RESUMO
piRNA (PIWI-interacting RNA) is a germ cell-specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.
Assuntos
Glicerol-3-Fosfato O-Aciltransferase/metabolismo , RNA Interferente Pequeno/metabolismo , Células-Tronco/metabolismo , Testículo/metabolismo , Animais , Animais Recém-Nascidos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Western Blotting , Proteínas de Ciclo Celular , Células Cultivadas , Técnicas de Silenciamento de Genes , Vetores Genéticos/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/genética , Imunoprecipitação , Lisofosfolipídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , MicroRNAs/genética , MicroRNAs/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Vírus Sendai/genética , Vírus Sendai/metabolismo , Células-Tronco/citologia , Testículo/citologiaRESUMO
We previously demonstrated that esculeogenin A, a new aglycone of the tomato sapogenol esculeoside A, inhibits both acyl coenzyme A:cholesterol acyl-transferase (ACAT)-1 and -2 and ameliorates the pathogenesis of atherosclerosis in apoE deficient mice. Although we believe that daily intake of esculeoside A from tomato products can play a beneficial role in preventing the pathogenesis of atherosclerosis, the compound is not being used for preventive medicine due to the lack of information on methods for quantitative analysis and the content and stability of the compound in tomato products. In the present study, we report the development of a high-performance liquid chromatography (HPLC) method using an instrument equipped with a refractive index (RI) detector for esculeoside A quantification. We used this method to measure the changes in esculeoside A content during maturation, its distribution in the fruit body, and its stability during the heating process. The contents of esculeoside A in cherry tomatoes and Momotaro tomatoes were 21- and 9-fold, respectively, higher than that of lycopene, which is the most well-known compound in tomatoes. Furthermore, the esculeoside A content in pericarp wall was higher than in the whole tomato fruit and increased in a time-dependent manner during maturation. Although the melting point of purified esculeoside A was 225 °C, the esculeoside A in crude tomato extract decreased in a temperature-dependent manner. Degradation due to the heating process was inhibited under a pH of 9. These results demonstrated that the esculeoside A content differs in the various types of tomatoes, during maturation, and during the heating process used for preservation.