• Clinical and Experimental Reproductive Medicine
• /
• 제43권1호
• /
• pp.9-14
• /
• 2016

Objective: Autophagy contributes to the clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified mouse oocytes show acute increases in autophagy during warming. Herein, we investigate the potential role of Atg7 in oocyte vitrification by using an oocyte-specific deletion model of the Atg7 gene, a crucial upstream gene in the autophagic pathway. Methods: Oocyte-specific Atg7 deficient mice were generated by crossing Atg7 floxed mice and Zp3-Cre transgenic mice. The oocytes were vitrified-warmed and then subjected to in vitro fertilization and development. The rates of survival, fertilization, and development were assessed in the Atg7 deficient oocytes in comparison with the wildtype oocytes. Light chain 3 (LC3) immunofluorescence staining was performed to determine whether this method effectively evaluates the autophagy status of oocytes. Results: The survival rate of vitrified-warmed $Atg7^{f/f}$;Zp3-Cre ($Atg7^{d/d}$) metaphase II (MII) oocytes was not significantly different from that of the wildtype ($Atg7^{f/f}$) oocytes. Fertilization and development in the $Atg7^{d/d}$ oocytes were significantly lower than the $Atg7^{f/f}$ oocytes, comparable to the $Atg5^{d/d}$ oocytes previously described. Notably, the developmental rate improved slightly in vitrified-warmed $Atg7^{d/d}$ MII oocytes when compared to fresh $Atg7^{d/d}$ oocytes. LC3 immunofluorescence staining showed that this method can be reliably used to assess autophagic activation in oocytes. Conclusion: We confirmed that the LC3-positive signal is nearly absent in $Atg7^{d/d}$ oocytes. While autophagy is induced during the warming process after vitrification of MII oocytes, the Atg7 gene is not essential for survival of vitrified-warmed oocytes. Thus, induction of autophagy during warming of vitrified MII oocytes seems to be a natural response to manage cold or other cellular stresses.

• Journal of Microbiology and Biotechnology
• /
• 제20권12호
• /
• pp.1647-1652
• /
• 2010

Autophagy refers to the bulk degradation of cellular proteins and organelles through an autophagosome and plays a pivotal role in the development, cellular differentiation, aging, and elimination of aberrant structures. A failure of autophagy has been implicated in a growing list of mammalian disease states, including cancer and cardiomyopathy. Two ubiquitin-like systems are highly involved in autophagy, especially in the formation of autophagosomes. Here, we purified and characterized Atg7 (an E1-like enzyme), and Atg3 and Atg10 (E2-like enzymes) in order to gain an insight into the role played by ubiquitin-like systems in the formation of autophagosomes. Interestingly, we observed that Atg7 forms a homodimer to construct an active conformation, unlike other E1-like enzymes. Although Atg3 was detected as a monomer under physiological conditions, Atg10 existed in an oligomeric form, indicating that the mechanism by which Atg10 functions may differ from that of Atg3.

• Molecules and Cells
• /
• 제41권1호
• /
• pp.50-54
• /
• 2018

Atg5 and Atg7 have long been considered as essential molecules for autophagy. However, we found that cells lacking these molecules still form autophagic vacuoles and perform autophagic protein degradation when subjected to certain stressors. During this unconventional autophagy pathway, autophagosomes appeared to be generated in a Rab9-dependent manner by the fusion of vesicles derived from the trans-Golgi and late endosomes. Therefore, mammalian autophagy can occur via at least two different pathways; the Atg5/Atg7-dependent conventional pathway and an Atg5/Atg7-independent alternative pathway.

• Molecules and Cells
• /
• 제37권5호
• /
• pp.399-405
• /
• 2014

Autophagy targets cytoplasmic cargo to a lytic compartment for degradation. Autophagy-related (Atg) proteins, including the transmembrane protein Atg9, are involved in different steps of autophagy in yeast and mammalian cells. Functional classification of core Atg proteins in plants has not been clearly confirmed, partly because of the limited availability of reliable assays for monitoring autophagic flux. By using proUBQ10-GFP-ATG8a as an autophagic marker, we showed that autophagic flux is reduced but not completely compromised in Arabidopsis thaliana atg9 mutants. In contrast, we confirmed full inhibition of auto-phagic flux in atg7 and that the difference in autophagy was consistent with the differences in mutant phenotypes such as hypersensitivity to nutrient stress and selective autophagy. Autophagic flux is also reduced by an inhibitor of phosphatidylinositol kinase. Our data indicated that atg9 is phenotypically distinct from atg7 and atg2 in Arabidopsis, and we proposed that ATG9 and phosphatidylinositol kinase activity contribute to efficient autophagy in Arabidopsis.

• Journal of the Korea Academia-Industrial cooperation Society
• /
• 제12권8호
• /
• pp.3541-3546
• /
• 2011

Autophagy is an evolutionarily conserved lysosomal pathway for degrading cytoplasmic proteins, macromolecules, and organelles in addition to recycling protein and ATP synthesis. Although autophagy is very important during embryogenesis, the mechanism underlying the dynamic development during this process remains largely unknown. In order to obtain insights into autophagy in early embryo development, we analyzed gene expression levels of autophagy-related genes (ATGs) in mouse embryos developing in vitro. Using real time RT-PCR technique, ATGs including Atg2a, Atg3, Atg4b, Atg5, Atg6, Atg7, Atg9a, and Wipi3, as maternal transcripts, were only up-regulated in 1-cell embryo stage before zygotic genomic activation (ZGA), and then expression decreased from 2-cell to blastocyst embryo stage. ATGs including Dram and Atg9b were expressed abundantly in 1-cell embryo state and in blastocyst embryo stage, athough Atg8 and Ulk1 were constantly expressed during preimplantation stage. However, Atg4d were only up-expressed from 4-cell to blastocyst stage. These results suggest that autophagy is related in mouse embryo, which possibly gives an important role for early development.

• Journal of Microbiology and Biotechnology
• /
• 제29권6호
• /
• pp.989-998
• /
• 2019

Autophagy is crucial for immune defense against Mycobacterium tuberculosis (Mtb) infection. Mtb can evade host immune attack and survival within macrophages by manipulating the autophagic process. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in regulating vital genes during Mtb infection. The precise role of miRNAs in autophagy with the exits of Mtb remains largely unknown. In this study, we found miR-1958, a new miRNA that could regulate autophagy by interacting with 3'UTR of autophagy-related gene 5 (Atg5). In addition, Mtb infection triggered miR-1958 expression in RAW264.7 cells. What's more, miR-1958 overexpression blocked autophagic flux by impairing the fusion of autophagosomes and lysosomes. Overexpression of miR-1958 reduced Atg5 expression and LC3 puncta while inhibition of miR-1958 brought an increase of Atg5 and LC3 puncta; the opposite results were observed in detection of p62. The survival of Mtb in RAW264.7 cells transfected with mimic of miR-1958 was enhanced. Taken together, our research demonstrated that a novel miR-1958 could inhibit autophagy by interacting with Atg5 and favored intracellular Mtb survival in RAW264.7 cells.

• Clinical and Experimental Reproductive Medicine
• /
• 제50권3호
• /
• pp.170-176
• /
• 2023

Objective: Autophagy is highly active in ovariectomized mice experiencing hormone deprivation, especially in the uterine mesenchyme. Autophagy is responsible for the turnover of vasoactive factors in the uterus, which was demonstrated in anti-Müllerian hormone receptor type 2 receptor (Amhr2)-Cre-driven autophagy-related gene 7 (Atg7) knockout (Amhr-Cre/Atg7^f/f mice). In that study, we uncovered a striking difference in the amount of sequestosome 1 (SQSTM1) accumulation between virgin mice and breeder mice with the same genotype. Herein, we aimed to determine whether repeated breeding changed the composition of mesenchymal cell populations in the uterine stroma. Methods: All female mice used in this study were of the same genotype. Atg7 was deleted by Amhr2 promoter-driven Cre recombinase in the uterine stroma and myometrium, except for a triangular stromal region on the mesometrial side. Amhr-Cre/Atg7^f/f female mice were divided into two groups: virgin mice with no mating history and aged between 11 and 12 months, and breeder mice with at least 6-month breeding cycles with multiple pregnancies and aged around 12 months. The uteri were used for Western blotting and immunofluorescence staining. Results: SQSTM1 accumulation, representing Atg7 deletion and halted autophagy, was much higher in virgin mice than in breeders. Breeders showed reduced accumulation of several vasoconstrictive factors, which are potential autophagy targets, in the uterus, suggesting that the uterine stroma was repopulated with autophagy-intact cells during repeated pregnancies. Conclusion: Multiple pregnancies seem to have improved the uterine environment by replacing autophagy-deficient cells with autophagy-intact cells, providing evidence of cell mixing.

• Journal of Life Science
• /
• 제24권11호
• /
• pp.1180-1186
• /
• 2014

Autophagy, the lysosomal degradation pathway, is an intracellular recycling system that is necessary for the metabolic benefits of exercise and for producing lasting beneficial effects of exercise in various diseases. However, the most recent studies have only examined the effect of a single bout of exercise or resistance exercise on autophagic responses. To determine the differential effects of acute and chronic exercise on the expression of autophagy-related genes in D. melanogaster, white-eyed mutant D. melanogaster were assigned randomly to four groups: control, acute exercise, 2 hr chronic exercise, and 3 hr chronic exercise. The flies were exercised using a mechanized platform known as the Power Tower. Our results revealed that a single bout of exercise resulted in increased mRNA levels of the Atg8a gene (~20%, p<0.05). However, Atg1 and Atg6 mRNA expression were not induced by acute exercise. Transcript levels of Atg6 (~29%, p<0.05) related to the nucleation of autophagosomes were significantly induced by 2 hr of chronic exercise. However, this chronic exercise was not enough to increase Atg1 and Atg8a mRNA expression. On the other hand, 3 hr of exercise for 7 days significantly increased Atg1, Atg6, and Atg8a gene expression-about 57%, 37%, and 71%, respectively (p<0.05). These results suggest that a single bout of exercise is not enough to induce full activation of selected autophagy-related genes in D. melanogaster. Our results demonstrated that chronic regular exercise induced autophagy-related gene expression, suggesting that chronic regular exercise training might be required to activate autophagic responses important for producing beneficial effects of exercise in various diseases.

• Journal of the Korean Ceramic Society
• /
• 제35권10호
• /
• pp.1061-1069
• /
• 1998

The thermal instability of Al2TiO5 Ceramics was contrlled by solid solution with MgO SiO2 and ZrO2 through electrofusion in an arc furnace. The thermal expansion properties of Al2TiO5 composites show the hysteresis due to the strong anisotropy of The crystal axes of these material. These phenomena are ex-plained by the opening and closing of microcracks. The difference in microcracking temperatures e.g 587.6(ATG2), 405.9(ATG3) and 519.7$^{\circ}C$(ATG4) is caused by the difference in grain size and stabilizer type. The thermal shock behaviour under cyclic conditions between 750-1400-75$0^{\circ}C$ show no change in mi-crostructure and phase assemblage for all three stabilized specimens. After the thermal loading test at 110$0^{\circ}C$ for 100hrs. ATG1 and ATG2 materials decomposes completely to its components corundum and ru-tile in both cases. However with approximatelly 20% retention of the Al2TiO5 Thus in order to prevent decomposition of the stabilized material in the critical temperature range 800-130$0^{\circ}C$ it must be traversed within a short period of time.

• Clinical and Experimental Pediatrics
• /
• 제58권7호
• /
• pp.267-269
• /
• 2015

Antithymocyte globulin (ATG) is used as an immunosuppressive treatment (IST) to deplete clonal suppressor T cells in patients with severe aplastic anemia (SAA). The depletion of suppressor T cells by ATG may affect the activation of B cells, which results in an increased risk for autoimmune conditions. A 12-year-old boy was diagnosed with idiopathic SAA. As he did not have an human leukocyte antigen-matched sibling, he was treated with rabbit ATG (3.5 mg/kg/day for 5 days) and cyclosporine. Five months later, he became transfusion independent. However, 23 months after IST, he complained of mild hand tremors, sweating, weight loss, palpitations, and goiter. Results of thyroid function tests revealed hyperthyroidism (free thyroxine, 3.42 ng/dL; thyroid stimulating hormone [TSH], <0.01 nIU/mL; triiodothyronine, 3.99 ng/mL). Results of tests for autoantibodies were positive for the antimicrosome antibody and TSH-binding inhibitory immunoglobulin, but negative for the antithyroglobulin antibody and antinuclear antibody. He was treated with methimazole, and his symptoms improved. The patient has been disease free for 39 months after IST and 9 months after methimazole treatment. This case report suggests that although rare, rabbit ATG may have implications in the pathogenesis of autoimmune hyperthyroidism. Our findings suggest that thyroid function tests should be incorporated in the routine follow-up of SAA patients treated with ATG.