• Journal of Microbiology and Biotechnology
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• v.34 no.4
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• pp.783-794
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• 2024

The antifungal activity of fisetin against Candida albicans is explored, elucidating a mechanism centered on membrane permeabilization and ensuing disruption of pH homeostasis. The Minimum Inhibitory Concentration (MIC) of fisetin, indicative of its interaction with the fungal membrane, increases in the presence of ergosterol. Hoechst 33342 and propidium-iodide staining reveal substantial propidium-iodide accumulation in fisetin-treated C. albicans cells at their MIC, with crystal violet uptake assays confirming fisetin-induced membrane permeabilization. Leakage analysis demonstrates a significant release of DNA and proteins in fisetin-treated cells compared to controls, underscoring the antifungal effect through membrane disruption. Green fluorescence, evident in both the cytoplasm and vacuoles of fisetin-treated cells under BCECF, AM staining, stands in contrast to controls where only acidic vacuoles exhibit staining. Ratiometric pH measurements using BCECF, AM reveal a noteworthy reduction in intracellular pH in fisetin-treated cells, emphasizing its impact on pH homeostasis. DiBAC4(3) uptake assays demonstrate membrane hyperpolarization in fisetintreated cells, suggesting potential disruptions in ion flux and cellular homeostasis. These results provide comprehensive insights into the antifungal mechanisms of fisetin, positioning it as a promising therapeutic agent against Candida infections.

• Molecular & Cellular Toxicology
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• v.1 no.2
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• pp.111-115
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• 2005

Ultraviolet (UV) radiation to mammalian skin is known to alter cellular function via generation of Reactive Oxygen Species (ROS), DNA damage and DNA lesions, such as pyrimidine dimmers and photoproducts, which could lead to DNA mutation if they are not repaired. In this study, we have investigated the reduction of DNA damage and of apoptosis with a particular attention to genetic effect of paeoniflorin in Normal Human Epidermal Keratinocytes (NHEK). After UVB irradiation from $10\;to\;500mJ/cm^{2}$ to NHEK, Mean Tail Moments (MTM) were increased with UVB dose increase. The greatest amount of strand breaks was induced at $500mJ/cm^{2}$ of UVB. Even at the lowest dose of UVB ($10mJ/cm^{2}$), change in MTM was detected (P<0.0001). Pretreated cell with 0.1% paeoniflorin maximally reduced the level of DNA damage to about 21.3%, compared to untreated cell. In the lower concentrations less than 0.01% of paeoniflorin, MTM had a small increase but paeoniflorin still had reductive effects of DNA damage. We measured the apoptosis suppression of paeoniflorin with annexin V flous staining kit. As we observed under the fluorescence microscopy to detect apoptosis in the irradiated cell, the fluorescence intensity was clearly increased in the untreated cell, but decreased in treated cells with paeoniflorin. These results suggest that paeoniflorin reduces the alteration of cell membranes and prevents DNA damage. Therefore, the use of paeoniflorin as a free radical scavenger to reduce the harmful effects of UV lights such as chronic skin damage, wrinkling and skin cancer can be useful to prevent the formation of photooxidants that result in radical damage.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.1
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• pp.17-24
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• 2018

Objective: To investigate sperm chromatin/DNA integrity, global DNA methylation, and DNMT mRNA transcription in men with oligoasthenoteratozoospermia (OAT) compared with normozoospermic men. Methods: Semen samples from 32 OAT patients who comprised the case group and 32 normozoospermic men who comprised the control group were isolated and purified using a standard gradient isolation procedure according to World Health Organization criteria. DNMT1, DNMT3A, and DNMT3B transcripts were then compared between groups using real-time quantitative reverse-transcription polymerase chain reaction. Global DNA methylation in sperm was determined by an enzyme-linked immunosorbent assay. Protamine deficiency and the proportion of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3), aniline blue (AB), and toluidine blue (TB) staining, as well as the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The p-values < 0.05 were considered to indicate statistical significance. Results: Significantly higher proportions of AB+, TB+, CMA3+, and TUNEL+ spermatozoa, as well as DNMT3A and DNMT3B transcription, were found in the OAT group. Positive correlations were detected between sperm parameters, DNA/chromatin damage, and DNMT3A and DNMT3B transcripts. Global DNA methylation was significantly higher in the OAT patients and had a significant correlation with abnormal results of all sperm chromatin integrity tests, but was not associated with DNMT1, DNMT3A, or DNMT3B expression. Conclusion: Oligoasthenoteratozoospermic men showed abnormal sperm parameters, abnormal chromatin/DNA integrity, and a higher global DNA methylation rate, as well as overexpression of DNMT mRNA.

• Korean Journal of Clinical Pharmacy
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• v.9 no.2
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• pp.103-108
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• 1999

The anti-proliferating effects of some plants on hepatoma cell lines were studied by the 3-[4,5-dim-ethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide (MTT assay), to investigate the anticancer effect with some plants around here. As the result, we saw that the anti-prolferating effect to the plants. Among the plants, Equisetum arvense L. and Lactuca dentata Makino. var, flaviflora Makino of them relatively showed a good ant-proliferating effect. Capsicum annuum L. var. angulosum Mill (Leaf) was the best among them. We also examined morphological changes on the hepatoma cells in this process. In case of Capxicum annuum L. var. angulosum Mill, the tells become vague after 2 days, and then destroyed faster than others. We can fee also the condensated chromosome on the treated cells with Capxicum annuum L. var. angulosum Mill. And we also observed condensation through using a fluorescent microscope by PI staining, and observed DNA fragmentation.

• Proceedings of the PSK Conference
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• 2003.10b
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• pp.106.1-106.1
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• 2003

Cadmium (Cd) is an inorganic toxicant of great environmental and occupational concern which was classified as a human carcinogen in 1993. Occupational cadmium exposure is associated with lung cancer in human. In the present study, we established the mechanistic basis of apoptotic cell death induced by Cd in WI38 human lung fibroblast. Cd at 20-80 ${\mu}$M decreased viablility of cells in a concentration-dependent manner. PI staining, TUNEL staining and DNA fragmentation analysis demonstrated the apoptotic cell death by Cd. (omitted)

• Journal of Microbiology
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• v.40 no.4
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• pp.319-326
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• 2002

Cellular DNA in prokaryotes is organized in nucleic acid-protein self-assemblies referred to as the nucleoid. The physical forces responsible for its stability inside the poor solvent properties of the cytoplasm and their functional implications are not understood. Studies on the organisation and functioning of the cytosol of cells largely rely on experimental protocols performed in highly dilute solutions using biochemically purified molecules, which is not a reliable substitute for the situation existing in vivo. Our current research interest is focused on the characterization of biological and physical forces determining the compaction and phase separation of DNA in Escherichia coli cytoplasm. We have emphasized the effect of excluded volume in solutions with high macromolecular concentrations (macromolecular crowding) upon self-association patterns of reactions. The prokaryotic cytosol was simulated by addition of inert polymer polyethylene glycol (PEG) (average molecular weight 20000), as an agent which afterwards facilitates the self-association of macromolecules. Fluorescence microscopy was used for direct visualization of nucleoids in intact cells, after staining with DAPI (4',6-diamidino-2-phenylindole dihydrochloride). Addition of the crowding agent PEG 20,000, in increasing concentrations generated progressively enhanced nucleoid compaction, the effect being stronger in the presence of 0.2 M NaCl and 5 mM MgCl$\_$2/. Under these conditions, the nucleoids were compacted to volumes of around 2 ㎛$\^$3/ or comparable sizes with that of living cells.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.16
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• pp.6627-6632
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• 2014

Purpose: The present study concerns molecular mechanisms involved in induction of apoptosis by a fungal taxol extracted from the fungus Cladosporium oxysporum in T47D human breast cancer cells. Materials and Methods: Apoptosis-induced by the fungal taxol was assessed by MTT assay, nuclear staining, DNA fragmentation, flow cytometry and pro- as well as anti-apoptotic protein expression by Western blotting. Results: Our results showed inhibition of T47D cell proliferation with an $IC_{50}$ value of $2.5{\mu}M/ml$ after 24 h incubation. It was suggested that the extract may exert its anti-proliferative effect on human breast cancer cell line by suppressing growth, arresting through the cell cycle, increase in DNA fragmentation as well as down-regulation of the expression of NF-${\kappa}B$, Bcl-2 and Bcl-XL and up-regulation of pro-apoptotic proteins like Bax, cyt-C and caspase-3. Conclusions: We propose that the fungal taxol contributes to growth inhibition in the human breast cancer cell through apoptosis induction via a mitochondrial mediated pathway, with possible potential as an anticancer therapeutic agent.

• Korean Journal of Medicinal Crop Science
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• v.18 no.3
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• pp.186-190
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• 2010

The chromosome number was identified and fluorescence in situ hybridization(FISH) mapping of 5S and 45S rDNAs were conducted for C. minima and C. lanceolata in the genus Codonopsis from Jeju island. In this study, we have confirmed that the somatic metaphase chromosome number determined as 2n=2x=16 was the same as the findings from the previous studies. While the conventional staining method makes it rather difficult to distinguish satellite chromosomes due to high degree of variability, FISH analysis produced the exact number and location of 5S and 45S rDNAs. Both species in the genus Codonopsis have a pair of 5S rDNA and their gene loci were observed on chromosome 3. Although two pairs of 45S rDNAs (one on chromosome 1 and the other on chromosome 8) were identified in both species, the 45S rDNA signals on chromosome 8 in C. minima were significantly weaker than those on chromosome 1. In addition, the 45S rDNA signals on chromosome 1 in C. lanceolata showed that the chromosome is non-homologus. In this study, we have determined cytogenetic characteristics of C. minima and C. lanceolata according to their gene replication patterns.

• Journal of Photoscience
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• v.9 no.2
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• pp.479-481
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• 2002

UV-induced DNA damage causes cell killing and mutations leading to carcinogenesis. In normal human cells, UV damage such as cyclobutane pyrimidine dimers (CPDs) and primidine-prymidone (6-4) photoproducts are mainly repaired by nucleotide excision repair mechanism. The molecular processes have been well characterized recently. To know the influence of mitochondrial genome on the nucleotide excision repair mechanism against CPDs, we comparatively examined the production of CPDs by UVC irradiation and their repair kinetics in human cells completely lacking mitochondrial DNA (mtDNA) and the parental HeLa S cells. Whole DNA extracted from the cells exposed to UVC was treated with T4-endonuclease V to break the phosphodiester bond adjacent to CPDs. The DNA was electrophoresed in a denaturing agarose gel, which was visualized by ethidium bromide staining. The relative amount of CPDs was determined by image analysis using NIH Image software. MtDNA- less (rho-O) cells were apparently more sensitive to UVC than HeLa S cells, while the level of induction of CPDs in rho-O and HeLa cells was comparable. The repair of CPDs was less efficient in rho-O cells compared with HeLa cells. The residual amount of CPDs after 24-h repair was larger in rho-O cells than in HeLa cells where more than 90 % of CPDs were repaired by then. The non-repaired CPDs would lead to apoptosis in rho-O cells. These results suggest that mitochondrial genome may contribute to some ATP-dependent steps in nucletide excision repair by supplying sufficient ATP which is generated through a respiratory chain in mitochondria.

• Journal of Korean Neurosurgical Society
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• v.30 no.7
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• pp.861-869
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• 2001

Objective : In this study, we investigated the relationship between the histologic grading of meningiomas and proliferative potentials determined by the Ki-67, proliferating cell nuclear antigen(PCNA) and flow cytometry (FCM) with the aim of determining whether these potentials can be used as a parameter to the proliferative activity, in particular of atypical and malignant meningiomas. Methods : This study consisted of 47 meningiomas(6 malignant, 14 atypical, and random sampled 27 benign meningiomas). By immunohistochemical staining of Ki-67 and PCNA on formalin-fixed, paraffin-embedded sections, the anti-human rabbit polyclonal antibody against Ki-67 antigen and anti-PCNA monoclonal antibody(PC10) scores were counted. FCM was also performed on paraffin-embedded tissue using a selective staining technique for DNA. DNA ploidy, S-phase fraction, and proliferative index(PI)) were determined. Results : The results are summarized as follows ; 1) Proliferation rates as assessed by Ki-67 and PCNA closely correlated with the degree of anaplastic histologic features. 2) Proliferative potentials determined by FCM(S-phase fraction and PI) were not able to distinguish between benign and atypical/malignant meningiomas. 3) DNA ploidy was not a useful indicator of histologic grade in these tumors. 4) Proliferative potentials such as Ki-67 staining index(SI) and PCNA SI did not correlate with the ploidy pattern. 5) There was a linear correlation between Ki-67 SI and PCNA SI, but we could not find a correlation between Ki-67 SI and S-phase fraction or PI. Our results also did not show a statistically signficant correlation between PCNA SI and S-phse fraction or PI. Conclusions : We conclude that evaluation of the proliferative potentials with Ki-67 and PCNA is important as an additional factor for the prediction of malignancy in meningiomas. A dual study of Ki-67 and PCNA SIs on the same tissue might improve the accuracy with which the proliferative potential of a tumor can be predicted. We demonstrated that FCM in meningiomas is not valuable in predicting the behavior of these neoplasms, but we did observe a trend toward more malignancy with higher percent S-phase fraction and higher PI. Analysis of the S-phase fraction and PI might therefore be a useful tool to discriminate among histologic grades of meningiomas.