• Microbiology and Biotechnology Letters
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• v.19 no.6
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• pp.610-613
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• 1991

Some of the Kinetic properties of crystallic xanthine dehydrogenase form Pseudomonas synxantha A3 were studied. The enzyme activity was strongly inhibited by adenine, 8-azaadenine, 2-methyladenine, guanine, and 8-azaguanine, but not by caffeine, and the inhibitions by adenine and guanine were observed to be of noncompetitive type. The $K_i$ values for adenine and guanine were 0.037 and 0.098 mM, respectively. Michaelis constants were found to be 0.33 and 0.06 rnM for hypoxanthine and xanthine with $NAD^+$ as the second substrate, respectively, and 0.1 rnM for $NAD^+$ with either hypoxanthine or xanthine as the second substrate.

• Journal of Ginseng Research
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• v.47 no.3
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• pp.440-447
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• 2023

Background: The human hair follicle undergoes cyclic phases-anagen, catagen, and telogen-throughout its lifetime. This cyclic transition has been studied as a target for treating hair loss. Recently, correlation between the inhibition of autophagy and acceleration of the catagen phase in human hair follicles was investigated. However, the role of autophagy in human dermal papilla cells (hDPCs), which is involved in the development and growth of hair follicles, is not known. We hypothesized that acceleration of hair catagen phase upon inhibition of autophagy is due to the downregulation of Wnt/β-catenin signaling in hDPCs, and that components of Panax ginseng extract can increase the autophagic flux in hDPCs. Methods: We generated an autophagy-inhibited condition using 3-methyladenine (3-MA), a specific autophagy inhibitor, and investigated the regulation of Wnt/β-catenin signaling using the luciferase reporter assay, qRT-PCR, and western blot analysis. In addition, cells were cotreated with ginsenoside Re and 3-MA and their roles in inhibiting autophagosome formation were investigated. Results: We found that the unstimulated anagen phase dermal papilla region expressed the autophagy marker, LC3. Transcription of Wnt-related genes and nuclear translocation of β-catenin were reduced after treatment of hDPCs with 3-MA. In addition, treatment with the combination of ginsenoside Re and 3-MA changed the Wnt activity and hair cycle by restoring autophagy. Conclusions: Our results suggest that autophagy inhibition in hDPCs accelerates the catagen phase by downregulating Wnt/β-catenin signaling. Furthermore, ginsenoside Re, which increased autophagy in hDPCs, could be useful for reducing hair loss caused by abnormal inhibition of autophagy.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.287-294
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• 2011

Autophagy is a process of intracellular bulk protein degradation, in which the accumulated proteins and cytoplasmic organelles are degraded. It plays important roles in cellular homeostasis, apoptosis, and development, but its role during early embryo development remains contentious. Therefore, in the present study, we investigated the effects of 3-methyladenine (3-MA) on early embryonic development in pigs, we also investigated several indicators of developmental potential, including mitochondrial distribution, genes expressions (autophagy-, apoptosis- related genes), apoptosis and ER-stress, which are affected by 3-MA. After in vitro maturation and fertilization, presumptive pig embryos were cultured in PZM-3 medium supplemented with 3-MA for 2 days at $39^{\circ}C$ 5% $CO_2$ in air. Developmental competence to the blastocyst stage in the presence of 3-MA was gradually decreased according to increasing concentration. Thus, all further experiments were performed using 2 mM 3-MA. Blastocysts that developed in the 3-MA treated group decreased LC3-II intensity and expressions of autophagy related genes than those of the untreated control, resulting in down-regulates the autophagy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 3-MA treated group compared with control ($6.0{\pm}1.0$ vs $3.3{\pm}0.6$, p<0.05). Also, the expression of the pro-apoptotic gene Bax increased in 3-MA treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. Mito Tracker Green FM staining showed that blastocysts derived from the 3-MA treated group had lower mitochondrial integrity than that of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. Then, the expression of the spliced form of pXBP-1 product (pXBP-1s) increased in 3-MA treated group, resulting increase of ER-stress. Taken together, these results indicate that inhibition of autophagy by 3-MA is closely associated with apoptosis and ER-stress during preimplantation periods of porcine embryos.

• Korean Journal of Microbiology
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• v.25 no.1
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• pp.57-66
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• 1987

Mutants of E. coli which showed increased sensitivity to MMS(methylmethane sulfonate)were isolated by MNNG mutagenesis and characterized by enzymatic assay, survival of simple alkylating agents and host-cell reactivation. E.coli mutant, 5-62, which showed absolute deficiency in 3-methyladenine DNA glycosylase II activity and had low capability of reactivating MMS-treated phage charon 35 was very sensitive to MMS and MNNG. NNS gene which confered resistance to the lethal effects of MMS was cloned in 5-62 strain. 5-62 mutants carrying recombinant plasmid, pMRG 1, which acquired resistance to the lethal effects of MMS had normal sensitivity to MNNG. Resistance to MMS was somewhat increased after they were treated with 0.5.$\mu$g MNNG/ml for 2 hours at $37^{\circ}C$. Although recombinant plasmid, pMRG 1, did not complement alk A mutation in 5-62 and ada mutation in 1-27 mutnat, mutnats transformed with this plasmid showed more capability of reactivating MMS treated phage than mutants.

• Korean Journal of Food Science and Technology
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• v.30 no.6
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• pp.1464-1469
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• 1998

SOS Chromotest and Ames test were carried out to evaluate the mutagenicity of three chloropropanols. In the SOS Chromotest, 3-monochloro-l,2-propanediol (3-MCPD) and 2,3-dichloro-1-propanol (2,3-DCP) except for 1,3-dichloro-2-propanol (1,3-DCP) induced SOS response in Escherichia coli PQ37 with dose-response relationship and 2,3-DCP was far more genotoxic than 3-MCPD. The genotoxic activities of both compounds, however, were very lower in E. coli PQ35 (PQ37 $uvrA^+)$ as compared to them in E. coli PQ37, whereas much higher in E. coli PQ243 (PQ37 tagA alkA). These results indicate that there are at least two types of DNA lesions caused by these compounds; one is a excision-repairable and the other is 3-methyladenine or any similar lesion which is excision-unrepairable and can induce adaptive response. In Salmonella typhimurium TA100, all the compounds showed strong mutagenicities, establishing the following genotoxic order: 2,3-DCP>3-MCPD>1,3-DCP. But the mutagenic activities were very low in S. typhimurium TA98 and TA97a. These results suggest that the mutation by chloropropanols can be induced by the DNA lesions causing base-pair substitutions. From the result that the mutagenicities of 3-MCPD and 2,3-DCP in S. typhimurium TA1535 were very low as compared to those in S. typhimurium TA100, it was appeared that the mutations by both compounds necessitate error-prone SOS repair.

• Molecules and Cells
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• v.40 no.11
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• pp.880-887
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• 2017

We hypothesized that inflammation affects number and activity of osteoclasts (OCs) via enhancing autophagy. Lipopolysaccharide (LPS) induced autophagy, osteoclastogenesis, and cytoplasmic reactive oxygen species (ROS) in bone marrow-derived macrophages that were pre-stimulated with receptor activator of nuclear $factor-{\kappa}B$ ligand. An autophagy inhibitor, 3-methyladenine (3-MA) decreased LPS-induced OC formation and bone resorption, indicating that autophagy is responsible for increasing number and activity of OCs upon LPS stimulus. Knockdown of autophagy-related protein 7 attenuated the effect of LPS on OC-specific genes, supporting a role of LPS as an autophagy inducer in OC. Removal of ROS decreased LPS-induced OC formation as well as autophagy. However, 3-MA did not affect LPS-induced ROS levels, suggesting that ROS act upstream of phosphatidylinositol-4,5-bisphosphate 3-kinase in LPS-induced autophagy. Our results suggest the possible use of autophagy inhibitors targeting OCs to reduce inflammatory bone loss.

• Korean Journal of Microbiology
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• v.50 no.1
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• pp.27-32
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• 2014

Autophagy is one of the lysosomal degradation pathways to maintain cellular homeostasis. The damaged proteins or organelles are uptaken through extra- and intra-cellular stress, starvation and infected pathogens, subsequently, autophagosomes are fused with lysosomes to break down the molecules. Salmonella enterica serovar Typhimurium (S. Typhimurium), intracellular bacteria, cause acute gastroenteritis and food poisoning. Given that autophagy induced by S. Typhimurium plays an important role in the cells to control the infection, we identify whether the induction of autophagy with rapamycin, chemical inducer of autophagy, before infection regulates S. Typhimurium infection. After treatment of rapamycin or 3-methyladenine (3-MA), autophagy inhibitor, RAW264.7 cells were infected with S. Typhimurium. Pretretment of rapamycin decreased the growth rate of S. Typhimurium in the cells; otherwise, pretreatment of 3-MA increased the growth rate of S. Typhimurium. The expression of autophagy-related genes was significantly increased in the S. Typhimurium-infected cells pretreated with rapamycin. To examine whether induced autophagy by rapamycin control the infection with increase the production of reactive oxygen species (ROS) and nitric oxide (NO), antibacterial radical substrates were measured in infected cells followed by the treatment with either rapamycin or 3-MA. NO production increased in RAW264.7 cells; otherwise, ROS production remained unchanged during the infection. These findings suggest that inducing autophagy with rapamycin reveals antimicrobial activity as producing NO against S. Typhimurium infection in mouse macrophages.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.3
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• pp.137-144
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• 2021

In this study, to analyze whether artificial regulation of apoptosis in the development of somatic cells can affect the stable growth and development of cells, 20 alpha-hydroxysteroid dehydrogenase (20α-HSD) and rapamycin were treated to induce apoptosis and autophagy in the both skin and muscle cells. Respectively, and 3-methyladenine was supplemented to inhibit cell death. Our results show that stimulation with rapamycin activated autophagy in both types of cells, but increased apoptosis more than autophagy in the case of skin cells. These results indicate that there was a difference in the expression of survival factors and cell development depending on the type of cell. In particular, in the expression of autophagy-related gene (MAP1LC3A) was higher than that of Casp-3, an apoptosis factor. Furthermore, cell development was the highest in all cell groups cultured by artificially inducing autophagy, however the lowest in the apoptosis-inhibited group. Especially, the noteworthy result of this study was that when apoptosis was induced using 20α-HSD, it was possible to induce apoptosis in both skin and muscle cells. Therefore, the main point of this study is that apoptosis induced during cell culture plays a pivotal role in cell remodeling.

• Journal of Life Science
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• v.30 no.1
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• pp.88-95
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• 2020

Using a random arbitrarily primed polymerase chain reaction, messenger RNA expression levels were assessed after exposure to 10% (v/v) toluene for 8 hr in solvent-tolerant Pseudomonas sp. BCNU 106. Among the 100 up-expressed products, 50 complementary DNA fragments were confirmed to express repeatedly; these were cloned and then sequenced. Blast analysis revealed that toluene stimulated an adaptive increase in the gene expression level in association with transcriptions such as LysR family of transcriptional regulators and RNA polymerase factor sigma-32. The expression of catalase and Mn2+/Fe2+ transporter genes functionally associated with inorganic ion transport and metabolism increased, and the increased expression of type IV pilus assembly PilZ and multi-sensor signal transduction histidine kinase genes, functionally categorized into signal transduction and mechanisms, was also demonstrated under toluene stress. The gene expression level of beta-hexosaminidase in association with carbohydrate transport and metabolism increased, and those of DNA polymerase III subunit epsilon, DNA-3-methyladenine glycosylase II, DEAD/DEAH box helicase domain-containing protein, and ABC transporter also increased after exposure to toluene in DNA replication, recombination, and repair, and even in defense mechanism. In particular, the RNAs corresponding to the ABC transporter, Mn2+/Fe2+ transporter, and the β-hexosaminidase gene were confirmed to be markedly induced in the presence of 10% toluene. Thus, defense mechanism, cellular ion homeostasis, and biofilm formation were shown as essential for toluene tolerance in Pseudomonas sp. BCNU 106.

• Herbal Formula Science
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• v.29 no.4
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• pp.155-165
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• 2021

Objectives : Recent studies have suggested that Platycodonis Radix has various pharmacological effects such as anti-cancer, antioxidant, anti-asthma, anti-diabetes, anti-obesity, hepatoprotective, and cardiovascular protection effects. The aim of this study was to investigate the role of water extract of Platycodonis Radix (WPR)-induced autophagy in H460 human lung cancer cells. Methods : H460 cells were treated with WPR and cell viability was calculated by an MTT assay. To evaluate changes in apoptosis- and autophagy-related genes, Western blotting was performed. Two kinds of autophagy inhibitors, 3-Methyladenine (3-MA) and bafilomycin A1, were pretreated to confirm the role of WPR-induced autophagy. Results : WPR reduced the viability of H460 cells in a treatment concentration-dependent manner, which was associated with induction of apoptosis. It was also confirmed that WPR induced autophagy based on the formation of specific intracellular vacuoles and changes in the expression of autophagy-related genes. Interestingly, pretreatment with 3-MA and bafilomycin A1 increased WPR-induced cytotoxicity and apoptosis. Conclusions : WPR induced autophagy at low concentrations and early stages of treatment, but promoted apoptosis at high concentrations and late stages. Moreover, WPR-induced autophagy had a cytoprotective role in H460 cells.