CR-11 strain were 0.31 hr$^{-1}$ , 2.24 hr at 2$0^{\circ}C$, 0.64 hr$^{-1}$ , 1.09 hr at 3$0^{\circ}C$ and 0.78 hr$^{-1}$ , 0.89 hr at 35$^{\circ}C$. Heat resistance value of the spores of CR-11 strain suspended in phosphate buffer solution was D$_{85}$ $^{\circ}C$=41.9 min, D$_{90}$ $^{\circ}C$=27.9 min, D$_{95}$ $^{\circ}C$=10.2 min, D$_{100}$ $^{\circ}C$=4.3 min (Z=13.8$^{\circ}C$)

• Journal of Life Science
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• v.27 no.10
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• pp.1111-1120
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• 2017

This study was conducted to select pepper lines that were tolerant to excessive water injury among the pepper germplasm and investigate the genetic characteristics of those lines to contribute to the breeding of pepper cultivars with stable productivity in abnormal weather. Each of the tolerant and susceptible lines went through immersion treatment, and differentially expressed genes between them were analyzed. The tolerant line showed increased expression of the CA02g26670 gene, which is involved in the CONSTANS protein pathway and regulation of flowering by day length, but it exhibited decreased expressions of CA01g21450, CA01g22480, CA01g34470, CA02g00370 and CA02g00380. The susceptible line showed increased gene expressions of CA02g09720, CA02g21290, CA03g16520, CA07g 02110, and CA12g17910, which are involved in the inhibition of proteolytic enzyme activity, DNA binding, inhibition of cell wall-degrading enzyme, and inhibition of nodulin, respectively. Meanwhile the expressions of CA02g02820, CA03g21390, CA06g17700 and CA07g18230 decreased in the susceptible line, in relation to calcium-ion binding, high temperature, synthesis of phosphocholine and cold stress, respectively. The expressions of genes related to apoptosis and peroxidase increased, while that of CA02g16990, which functions as a nucleoside transporter, decreased in both the tolerant and susceptible lines. Based on the different gene expressions between the tolerant and susceptible lines, further studies are needed on breeding abiotic stress-tolerant lines.