• The Journal of Internal Korean Medicine
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• v.35 no.3
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• pp.317-331
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• 2014

Objectives: The root of Platycodon grandiflorum (PG) has been known to possess a range of pharmacological activities including anti-cancer, anti-inflammatory, and anti-oxidant effects. The present study was designed to investigate whether or not PG-induced cell death was connected with autophagy and apoptosis in NCI-H460 human lung cancer cells. Methods: Effects on the cell viability and apoptotic activity were quantified using MTT assays and flow cytometry analysis, respectively. Protein activation was measured by immunoblotting. Autophagy was measured by LC3 immunofluorescence and immunoblotting. ROS production and loss of mitochondria membrane potential (MMP) were checked with flow cytometry analysis. Results: Following exposure to PG, NCI-H460 cell proliferation decreased simultaneously inducing autophagic vacuoles and up-regulation of microtubule-associated protein 1 light chain 3 and beclin-1 protein expressions. Interestingly, pre-treated with autophagy inhibitors, 3-methyladenin or bafilomycin A1 further triggered reduction of cell viability. PG treatment also induced apoptosis that was related modulation of Bcl-2 family proteins, death receptors and activation of caspases. In addition, PG stimulation clearly enhanced loss of MMP and reactive oxygen species (ROS) generation. Conclusions: Our results suggest that PG elicited both autophagy and apoptosis by increasing loss of MMP and ROS production. PG induced-autophagy may play a cell protective role.

• The Journal of Korean Academy of Prosthodontics
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• v.44 no.5
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• pp.574-583
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• 2006

Statement of problem : The use of permanent magnetics is increasing in implant dentistry. Purpose : This study is to know the effect of permanent magnetics on bone matrix formation of osteoblasts. Materials and methods : The konus abutment-shaped permanent magnetics were connected to the implant fixture, and placed on the culture plate. The osteoblast-like cell Mc3T3E1 were used for cell culture. As the control group, the implants were connected to titanium healing caps, and cultured in the same conditions of experimental group. After 3. 7, 14 days, cells were cultured, and we measured and compared the amount of collagen type I, osteocalcin, which is bone matrix protein by Western immunoblotting analysis. Results: As a result of Western immunoblotting analysis for estimating the amount of bone extracellular matrix, there was no difference between osteoblast of the experimental group and the control group during 3 and 7day-osteoblast culturing. However when cells were cultured for 14days, the amount of bone extracellular matrix was increased, on the experimental group. Conclusion: From these results, magnetic field of permanent magnetics might have effect on bone formation of osteoblast, especially at initial stage of implant placement. Therefore, their clinical application for implant or bone graft could be possible.

• Korean Journal of Veterinary Research
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• v.33 no.2
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• pp.249-254
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• 1993

The etiological survey on porcine epidemic diarrhea virus(PEDV) by immunofluorescence antibody test(IFA) showed the positive rusult from the intestines of piglet died from acute diarrhea. The viral agent of PED was also isolated from intestine, which showed positive reaction by immunofluorescence test. After passage in Vero cell, the viral agent was further cloned by plaque purification and designated as KPEDV-9. The immunoblotting analysis using hyperimmune sera and porcine sera revealed the presence of several polypetide bands with molecular weight(M.W.) of 88K, 74K, 70K, 58~54, 54~46K, 44~40K and 33~32K, respectively.

• Korean Journal of Veterinary Research
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• v.32 no.1
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• pp.83-90
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• 1992

Monoclonal antibodies against structural proteins of bovine viral diarrhea virus(BVDV) were derived by classical hybridoma techniques. These antibodies were characterized by serum neutralization, immunoblotting and immunoprecipitation. The neutralizing monoclonal antibody reacted with the 56kd to 54kd(M.W.) viral protein in western blotting and immunoprecipitation analysis. Although there was no neutralizing activity, another monoclanal antibody reacted with the 45kd protein by immunoprecipitation and with both the 45kd and 36kd proteins in immunoblotting analysis. respectively. Densitometer scanning of purified BVDV and the immunopreipitation of whole virus particles with neutralizing monoclonal antibody revealed the presence of more than twelve viral polypeptides. Although no possible precursor form of protein was identified with the neutralizing monoclonal antibody. the presence of intact virion was detected in the infected cell supernatant immediatelty after pulse labeling, indicating rapid translational processing as well as packaging of the virus. The partial peptide mapping of 45kd and 36kd proteins with Staphylococcus aureus V 8 protease showed that these two proteins are related.

• Journal of Ginseng Research
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• v.42 no.4
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• pp.496-503
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• 2018

Background: Korean ginseng (Panax ginseng) plays an anti-inflammatory role in a variety of inflammatory diseases such as gastritis, hepatitis, and colitis. However, inflammation-regulatory activity of the calyx of the P. ginseng berry has not been thoroughly evaluated. To understand whether the calyx portion of the P. ginseng berry is able to ameliorate inflammatory processes, an ethanolic extract of P. ginseng berry calyx (Pg-C-EE) was prepared, and lipopolysaccharide-activated macrophages and HEK293 cells transfected with inflammation-regulatory proteins were used to test the anti-inflammatory action of Pg-C-EE. Methods: The ginsenoside contents of Pg-C-EE were analyzed by HPLC. Suppressive activity of Pg-C-EE on NO production, inflammatory gene expression, transcriptional activation, and inflammation signaling events were examined using the Griess assay, reverse transcription-polymerization chain reaction, luciferase activity reporter gene assay, and immunoblotting analysis. Results: Pg-C-EE reduced NO production and diminished mRNA expression of inflammatory genes such as cyclooxygenase-2, inducible NO synthase, and tumor necrosis factor-${\alpha}$ in a dose-dependent manner. This extract suppressed luciferase activity induced only by nuclear factor-${\kappa}B$. Interestingly, immunoblotting analysis results demonstrated that Pg-C-EE reduced the activities of protein kinase B (AKT)1 and AKT2. Conclusion: These results suggest that Pg-C-EE may have nuclear-factor-${\kappa}B$-targeted anti-inflammatory properties through suppression of AKT. The calyx of the P. ginseng berry is an underused part of the ginseng plant, and development of calyx-derived extracts may be useful for treatment of inflammatory diseases.

• Journal of Microbiology
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• v.43 no.4
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• pp.354-360
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• 2005

Heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli (ETEC) is both a strong mucosal adjuvant and immunogen. It is a subunit vaccine candidate to be used against ETEC-induced diarrhea. It has already been expressed in several bacterial and plant systems. In order to construct yeast expressing vector for the LTB protein, the eltB gene encoding LTB was amplified from a human origin enterotoxigenic E. coli DNA by PCR. The expression plasmid pLTB83 was constructed by inserting the eltB gene into the pYES2 shuttle vector immediately downstream of the GAL1 promoter. The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The LTB protein was detected in the total soluble protein of the yeast by SDS-PAGE analysis. Quantitative ELISA showed that the maximum amount of LTB protein expressed in the yeast was approximately $1.9\%$ of the total soluble protein. Immunoblotting analysis showed the yeast-derived LTB protein was antigenically indistinguishable from bacterial LTB protein. Since the whole-recombinant yeast has been introduced as a new vaccine formulation the expression of LTB in S. cerevisiae can offer an inexpensive yet effective strategy to protect against ETEC, especially in developing countries where it is needed most.

• Journal of Ginseng Research
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• v.43 no.3
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• pp.475-481
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• 2019

Background: The ginsenoside Rg1 has been shown to exert various pharmacological activities with health benefits. Previously, we have reported that Rg1 promoted myogenic differentiation and myotube growth in C2C12 myoblasts. In this study, the in vivo effect of Rg1 on fiber-type composition and oxidative metabolism in skeletal muscle was examined. Methods: To examine the effect of Rg1 on skeletal muscle, 3-month-old mice were treated with Rg1 for 5 weeks. To assess muscle strength, grip strength tests were performed, and the lower hind limb muscles were harvested, followed by various detailed analysis, such as histological staining, immunoblotting, immunostaining, and real-time quantitative reverse transcription polymerase chain reaction. In addition, to verify the in vivo data, primary myoblasts isolated from mice were treated with Rg1, and the Rg1 effect on myotube growth was examined by immunoblotting and immunostaining analysis. Results: Rg1 treatment increased the expression of myosin heavy chain isoforms characteristic for both oxidative and glycolytic muscle fibers; increased myofiber sizes were accompanied by enhanced muscle strength. Rg1 treatment also enhanced oxidative muscle metabolism with elevated oxidative phosphorylation proteins. Furthermore, Rg1-treated muscles exhibited increased levels of anabolic S6 kinase signaling. Conclusion: Rg1 improves muscle functionality via enhancing muscle gene expression and oxidative muscle metabolism in mice.

• Parasites, Hosts and Diseases
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• v.51 no.6
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• pp.751-754
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• 2013

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.

• Nutrition Research and Practice
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• v.8 no.3
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• pp.278-283
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• 2014

BACKGROUND/OBJECTIVES: Due to its beneficial health effects, use of buckwheat has shown a continuous increase, and concerns regarding the allergic property of buckwheat have also increased. This study was conducted for evaluation of the hydrolytic effects of seven commercial proteases on buckwheat allergens and its allergenicity. MATERIALS/METHODS: Extracted buckwheat protein was hydrolyzed by seven proteolytic enzymes at individual optimum temperature and pH for four hours. Analysis was then performed using SDS-PAGE, immunoblotting, and competitive inhibition ELISA (ciELISA) with rabbit antiserum to buckwheat protein, and direct ELISA with pooled serum of 21 buckwheat-sensitive patients. RESULTS: Alkaline protease, classified as serine peptidase, was most effective in reducing allergenicity of buckwheat protein. It caused decomposition of the whole buckwheat protein, as shown on SDS-PAGE, and results of immunoblotting showed that the rabbit antiserum to buckwheat protein no longer recognized it as an antigen. Allergenicity showed a decrease of more than 50% when pooled serum of patients was used in ELISA. Two proteolytic enzymes from Aspergillus sp. could not hydrolyze buckwheat allergens effectively, and the allergenicity even appeared to increase. CONCLUSIONS: Serine-type peptidases appeared to show a relatively effective reduction of buckwheat allergenicity. However, the antigenicity measured using rabbit antiserum did not correspond to the allergenicity measured using sera from human patients. Production of less allergenic buckwheat protein may be possible using enzymatic hydrolysis.

• International Journal of Oral Biology
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• v.45 no.1
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• pp.8-14
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• 2020

Bilobalide isolated from the leaves of Ginkgo biloba has several pharmacological activities such as neuroprotective, anti-inflammatory, and anticonvulsant. However, the effect of bilobalide on cancer has not been clearly established. The main purpose of this study was to investigate the effect of bilobalide on cell growth and apoptosis induction in FaDu human pharyngeal squamous cell carcinoma. This was examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, nuclear 4′,6-diamidino-2-phenylindole dihydrochloride staining, DNA fragmentation analysis, and immunoblotting. Bilobalide inhibited the growth of FaDu cells in dose- and time-dependent manners. Treatment with bilobalide resulted in nuclear condensation and DNA fragmentation in FaDu cells. Furthermore, it promoted the proteolytic cleavage of procaspase-3/-7/-8/-9 with increase in the amount of cleaved caspase-3/-7/-8/-9. Bilobalide-induced apoptosis in FaDu cells was mediated by the expression of Fas and the activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase. Immunoblotting revealed that the antiapoptotic mitochondrial protein Bcl-2 was downregulated, but the proapoptotic protein Bax was upregulated by bilobalide in FaDu cells. Bilobalide significantly increased Bax/Bcl-2 ratio. These results suggest that bilobalide inhibits cell proliferation and induces apoptosis in FaDu human pharyngeal squamous cell carcinoma via both the death receptor-mediated extrinsic apoptotic pathway and the mitochondrial-mediated intrinsic apoptotic pathway.