• Microbiology and Biotechnology Letters
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• v.48 no.4
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• pp.429-438
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• 2020

The emergence of multi-drug resistant, pathogenic methicillin-resistant Staphylococcus aureus (MRSA) is a threat to global health and has created a need for novel functional therapeutic agents. In this study, we evaluated the underlying mechanisms of the anti-MRSA effect of an azaphilone pigment, sclerotiorin (SCL) from Penicillium sclerotiorum. The antimicrobial activity of SCL was evaluated using agar disc diffusion, broth microdilution, time-kill assays and biophysical studies. SCL exhibits selective activity against Gram positive bacteria including MRSA (range, MIC = 128-1028 ㎍/ml) and exhibited rapid bactericidal action against MRSA with a > 4 log reduction in colony forming units within three hours of administration. Biophysical studies, using fluorescent probes and laser or electron microscopy, demonstrated a SCL dose-dependent alternation in membrane potential (62.6 ± 5.0.4% inhibition) and integrity (> 95 ± 2.3%), and the release of UV260 absorbing materials within 60 min (up to 3.2 fold increase, p < 0.01) of exposure. Further, SCL localized to the cytoplasm and hydrolyzed plasmid DNA. While in vitro checkerboard studies revealed that SCL potentiated the antimicrobial activity of topical antimicrobials such as polymixin, neomycin, and bacitracin (Fractional Inhibitory Concentration Index range, 0.26-0.37). Taken together these results suggest that SCL targets the membrane and DNA of MRSA to facilitate its anti-MRSA antimicrobial effect.

• Parasites, Hosts and Diseases
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• v.56 no.5
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• pp.419-427
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• 2018

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, $FAM^{TM}$, $HEX^{TM}$, $Cy5^{TM}$, and CAL Fluor $Red^{(R)}$ 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was $2{\times}10$ copies for C. parvum and for C. cayetanensis, while it was $2{\times}10^3$ copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.

• Korean Journal of Microbiology
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• v.33 no.4
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• pp.257-261
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• 1997

To scrutinize the bacterial community composition of Lake Soyang in winter, bacterial numbers belonging to Eubacteria, Proteobacteria and Cytophaga-Flavobacterium group were estimated by using 16S and 23S rRNA targeted oligonucleotide probes. Total bacterial numbers ranged from $0.7{\times}10^6$ to $1.1{\times}10^6cells{\cdot}ml^{-1}$, and vertical profile of total bacteria showed a peak at 5 m depth. The ratio of eubacteria to total bacteria were 34~90% and at 5 m and 10 m depths those were low exhibiting, 39 and 34%, respectively. The percentage of proteobacteria ${\alpha}$-group ranged 10.8~28.7%, ${\beta}$-group 4.5~53.5%, ${\gamma}$-group 4.9~35.5% and Cytophaga-Flavobacterium group 6.1~21.1%. The dominant groups were ${\beta}$-group at 0, 2 and 5 m, ${\gamma}$-group at 10 m, ${\alpha}$-group at 30 m and Cytophaga-Flavobacterium group at 50 m depth. In winter season, Lake Soyang can be divided into three layer, 0~2 m, 5~10 m and 30~50 m, by the bacteria community composition. By this method, new informations about aquatic ecosystem were developed.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.192-197
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• 2002

Fluorescent in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to compare the community structures of free-living and aggregated bacteria at thawing period in Lake Baikal. Targeted groups were Eubacteria, $\alpha$-, $\beta$-, $\gamma$- proteobacteria groups, Cytophaga-Flavobacterium group and Planctomycetales. Total bacterial numbers of free-living bacteria were ranged from $0.2{\times}10^6\cells{\cdot}ml^-1$ to $3.2{\times}10^6\cells{\cdot}ml^-1$, which were decreasing with depth, while the aggregated bacterial numbers were dramatically increasing from $0.4{\times}10^4 to 3.3{\times}10^4 \cells{\cdot}ml^-1$ with depth. The ratios of EUB probe binding cells to DAPI counts were ranged from 52.3 to 74.1% in free-living bacteria, and from 39.6 to 66.7% in the aggregated bacteria, respectively. Community structures of the aggregated bacteria were very different from each free-living bacteria at every depth. At 25 m depth, where the chlorophyll a concentration was highest, both structures were quite different from those of surface layers, rendering the fact that the community structures might be affected by phytoplankton. The vertical profile of community structure of aggregated bacteria is particular. The proportion of $\beta$-proteobacteria group was increasing with depth and it was 51.8% at 100 m, but the dominant group was $\gamma$-pro-teobacteria group at 250 m. Taken together, the biodiversity and succession of aggregated bacteria are quite different from free-living bacteria.

• Bulletin of the Korean Chemical Society
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• v.27 no.5
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• pp.613-630
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• 2006

Nucleic acids are virtually omnipresent; they exist in every living being. These macromolecules constitute the most important genetic storage material: the genes. Genes are conserved throughout the evolution of all living beings; they are transmitted from the parents to their offspring. Many interdisciplinary research groups are interested in modifying nucleic acids for use in a wider variety of applications. These modified oligonucleotides are used in many diverse fields, including diagnostics, detection, and therapeutics. In this account, we summarize our research efforts related to modified nucleic acid systems. First, we discuss our syntheses of modified oligonucleotides containing fluorescent tags for use as molecular probes (molecular beacons) to detect single-nucleotide polymorphisim (SNP) in nucleic acids and to distinguish between the B and Z forms of DNA. We also describe our research efforts into oligonucleotides functionalized with steroid derivatives to enhance their cell permeability, and the synthesis of several calix[4]arene-oligonucleotide conjugates possessing the ability to form defined triplexes. In addition, we have performed systematic studies to have an understanding about the functional groups necessary for a given nucleoside to behave as an organo or hydrogelator. The aggregation properties of a number of nucleoside-based phospholipids have been examined in different solvents; some of these derivatives are potential candidates for use as nucleoside-based liposomes. Finally, we also describe our research efforts toward the preparation of isoxazole- and isoxazoline-containing nucleoside derivatives and the determination of their antiviral activities.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.6
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• pp.1475-1481
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• 2008

This study was to investigate the inhibitory effects of Mori Fructus on the generation of peroxynitrite ($ONOO^-$), nitric oxide (NO) and superoxide anion radical (${\cdot}O_2{^-}$) in the endothelial cells of rat vessels. The aim of this study was to investigate the $ONOO^-$, NO, ${\cdot}O_2{^-}$ scavenging and anti-inflammatory activitives of Mori Fructus. For this study, the fluorescent probes, namely dihydrorhodamine 123 (DHR 123), 4,5-diaminofluorescein (DAF-2) and 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) were used. Western blotting was performed using anti-NF-${\kappa}B$ (p50, p65), anti-COX-2, anti-iNOS antibodies, respectively. Mori Fructus prevented lipopolysaccharide (LPS)-induced cell death in YPEN cells. Mori Fructus inhibited the generation of $ONOO^-$, NO and ${\cdot}O_2{^-}$ in the LPS-treated cells. Mori Fructus inhibited the expression of COX-2 and iNOS genes by means of decreasing the NF-${\kappa}B$ activation. These results suggest Mori Fructus is effective on inhibiting the generation of $ONOO^-$, NO and ${\cdot}O_2{^-}$, and that therefore it might have a potential role as a treatment for the inflammatory process and inflammation-related diseases.

• The Journal of Korean Medicine
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• v.29 no.1
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• pp.106-116
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• 2008

Objectives : Peroxynitrite $(ONOO^-)$, superoxide anion radical $({\cdot}O_2^-)$ and nitric oxide (NO) are cytotoxic because they can oxidize several cellular components such as proteins, lipids and DNA. They have been implicated in the aging processes, and age-related diseases such as Alzheimer's disease, rheumatoid arthritis, cancer and atherosclerosis. The aim of this study was to investigate the $ONOO^-$, NO, and $({\cdot}O_2^-)$ scavenging and anti-inflammatory activities of Mori Fructus in ob/ob mice. Methods : Mice were grouped and treated for 5 weeks as follows. Both the normal lean (C57/BL6J black mice) and control obese (ob/ob mice) groups received the standard chow. The experimental groups were fed a diet of chow supplemented with 7.5, 15 and 30 mg Mori Fructus per 1 kg of body weight for 14 days. For this study, the fluorescent probes, namely 2',7'-dichlorodihydrofluorescein diacetate (DCFDA), 4,5-diaminofluorescein (DAF-2) and dihydrorhodamine 123 (DHR 123) were used. Western blotting was performed using anti-phospho $I{\kappa}B-\alpha$, anti-IKK-$\alpha$, anti-NF-${\kappa}B$ (p50, p65), anti-COX-2 and anti-iNOS respectively. Results : Mori Fructus inhibited the generation of $ONOO^-$, NO and $({\cdot}O_2^-)$ in the lipopolysaccharide (LPS)-treated mouse kidney postmitochondria in vitro. The generation of $ONOO^-$, NO and $({\cdot}O2^-)$ were inhibited in the Mori Fructus-administered ob/ob mice groups. The GSH/GSSG ratio decreased in the ob/ob mice, whereas they improved in the Mori Fructus-administered groups. Mori Fructus inhibited the expression of phospho $I{\kappa}B-\alpha$, IKK-$\alpha$, COX-2, iNOS genes, and thereby the activation of NF-$I{\kappa}B$. Conclusions : These results suggest that Mori Fructus is an effective $ONOO^-$, $({\cdot}O_2^-)$ and NO scavenger, and therefore it might be a potential therapeutic drug against the inflammation process and inflammation-related diseases.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.645-650
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• 2019

Despite differences in virulence between strains of Toxoplasma gondii, rapid and accurate genotyping methods are lacking. In this study, a method was developed to detect and genotype T. gondii in food and environmental samples using PCR and a novel peptide nucleic acid (PNA) melting array. An alignment of genome sequences for T. gondii type I, II, and III obtained from NCBI was generated, and a single nucleotide polymorphism analysis was performed to identify targets for PCR amplification and a PNA melting array. Prior to the PNA melting array, conventional PCR was used to amplify GRA6 of T. gondii. After amplification, the PNA melting array was performed using two different PNA hybridization probes with fluorescent labels (FAM and HEX) and quenchers. Melting curves for each probe were used to determine genotypes and identify mutations. A 214-bp region of the GRA6 gene of T. gondii was successfully amplified by PCR. For all T. gondii strains (type I, II, and III) used to evaluate specificity, the correct genotypes were determined by the PNA melting array. Non-T. gondii strains, including 14 foodborne pathogens and 3 protozoan parasites, such as Giardia lamblia, Cryptosporidium parvum, and Entamoeba histolytica, showed no signal, suggesting that the assay has a high specificity. Although this is only a proof-of-concept study, the assay is promising for the fast and reliable genotyping of T. gondii from food and environmental samples.

• Journal of Pharmaceutical Investigation
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• v.21 no.3
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• pp.133-141
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• 1991

The fluorescence characteristics of l-anilinonaphthalene-8-sulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) made the dyes useful probes for the determination of the polarity at the binding sites of several water-soluble polyparacyclophanes. Polyparacyclophanes used were 1,6,20,25-tetraaza[ 6.1.6.1]paracyclophane (CPM 44), 1,7,21,27 -tetraaza[7.1.7.1]paracyclophane (CPM 55). 1,7,21,27 -tetraaza-14,34-dioxa[7.1.7.1]paracyclophane (CPE 55) and 1,8,22,29-tetraaza-15,36-dioxa[8.1.8.1] paracyclophane (CPE 66). The fluorescence quantum yield, emission maximum, and half bandwidth of ANS or TNS obtained in a variety of solvent systems were plotted as a function of four kinds of empirical solvent polarity scales such as dielectric constant (D), (D-l)/(2D+1). Y and Z values. It was found that the Z-value-emission maximum $(\overline}V_F,\;cm^{-1})$ profile showed the most reliable linearity. ANS and TNS interacted with CPM 44, CPM 55, CPE 55. CPE 66. ${\alpha}-cyclodextrin$ (CyD) and ${\beta}-CyD$ in the aqueous solution, and from the emission maxima the polarities (Z-value) of their binding sites were calculated to be 92.65, 87.50, 93.35, 84.52, 94.36, and 90.48 for ANS, respectively. and 91.07, 89.68, 85.44, 86.74 and 87.6 for TNS except for ${\alpha}-CyD$, respectively.

• Biomaterials and Biomechanics in Bioengineering
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• v.3 no.3
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• pp.129-140
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• 2016

This article reports the development of biodegradable photoluminescent polymer (BPLP)-based nanoparticles (NPs) incorporating either magnetic nanoparticles (BPLP-MNPs) or gadopentate dimeglumine (BPLP-Gd NPs), for cancer diagnosis and treatment. The aim of the study is to compare these nanoparticles in terms of their surface properties, fluorescence intensities, MR imaging capabilities, and in vitro characteristics to choose the most promising dual-imaging nanoprobe. Results indicate that BPLP-MNPs and BPLP-Gd NPs had a size of $195{\pm}43nm$ and $161{\pm}55nm$, respectively and showed good stability in DI water and 10% serum for 5 days. BPLP-Gd NPs showed similar fluorescence as the original BPLP materials under UV light, whereas BPLP-MNPs showed comparatively less fluorescence. VSM and MRI confirmed that the NPs retained their magnetic properties following encapsulation within BPLP. Further, in vitro studies using HPV-7 immortalized prostate epithelial cells and human dermal fibroblasts (HDFs) showed > 70% cell viability up to $100{\mu}g/ml$ NP concentration. Dose-dependent uptake of both types of NPs by PC3 and LNCaP prostate cancer cells was also observed. Thus, our results indicate that BPLP-Gd NPs would be more appropriate for use as a dual-imaging probe as the contrast agent does not mask the fluorescence of the polymer. Future studies would involve in vivo imaging following administration of BPLP-Gd NPs for biomedical applications including cancer detection.