RESUMEN
Background and aims: Research on the psychological impact of the coronavirus disease 2019 pandemic has highlighted its negative and positive effects on children with autism spectrum disorder and their families. However, little is known about the neutral effects that remain the same, even in particular circumstances, and how children with autism spectrum disorder and their parents perceive each other. We explored how children with autism spectrum disorder and their mothers perceived and experienced the pandemic in Japan. Methods: A mixed-methods design was employed. Thirteen children with autism spectrum disorder and 12 mothers participated. Data were collected through online semi-structured interviews and analyzed using thematic analysis. Similarities and differences in perceptions were compared. Results: The results revealed six broad themes and 27 categories. Regarding neutral effects, some mothers reported no substantial impact because there were no changes in their jobs or other dramatic life changes. In addition, some children were not affected because they had had no social contact before the pandemic or because their lives had not changed dramatically. Regarding the perceptions of children/mothers, most expressed that they/their children enjoyed spending time with their families. At home, mothers made various efforts to interact with their children. However, mothers and children differed in their perceptions, such as regarding the emergence of anxiety about conducting school events and the resolution of study-related concerns. Conclusions: There were negative, neutral, and positive effects on both children with autism spectrum disorder and their mothers; specifically, they were striving to move forward to overcome the problems posed by the pandemic. Both parties tapped into their resilience by enhancing family interactions, such as cooking together or discussing children's interests. Implications: These findings have important implications for developing more creative solutions to the challenges of coping and resilience in future crises.
RESUMEN
Antithrombin (AT) is a major plasma protease inhibitor with three intramolecular disulfide bonds and a deficiency of it is associated with venous thrombosis. Recently, we prepared CHO cells overexpressing a novel mutant, AT(C95R), with a disulfide bond removed, and revealed that this mutant remained for a long time in the endoplasmic reticulum (ER) without being degraded and also accumulated in newly formed membrane structures that resembled Russell bodies (RB) [Tanaka, Y. et al. (2002) J. Biol. Chem. 277, 51058-51067]. In this study, we replaced each of the individual cysteine residues of AT with an arginine and also two paired cysteine residues with arginines. We stably expressed these mutant ATs in CHO cells, and examined the roles of each cysteine residue or disulfide bond in the accumulation of mutant ATs and the formation of RB-like structures. In pulse-chase experiments, the secretion of mutant ATs with single mutations decreased approximately 1/5-1/50 times compared to that of the wild type AT. All of the mutant ATs were retained in the ER and were also found to accumulate in the RB-like structures. On the other hand, the fates of mutant ATs with double mutations fell into two categories. Secretion of mutant AT(C8R,C128R) decreased only approximately 1/2 times and no RB-like structures appeared. Mutants AT(C21R,C95R) and AT(C247R,C430R) exhibited similar secretion kinetics to the mutant ATs with the single mutations and were found in RB-like structures. On a sucrose gradient, all of the mutant ATs that induced RB-like structures migrated as oligomeric structures, whereas wild type AT and AT(C8R,C128R) migrated as monomers. Further, to clarify the morphological pathway through which RB-like structures are formed, we prepared CHO cells in which the expression of AT(C95R) was controlled by the Tet-On system. During expression of AT(C95R), RB-like structures formed through expansion of the ER. These results suggest that the correct folding with each disulfide bond is essential for the secretion of AT and oligomerization of mutant ATs in the ER is involved in the formation of RB-like structures.
Asunto(s)
Antitrombinas/química , Antitrombinas/genética , Disulfuros/química , Cuerpos de Inclusión/ultraestructura , Animales , Proteínas Antitrombina , Antitrombinas/aislamiento & purificación , Antitrombinas/metabolismo , Células CHO , Centrifugación por Gradiente de Densidad , Cricetinae , Cisteína/genética , Retículo Endoplásmico/metabolismo , Immunoblotting , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Peso Molecular , Mutación Missense/genéticaRESUMEN
Antithrombin (AT) is a major plasma protease inhibitor with three intramolecular disulfide bonds, and its deficiency is associated with increased venous thrombosis. Recently, we found a novel missense mutation named AT Morioka (C95R), which causes the loss of one of the three disulfide bonds. In this study, we prepared Chinese hamster ovary cells stably overexpressing wild type or mutant AT and examined the intracellular fate of the ATs. In pulse-chase experiments, newly synthesized wild type AT was secreted into the medium with a half-life of approximately 1.5 h. In contrast, most of the mutant type AT was not secreted during the chase period of 9 h and, surprisingly, was not degraded in the cells. The kinetics of the secretion suggests that the mutant was secreted about 50 times more slowly into the medium. Most of the mutant AT in the cells had high mannose type oligosaccharides, suggesting that it was retained in the endoplasmic reticulum (ER). In addition, half of the mutant AT existed in a dimeric form with an intermolecular disulfide bond. On immunoelectron microscopy, the mutant AT was found to have accumulated in variously sized structures surrounded by a single membrane in the cytoplasm. Immunogold particles exhibiting calnexin immunoreactivity were detected on the membranes. Ribosomes were attached to some of the small structures that had accumulated the mutant AT. Further, we prepared Chinese hamster ovary cells stably overexpressing another mutant AT in which two cysteine residues at 21 and 95, responsible for disulfide bond formation, were substituted for arginines. In pulse-chase experiments, the mutant AT (C21C,C95R) was secreted faster than that of AT Morioka (C95R) into the medium. These results suggest that AT Morioka remained for a long time in ER without being degraded and accumulated in newly formed membrane structures derived from the ER. The dimerization of AT Morioka (C95R) through Cys-21 seems to be critical for its intracellular accumulation.