• Biomolecules & Therapeutics
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• v.29 no.2
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• pp.195-204
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• 2021

Cereblon (CRBN), a substrate receptor of cullin 4-RING E3 ligase (CRL4) regulates the ubiquitination and degradation of c-Jun, mediating the lipopolysaccharide-induced cellular response. However, the upstream signaling pathway that regulates this process is unknown. In this study, we describe how endoplasmic reticulum (ER) stress reversely regulates sequestosome-1 (p62)and c-Jun protein levels. Furthermore, our study reveals that expression of p62 attenuates c-Jun protein levels through the ubiquitinproteasome system. Conversely, siRNA knockdown of p62 elevates c-Jun protein levels. Immunoprecipitation and immunoblotting experiments demonstrate that p62 interacts with c-Jun and CRBN to form a ternary protein complex. Moreover, we find that CRBN knockdown completely abolishes the inhibitory effect of p62 on c-Jun. Using brefeldin A as an inducer of ER stress, we demonstrate that the p62/c-Jun axis participates in the regulation of ER stress-induced apoptosis, and that CRBN is required for this regulation. In summary, we have identified an upstream signaling pathway, which regulates p62-mediated c-Jun degradation. Our findings elucidate the underlying molecular mechanism by which p62/c-Jun axis regulates the ER stress-induced apoptosis, and provide a new molecular connection between ER stress and apoptosis.

• Journal of Physiology & Pathology in Korean Medicine
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• v.34 no.4
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• pp.201-208
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• 2020

The effects of Gamisoyo-san (GMSYS) co-administration within 5 min on the pharmacokinetics (PK) of tamoxifen were observed. After 50 mg/kg of tamoxifen oral treatment, GMSYS 100 mg/kg was orally administered within 5 min to 7-wk old male SPF.VAF Outbred Crl:CD [Sprague-Dawley (SD)] rats. The plasma were collected at 30 min before administration, 30 min, 1, 2, 3, 4, 6, 8 and 24 hrs after end of GMSYS treatment, and plasma concentrations of tamoxifen were analyzed using LC-MS/MS methods. Tmax, Cmax, AUC, t1/2 and MRTinf of tamoxifen were analysis as compared with tamoxifen single administered rats. Although co-administration with GMSYS did not critically influenced on the pharmacokinetic parameters of oral tamoxifen, they induced increased trends of plasma tamoxifen concentrations, especially significant (p<0.05) increases of plasma tamoxifen concentrations were demonstrated at 0.5 hr after end of co-administration with GMSYS as compared with tamoxifen single formula treated rats, at dosage levels of tamoxifen 10 mg/kg and GMSYS 100 mg/kg within 5 min. It is considered that pharmacokinetic studies should be tested like the effects of GMSYS on the pharmacokinetics of tamoxifen, when they were co-administered with prolonger intervals than Tmax of tamoxifen oral administration (about 2.5 hr-intervals), to achieve the optimal dosing regimen of GMSYS and tamoxifen co-administration.

• The Journal of Korean Obstetrics and Gynecology
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• v.30 no.2
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• pp.49-70
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• 2017

Objectives: The present study is to observe the skin-regeneration, anti-wrinkle, whitening and skin moisturizing effects of Cheongsangbangpung-tang (CSBPT) with cytotoxicity. Methods: In the present study, cytotoxicity of CSBPT lyophilized aqueous extracts (yield=18.71%) was experimented against human normal fibroblast cells and B16F10 murine melanoma cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) assay, and skin regeneration and anti-wrinkle effects were also showed through the assay of collagen type I synthesis by an enzyme immunoassay (EIA) kit as comparing with transforming growth factor (TGF)-${\beta}1$, hyaluronidase, collagenase and matrix metalloproteinase (MMP)-1 inhibitory assays as comparing with oleanolic acid (OA), and elastase inhibitory effects as comparing with phosphoramidon disodium salt (PP). In addition, whitening effects of CSBPT were observed by tyrosinase inhibitory assay and melanin formation test in B16/F10 melanoma cells as comparing with arbutin, and skin moisturizing effects were measured through mouse skin water contents test, respectively. Results: No CSBPT treatment related cytotoxic effects were demonstrated against human normal fibroblast cells and B16/F10 murine melanoma cells. CSBPT concentration-dependent increased collagen type I synthesis at human normal fibroblast cells. It also effectively suspreessed hyaluronidase, collagenase, elastase and MMP-1 activities, which were enzymes that related to declining of ECM and formation of wrinkle. CSBPT supressed B16/F10 melanoma cells's melanin productions with tyrosinase activity, which was an enzyme connected with melanin formation, and dose-dependent and significant increases of skin water contents were detected in CSBPT treated mouse skin as compared with vehicle control skins. Conclusions: CSBPT showed favorable and enough skin regeneration, anti-wrinkle, whitening and skin moisturizing effects at least in a condition of this experiment. However, more detail mechanism and in vivo skin protective efficacy studies should be conducted in future with the screening of the biological active compounds in individual herbs of Cheongsangbangpung-tang.

• The Journal of Korean Obstetrics and Gynecology
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• v.29 no.1
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• pp.14-34
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• 2016

Objectives: The objective of this study was to evaluate the effects of cytotoxicity, skin regeneration, anti-wrinkle, whitening and skin moisturizing of Oncheongeum (OCE).Methods: The cytotoxicity of OCE lyophilized aqueous extracts (yield=13.82%) was observed against human normal fibroblast cells and B16/F10 murine melanoma cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium Bromide (MTT) assay, and skin regeneration and anti-wrinkle effects were also evaluated through the assay of collagen type I synthesis compared to the transformation of the growth factor (TGF)-β1, hyaluronidase, collagenase and matrix metalloproteinase (MMP)-1 inhibitory assays compared to oleanolic acid (OA), and elastase inhibitory effects compared to phosphoramidon disodium salt (PP). In addition, OCE’s whitening effects were measured by a tyrosinase inhibitory assay and melanin formation test in B16/F10 murine melanoma cells compared to arbutin, and skin moisturizing effects were observed through a mouse skin water content test, respectively. Results: No OCE treatment-related cytotoxic effects appeared on human normal fibroblasts and B16/F10 murine melanoma cells. OCE concentration-dependently increased the collagen Type I synthesis on human normal fibroblast cells, and also effectively inhibited hyaluronidase, elastase, collagenase and MMP-1 activities. In addition, OCE inhibited melanin production of B16/F10 murine melanoma cells and activity of tyrosinase. And significant and dose-dependent increases of skin water content were detected in OCE-treated mouse skin compared to vehicle control skins. Conclusions: OCE showed favorable and sufficient effects in skin regeneration, anti-wrinkle, whitening and skin moisturizing in this experiment. But more detail mechanisms and studies on the skin protective efficiency of in vivo are needed with the screening of active biological compounds in individual OCE herbs.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.169-177
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• 2016

There is a growing interest in the application of primary hepatocytes for treatment of liver diseases in humans and for drug development. Several studies have focused on long-term survival and di-differentiation blocking of primary hepatocytes in an in vitro culture system. Therefore, the present study also aimed to optimize an in vitro culture system using primary rat hepatocytes. Primary rat hepatocytes from 6-week-old male Crl:CD rats were isolated using a modified two-step collagenase perfusion. Healthy $3.5{\times}10^6$ primary rat hepatocytes were seeded into a 2 dimensional (2D) culture in a 25T culture flask coated with collagen type I or into a 3D culture in a 125-ml spinner flask for 7 days. Production of plasma protein (ALB and TF), apoptosis (BAX and BCL2), and CYP (CYP3A1) related genes were compared between the 2D and 3D culture systems. The 3D culture system had an advantage over the 2D system because of the relatively high expression of ALB and low expression of BAX in the 3D system. However, the level of CYP3A1 did not improve in the 3D culture with and without the presence of a dexamethasone inducer. Therefore, 3D culture has an advantage for albumin production and primary rat hepatocyte survivability, but a low expression of CYP3A1 indicated that primary rat hepatocytes require a high-density culture for stress reduction by continuous flow.

• The KIPS Transactions:PartC
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• v.13C no.2 s.105
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• pp.147-154
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• 2006

The certificate status validation mechanism is a critical component of a public key infrastructure based on certificate system. The most generally mechanisms used these days are the use of the certificate revocation list and the real-time certificate status protocol. But the certificate revocation list can not give the real-time certificate status because the certificate is being delivered periodically, and the real-time certificate status protocol method will generate a concentrated load to the server because the protocol in the central server will be accessed whenever a certification is necessary. It will also take a long time to validate the certificate because each trade has to send mass information through the network. This paper will present that real-time validation is guaranteed as the real-time certificate status protocol method and the traffic congestion in the network Is reduced in a way that the certification would be requested using the user information hash value and would be validated using the user information kept in the certification authorities and the service providers. Based on the this study, we suggest a real-time certificate status validation mechanism which can reduce the certificate status validation time using the signed user information hash value. And we confirm speed of certificate status verification faster than existing CRL(Certificate Revocation List) and OCSP(Online Certificate Status Protocol) method by test.

• Journal of Veterinary Clinics
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• v.18 no.2
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• pp.146-151
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• 2001

This study was carried out to find if the X-irradiation being used for clinical diagnosis during pregnancy would affect fetal development and cause fetal malformation in rats or not. To determine the dose and irradiation frequency of X-irradiation and gestation period by which fetal development would be affected when irradiated during pregnancy, seventy-two Sprague Dawley female rats (8 weeks old) were used for the experiment and grouped into three according to different gestation period of 5-8 days, and 6-12 days of gestation. Experimental rats were irradiated on the daily irradiation conditions of 40, 60, 80 kvp(kilo volt peak), 150 mA(milliampere), 0.25 sec and 4 times/day for both 5-8 days and 10-13 days of gestation, and 100 kvp, 100 mA, 2 min. and 4 times/day for 6-12 days of gestation. Rats were put in a small dark box when irradiated, which animals were sacrificed on the 20th day of gestation and mean litter size, fetal body weight, fetal crown-rump length(CRL) were investigated along with pathological findings. 1. Litter size were significantly decreased in the rats which were irradiated by both 60 and 80 kvp during 5 to 8 days of gestation and by 100 kvp during 6-12 days of gestation compared to those from the control rats(p<0.05) 2. Fetal body weight was significantly decreased in the fetus from the rats which were irradiated by both 60-80 kvp during 5-8 days of gestation and by 100 kvp during 6-12 days of gestation compared to those from the control rats(p<0.05). 3. There was no significant difference of fetal crown-rump length between all the experimental rats and the controls. 4. Fetal absorption, fetal death, and fetal malformation were not observed in the fetus form the rats irradiated by 40-80 kvp during 5-8 and 10-13 days of gestation, however, the pathological findings were found in those from the rats irradiated by 100 kvp during 6-12 days of gestation. 5. The harmful effect of x-irradiation on fetal development was estimated to occur when irradiated during 5-8 days of gestation. These results indicated that even X-irradiation for clinical diagnosis could affect fetal development in the early embryonic stage and when the fetus were exposed to frequent and prolonged x-irradiation with over dose.

• The KIPS Transactions:PartC
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• v.10C no.4
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• pp.433-440
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• 2003

According as the real economic activities are carried out in the cyber world and the identity problem of a trade counterpart emerges, digital signature has been diffused. Due to the weakness for real-time validation using the validation method of digital signature, Certificate Revocation List, On-line Certificate Status Protocol was introduced. In this case, every transaction workload requested to verify digital signature is concentrated of a validation server node. Currently this method has been utilized on domestic financial transactions, but sooner or later the limitation will be revealed. In this paper, the validation method will be introduced which not only it can guarantee real-time validation but also the requesting node of certificate validation can maintain real-time certificate status information. This method makes the revocation management node update the certificate status information in real-time to the validation node while revoking certificate. The characteristic of this method is that the revocation management node should memorize the validation nodes which a certificate holder uses. If a certificate holder connects a validation node for the first time, the validation node should request its certificate status information to the above revocation management node and the revocation management node memorizes the validation node at the time. After that, the revocation management node inform the revocation information in real-time to all the validation node registered when a request of revocation happens. The benefits of this method are the fact that we can reduce the validation time because the certificate validation can be completed at the validation node and that we can avoid the concentration of requesting certificate status information to a revocation node.

• Journal of Nutrition and Health
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• v.45 no.5
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• pp.429-436
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• 2012

This study was performed to examine the characteristics of protein of red crab (Chionoecetes japonicus) shell powder hydrolyzed by commercial proteases. Red crab shell was digested by commercial proteases, such as Protamex (P), Neutrase (N), Flavourzyme (F), Alcalase (A), Protease M (PM) and Protease A (PA). Protein yield analyzed by Biuret assay, absorbance at 280 nm and brix revealed that PA was the enzyme having the highest proteolytic activity. SDS PAGE showed that molecular weight of proteins produced by protease treatments was various and below 150 kDa. Combinational treatment of proteases (PA + P, PA + PM, PA + F, PA + A) was tried whether these increase protein hydrolysis from red crab shell powder compared to a PA single treatment. Soluble protein content was similar, but amino acid concentration by combinational treatments was higher than PA single treatment [PA + P 247.4 mg/g > PA + F (206.4 mg/g) > PA + A (133.4 mg/g) > PA + PM (59.1 mg/g) > PA (54.9 mg/g)]. Amino acid composition by combinational treatments was slightly different. Most abundant essential amino acids were phenylalanine, glycine, alanine, and leucine, whereas tyrosine and cystine were not detected.

• Toxicological Research
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• v.35 no.2
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• pp.167-179
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• 2019

Ovarian cancer is the fifth main cause of pre-senescent death in women. Although chemotherapy is generally an efficient treatment, its side effects and the occurrence of chemotherapeutic resistance have prompted the need for alternative treatments. In this study, ${\alpha}$-mangostin and apigenin were evaluated as possible anticancer alternatives to the chemotherapeutic drug doxorubicin, used herein as a positive control. The ovarian adenocarcinoma cell line SKOV-3 (ATCC No. HTB77) was used as model ovarian cancer cells, whereas the skin fibroblast line CCD-986Sk (ATCC No. CRL-1947) and lung fibroblast line WI-38 (ATCC No. CCL-75) were used as model untransformed cells. Apigenin and doxorubicin inhibited the growth of SKOV-3 cells in a dose- and time-dependent manner. After 72 hr exposure, doxorubicin was mostly toxic to SKOV-3 cells, whereas apigenin was toxic to SKOV-3 cells but not CCD-986Sk and WI-38 cells. ${\alpha}$-Mangostin was more toxic to SKOV-3 cells than to CCD-986Sk cells. A lower cell density, cell shrinkage, and more unattached (floating round) cells were observed in all treated SKOV-3 cells, but the greatest effects were observed with ${\alpha}$-mangostin. With regard to programmed cell death, apigenin caused early apoptosis within 24 hr, whereas ${\alpha}$-mangostin and doxorubicin caused late apoptosis and necrosis after 72 hr of exposure. Caspase-3 activity was significantly increased in ${\alpha}$-mangostin-treated SKOV-3 cells after 12 hr of exposure, whereas only caspase-9 activity was significantly increased in apigenin-treated SKOV-3 cells at 24 hr. Both ${\alpha}$-mangostin and apigenin arrested the cell cycle at the $G_2/M$ phase, but after 24 and 48 hr, respectively. Significant upregulation of BCL2 (apoptosis-associated gene) and COX2 (inflammation-associated gene) transcripts was observed in apigenin- and ${\alpha}$-mangostin-treated SKOV-3 cells, respectively. ${\alpha}$-Mangostin and apigenin are therefore alternative options for SKOV-3 cell inhibition, with apigenin causing rapid early apoptosis related to the intrinsic apoptotic pathway, and ${\alpha}$-mangostin likely being involved with inflammation.