Physiology of Insect Amino Acid Transport

昆虫氨基酸运输的生理学

基本信息

批准号：
7344844
负责人：
DMITRI Y. BOUDKO
金额：
$ 28.65万
依托单位：
ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-12-01 至 2010-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7344844
关键词：
ATP phosphohydrolase Acids Adult Amino Acid Transporter Amino Acids Animals Anions Anopheles gambiae Anterior Antibodies Apical Biological Assay Capillary Electrophoresis Cations Cell Line Cell membrane Cells Cloning Code Collection Confocal Microscopy Culicidae Data Databases Disease Vectors Electrochemistry Electrodes Endosome Proton Epithelial Cells Epithelium Evaluation Genes Genetic Genome Genomics Goals In Situ Hybridization Insecta Ions Isotope Labeling Label Larva Life Ligation Lysosome Proton Measures Mediating Messenger RNA Microelectrodes Midgut Modeling Molecular Molecular Genetics Molecular Profiling Mosquito Control Nutrient Open Reading Frames Organism Physiological Physiology Plasmids Polymerase Chain Reaction Population Preparation Progress Reports Property Proteins Proton Pump Pump Relative (related person)Resolution Screening procedure Site System Testing Time Tissues Transcript Xenopus oocyte base cDNA Library driving force expression cloning gene cloning genome sequencing human CXCL5 protein immunocytochemistry in vivo tool uptake vacuolar H+-ATPase vector mosquito voltage clamp

项目摘要

DESCRIPTION (provided by applicant): Mosquito larvae have evolved efficient systems to take up amino acids across the midgut epithelium. This uptake appears to be mediated by secondary active amino acid transporters (AATs) and to be energized by primary H+ V-ATPases in synergy with secondary inorganic ion exchangers; the entire protein ensemble constitutes an "amino acid uptake metabolon". The completed Anopheles gambiae genome provides a tool with which to study the metabolon. Cyber-screening of the genome confirmed that the V-ATPase subunits are all present, and revealed putative genes for 3 anion exchangers, 5 cation transporters, and 92 amino acid transporters. Although not all of these genes will be active in larvae, this relatively simple amino acid uptake metabolon provides an excellent subject for genetic and molecular evaluation. The larval midgut epithelium consists of a few thousand large (50 - 100 micron) cells, is simple and accessible, and provides a unique opportunity to analyze the physiology/electrochemistry of the amino acid transport metabolon in vivo. The goal of this proposal is to explore the amino acid transport metabolon in mosquito larvae by identifying, cloning, and characterizing the entire population of putative nutrient AATs in An. gambiae larvae and quantifying their physiological interplay with the ATPase and inorganic ion transporters in vivo. Aim 1 is to identify genes and clone transcripts encoding nutrient AATs by screening tissue specific midgut cDNA libraries with exact primers for putative AAT genes in the An. gambiae genome. Aim 2 is to characterize the ATTs by heterologous expression in Xenopus oocytes or cell lines, followed by labeled amino acid uptake assays and electrochemical analysis of the uptake properties. Aim 3 is to define the distribution of these nutrient AATs in larval An gambiae midgut using in situ hybridization, real time PCR, and immunocytochemistry. Aim 4 is to characterize the electrochemical properties of nutrient amino acid uptake in vivo using high-resolution capillary electrophoresis as well as conventional and self-referencing ion selective microelectrodes. The characterized An. gambiae amino acid uptake metabolon will serve as a model for amino acid uptake in other animals and will provide specific targets for developing mosquito control agents.

描述（由申请人提供）：蚊子幼虫已经进化了有效的系统，可以在整肠上皮上占用氨基酸。这种摄取似乎是由次级活性氨基酸转运蛋白（AAT）介导的，并通过与二次无机离子交换剂协同作用中的原代H+ V-ATP酶进行了能量。整个蛋白质合奏构成了“氨基酸摄取代谢剂”。冈比亚冈比亚基因组完整的蚊子提供了一种研究化学物质的工具。基因组的网络筛查证实了V-ATPase亚基都存在，并显示了3个阴离子交换器，5个阳离子转运蛋白和92个氨基酸转运蛋白的推定基因。尽管并非所有这些基因都会在幼虫中活跃，但这种相对简单的氨基酸摄取代谢子为遗传和分子评估提供了极好的主题。幼虫中肠上皮由几千个大型（50-100微米）细胞组成，简单易用，并提供了一个独特的机会，可以分析体内氨基酸转运物质的生理/电化学。该提案的目的是通过鉴定，克隆和表征AN中推定的养分AAT的全部人群来探索蚊子幼虫中的氨基酸转运代谢。冈比亚幼虫并量化其与ATPase和无机离子转运蛋白体内的生理相互作用。目的1是通过筛选组织特异性中肠cDNA文库的基因和克隆转录本，并用AN中的假定AAT基因进行精确的引物。冈比亚基因组。目的2是通过异爪蟾卵母细胞或细胞系中的异源表达来表征ATT，然后是标记的氨基酸摄取测定法和摄取性能的电化学分析。目的3是使用原位杂交，实时PCR和免疫细胞化学定义这些营养AAT在幼虫中gambiae Midgut中的分布。目的4是使用高分辨率毛细管电泳以及常规和自我引用的离子选择性微电极来表征体内营养氨基酸摄取的电化学特性。特征是。冈比亚氨基酸的摄取代谢子将作为其他动物中氨基酸摄取的模型，并将为开发蚊子控制剂提供特定的靶标。