• The Korea Journal of Herbology
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• v.38 no.5
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• pp.9-19
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• 2023

Objectives : Osteoporosis is a systemic skeletal disorder characterized by reduced bone mineral density and increased risk of fractures. Bisphosphonates and selective estrogen receptors, which are bone resorption inhibitors that are currently widely used as osteoporosis treatments, show serious side effects when administered for a long time. Research on bone resorption inhibitors that complement the problems of existing treatments is needed. The purpose of this study was to investigate the effect of inhibiting osteoclast differentiation and activity on the tuberous root of Ampelopsis japonica (Thunb.) Makino (AM). Methods : After extracting AM using distilled water and ethanol, the inhibitory effects of the two solvents on osteoclast differentiation were compared using the RANKL-induced in vitro experimental model and the TRAP assay kit. The impact of AM on bone resorption was investigated through the pit formation assay, and its effect on F-actin formation was assessed through fluorescent staining. Additionally, protein and mRNA expression levels of osteoclast differentiation markers (NFATc1, c-Fos, TRAP and ATP6v0d2) and resorption markers (MMP-9, CTK, and CA2) were analyzed via western blot and RT-PCR. Results : AM treatment significantly decreased the number of TRAP-positive cells and pit formation area. Furthermore, AM suppressed both the protein and mRNA expression of NFATc1 and c-Fos, key transcription factors involved in osteoclast differentiation and it downregulated the expression of osteoclast-associated genes such as TRAP, CTK, MMP-9, CA2, and ATP6v0d2. Conclusions : These results suggest that AM can inhibit bone resorption and osteoclast differentiation, indicating its potential for use in the treatment and prevention of osteoporosis.

• Journal of Life Science
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• v.22 no.7
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• pp.912-919
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• 2012

A Bacillus sp. strain producing celluase and xylanase was isolated from environmental soil with LB agar plate containing carboxymethylcellulose (CM-cellulose) and beechwood xylan stained with trypan blue as substrates, respectively. Based on the 16S rRNA gene sequence and API 50 CHL test, the strain was identified as B. subtilis and named B. subtilis NC1. The cellulase and xylanase from B. subtilis NC1 exhibited the highest activities for CM-cellulose and beechwood xylan as substrate, respectively, and both enzymes showed the maximum activity at pH 5.0 and $50^{\circ}C$. We cloned and sequenced the genes for cellulase and xylanase from genomic DNA of the B. subtilis NC1 by the shot-gun cloning method. The cloned cellulase and xylanase genes consisted of a 1,500 bp open reading frame (ORF) encoding a 499 amino acid protein with a calculated molecular mass of 55,251 Da and a 1,269 bp ORF encoding a 422 amino acid protein with a calculated molecular mass of 47,423 Da, respectively. The deduced amino acid sequences from the genes of cellulase and xylanase showed high identity with glycosyl hydrolases family (GH) 5 and 30, respectively.

• IMMUNE NETWORK
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• v.3 no.1
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• pp.53-60
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• 2003

Background: The role of macrophages in tumor angiogenesis is known to be the production of angiogenic cytokines and growth factors including TNF-${\alpha}$. Recently, macrophage also can produce the INF-${\gamma}$ that is being studied to be involved in angiogenic inhibition. Thus, the importance of macrophages in tumor angiogenesis is might being an angiogenic switch. Thus, the hypothesis tested here is that TNF-${\alpha}$ can modulate the INF-${\gamma}$ production in the macrophages from tumor environment as a part of tumor angiogenic switch. Methods: Macrophages in tumor environment were obtained from the peritoneal cavity of C57BL/6 mice injected with B16F10 melanoma cell line for 6 or 11 days. $Mac1^+$-macrophages were purified using magnetic bead ($MACs^{TM}$; Milteny Biotech, Germany) and cultured with various concentrations of TNF-${\alpha}$ for various time points at $37^{\circ}C$. The supernatants were analyzed for IFN-${\gamma}$ or VEGF by ELISA kit (Endogen, Woburn, MA). Results: Residential macrophages from the peritoneal cavity did not respond to LPS or TNF-${\alpha}$ to produce INF-${\gamma}$. However, the cells from tumor environment produced IFN-${\gamma}$ as well as VEGF and upregulated by the addition of LPS or TNF-${\alpha}$. RT-PCR analysis revealed the external TNF-${\alpha}$-induced IFN-${\gamma}$ gene expression in the macrophages from tumor environment. Conclusion: The overall data suggest that the macrophages in tumor environment might have an important role not only in angiogenic signal but also in anti-angiogenic signal by producing related cytokines. And TNF-${\alpha}$ might be a key cytokine in tumor angiogenic switch.

• Korean Journal of Microbiology
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• v.41 no.3
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• pp.168-176
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• 2005

Vibrio vulnificus, one of the marine bacterial pathogens causing septicemia, was detected using molecular methods, namely, PCR and/or Southern hybridization, and real-time PCR. Extracted and purified total DNAs by using commercial kits were used as templates for PCR. Multiplex-PCR was conducted by employing three sets of primers for the genes, hemolysin (vvhA), phosphomannomutase (pmm), and metalloprotease (vvpE), for V vulnificus virulence. The presence of DMSO ($5\%$) and BSA ($0.1\%$) in PCR reaction mixture improved a detection efficiency by higher PCR band intensities. TaqMan real-time PCR was carried out by using gene segment of vvhA as a target. Detection limit of PCR/Southern hybridization without enrichments was to be around $10^2\;cells\;g^{-1}$ of sample. However, those three methods using the enrichment at $35^{\circ}C$ in APW showed high sensitivity ($2\~10\;cells\;g^{-1}$ of sediments). Highly sensitive detection of V vulnificus by real-time PCR was achieved within $5\~6$ hr, whereas the detection by PCR/Southern hybridization required about 36 hr. Thus, it was evident that real-time PCR is the most rapid and efficient method for detecting V vulnificus in tidal flat sediments.

• Journal of Mushroom
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• v.13 no.4
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• pp.330-333
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• 2015

This study was carried out to compare the anti-inflammation effects of various fruiting body of Ganoderma species and Cordyceps militaris, Phelinus linteus extracts. We concentrated Ganoderma species and other medicinal mushrooms by extracting with ethanol. And We made it $100{\mu}g/ml$ concentration. As a result of nitrite scavenging activity, in the contrast to the positive control; Ascorbic acid was 25%, ASI 7080 of Ganoderma species was disappeared up to around 40%. And in the contrast to Ascorbic acid was 55%, ASI 7002 was 78.5% that was the highest anti-inflammation effect in the result of "No assay test". The Cordyceps militaris showed 75% and Hericium erinaceus showed 59.7% of anti-inflammation effect. As a result of the fungus yield control test of $TNF-{\alpha}$ through ELISA method to ASI 7002 of Ganoderma species that showed the highest anti-inflammation, it was reduced as same as LPS non-treatment. We extracted RNA from ASI 7002 Ganoderma species 10, 50, $100{\mu}g/ml$ concentration and LPS $10{\mu}g/ml$ of Raw 264.7 cell. And we tested the expression of iNOS, COX-2 and TNF-a that are kinds of inflammation gene after synthesizing RNA with cDNA. Finally we could find that iNOS, COX-2 and TNF-a were all controlled expression in the result of above experiment.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.198-203
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• 2007

DNA transfection is a powerful tool for studying gene functions. The $Ca^{2+}$-phosphate precipitation remains one of the most popular and cost-effective transfection techniques. Mature neurons are more resistant to transfection than young ones and most other cell types, and easy to die if microenvironment changes. Here, we report a transfection protocol for mature neurons. The critical modifications are inclusion of glial cells in culture and careful control of $Ca^{2+}$-phosphate precipitation under microscope. Cerebral glial cells were grown until ${\sim}70-80%$ confluence in DMEM/10% horse serum, which was thereafter replaced with serum-free Neurobasal/Ara-C, and 319 hippocampal neurons were plated onto the glial layer Formation of fine $DNA/Ca^{2+}$-phosphate precipitates was induced using Clontech $CalPhos^{TM}$ Mammalian Transfection Kit, and the size ($0.5-1\;{\mu}m$ in diameter) and density(about 10 particles/$100\;{\mu}m^2$) were carefully controlled by the time of incubation in the medium. This modified protocol can be reliably applied for transfection of mature neurons that are maintained longer than two weeks in vitro, resulting in 10-15 healthy transfected neurons per a well of 24-well plates. The efficacy of the protocol was verified by punctate expression of $pEGFP-CaMKII{\alpha}$, a synaptic protein, and diffuse expression of pDsRed2. Our protocol provides a reliable method for transfection of mature neurons in vitro.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.4
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• pp.2325-2331
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• 2013

Objective: To detect effects of plumbagin on proliferation and apoptosis in non-small cell lung cancer cell lines, and investigate the underlying mechanisms. Materials and Methods: Human non-small cell lung cancer cell lines A549, H292 and H460 were treated with various concentrations of plumbagin. Cell proliferation rates was determined using both cell counting kit-8 (CCK-8) and clonogenic assays. Apoptosis was detected by annexin V/propidium iodide double-labeled flow cytometry and TUNEL assay. The levels of reactive oxygen species (ROS) were detected by flow cytometry. Activity of NF-${\kappa}B$ was examined by electrophoretic mobility shift assay (EMSA) and luciferase reporter assay. Western blotting was used to assess the expression of both NF-${\kappa}B$ regulated apoptotic-related gene and activation of p65 and $I{\kappa}B{\kappa}$. Results: Plumbagin dose-dependently inhibited proliferation of the lung cancer cells. The IC50 values of plumbagin in A549, H292, and H460 cells were 10.3 ${\mu}mol/L$, 7.3 ${\mu}mol/L$, and 6.1 ${\mu}mol/L$ for 12 hours, respectively. The compound concentration-dependently induced apoptosis of the three cell lines. Treatment with plumbagin increased the intracellular level of ROS, and inhibited the activation of NK-${\kappa}B$. In addition to inhibition of NF-${\kappa}B$/p65 nuclear translocation, the compound also suppressed the degradation of $I{\kappa}B{\kappa}$. ROS scavenger NAC highly reversed the effect of plumbagin on apoptosis and inactivation of NK-${\kappa}B$ in H460 cell line. Treatment with plumbagin also increased the activity of caspase-9 and caspase-3, downregulated the expression of Bcl-2, upregulated the expression of Bax, Bak, and CytC. Conclusions: Plumbagin inhibits cell growth and induces apoptosis in human lung cancer cells through an NF-${\kappa}B$-regulated mitochondrial-mediated pathway, involving activation of ROS.

• Journal of Ginseng Research
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• v.44 no.3
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• pp.475-482
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• 2020

Background: Active natural ingredients, especially small molecules, have recently received wide attention as modifiers used to treat neurodegenerative disease by promoting neurogenic regeneration of neural stem cell (NSC) in situ. 20(S)-protopanaxadiol (PPD), one of the bioactive ingredients in ginseng, possesses neuroprotective properties. However, the effect of PPD on NSC proliferation and differentiation and its mechanism of action are incompletely understood. Methods: In this study, we investigated the impact of PPD on NSC proliferation and neuronal lineage differentiation through activation of the Wnt/glycogen synthase kinase (GSK)-3β/β-catenin pathway. NSC migration and proliferation were investigated by neurosphere assay, Cell Counting Kit-8 assay, and EdU assay. NSC differentiation was analyzed by Western blot and immunofluorescence staining. Involvement of the Wnt/GSK3β/β-catenin pathway was examined by molecular simulation and Western blot and verified using gene transfection. Results: PPD significantly promoted neural migration and induced a significant increase in NSC proliferation in a time- and dose-dependent manner. Furthermore, a remarkable increase in anti-microtubule-associated protein 2 expression and decrease in nestin protein expression were induced by PPD. During the differentiation process, PPD targeted and stimulated the phosphorylation of GSK-3β at Ser9 and the active forms of β-catenin, resulting in activation of the Wnt/GSK-3β/β-catenin pathway. Transfection of NSCs with a constitutively active GSK-3β mutant at S9A significantly hampered the proliferation and neural differentiation mediated by PPD. Conclusion: PPD promotes NSC proliferation and neural differentiation in vitro via activation of the Wnt/GSK-3β/β-catenin pathway by targeting GSK-3β, potentially having great significance for the treatment of neurodegenerative diseases.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.24
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• pp.10577-10583
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• 2015

Background: Triple-negative breast cancer (TNBC), characterized by the lack of expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2, is typically associated with a poor prognosis. The majority of TNBCs show the expression of basal markers on gene expression profiling and most authors accept TNBC as basal-like (BL) breast cancer. However, a smaller fraction lacks a BL phenotype despite being TNBC. The literature is silent on non-basal-like (NBL) type of TNBC. The present study was aimed at defining behavioral differences between BL and NBL phenotypes. Objectives: i) Identify the TNBCs and categorize them into BL and NBL breast cancer. ii) Examine the behavioral differences between two subtypes. iii) Observe the pattern of treatment failure among TNBCs. Materials and Methods: All TNBC cases during January 2009-December 2010 were retrieved. The subjects fitting the inclusion criteria of study were differentiated into BL and NBL phenotypes using surrogate immunohistochemistry with three basal markers $34{\beta}E12$, c-Kit and EGFR as per the algorithm defined by Nielsen et al. The detailed data of subjects were collated from clinical records. The comparison of clinicopathological features between two subgroups was done using statistical analyses. The pattern of treatment failure along with its association with prognostic factors was assessed. Results: TNBC constituted 18% of breast cancer cases considered in the study. The BL and NBL subtypes accounted for 81% and 19% respectively of the TNBC group. No statistically significant association was seen between prognostic parameters and two phenotypes. Among patients with treatment failure, 19% were with BL and 15% were with NBL phenotype. The mean disease free survival (DFS) in groups BL and NBL was 30.0 and 37.9 months respectively, while mean overall survival (OS) was 31.93 and 38.5 months respectively. Treatment failure was significantly associated with stage (p=.023) among prognostic factors. Conclusions: Disease stage at presentation is an important prognostic factor influencing the treatment failure and survival among TNBCs. Increasing tumor size is related to lymph node positivity. BL tumors have a more aggressive clinical course than that of NBL as shown by shorter DFS and OS, despite having no statistically significant difference between prognostic parameters. New therapeutic alternatives should be explored for patients with this subtype of breast cancer.

• Radiation Oncology Journal
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• v.17 no.2
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• pp.151-157
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• 1999

Purpose : By sequencing the Erpressed Sequence Tags of human 걸ermal papilla CDNA library, we identified a clone named K872 of which the expression decreased during differentiation of HL6O cell line. Materials and Methods : K872 plasmid DNA was isolated according to QIA plasmid extraction kit (Qiagen GmbH, Germany). The nucleotide sequencing was performed by Sanger's method with K872 plasmid DNA. The most updated GenBank EMBL necleic acid banks were searched through the internet by using BLAST (Basic Local Alignment Search Tools) program. Nothern bots were performed using RNA isolated from various human tissues and cancer cell lines. The gene expression of the fusion protein was achieved by His-Patch Thiofusicn expression system and the protein product was identified on SDS-PAGE. Results : K872 clone is 1006 nucleotides long, and has a coding region of 675 nucleotides and a 3' non-coding region of 280 nucleotides. The presumed open reading frame starting at the 5' terminus of K872 encodes 226 amino acids, including the initiation methionine residue. The amino acid sequence deduced from the open reading frame of K872 shares $70\%$, identity with that of rat glutathione 5-transferase kappa 1 (rGSTKl). The transcripts were expressed in a variety of human tissues and cancer cells. The levels of transcript were relatively high in those tissues such as heart, skeletal muscle, and peripheral blood leukocyte. It is noteworthy that K872 was found to be abundantly expressed in coloreetal cancer and melanoma cell lines. Conclusion : Homology search result suggests that K872 clone is the human homolog of the rGSTK1 which is known to be involved in the resistance of cytotoxic therapy. We propose that meticulous functional analysis should be followed to confirm that.