Copper Transport in Lactation

哺乳期的铜转运

• 批准号: 7173457
• 负责人: MARIA C LINDER
• 金额: $ 26.13万
• 依托单位: CALIFORNIA STATE UNIVERSITY FULLERTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-10 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7173457
• 关键词: ATP phosphohydrolase; Address; Affect; Affinity; Animal Model; Animals; Antibodies; Apical; Appearance; Back; Binding; Binding Sites; Blood; Blood Cells; Breast; Cell membrane; Cell model; Cell surface; Cells; Ceruloplasmin; Complex; Condition; Confocal Microscopy; Copper; Cultured Cells; Diet; Drug or chemical Tissue Distribution; Endocytosis; Endopeptidases; Enterocytes; Epithelial; Epithelial Cells; Epithelium; Exocytosis; Gastrointestinal tract structure; Goals; Hepatocyte; Hepatolenticular Degeneration; Hereditary Disease; Heterozygote; Homeostasis; Hormones; Human; Human Milk; Immunoassay; Immunofluorescence Immunologic; Immunoprecipitation; Infant; Intestines; Kidney; Knock-out; Knowledge; Laboratories; Lactation; Ligands; Liquid substance; Liver; Location; Macroglobulins; Mammary gland; Measures; Mediating; Membrane; Membrane Transport Proteins; Menkes Kinky Hair Syndrome; Messenger RNA; Metabolism; Milk; Modeling; Molecular Chaperones; Mothers; Mus; Mutant Strains Mice; Mutation; Numbers; Pattern; Peptide Hydrolases; Personal Satisfaction; Physiological; Plasma; Polymerase Chain Reaction; Pregnancy; Principal Investigator; Process; Production; Protein Overexpression; Proteins; Pump; Radioactive; Radioactive Tracers; Rattus; Relative (related person); Rest; Rodent; Rodent Model; Role; Route; SLC11A2 gene; Side; Small Interfering RNA; Testing; Time; Tissues; Transfection; Transgenic Mice; Translations; Vesicle; Wild Type Mouse; Wilson disease protein; cell type; hypocupremia; inhibitor/antagonist; mammary epithelium; monolayer; mutant; neonate; polarized cell; prevent; pup; receptor; receptor expression; response; trafficking; trans-Golgi Network; uptake

DESCRIPTION (provided by applicant): Many aspects of the mechanisms by which Cu is taken up from the blood plasma by different kinds of cells (including the transporters and delivery proteins involved), and just how (after cell entry) Cu finds its way to specific secretions, remain to be defined. We have obtained evidence that lactation profoundly alters the tissue distribution of newly absorbed Cu, diverting most of it from the liver and kidney to the mammary gland, where it rapidly appears in the milk and in milk ceruloplasmin. Cu in milk ceruloplasmin may be more bio-available to the infant than the other forms of Cu in the milk, which have not been well defined. A polarized cell culture model responsive to lactational hormones has been developed for studying milk production by mammary epithelial cells. Mutant rodent models lacking specific proteins that could be involved in the steps required for uptake of Cu by mammary gland and its delivery to developing milk are available. The long term objective is to understand how Cu distribution and transport are regulated under normal conditions and in gestation and lactation. The immediate objectives are to determine how Cu from the exchangeable plasma pool enters mammary (and hepatic) cells, under normal conditions and in lactation, to explain the marked changes in distribution observed in the latter condition (what potential membrane transporters are involved, and how their expression and/or disposition is changed by lactation). The objectives are also to determine how, after cell entry, copper is routed to the milk and milk cemloplasmin; the role(s) of ATOX1, WND and/or MNK in this process and in regulating the copper content of the milk; and to further define the copper components of mammalian milk. Expression of potential transporters will be measured at the mRNA and protein levels, by Real Time PCR and immunoassays, respectively; and localization will be followed with immunofluorescence and confocal microscopy. Alterations in expression will be induced in the cell culture model with lactational hormones, transfection, and antisense oligos. Effects of natural and knockout mutations of specific transporters in whole animal models on copper distribution and transport will be determined. Cu uptake and its emergence in milk and milk ceruloplasmin will be followed with 67Cu/64Cu and immunoprecipitation. Effects on Cu uptake of specific antibodies against membrane transporters will be measured. Cu binding components in milk secretions will be characterized. It is expected that the proposed studies will greatly advance our knowledge of how Cu is distributed to cells from the blood and particularly how mammary epithelial cells put Cu into components of the milk that may have specific effects on the infant.

描述（由申请人提供）：不同种类的细胞（包括所涉及的转运蛋白和递送蛋白）从血浆中摄取铜的机制的许多方面，以及铜（在进入细胞后）如何找到特定分泌物的方式，仍有待确定。我们获得的证据表明，哺乳期极大地改变了新吸收的铜的组织分布，将大部分铜从肝脏和肾脏转移到乳腺，并迅速出现在乳汁和乳铜蓝蛋白中。牛奶铜蓝蛋白中的铜可能比牛奶中其他形式的铜更容易被婴儿生物利用，但其他形式的铜尚未明确。已开发出一种对哺乳激素敏感的极化细胞培养模型，用于研究乳腺上皮细胞的产奶量。突变啮齿动物模型缺乏特定的蛋白质，这些蛋白质可能参与乳腺吸收铜并将其输送到发育中的乳汁所需的步骤。长期目标是了解正常条件下以及妊娠和哺乳期铜的分布和运输是如何调节的。直接目标是确定在正常条件下和哺乳期，可交换血浆库中的铜如何进入乳腺（和肝）细胞，以解释在后一种情况下观察到的分布的显着变化（涉及哪些潜在的膜转运蛋白，以及它们的表达和/或处置如何因哺乳而改变）。目标还在于确定进入细胞后，铜如何进入牛奶和牛奶铜蓝蛋白； ATOX1、WND 和/或 MNK 在此过程以及调节牛奶铜含量中的作用；并进一步明确哺乳动物乳汁中的铜成分。潜在转运蛋白的表达将分别通过实时 PCR 和免疫测定在 mRNA 和蛋白质水平进行测量；定位将通过免疫荧光和共聚焦显微镜进行。使用哺乳激素、转染和反义寡核苷酸在细胞培养模型中诱导表达的改变。将确定整个动物模型中特定转运蛋白的自然突变和敲除突变对铜分布和转运的影响。 Cu 的吸收及其在牛奶和牛奶铜蓝蛋白中的出现将通过 67Cu/64Cu 和免疫沉淀进行跟踪。将测量针对膜转运蛋白的特异性抗体对铜吸收的影响。将表征乳汁分泌物中的铜结合成分。预计拟议的研究将极大地增进我们对铜如何从血液分配到细胞的认识，特别是乳腺上皮细胞如何将铜放入可能对婴儿产生特定影响的牛奶成分中。