• International Journal of Stem Cells
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• v.16 no.2
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• pp.234-243
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• 2023

The recent advances in human pluripotent stem cells (hPSCs) enable to precisely edit the desired bases in hPSCs to be used for the establishment of isogenic disease models and autologous ex vivo cell therapy. The knock-in approach based on the homologous directed repair with Cas9 endonuclease, causing DNA double-strand breaks (DSBs), produces not only insertion and deletion (indel) mutations but also deleterious large deletions. On the contrary, due to the lack of Cas9 endonuclease activity, base editors (BEs) such as adenine base editor (ABE) and cytosine base editor (CBE) allow precise base substitution by conjugated deaminase activity, free from DSB formation. Despite the limitation of BEs in transition substitution, precise base editing by BEs with no massive off-targets is suggested to be a prospective alternative in hPSCs for clinical applications. Considering the unique cellular characteristics of hPSCs, a few points should be considered. Herein, we describe an updated and optimized protocol for base editing in hPSCs. We also describe an improved methodology for CBE-based C to T substitutions, which are generally lower than A to G substitutions in hPSCs.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.6
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• pp.817-824
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• 1997

Extracellular ATP elicits various biological responses and plays a significant role in physiological regulation. Recently, ATP-induced growth inhibitions were reported in some tumor cell lines, but these effects and mechanisms are not well hewn. This study was conducted to investigate ATP-induced growth inhibition in mouse $leukemic(P388D_1)$ cells. ATP inhibited cell growth in a dose-dependent manner as analyzed by MTS assay$(IC_{50}: 33.1\;{\mu}M)$. Nucleotides other than ATP, such as ADP$(37.5;{\mu}M)$ and AMP$(33.2;{\mu}M)$ had the same effects as ATP but adenosine$(57.8;{\mu}M)$ showed less effect than ATP. ATP attenuated the cells in $G_0/G_l\;and\;G_2/M$ phases but increased those in S phase in flow cytometric analysis. Hypodiploid cells$(A_0)$, the presumptive findings of apoptosis, were found among the ATP-treated cells. ATP induced DNA fragmentation into $180{\mu}200\;bps $as measured by electrophoresis. some apoptotic cells were stained by TUNEL method. ATP increased the intracellular free $Ca^{++}$concentration$([Ca^{++}]_i)$ and the increment of $([Ca^{++}]_i)$ was caused by influx from the extracellular space. These results suggest that extracellular ATP induces growth inhibition through apoptosis.

• Biomolecules & Therapeutics
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• v.26 no.4
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• pp.409-416
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• 2018

Vasicinone, a quinazoline alkaloid from Adhatoda vasica Nees. is well known for its bronchodilator activity. However its anti-proliferative activities is yet to be elucidated. Here-in we investigated the anti-proliferative effect of vasicinone and its underlying mechanism against A549 lung carcinoma cells. The A549 cells upon treatment with various doses of vasicinone (10, 30, 50, $70{\mu}M$) for 72 h showed significant decrease in cell viability. Vasicinone treatment also showed DNA fragmentation, LDH leakage, and disruption of mitochondrial potential, and lower wound healing ability in A549 cells. The Annexin V/PI staining showed disrupted plasma membrane integrity and permeability of PI in treated cells. Moreover vasicinone treatment also lead to down regulation of Bcl-2, Fas death receptor and up regulation of PARP, BAD and cytochrome c, suggesting the anti-proliferative nature of vasicinone which mediated apoptosis through both Fas death receptors as well as Bcl-2 regulated signaling. Furthermore, our preliminary studies with vasicinone treatment also showed to lower the ROS levels in A549 cells and have potential free radical scavenging (DPPH, Hydroxyl) activity and ferric reducing power in cell free systems. Thus combining all, vasicinone may be used to develop a new therapeutic agent against oxidative stress induced lung cancer.

• The Korean Journal of Mycology
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• v.46 no.3
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• pp.315-323
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• 2018

The goal of this study was to isolate wild yeasts from the fields of medicinal plants and investigate its tyrosinase inhibitory activities. Wild yeasts isolated from soil in the ginseng and Korean angelica fields of Geumsan, Chungcheongnam-do, Korea were identified by comparing the nucleotide sequences of the D1/D2 domain of 26S rDNA. In total, 43 yeast strains belonging to 21 species were isolated from 50 soil samples obtained from two medicinal plant fields. From the ginseng field, six strains of Rhodotorula glutinis and four strains of Sampaiozyma ingeniosa were isolated, out of which Rhodotorula glutinis strains were dominant. In the Korean angelica field, six strains of Cyberlindnera saturnus, three strains of Piskurozyma taiwanensis, and three strains of Saitozyma podzolica were isolated, out of which Cyberlindnera saturnus strains were dominant. We prepared cell-free extracts of the isolated wild yeasts and their tyrosinase inhibitory activities were investigated. Among 43 yeast strains, cell-free extracts of Naganishia globosa G1-7 showed the highest tyrosinase inhibitory activity (28.0%).

• Molecules and Cells
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• v.24 no.1
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• pp.113-118
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• 2007

The brains of Alzheimer's disease (AD) patients are characterized by large deposits of amyloid beta peptide ($A{\beta}$). $A{\beta}$ is known to increase free radical production in nerve cells, leading to cell death that is characterized by lipid peroxidation, free radical formation, protein oxidation, and DNA/RNA oxidation. In this study, we selected an extract of Gardenia jasminoides by screening, and investigated its ameliorating effects on $A{\beta}$-induced oxidative stress using PC12 cells. The effects of the extract were evaluated using the 2',7'-dichlorofluorescein diacetate (DCF-DA) assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. To find the active component, the ethanol extract was partitioned with hexane, chloroform, and ethyl acetate, respectively, and the active component was purified by silica-gel column chromatography and HPLC. The results suggested that Gardenia jasminoides extract can reduce the cytotoxicity of $A{\beta}$ in PC 12 cells, possibly by reducing oxidative stress.

• BMB Reports
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• v.34 no.3
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• pp.250-258
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• 2001

For a long time Rhus verniciflua Stokes (RVS) has traditionally been used as a herbal plant. It is known to contain various biological activities. Previously, a crude ethanol extract from RVS was reported to have antioxidant effects, and antiproliferative activities, on human cancer cell lines. In this report, we prepared a highly purified ethanol extract from RVS, which did not contain the urushiol derivatives, named REEE-1 ($\underline{R}$hus $\underline{e}$thanol $\underline{e}$luted $\underline{e}$xtract-1), to investigate the mechanisms of the scavenging activity of hydroxyl radicals using mouse thymocytes. The results from the deoxyribose, DNA nicking, and glucose/glucose oxidase enzyme assays showed that REEE-1 contained a strong scavenging activity of oxygen free radicals, especially of hydroxyl radicals. However, interestingly, REEE-1 also showed cytotoxicity against the thymocytes, although the effect was variable, depending on the concentrations and times of treatment. The REEE-1-mediated cytotoxicity against thymocytes, which has been used as one of the well-characterized models for apoptosis studies, was verified to be apoptotic. This was proven by the following: the appearance of DNA laddering, increases in DNA fragmentation, low fluorescence intensity in the nuclei after propidium iodide staining, and positive Annexin V staining of the cells. These results suggested that REEE-1 had both antioxidative activity and cytotoxicity against the thymocytes, although the effect of the cytotoxicity was variable, depending on the dose and time of the treatment.

• Journal of Physiology & Pathology in Korean Medicine
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• v.16 no.6
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• pp.1117-1121
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• 2002

To test the protective effect of herbal medicine on myocardial damage against oxygen free radical-induced myocardiotoxicity, cytotoxicity was examined using MTT, Beating rate and DNA synthesis assay in the presence of water extract of three kinds of Radix Rehmanniae. Myocardial toxicity was evaluated in neonatal rat myocardiocytes in cultures. The results of these experiments were obtained as follows : Xanthine oxydase/hypoxanthine resulted in a decrease in viability, beating rate and DNA synthesis in cultured myocardial cells. Radix Rehmanniae Recens(生地黃, RRR) water extract shows effects of protection from the cardiocyte toxicity induced by xanthine oxydase/hypoxanthine treatment such as increases in beating rate. Radix Rehmanniae Preparat(熟地黃, RRP) water extract shows effects of protection from the cardiocyte toxicity induced by xanthine oxydase/hypoxanthine treatment such as increases in DNA synthesis. These results show that xanthine oxydase/hypoxanthine elicits toxic effects in cultured myocardial cells derived from neonatal rat, and suggest that water extract of three kinds of Radix Rehmanniae is very effective in the prevention of xanthine oxydase/hypoxanthine-induced cardiotoxicity.

• IMMUNE NETWORK
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• v.1 no.2
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• pp.104-108
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• 2001

Aging is a senescence and defined as a normal physiologic and structural alterations in almost all organ systems with age. As Leonard Hayflick, one of the first gerontologists to propose a theory of biologic aging, indicated that a theory of aging or longevity satisfies the changes of above conditions to be universal, progressive, intrinsic and deleterious. Although a number of theories have been proposed, it is now clear that cell aging (cell senescence) is multifactorial. No single mechanism can account for the many varied manifestations of biological aging. Many theories have been proposed in attempt to understand and explain the process of aging. Aging is effected in individual by genetic factors, diet, social conditions, and the occurrence of age-related diseases as diabetes, hypertension, and arthritis. It involves an endogenous molecular program of cellular senescence as well as continuous exposure throughout life to adverse exogenous influences, leading to progressive infringement on the cell's survivability so called wear and tear. So we could say the basic mechanism of aging depends on the irreversible and universal processes at cellular and molecular level. The immediate cause of these changes is probably an interference in the function of cell's macromolecules-DNA, RNA, and cell proteins-and in the flow of information between these macromolecules. The crucial questions, unanswered at present, concerns what causes these changes in truth. Common theories of aging are able to classify as followings for the easy comprehension. 1. Biological, 1) molecular theories - a. error theory, b. programmed aging theory, c. somatic mutation theory, d. transcription theory, e. run-out-of program theory, 2) cellular theories - a. wear and tear theory, b. cross-link theory, c. clinker theory, d. free radical theory, e. waste product theory, 3) system level theory-a. immunologic/autoimmune theory, 4) others - a. telomere theory, b. rate of living theory, c. stress theory, etc. Prevention of aging is theoretically depending on the cause or theory of aging. However no single theory is available and no definite method of delaying the aging process is possible by this moment. The most popular action is anti-oxidant therapy using vitamin E and C, melatonin and DHEA, etc. Another proposal for the reverse of life-span is TCP-17 and IL-16 administration from the mouse bone marrow B cell line study for the immunoglobulin VDJ rearrangement with RAG-1 and RAG-2. Recently conclusional suggestion for the extending of maximum life-span thought to be the calory restriction.

• Journal of the Korean Chemical Society
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• v.59 no.1
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• pp.22-30
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• 2015

The effect of diphtheria toxin on cell membrane lipids was studied by examining the phospholipase D (PLD) activity and free fatty acids (FFA) release in HepG2 cells. The diphtheria toxin effects on lipid alteration show apparently maximal at pH 5.1, stimulating PLD activity nearly 3.5 fold and enhancing FFA release approximately 5 fold over the control. These results indicate that the membrane is perturbed and its lipid component is rearranged during the diphtheria toxin translocation. Digitonin, a random membrane perturbing detergent, exhibit about four-fold higher perturbation effect over the diphtheria toxin at neutral pH. This observation suggests that the membrane perturbation induced by diphtheria toxin appears to be rather selective. To investigate the cause of the membrane perturbation, Cibacron blue, an inhibitor of membrane pore formation, and hemagglutinin, an influenza virus with fusion peptide, were tested for their effects on diphtheria toxin action. Cibacron blue decreased the diphtheria toxin effect by almost 50%, but the lipid alteration induced by hemagglutinin was similar to the diphtheria toxin effect. These observations imply that the membrane perturbation induced by diphtheria toxin may be caused by a combination of pore formation and insertion of hydrophobic peptide of toxin to the membrane as well. Additionally, we found that the diphtheria toxin increased the HepG2 cells permeability but the cells viability was maintained at high level at the same time. DNA fragmentation which is related to apoptosis was not induced by the toxin. Under these conditions, we could demonstrate that the lipid alteration of HepG2 cells was brought about by diphtheria toxin at acidic pH.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.20
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• pp.8693-8698
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• 2014

Background: Up-regulation of hsp90 gene expression occurs in numerous cancers such as lung cancer. D,L-lactic-co-glycolic acid-poly ethylene glycol-17-dimethylaminoethylamino-17-demethoxy geldanamycin (PLGA-PEG-17DMAG) complexes and free 17-DMAG may inhibit the expression. The purpose of this study was to examine whether nanocapsulating 17DMAG improves the anti cancer effect over free 17DMAG in the A549 lung cancer cell line. Materials and Methods: Cells were grown in RPMI 1640 supplemented with 10% FBS. Capsulation of 17DMAG is conducted through double emulsion, then the amount of loaded drug was calculated. Other properties of this copolymer were characterized by Fourier transform infrared spectroscopy and H nuclear magnetic resonance spectroscopy. Assessment of drug cytotoxicity on the grown of lung cancer cell line was carried out through MTT assay. After treatment, RNA was extracted and cDNA was synthesized. In order to assess the amount of hsp90 gene expression, real-time PCR was performed. Results: In regard to the amount of the drug load, IC50 was significant decreased in nanocapsulated(NC) 17DMAG in comparison with free 17DMAG. This was confirmed through decrease of HSP90 gene expression by real-time PCR. Conclusions: The results demonstrated that PLGA-PEG-17DMAG complexes can be more effective than free 17DMAG in down-regulating of hsp90 expression by enhancing uptake by cells. Therefore, PLGA-PEG could be a superior carrier for this kind of hydrophobic agent.