• Journal of Microbiology and Biotechnology
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• v.27 no.4
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• pp.808-815
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• 2017

We demonstrated the quantitative detection of a toxin-producing Microcystis aeruginosa (M. aeruginosa) strain with the laboratory protocol of the NanoGene assay. The NanoGene assay was selected because its laboratory protocol is in the process of being transplanted into a portable system. The mcyD gene of M. aeruginosa was targeted and, as expected, its corresponding fluorescence signal was linearly proportional to the mcyD gene copy number. The sensitivity of the NanoGene assay for this purpose was validated using both dsDNA mcyD gene amplicons and genomic DNAs (gDNA). The limit of detection was determined to be 38 mcyD gene copies per reaction and 9 algal cells/ml water. The specificity of the assay was also demonstrated by the addition of gDNA extracted from environmental algae into the hybridization reaction. Detection of M. aeruginosa was performed in the environmental samples with environmentally relevant sensitivity (${\sim}10^5$ algal cells/ml) and specificity. As expected, M. aeruginosa were not detected in nonspecific environmental algal gDNA over the range of $2{\times}10^0$ to $2{\times}10^7$ algal cells/ml.

• Nutrition Research and Practice
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• v.14 no.5
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• pp.453-462
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• 2020

BACKGROUND/OBJECTIVES: Platycodon grandiflorum (PG), an oriental herbal medicine, has been known to improve liver function, and has both anti-inflammatory and antimicrobial properties. However, little is known about the immune-enhancing effects of PG and its mechanism. In this study, we aimed to investigate whether fermented PG extract (FPGE), which has increased platycodin D content, activates the immune response in a murine macrophage cell line, RAW 264.7. MATERIALS/METHODS: Cell viability was determined by Cell Counting Kit-8 assay and the nitric oxide (NO) levels were measured using Griess reagent. Cytokine messenger RNA levels of were monitored by quantitative reverse transcription polymerase chain reaction. To investigate the molecular mechanisms underlying immunomodulatory actions of FPGE in RAW 264.7 cells, we have conducted luciferase reporter gene assay and western blotting. RESULTS: We found that FPGE treatment induced macrophage cell proliferation in a dose-dependent manner. FPGE also modulated the expression of NO and pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. The activation and phosphorylation levels of nuclear factor kappa B (NF-κB) were increased by FPGE treatment. Moreover, 5-aminoimidazole-4-carboxamide ribonucleotide, an activator of AMP-activated kinase (AMPK), significantly reduced both lipopolysaccharides- and FPGE-induced NF-κB reporter gene activity. CONCLUSIONS: Taken together, our findings suggest that FPGE may be a novel immune-enhancing agent acting via AMPK-NF-κB signaling pathway.

• Molecular & Cellular Toxicology
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• v.5 no.2
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• pp.172-178
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• 2009

Nanoparticles are small-scale substances (<100 nm) with unique properties, complex exposure and health risk implications. Aluminum oxide ($Al_2O_3$) nanoparticles (NP) have been widely used as abrasives, wear-resistant coatings on propeller shafts of ships, to increase the specific impulse per weight of composite propellants used in solid rocket fuel and as drug delivery systems to increase solubility. However, recent studies have shown that nano-sized aluminum (10 nm in diameter) can generate adverse effects, such as pulmonary response. The cytotoxicity and genotoxicity of $Al_2O_3$ NP were investigated using the dye exclusion assay, the comet assay, and the mouse lymphoma thymidine kinase (tk$^{+/-}$) gene mutation assay (MLA). IC$_{20}$ values of $Al_2O_3$ NP in BEAS-2B cells were determined the concentration of 273.44 $\mu$g/mL and 390.63 $\mu$g/mL with and without S-9. However IC$_{20}$ values of $Al_2O_3$ NP were found nontoxic in L5178Y cells both of with and without S-9 fraction. In the comet assay, L5178Y cells and BEAS-2B cells were treated with $Al_2O_3$ NP which significantly increased 2-fold tail moment with and without S-9. Also, the mutant frequencies in the $Al_2O_3$ NP treated L5178Y cells were increased compared to the vehicle controls with S-9. The results of this study indicate that $Al_2O_3$ NP can cause primary DNA damage and cytotoxicity but not mutagenicity in cultured mammalian cells.

• KSBB Journal
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• v.32 no.1
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• pp.9-15
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• 2017

Chamomile (Matricaria chamomilla), a member of the Asteraceae family, is a well-known for medicinal plant and can be found in India and Europe. Chamomile is an effective sedative and various medical effects. But, the effects of acne treatment by chamomile were not investigated. Therefore, we assessed the anti-oxidant effects, anti-microbial activity and anti-inflammatory effects by chamomile extracts in HaCaT keratinocyte cells. Anti-oxidant effects of chamomile extracts were investigated by DPPH assay. Also, results of MTT assay was demonstrated that chamomile extracts did not have a cytotoxic effect in HaCaT cells. To assess the antimicrobial activity, we determined formation of inhibition zone of Propionibacterium acnes by extracts from chamomile. Tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) induces production of inflammatory cytokines such as interleukin-$1{\beta}$ (IL-$1{\beta}$), IL-6 and IL-8 and expression of COX-2. Chamomile extracts could inhibit TNF-${\alpha}$-induced mRNA expression levels of IL-$1{\beta}$, IL-6, IL-8 and COX-2 gene. These results demonstrated the possibility of chamomile for prevention and treatment of skin inflammatory diseases such as acne.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4751-4756
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• 2012

Colon cancer continues to be one of the most common cancers, and the importance and necessity of new therapies needs to be stressed. The most important proto-oncogen factors for colon cancer appear to be epidermal growth factor receptor, EGFR, and c-Src with high expression and activity leading to tumor growth and ultimately to colon cancer progression. Application of c-Src and EGFR antisense agents simultaneously should theoretically therefore have major benefit. In the present study, anti-EGFR and c-Src specific antisense oligodeoxynucleotides were combined in a formulation using PAMAM dendrimers as a carrier. Nano drug entry into cells was confirmed by flow cytometry and fluorescence microscopy imaging and real time PCR showed gene expression of c-Src and EGFR, as well as downstream STAT5 and MAPK-1 with the tumor suppressor gene P53 to all be downregulated. EGFR and c-Src protein expression was also reduced when assessed by western blotting techniques. The effect of the antisense oligonucleotide on HT29 cell proliferation was determined by MTT assay, reduction beijng observed after 48 hours. In summary, nano-drug, anti-EGFR and c-Src specific antisense oligodeoxynucleotides were effectively transferred into HT-29 cells and inhibited gene expression in target cells. Based on the results of this study it appears that the use of antisense EGFR and c-Src simultaneously might have a significant effect on colon cancer growth by down regulation of EGFR and its downstream genes.

• Advances in nano research
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• v.4 no.2
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• pp.145-156
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• 2016

Dendrimers are one of the most appropriate nanocaries for imaging moieties in imaging applications.The purpose of this study was the evalution of cytotoxicity and inducing apoptosis of dendrimers. This study was conducted in order to investigate the metastasis suppression effect of dendrimer in human breast MCF-7 cell line and finding the nanoparticle protein corona in biological enviromental. Dendrimer cytotoxicity effect was assessed by MTT assay. The mRNA experession level of KAI1 as a metastasis suppressor gene, Bax as Pro- apoptotic gene, Bcl-2 as an anti-apoptotic gene and GAPDH as a housekepping gene were determined by real-time PCR assays.concentration-dependent nanoparticle cytotoxicity effect was proofed at range of 1-2 mg/mL in 24 hours, significant upregulation of mRNA expression of Bax, was observed whereas expression of anti-apoptotic Bcl-2 was down-regulated, also expression of metastasis suppressor gene KAI1 was up-regulated. So far a few studies confirmed apoptosis enhancement effect of dendrimers in MCF-7 cell line via bax/bcl-2 pathways. dendrimer nanoparticles was able to act as metastase inhibitor via upregulation of KAI1 gene.

• Journal of Life Science
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• v.29 no.9
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• pp.949-954
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• 2019

Membrane topology is a key characteristic of membrane proteins. We previously reported the cloning of the chromosome 4 open-reading frame 32 (C4orf32) gene as a potential membrane protein; however, the cellular localization and membrane topology of C4orf32 was as yet unknown. In this study, we found that green fluorescent protein (GFP) fused to the C-terminus of C4orf32 (C4orf32-GFP) was localized to the endoplasmic reticulum (ER). We applied three tools to identify determinants of C4orf32 topology: protease protection, fluorescence protease protection (FPP), and an inducible system using the ternary complex between FK506 binding protein 12 (FKBP), rapamycin, and the rapamycin-binding domain of mTOR (FRB) (the FRB-rapamycin-FKBP system). Using protease protection and FPP assays, we found that the GFP tag in C4orf32-GFP was localized to the cytoplasmic surface of the ER membrane of HeLa cells. Protease protection and FPP assays are useful and complimentary tools for identifying the topology of GFP fusion membrane proteins. The FRB-rapamycin-FKBP system was also used to study the topology of C4orf32. In the absence of rapamycin, a monomeric red fluorescent protein-FKBP fusion (mRFP-FKBP) and C4orf32-GFP-FRB were localized to the cytoplasm and the ER membrane, respectively. However, in the presence of rapamycin, the mRFP-FKBP was shifted from the cytoplasm to the ER and colocalized with the C4orf32-GFP-FRB. These results indicate that the FRB moiety is facing the cytoplasmic surface of ER membrane. Overall, our results clearly suggest that C4orf32 belongs to the family of type I ER resident membrane proteins.

• Korean Journal of Clinical Laboratory Science
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• v.49 no.4
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• pp.323-328
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• 2017

Angiogenesis is a process including members of the angiogenic factors. In particular, fibroblast growth factor 2 (FGF2) is considered the most potent angiogenic factor because it promotes cell proliferation and tube formation. A recent study reported that fucoidan derived from marine plant potentiated FGF-2 induced tube formation in human endothelial cells. On the other hand, the molecular mechanisms involved in the angiogenic activity of fucoidan and FGF2 are unknown. In this study, a fucoidan treatment promoted angiogenesis induced by FGF2. The effects of fucoidan on FGF2-induced angiogenesis were confirmed by a proliferation assay using a CellTiter96 Aqueous One solution after a treatment with fucoidan and FGF2. The tube formation and wound healing assay for the angiogenic activity were also confirmed. Reverse transcription PCR showed a change in the mRNA of vascular endothelial growth factor-A (VEGF-A), intercellular adhesion molecule-1 (ICAM-1), matrix metallopeptidase9 (MMP9), and the signal transducer and activator of transcription3 (STAT3). In summary, the Fucoidan/FGF2 treatment induced an increase in cell proliferation, improved the tube formation and wound healing activity, and altered the STAT3, VEGF-A, ICAM-1, and MMP9 mRNA expression levels. Further research will be needed to provide a scientific explanation in terms of cell-signaling and confirm the present findings.

• Advances in nano research
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• v.12 no.5
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• pp.501-514
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• 2022

This study aimed to investigate the effects and potential mechanisms of Chikusetsusaponin V (CsV) on endothelial nitric oxide synthase (eNOS) and vascular endothelial cell functions. Different concentrations of CsV were added to animal models, bovine aorta endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs) cultured in vitro. qPCR, Western blotting (WB), and B ultrasound were performed to explore the effects of CsV on mouse endothelial cell functions, vascular stiffness and cellular eNOS mRNA, protein expression and NO release. Bioinformatics analysis, network pharmacology, molecular docking and protein mass spectrometry analysis were conducted to jointly predict the upstream transcription factors of eNOS. Furthermore, pulldown and ChIP and dual luciferase assays were employed for subsequent verification. At the presence or absence of CsV stimulation, either overexpression or knockdown of purine rich element binding protein A (PURA) was conducted, and PCR assay was employed to detect PURA and eNOS mRNA expressions, Western blot was used to detect PURA and eNOS protein expressions, cell NO release and serum NO levels. Tube formation experiment was conducted to detect the tube forming capability of HUVECs cells. The animal vasodilation function test detected the vasodilation functions. Ultrasonic detection was performed to determine the mouse aortic arch pulse wave velocity to identify aortic stiffness. CsV stimulus on bovine aortic cells revealed that CsV could upregulate eNOS protein levels in vascular endothelial cells in a concentration and time dependent manner. The expression levels of eNOS mRNA and phosphorylation sites Ser1177, Ser633 and Thr495 increased significantly after CsV stimulation. Meanwhile, CsV could also enhance the tube forming capability of HUVECs cells. Following the mice were gavaged using CsV, the eNOS protein level of mouse aortic endothelial cells was upregulated in a concentration- and time-dependent manner, and serum NO release and vasodilation ability were simultaneously elevated whereas arterial stiffness was alleviated. The pulldown, ChIP and dual luciferase assays demonstrated that PURA could bind to the eNOS promoter and facilitate the transcription of eNOS. Under the conditions of presence or absence of CsV stimulation, overexpression or knockdown of PURA indicated that the effect of CsV on vascular endothelial function and eNOS was weakened following PURA gene silence, whereas overexpression of PURA gene could enhance the effect of CsV upregulating eNOS expression. CsV could promote NO release from endothelial cells by upregulating the expression of PURA/eNOS pathway, improve endothelial cell functions, enhance vasodilation capability, and alleviate vessel stiffness. The present study plays a role in offering a theoretical basis for the development and application of CsV in vascular function improvement, and it also provides a more comprehensive understanding of the pharmacodynamics of CsV.

• Korean Journal of Microbiology
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• v.43 no.1
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• pp.19-22
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• 2007

Acetohydroxyacid synthase (E.C.2.2.1.6., AHAS) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine. The AHAS gene (TIGR access code HI2585) from Heamophilus influenzae was cloned into the bacterial expression vector pET-28a and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by $Ni^{2+}-charged$ HiTrap chelating HP column. The purified enzyme appears as a single band on SDS-PAGE with a molecular mass of about 63.9 kDa. The enzyme exhibits absolute dependence on the three cofactors FAD, $MgCl_{2}$ and thiamine diphosphate for activity. Specific activity of purified enzyme has 3.22 unit/mg and optimum activity in the pH 7.5 at $37^{\circ}C$. This enzyme activity has an effect on the buffer. When comparing the enzyme activity against the organic solvent, it followed in type and the difference it is but even from the aqueous solution where the organic solvent is included with the fact that the enzyme activity is maintained.