• Proceedings of the SCSK Conference
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• 2003.09b
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• pp.12-25
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• 2003

L-camitine ($\beta$ -hydroxy-${\gamma}$ -trimethyl-ammoniumbutyric acid) is a small water-soluble molecule important in mammalian fat metabolism. It is essential for the normal oxidation of fatty acids by the mitochondria, and is involved in the trans-esterification and excretion of acyl-CoA esters. In this paper, to investigate the relationship between aging and L-camitine, we investigated the effects of in vitro MMP inhibition and activity and expression of UVA-induced MMP 1 in human skin fibroblasts. Fluorometric assays of the proteolytic activities of MMP-l were performed using fluorescent collagen substrates. ELISA (enzyme linked immuno sorbent assay), gelatin-substrate zymography, and RT-PCR ELISA techniques were used for the effects of L-camitine on MMP expression and activity, MMP mRNA expression in UVA irradiated fibroblast. L-camitine inhibited the activities of MMP-l in a dose-dependent manner and the $IC_{50}$/ values calculated from semi-log plots were 2.45mM, and L-carnitine showed strong inhibition on MMP-2 (gelatinase) activity in UVA irradiated fibroblast by zymography. Also, UVA induced MMP expression was reduced 40% by treated with L-carnitine, and MMP-l mRNA expression was reduced dose-dependent manner. Therefore L-carnitine was able to significantly inhibition the MMP activity, regulation of MMP expression in protein and mRNA level. All these results suggest that L-carnitine may be useful as new anti-aging cofactor for protection against UVA induced MMP expression and activity.

• Osong Public Health and Research Perspectives
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• v.8 no.6
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• pp.389-396
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• 2017

Objectives: To circumvent the limitations of the current golden standard method, colony-forming unit (CFU) assay, for viability of Bacille Calmette-$Gu{\acute{e}}rin$ (BCG) vaccines, we developed a new method to rapidly and accurately determine the potency of BCG vaccines. Methods: Based on flow cytometry (FACS) and fluorescein diacetate (FDA) as the most appropriate fluorescent staining reagent, 17 lots of BCG vaccines for percutaneous administration and 5 lots of BCG vaccines for intradermal administration were analyzed in this study. The percentage of viable cells measured by flow cytometry along with the total number of organisms in BCG vaccines, as determined on a cell counter, was used to quantify the number of viable cells. Results: Pearson correlation coefficients of FACS and CFU assays for percutaneous and intradermal BCG vaccines were 0.6962 and 0.7428, respectively, indicating a high correlation. The coefficient of variation value of the FACS assay was less than 7%, which was 11 times lower than that of the CFU assay. Conclusion: This study contributes to the evaluation of new potency test method for FACS-based determination of viable cells in BCG vaccines. Accordingly, quality control of BCG vaccines can be significantly improved.

• Biomolecules & Therapeutics
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• v.29 no.6
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• pp.615-629
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• 2021

An active compound, triterpene saponin, astersaponin I (AKNS-2) was isolated from Aster koraiensis Nakai (AKNS) and the autophagy activation and neuroprotective effect was investigated on in vitro and in vivo Parkinson's disease (PD) models. The autophagy-regulating effect of AKNS-2 was monitored by analyzing the expression of autophagy-related protein markers in SH-SY5Y cells using Western blot and fluorescent protein quenching assays. The neuroprotection of AKNS-2 was tested by using a 1-methyl-4-phenyl-2,3-dihydropyridium ion (MPP⁺)-induced in vitro PD model in SH-SY5Y cells and an MPTP-induced in vivo PD model in mice. The compound-treated SH-SY5Y cells not only showed enhanced microtubule-associated protein 1A/1B-light chain 3-II (LC3-II) and decreased sequestosome 1 (p62) expression but also showed increased phosphorylated extracellular signal-regulated kinases (p-Erk), phosphorylated AMP-activated protein kinase (p-AMPK) and phosphorylated unc-51-like kinase (p-ULK) and decreased phosphorylated mammalian target of rapamycin (p-mTOR) expression. AKNS-2-activated autophagy could be inhibited by the Erk inhibitor U0126 and by AMPK siRNA. In the MPP⁺-induced in vitro PD model, AKNS-2 reversed the reduced cell viability and tyrosine hydroxylase (TH) levels and reduced the induced α-synuclein level. In an MPTP-induced in vivo PD model, AKNS-2 improved mice behavioral performance, and it restored dopamine synthesis and TH and α-synuclein expression in mouse brain tissues. Consistently, AKNS-2 also modulated the expressions of autophagy related markers in mouse brain tissue. Thus, AKNS-2 upregulates autophagy by activating the Erk/mTOR and AMPK/mTOR pathways. AKNS-2 exerts its neuroprotective effect through autophagy activation and may serve as a potential candidate for PD therapy.

• Journal of Microbiology and Biotechnology
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• v.27 no.3
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• pp.480-491
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• 2017

Fluorescent pseudomonads have been isolated from halophytes, mesophytes, and xerophytes of Pakistan. Among these, eight isolates, GS-1, GS-3, GS-4, GS-6, GS-7, FS-2 (cactus), ARS-38 (cotton), and RP-4 (para grass), showed antifungal activity and were selected for detailed study. Based on biochemical tests and 16S rRNA gene sequences, these were identified as strains of P. chlororaphis subsp. chlororaphis and aurantiaca. Secondary metabolites of these strains were analyzed by LC-MS. Phenazine-1-carboxylic acid (PCA), 2-hydroxy-phenazine, Cyclic Lipopeptide (white line-inducing principle (WLIP)), and lahorenoic acid A were detected in variable amounts in these strains. P. aurantiaca PB-St2 was used as a reference as it is known for the production of these compounds. The phzO and PCA genes were amplified to assure that production of these compounds is not an artifact. Indole acetic acid production was confirmed and quantified by HPLC. HCN and siderophore production by all strains was observed by plate assays. These strains did not solubilize phosphate, but five strains were positive for zinc solubilization. Wheat seedlings were inoculated with these strains to observe their effect on plant growth. P. aurantiaca strains PB-St2 and GS-6 and P. chlororaphis RP-4 significantly increased both root and shoot dry weights, as compared with uninoculated plants. However, P. aurantiaca strains FS-2 and ARS-38 significantly increased root and shoot dry weights, respectively. All strains except PB-St2 and ARS-38 significantly increased the root length. This is the first report of the isolation of P. aurantiaca from cotton and cactus, P. chlororaphis from para grass, WLIP and lahorenoic acid A production by P. chlororaphis, and zinc solubilization by P. chlororaphis and P. aurantiaca.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.1
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• pp.7-13
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• 2004

As a novel anti-wrinkle agent, 3,9-diferuloyl-6-oxopterocarpen (Tensolin-F$^{(R)}$) has been synthesized and its anti-aging effects have been investigated. In the present study, to investigate the relationship between aging and Tensolin-F$^{(R)}$, we examined its effect on scavenging activities of radicals and reactive oxygen species (ROS), in vitro inhibition activity of matrix-metalloproteinase (MMP) and expression of UVA-induced MMPs in human dermal fibroblasts (HDF). Tensolin-F$^{(R)}$ was found to show activities of scavenging radicals and ROS with the $IC_{50}$/ values of 0.2 mM and 0.95 mM against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide radicals, respectively, in the xanthine/xanthine oxidase system. Fluorometric assays for the proteolytic activities of MMP-l (collagenase) were performed using fluorescent collagen substrates. Tensolin-F$^{(R)}$ inhibited the activities of MMP-l in a dose-dependent manner and the $IC_{50}$/ values calculated from semi-log plots were 0.025 mM. Also, UVA induced MMP-1 expression was reduced 85％ by treatment with Tensolin-F$^{(R)}$ at 0.8 uM, which was reduced dose-dependent manner. The results of clinical study showed that 4.8 mM Tensolin-F$^{(R)}$ treated group reduced wrinkle significantly compared with placebo treated group (P 〈 0.05). Taken together, these result suggest that Tensolin-F$^{(R)}$ act as an anti-wrinkle agent by taking effects to antioxidation and reducing UVA-induced MMP-l production.-l production.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.8
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• pp.3213-3222
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• 2015

Background: Cancer metastasis depends on cell motility which is driven by cycles of actin polymerization and depolymerization. Reactive oxygen species (ROS) and metabolic oxidative stress have long been associated with cancer. ROS play a vital role in regulating actin dynamics that are sensitive to oxidative modification. The current work aimed at studying the effects of sub-lethal metabolic oxidative stress on actin cytoskeleton, focal adhesion and cell migration. Materials and Methods: T47D human breast cancer cells were treated with 2-deoxy-D-glucose (2DG), L-buthionine sulfoximine (BSO), or doxorubicin (DOX), individually or in combination, and changes in intracellular total glutathione and malondialdehyde (MDA) levels were measured. The expression of three major antioxidant enzymes was studied by immunoblotting, and cells were stained with fluorescent-phalloidin to evaluate changes in F-actin organization. In addition, cell adhesion and degradation ability were measured. Cell migration was studied using wound healing and transwell migration assays. Results: Our results show that treating T47D human breast cancer cells with drug combinations (2DG/BSO, 2DG/DOX, or BSO/DOX) decreased intracellular total glutathione and increased oxidized glutathione, lipid peroxidation, and cytotoxicity. In addition, the drug combinations caused a reduction in cell area and mitotic index, prophase arrest and a decreased ability to form invadopodia. The formation of F-actin aggregates was increased in treated T47D cells. Moreover, combination therapy reduced cell adhesion and the rate of cell migration. Conclusions: Our results suggest that exposure of T47D breast cancer cells to combination therapy reduces cell migration via effects on metabolic oxidative stress.

• Journal of Ginseng Research
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• v.45 no.2
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• pp.228-235
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• 2021

Backgroud: Ginsenoside compound K (GK) is a major metabolite of protopanaxadiol-type ginsenosides and has remarkable anticancer activities in vitro and in vivo. This work used an ionic cross-linking method to entrap GK within O-carboxymethyl chitosan (OCMC) nanoparticles (Nps) to form GK-loaded OCMC Nps (GK-OCMC Nps), which enhance the aqueous solubility and stability of GK. Methods: The GK-OCMC Nps were characterized using several physicochemical techniques, including x-ray diffraction, transmission electron microscopy, zeta potential analysis, and particle size analysis via dynamic light scattering. GK was released from GK-OCMC Nps and was conducted using the dialysis bag diffusion method. The effects of GK and GK-OCMC Nps on PC3 cell viability were measured by using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Fluorescent technology based on Cy5.5-labeled probes was used to explore the cellular uptake of GK-OCMC Nps. Results: The GK-OCMC NPs had a suitable particle size and zeta potential; they were spherical with good dispersion. In vitro drug release from GK-OCMC NPs was pH dependent. Moreover, the in vitro cytotoxicity study and cellular uptake assays indicated that the GK-OCMC Nps significantly enhanced the cytotoxicity and cellular uptake of GK toward the PC3 cells. GK-OCMC Nps also significantly promoted the activities of both caspase-3 and caspase-9. Conclusion: GK-OCMC Nps are potential nanocarriers for delivering hydrophobic drugs, thereby enhancing water solubility and permeability and improving the antiproliferative effects of GK.

• Horticultural Science & Technology
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• v.33 no.6
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• pp.891-899
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• 2015

The lily symptomless virus (LSV) is the most common virus in Korean native lilies and causes various types of damage to overall plant growth. This study was carried out to investigate the elimination rate of the LSV by the in vitro scale culture (scaling) method in Korean native lilies and to test reinfection rates of the LSV under several field culture conditions of bulb production. Four Korean native lilies (Lilium dauricum, L. distichum, L. lancifolium, and L. maximowitzii) were used and their scales were cultured in vitro for micro-scale formation. The micro-scales were subcultured repeatedly using MS culture medium supplemented with 30 or $90g{\cdot}L^{-1}$ sucrose. The culture conditions were $24{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$ PPFD with 16 hour daylength using fluorescent lamps and maintained at $22{\pm}2^{\circ}C$. The virus-free bulblets were grown for one to three years in the greenhouse and transplanted to the field in October or March. Virus infection rates were investigated by direct tissue blotting immunobinding assays and measurement of chlorophyll and protein contents. Virus-free plants could be obtained from the 5th subculture of micro-scales in L. lancifolium and L. maximowitzii or from primary culture in L. dauricum and L. distichum. LSV-free plants were reinfected during bulb production in the field. Reinfection rates were higher at older bulb ages and under higher planting density. The plants planted in October and at inland Gyeongsan had higher infection rates than those planted in March and at coastal area Pohang. The reinfection rate of L. maximowitzii was higher than those of L. dauricum and L. lancifolium. The LSV-infected plants had lower chlorophyll contents and unchanged protein contents compared to virus-free plants.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.1
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• pp.63-71
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• 2004

The human skin is constantly exposed to environmental irritants such as ultraviolet, smoke, chemicals. Free radicals and reactive oxygen species (ROS) caused by these environmental facts play critical roles in cellular damage. These irritants are in themselves damaging to the skin structure but they also participate the immensely complex inflammatory reaction. The purpose of this study was to investigate the skin cell protective effect of Juniperus chinensis xylem extract on the UV and SLS-induced skin cell damages. We tested free radical and superoxide scavenging effect in vitro. We found that Juniperus chinensis xylem extracts had potent radical scavenging effect by 98％ at 100 $\mu\textrm{g}$/mL. Fluorometric assays of the proteolytic activities of matrix metalloproteinase-l(MMP-1, collagenase) were performed using fluorescent collagen substrates. UV A induced MMP-1 synthesis and activity were analyzed by enzyme-linked immunosorbent assay (ELISA) and gelatin-based zymography in skin fibroblasts. The extract of Juniperus chinensis showed strong inhibitory effect on MMP-1 activities by 97％ at 100 $\mu\textrm{g}$/mL and suppressed the UVA induced expression of MMP-1 by 79％ at 25 $\mu\textrm{g}$/mL. This extract also showed strong inhibition on MMP-2 activity in UVA irradiated fibroblast by zymography. We also examined anti-inflammatory effects by the determination test of proinflammatory cytokine, interleukin 6 in HaCaT keratinocytes. In this test Juniperus chinensis decreased expression of interleukin 6 about 30％. Expression of prostaglandin E$_2$, (PGE$_2$) after UVB irradiation was measured by competitive enzyme immunoassay (EIA) using PGE$_2$ monoclonal antibody. At the concentrations of 5-50 $\mu\textrm{g}$/mL of the extracts, the production of PGE$_2$ by HaCaT keratinocytes (24 hours after 10 mJ/$\textrm{cm}^2$ UVB irradiation) was significantly inhibited in culture supernatants (p〈0.05). The viability of cultured HaCaT keratinocytes was significantly reduced at the doses of above 10 mJ/$\textrm{cm}^2$ of UVB irradiation, but the presence of these extracts improved cell viability comparing to control after UVB irradiation. We also investigated the protective effect of this extract in sodium lauryl sulfate (SLS)-induced irritant skin reactions from 24 hour exposure. Twice a day application of the extract for reducing local inflammation in human skin was done. Irritant reactions were assessed by various aspects of skin condition, that is, erythema (skin color reflectance) and transepidermal water loss (TEWL). After 5 days the extract was found to reduce SLS-induced skin erythema and improve barrier regeneration when compared to untreated symmetrical test site. In conclusion, our results suggest that Juniperus chinensis can be effectively used for the prevention of UV and SLS-induced adverse skin reactions such as radical production, inflammation and skin cell damage.

• Microbiology and Biotechnology Letters
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• v.33 no.2
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• pp.148-153
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• 2005

Malaria is a re-emerging infectious disease that is spreading to areas where it had been eradicated, such as Eastern Europe and Central Asia. To avoid the mortality from malaria, early detection of the parasite is a very important issue. The peripheral blood smear has been the gold standard method for the diagnosis of malaria infection. Recently, several other methods have been introduced for quantitative detection of malaria parasites. Real time PCR that employs fluorescent labels to enable the continuous monitoring of PCR product formation throughout the reaction has recently been used to detect several human malaria parasites. 18S rRNA sequences from malaria parasites have been amplified using Taqman real time PCR assay. Here, a SYBR Green-based real time quantitative PCR assay for the detection of malaria parasite-especially, Plasmodium vivax - was applied for the evaluation of 26 blood samples from Korean malaria patients. Even though SYBR Green-based real time PCR is easier and cheaper than Taqman-based assay, SYBR Green-based assay cannot be used because 18S rRNA cannot be specifically amplified using 1 primer set. Therefore, we used DBP gene sequences from Plasmodium vivax, which is specific for the SYBR Green based assays. We amplified the DBP gene from the 26 blood samples of malaria patients using SYBR Green based assay and obtained the copy numbers of DBP genes for each sample. Also, we selected optimal reference gene between ACTB and B2M using real time assay to get the stable genes regardless of Malaria titer. Using selected ACTB reference genes, we successfully converted the copy numbers from samples into titer, ${\sharp}$ of parasites per microliter. Using the resultant titer from DBP based SYBER Green assay with ACTB reference gene, we compared the results from our study with the titer from Taqman-based assay. We found that our results showed identical tendency with the results of 18S rRNA Taqman assay, especially in lower titer range. Thus, our DBP gene-utilized real time assay can detect Plasmodium vivax in Korean patient group semi-quantitatively and easily.