• Journal of Life Science
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• v.9 no.4
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• pp.439-452
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• 1999

This study was designed to investigate the effects of sea tangle (Laminaria japonica) extract and fucoidan components on anti-aging action. Sprague-Dawley(SD) male rats (210$\pm$5g) were fed experimental diets Dasi-Ex group: sea tangle extract powder of 4.0% added to control diet; Fuco-I, II and III groups: funcoidan powder of 1, 2 and 3% added to Dasi-Ex group for 45 days. Hydroxyl radical (.OH) formations were significantly inhibited (10-20% and 25-30%) in serum and brain mitochondria of Dasi-Ex and Fuco-I, II and III groups compared with control group. Significant differences in .OH formations of brain mitochondria in Dasi-Ex and Fuco-I groups could not be obtained, but.OH formations of brain microsomes resulted in a significant decrease (15-20%) in Fuco-II and III groups compared with control group. Basal oxygen radical (BOR) formations were significantly decreased about 10% and 13-15% in brain mitochondria of Dasi-Ex and Fuco-I group, and Fuco-II, III groups, and also decreased about 10% and 15-20% in brain microsomes of Dasi-Ex and Fuco-I groups, and Fuco-II, III groups. LPO levels of brain mitochondria and microsomes were significantly inhibited about 10% in Dasi-Ex and Fuco-I, II groups and 15% in Fuco-III groups. Oxidized proteins (>C=O) were significantly inhibited about 10% in serum of Dasi-Ex and Fuco-I, II, III groups and brain mitochondria of Dasi-Ex group, while remarkably inhibited (30~35%) in brain mitochondria of Fuco-I, II and III groups. Nitric oxide (NO) levels were significantly inhibited (12~15%) in serum of Fuco-I, II and III groups, but there no significant difference in serum NO levels of Dasi-Ex group. Superoxide dismutase (SOD) activities were remarkably increased (30~ 60%) in serum of Fuco-I, II and III groups, but there were no significant differences in SOD activities in serum of Dasi-Ex group. Catalase (CAT) activities were significantly increased about 20% in serum of Dasi-Ex and Fuco-I, II, III groups. Mn-SOD activities in brain mitochondria were significantly increased about 17% in Dasi-Ex group, while remarkably increased 26~36% in Fuco-I, II, III groups. Cu,Zn-SOD activities in brain cytosol were dose-dependently of fucoidan increased 10%, 12% and 18%, respectively, compared with control group. These results suggest that anti-aging effects of fucoidan may play a pivotal role in attenuating a various age-related changes such as chronic degenerative disease and senile dementia.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.186-186
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• 1994

Many agonists have been known to activate the hydrolysis of membrane phospholipids through the bindings with corresponding receptors on the various cells. Diacylglycerol and inositol 1,4,5-trisphosphate(IP3) generated by the action of phosphoinositide-specific phospholipase C (PI-PLC) are well known second messengers for the activation of protein kinase C and the mobilization of Ca2+ in many cells. Three types of PI-PLC isozyme (${\alpha}$,${\gamma}$, and $\delta$) and several subtrpes for each type have been identified from mammalian sources by purification of enzymes and cloning of their cDNAs. Each type PI-PLC isozyme is coupled to different receptors and mediators, for example, ${\beta}$-types are coupled to the seven-transmembrane-receptors via Gq family of G-proteins and ${\beta}$-types directly to the receptor tyrosine kinases. Specific modulators for the signaling pathway through each type of PI-PLC should be very useful as potential potential candidates for lend substances in developing novel drugs. To establish the sensitive and convenient screening systems for searching modulators on PI-PLC mediated signaling, two kinds of approaches have been tried. (1) Establishment of in vitro assay condition for each type of PI-PLC isozyme: Overexpression by using vaccinia virus and purification of each isozyme was carried out for the preparation of large amounts of enaymes. Optimum and sensitive assay condition for the measurements of PI-ELC activities were established. (2) Development of the cell lines in which each type of PI-PLC is permanently overexpressed: A fibroblast cell line (3T3${\gamma}$1-7) in which PI-PLC-${\gamma}$1 was overexpressed by using pZip-neo expression vector was developed and used for the measurement of PDGF-induced IP3 formation. The responses for IP3 formed in 3T3${\gamma}$1-7 cells by the treatment of PDGF is 8 times more sensitive than those in control cells. 3T3${\gamma}$l-7 cell is useful for the screening of the inhibitors on the PDGF-induced cellular responses from large number of samples in a small volume(50 ${\mu}$l) and short time(5-15 min). Using these systems, we screened hundreds of herb-extracts for the inhibition of PDGF-induced IP3 formation and selected several extracts that showed the inhibition as the candidates for isolation and characterization of active substances. The determination of the acting point of selected extracts or fractions in the PDGF signaling pathway has been analyzing.

• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.912-918
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• 2003

Mitotic centromere-associated kinesin (MCAK), which is a novel kinesin with a central motor domain, is believed to playa role in mitotic segregation of chromosome during the M phase of the cell cycle. In the present study, it is shown that a rabbit polyclonal antibody has been produced using the N-terminal region (187 aa) of human MCAK expressed in E. coli as the antigen. To express the N-terminal region in E. coli, the MCAK cDNA fragment encoding N-terminal 187 aa was obtained by PCR and was then inserted into the pET 3d expression vector. Molecular mass of the N-terminal region overexpressed in the presence of IPTG was 23.2 kDa on SDS-PAGE, and the protein was insoluble and mainly localized in the inclusion body that could be easily purified from the other cellular proteins. The N-terminal region was purified by electro-elution from the gel after the inclusion body was resolved on the SDS-PAGE. The antiserum obtained after tertiary immunization with the purified protein specifically recognized HsMCAK when subjected to Western blot analysis, and showed a fluctuation of the protein level during the cell cycle of human Jurkat T cells. Synchronization of the cell-cycle progression required for recovery of cells at a specific stage of the cell cycle was performed by either hydroxyurea or nocadazole, and subsequent release from each blocking at 2, 4, and 7 h. Northern and Western analyses revealed that both mRNA and protein of HsMCAK reached a maximum level in the S phase and declined to a basal level in the G1 phase. These results indicate that a polyclonal antibody raised against the N-terminal region (187 aa) of HsMCAK, overexpressed in E. coli, specifically detects HsMCAK (81 kDa), and it can analyze the differential expression of HsMCAK protein during the cell cycle.

• Herbal Formula Science
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• v.25 no.2
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• pp.179-191
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• 2017

Objectives : Increased oxidative stress by reactive oxygen species (ROS) has been suggested as a major cause of muscle fatigue. Although several studies have demonstrated the various biological properties of Sophora flavescens Aiton, Glycyrrhiza uralensis Fischer and Dictamnus dasycarpus Turcz, but the antioxidative potentials have not been clearly demonstrated. The present study was designed to investigate the protective effects of their water and ethanol extract mixtures (medicinal herbal mixtures, MHMIXs) on hydrogen peroxide ($H_2O_2$)-induced cell damage and apoptosis in C2C12 myoblasts. Methods : Cytotoxicity was assessed by an MTT assay. Quantitative evaluation of apoptosis induction and ROS production was evaluated by flow cytometry analysis. Expression levels of apoptosis regulatory and DNA-damage proteins were detected by Western blotting. Result : The inhibition of $H_2O_2$-induced cell proliferation was effectively blocked in extracts of 3: 1: 1 (EMHMIXs-1) or 2: 2: 1 (EMHMIXs-2) of S. flavescens, G. uralensis and D. dasycarpus Turcz, ethanol extracts from various complex extracts in C2C12 myoblasts. EMHMIXs-1 and EMHMIXs-2 also effectively attenuated $H_2O_2$-induced C2C12 cell apoptosis, which was associated with the restoration of the upregulation of Bad and death receptor 4, and downregulation of XIAP and cIAP-1 induced by $H_2O_2$. In addition, these herbal mixtures significantly blocked the $H_2O_2$-induced ROS generation and phosphorylation of $p-{\gamma}H2A.X$, which suggests that they can prevent $H_2O_2$-induced cellular DNA damage. Conclusions : The results suggest that EMHMIXs-1 and EMHMIXs-2 could block the DAN damage and apoptosis of C2C12 myoblasts by oxidative stress through blocking ROS generation.

• Microbiology and Biotechnology Letters
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• v.40 no.2
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• pp.117-127
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• 2012

The beneficial effects of adipose-derived stem cell conditioned media (ADSC-CM) for skin regeneration have previously been reported, despite the precise mechanism of how ADSC-CM promotes skin regeneration remaining unclear. ADSC-CM contains various secretomes and this may be a factor in it being a good resource for the treatment of skin conditions. It is also known that ADSC-CM produced in hypoxia conditions, in other words Advanced Adipose-Derived Stem cell Protein Extract (AAPE), has excellent skin regenerative properties. In this study, a human primary skin cell was devised to examine how AAPE affects human dermal fibroblast (HDF) and human keratinocyte (HK), which both play fundamental roles in skin regeneration. The promotion of collagen formation by HDFs was observed at 0.32 mg/ml of AAPE. AAPE treatment significantly stimulated stress fiber formation. DNA gene chips demonstrated that AAPE in HKs (p<0.05) affected the expression of 133 identifiable transcripts, which were associated with cell proliferation, migration, cell adhesion, and response to wounding. Twenty five identified proteins, including MMP, growth factor and cytokines such as CD54, FGF-2, GM-CSF, IL-4, IL-6, VEGF, TGF-${\beta}2$, TGF-${\beta}3$, MMP-1, MMP-10, and MMP-19, were contained in AAPE via antibody arrays. Thus, AAPE might activate the HK biological function and induce the collagen synthesis of HDF. These results demonstrate that AAPE has the potential to be used for clinic applications aimed at skin regeneration.

• Journal of Pharmaceutical Investigation
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• v.34 no.5
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• pp.369-377
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• 2004

The objective of this study was to investigate the patterns of protein leaching to an external phase during an ethyl acetate-based, double emulsion microencapsulation process. An aqueous protein solution (lactoglobulin, lysozyme, or ribonuclease; $W_1$) was emulsified in ethyl acetate containing poly-d,l-lactide-co-glycolide 75:25. The $W_1/O$ emulsion was transferred to a 0.5% polyvinyl alcohol solution saturated with ethyl acetate $(W_2)$. After the double emulsion was stirred for 5, 15, 30, or 45 min, additional 0.5% polyvinyl alcohol $(W_3)$ was quickly added into the emulsion. This so-called quenching step helped convert emulsion microdroplets into microspheres. After 2-hr stirring, microspheres were collected and dried. The degree of protein leaching to $W_2$ and/or $W_3$ phase was monitored during the microencapsulation process. In a separate, comparative experiment, the profile of protein leaching to an external phase was investigated during the conventional methylene chloride-based microencapsulation process. When ethyl acetate was used as a dispersed solvent, proteins continued diffusing to the $W_2$ phase, as stirring went on. Therefore, the timing of ethyl acetate quenching played an important role in determining the degree of protein microencapsulation efficiency. For example, when quenching was peformed after 5-min stirring of the primary $W_1/O$ emulsion, the encapsulation efficiencies of lactoglobulin and ribonuclease were $55.1{\pm}4.2\;and\;45.3{\pm}7.6%$, respectively. In contrast, when quenching was carried out in 45 min, their respective encapsulation efficiencies were $39.6{\pm}3.2\;and\;29.9{\pm}11.2%$. By sharp contrast, different results were attained with the methylene-chloride based process: up to 2 hr-stirring of the primary and double emulsions, less than 5% of a protein appeared in $W_2$. Afterwards, it started to partition from $W_1\;to\;W_2/W_3$, and such a tendency was affected by the amount of PLGA75:25 used to make microspheres. Different solvent properties (e.g., water miscibility) and their effect on microsphere hardening were to be held answerable for such marked differences observed with the two microencapsulation processes.

• Journal of Pharmaceutical Investigation
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• v.35 no.3
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• pp.193-199
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• 2005

Gabapentin is an antiepileptic drug that is structurally similar to ${\gamma}-aminobutyric$ acid (GABA), but does not interact with the GABA receptor. It does not bind significantly to plasma proteins, and is excreted to unchanged form in the urine. The purpose of the present study was to evaluate the bioequivalence of two gabapentin capsules, $Neurontin^{TM}$ capsule 300 mg (Pfizer Pharm. Co., Ltd.) and Kuhnil $Gabapentin^{TM}$ capsule 300 mg (Kuhnil Pharm. Co., Ltd), according to the guidelines of the Korea Food and Drug Administration (KFDA). The release of gabapentin from the two gabapentin formulations in vitro was tested using KP VIII Apparatus II method with various dissolution media (pH 1.2, 4.0, 6.8 buffer solution and water). Twenty six healthy male subjects, $22.46{\pm}1.86$ years in age and $67.64{\pm}7.24$ kg in body weight, were divided into two groups and a randomized $2{\times}2$ cross-over study was employed. After a single capsule containing 300 mg as gabapentin was orally administered, blood samples were taken at predetermined time intervals and the concentrations of gabapentin in serum were determined using HPLC with fluorescence detector. The dissolution profiles of two formulations were similar at all dissolution media. In addition, the pharmacokinetic parameters such as $AUC_t$, $C_{max}$ and $T_{max}$ were calculated and ANOVA test was utilized for the statistical analysis of the parameters using logarithmically transformed $AUC_t$, $C_{max}$ and untransformed $T_{max}$. The results showed that the differences between two formulations based on the reference drug, $Neurontin^{TM}$ capsule 300 mg, were -2.03, -0.43 and 4.29% for $AUC_t$, $C_{max}$ and $T_{max}$, respectively. There were no sequence effects between two formulations in these parameters. The 90% confidence intervals using logarithmically transformed data were within the acceptance range of log 0.8 to log 1.25 $(e.g.,\;log\;0.89{\sim}log\;1.09\;and\;log\;0.91{\sim}log\;1.09$ for $AUC_t$ and $C_{max}$, respectively). Thus, the criteria of the KFDA bioequivalence guideline were satisfied, indicating Kuhnil $Gabapentin^{TM}$ capsule 300 mg was bioequivalent to $Neurontin^{TM}$ capsule 300 mg.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.33-40
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• 2010

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked $\alpha$- and $\beta$-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (${\Delta}1$, ${\Delta}2$, ${\Delta}3$, ${\Delta}4$, and ${\Delta}5$) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2~3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (${\Delta}l$, ${\Delta}4$, and ${\Delta}5$) were transcripted, but not translated into proteins. Rec-eCG A2 was secreted in much lower amounts than the wild type. Only the rec-eCG ${\Delta}3$ ($\beta$-subunit: $Gln^{94}-Ile^{95}-Lys^{96}{\rightarrow}Ala^{94}-Ala^{95}-Ala^{96}$) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered $eCG{\beta\alpha}$. However, the FSH-like activity of rec-$eCG{\beta\alpha\Delta}3$ was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94~96 amino acid sequences in eCG $\beta$-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.47-53
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• 2010

Glycoprotein hormones have a common $\alpha$-subunit that is involved in the signaling pathway together with G protein, adenylcyclase and cAMP induction; however, it is an unclear how this common structure is related to hormonal action. To determine the biological functions of the COOH-terminal amino acids in the $\alpha$-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the $NH_2$-terminus of the $\alpha$-subunit to the COOH-terminus of the $\beta$-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form ${\Delta}96$, Lys was substituted at position 95 to form ${\Delta}95$, His was inserted at position 93 to form ${\Delta}93$ and Tyr was substituted at position 87 to form ${\Delta}87$. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and ${\Delta}96$, ${\Delta}95$, and ${\Delta}93$ mutants were efficiently secreted into the medium but the ${\Delta}87$ mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the ${\Delta}87$ mutant. However, the ${\Delta}87$ mutant protein was not detected in the medium or the intracellular fraction of the cell lysates. The LH- and FSH-like activities of the recombinant proteins were assayed in terms of cAMP production using rat LH/CG and rat FSH receptors. The metabolic clearance rate (MCR) was determined by injecting rec-eCG (2 IU) into the tail vein. The ${\Delta}95$ and ${\Delta}93$ mutants were completely inactive in both the LH- and FSH-like activity assays. The ${\Delta}96$ mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the ${\Delta}96$ mutant in the FSH-like activity assay was the highest among all the mutants. The MCR assay in which rec-eCG was injected showed a peak at 10 min in all the treatment groups, which disappeared 4 h after injection. These results imply a direct interaction between the receptor and the COOH-terminal region of the a-subunit. The data also reveal a significant difference in the mechanism by which the eCG hormone interacts with the rLH and rFSH receptors. The COOH-terminal region of the $\alpha$-subunit is very important for the secretion and functioning of this hormone.

• The Korea Journal of Herbology
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• v.30 no.4
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• pp.57-64
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• 2015

Objectives : Oxidative stress is one of the most causes of hepatocyte injury. Gleditsia spina, the thorns ofGleditsia sinensisLam., has been known for its anti-cancer and anti-inflammatory effects in Korean medicine. The present study investigated hepatoprotective effect of Gleditsia spina water extract (GSE) against oxidative stress induced by arachidonic acid (AA) + iron in HepG2 cells.Methods : To investigate cytoprotective effect of GSE, cells were pretreated with GSE and then subsequently exposed to 10 μM AA for 12 h, followed by 5 μM iron. Cell viability was monitored by MTT assay, and expression of apoptosis-related proteins was examined by immunoblot analysis. To identify responsible molecular mechanisms, reactive oxygen species (ROS) production, GSH contents, and mitochondrial membrane potential were measured. In addition, effect of GSE on nuclear factor erythroid 2-related factor 2 (Nrf2) activation was determined by immunoblot and antioxidant response element (ARE)-driven reporter gene assays.Results : GSE pretreatment prevented AA + iron-mediated cytotoxicity in concentration dependent manner. In addition, ROS production, glutathione depletion, and mitochondrial impairment by AA + iron were significantly inhibited by GSE. Furthermore, GSE promoted translocation of Nrf2 to nucleus, which acts as essential transcription factor for induction of antioxidant genes. Increased nuclear Nrf2 that caused by GSE treatment promoted transcriptional activity of ARE. Finally, GSE up-regulated sestrin-2 which was widely recognized as target gene of Nrf2.Conclusions : This study demonstrates that GSE protects hepatocytes from oxidative stress via activation of Nrf2 signaling pathway.