PLACENTAL AMINO ACID TRANSPORT: MOLECULAR ASPECTS

该项目的主要目标是获得一个清楚的了解 一种独特的氨基酸的运输机制， 牛磺酸，穿过人体胎盘。牛磺酸，虽然是一种非蛋白质氨基酸 酸是几种组织中最丰富的游离氨基酸(例如，大脑， 心脏和视网膜)。在动物体内，牛磺酸的诱导作用 母亲在怀孕期间缺乏营养会产生有害影响 对发育中的胎儿造成这些器官的功能损害 它积累了大量的牛磺酸。牛磺酸的作用 宫内缺陷包括视网膜变性、心肌病和 小脑发育障碍。这些研究表明， 牛磺酸在猪的生长发育中起着重要作用。 胎儿。然而，矛盾的是，无论是人类胎儿还是人类 胎盘可以内源性合成牛磺酸。母婴移植 穿过胎盘是胎儿唯一可用的机制 获取这种重要的营养物质。提出了一种新的输运模型。 牛磺酸穿过胎盘，根据这种情况，合体滋养细胞 表达两个相似但不同的牛磺酸转运蛋白，一个在 母体面刷缘膜，另一种在胎儿面上 基底膜和两者之间的功能协调 转运蛋白使牛磺酸从母亲体内转移到胎儿体内。 该项目寻求通过以下方式获得支持这一模式的证据 建立这两个菌株的分子和生化特性 传送者。这一目标将通过分离cDNA编码来实现 这些转运蛋白来自人胎盘cDNA文库，并通过 克隆的转运蛋白在哺乳动物细胞中的功能性表达 在非洲爪哇的卵母细胞中。抗多克隆和单克隆抗体 克隆的转运蛋白将被用于 免疫组织化学研究确定卵巢癌极化定位 合体滋养层细胞中的两种牛磺酸转运体。 牛磺酸在灌丛边缘和基底部的运输特性 膜也将用从正常情况下提纯的膜小泡进行 胎盘。这些研究将与完整的细胞系统相辅相成 胎盘绒毛膜癌细胞将生长在可渗透的 允许访问刷子边界和基面侧膜的过滤器 牛磺酸跨膜转运的差异性评价和表征 这些膜。该项目的另一个目标是调查 胎盘牛磺酸转运蛋白的激素调节(S)和 其中涉及的细胞机制。现建议本局 转运蛋白受翻译后修饰的调节，包括 磷酸化和蛋白激酶C和钙调神经磷酸酶(一种钙离子- 钙调素依赖的磷酸蛋白磷酸酶)参与了这一过程 进程。牛磺酸转运蛋白基因(S)及其反向转运蛋白 抗体将为这些研究提供极好的实验工具。 这个项目将导致对分子和 母体向胎儿移植的细胞机制 牛磺酸穿过人体胎盘。

The primary objective of the project is to obtain a clear understanding of the mechanisms involved in the transport of a unique amino acid, taurine, across the human placenta. Taurine, though a non-protein amino acid, is the most abundant free amino in several tissues (e.g. brain, heart, and retina) of the human fetus. In animals, induction of taurine deficiency in the mother during pregnancy produces deleterious effects on the developing fetus, causing functional impairment of those organs which accumulate high levels of taurine. The effects of taurine deficiency in utero include retinal degeneration, cardiac myopathy and impairment of cerebellar development. These studies indicate that taurine plays an important role in the growth and development of the fetus. Paradoxically however, neither the human fetus nor the human placenta can synthesize taurine endogenously. Maternal-to-fetal transfer across the placenta is the only mechanisms available for the fetus to obtain this important nutrient. A model is proposed for the transport of taurine across the placenta according to which the syncytiotrophoblast expresses two similar, yet distinct, taurine transporters, one in the maternal-facing brush border membrane and the other in the fetal-facing basal membrane and a functional coordination between these two transporters bring about the transfer of taurine from mother to fetus. The project seeks to obtain supporting evidence for this model, by establishing the molecular and biochemical identity of these two transporters. This goal will be achieved by isolating the cDNAs encoding these transporters from a human placental cDNA library and by functionally expressing the cloned transporters in mammalian cells and in Xenopus laevis oocytes. Polyclonal and monoclonal antibodies against the cloned transport proteins will be generated to be used in immunohistochemical studies to establish the polarized localization of the two taurine transporters in the syncytiotrophoblast. Characterization of taurine transport across the brush border and basal membranes will also be done with purified membrane vesicles from normal placentas. These studies will be complemented with an intact cell system in which placental choriocarcinoma cells will be grown on permeable filters allowing access to the brush border and basolateral membranes for differential assessment and characterization of taurine transport across these membranes. Another goal of the project is to investigate the hormonal regulation of the placental taurine transporter(s) and the cellular mechanisms involved therein. It is proposed that the transporter is regulated by posttranslational modification involving phosphorylation and that protein kinase C and calcineurin (a Ca2+- calmodulin-dependent phosphoprotein phosphatase) participate in this process. The taurine transporter cDNA(s) and the antitransporter antibodies will provide excellent experimental tools for these studies. This project will result in a thorough understanding of the molecular and cellular mechanisms responsible for the maternal-to-fetal transfer of taurine across the human placenta.