ABSTRACT
Objective: Autophagy is a major intracellular catabolic pathway governed by the sequential actions of proteins encoded by autophagy-related genes (Atg). ATG9, the only transmembrane protein involved in this process, regulates phospholipid translocation to autophagosomes during the early phases of autophagy. In mammals, two Atg9 isoforms have been reported: Atg9a and Atg9b. In this study, we examined whether the molecular and cellular characteristics of these two isoforms differed in mice. Methods: Whole uteri were collected on days 1, 4, and 8 of pregnancy and from ovariectomized mice injected with vehicle, progesterone, or 17ß-estradiol. Cells from reproductive tissues, such as granulosa cells, uterine epithelial cells (UECs), uterine stromal cells (USCs), and oocytes were collected. Two human uterine cell lines were also used in this analysis. Reverse transcription-polymerase chain reaction tests, Western blotting, and immunofluorescence staining were performed. Serum starvation conditions were used to induce autophagy in primary cells. Results: Atg9a and Atg9b were expressed in multiple mouse tissues and reproductive cells. Neither Atg9A nor Atg9B significantly changed in response to steroid hormones. Immunofluorescence staining of the UECs and USCs showed that ATG9A was distributed in a punctate-like pattern, whereas ATG9B exhibited a pattern of elongated tubular shapes in the cytoplasm. In human cancer cell lines, ATG9B was undetectable, whereas ATG9A was found in all cell types examined. Conclusion: The Atg9 isoforms exhibited distinct subcellular localizations in UECs and may play different roles in autophagy. Notably, human uterine cells exhibited reduced ATG9B expression, suggesting that this suppression may be due to epigenetic regulation.
ABSTRACT
BACKGROUND: Mesenchymal stem cells (MSCs) have gained significant attention for diverse biomedical applications, including cell-based therapy. Hence, in vitro expansion of MSCs is critical; however, in vitro MSC culture, especially long-term culture, inevitably leads to significant loss of stemness, growth, and differentiation potential. METHOD: Effects of mild heat treatment (HT) conditions (temperature, duration, and repetition) on the characteristics of adipose tissue-derived MSCs in vitro were systematically investigated. Characteristics of the MSCs subjected to the predetermined HT conditions (41 or 44ºC, 1 h, and 2X HT) were first analyzed in a single passage using various assays. In addition, the feasibility of HT for long-term MSC culture was studied. The RNA sequencing analyses were performed to elucidate the mechanism of HT effects on MSCs. RESULTS: A comprehensive exploration of various HT conditions revealed that specific mild HT at 41ºC or 44ºC for 1 h upregulated the expression of heat shock proteins and stemness markers and enhanced differentiation potentials. Furthermore, periodic mild HT extended the maintenance of growth rate and stemness of MSCs up to an additional 10 passages, which substantially retarded their spontaneous aging during subsequent in vitro culture. RNA sequencing analyses unveiled that HT downregulated genes associated with aging and apoptosis. CONCLUSION: Our study successfully demonstrated that mild HT of MSCs has positive effects on their application in various biomedical fields, enhancing their capabilities and slowing down the aging process.
ABSTRACT
The genes encoding dopamine receptor (DR) subtypes have received considerable attention for the past several years as a potential candidate that may affect susceptibility to addictive disorder, including alcoholism. The many association studies that compared the frequencies of alleles of the dopamine D2 receptor (DRD2) gene between alcoholics and control groups have produced results, but some have been equivocal. Dopamine D3 receptor genes (DRD3) are in the same class as DRD2 but with different pharmacological properties. So we compared the distribution of genotypes and frequencies of BalI polymorphism of the DRD3 gene in alcoholics and controls to assess the role of the DRD3 gene in Korean alcoholism. For this study, 67 male probands from alcoholics and 67 age-matched normal male controls were engaged. No evidence for an allelic association was found between the A1 allele of DRD3 and alcoholism in a Korean population. These results suggest that any role played by this receptor may account for only part of the variation in susceptibility to alcoholism.
