Transport Physiology of Disease Vector Mosquitoes

病媒蚊子的运输生理学

• 批准号: 6833446
• 负责人: WILLIAM R HARVEY
• 金额: $ 32.74万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-15 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6833446
• 关键词: Anopheles; Xenopus oocyte; acidity /alkalinity; adenosinetriphosphatase; bioinformatics; capillary electrophoresis; communicable disease transmission; confocal scanning microscopy; electrochemistry; functional /structural genomics; gastrointestinal epithelium; gastrointestinal function; gene expression; in situ hybridization; ion transport; larva; microelectrodes; nutrient intake activity; polymerase chain reaction

DESCRIPTION (provided by the applicant): Taking advantage of the complete genome as well as the morphological simplicity and cellular accessibility of larval midgut in An. gambiae, we will create a comprehensive physiological model that integrates molecular, cellular, and electrochemical mechanisms of epithelial transport. In particular, we wish to understand the integrative physiology of lumen alkalinization, which is critical for larval digestion and nutrient uptake. We postulate that alkalinization and nutrient uptake depend upon transepithelial and longitudinal ion gradients, which are generated by specific spatial distribution and electrochemical interaction between primary H + V-ATPases and secondary acid/base transporters in the larval midgut. We have identified eight genes in the An. gambiae genome, which encode the putative acid/base transporters and designed tissue-specific cDNA library from An. gambiae larvae, which dramatically increased the cloning efficiency. We have also developed unique nanoscale analytical approaches, which allow us to analyze epithelial tissue with subcellular resolution. To explore the integrative physiology and functional genomics of the mosquito midgut, we will complete four specific aims. Aim 1 uses preliminary bioinformatics data to clone acid/base transporters from An. gambiae larvae; the gene products will be evaluated by electrochemical analysis of transcript expression in Xenopus oocytes. Aim 2 localizes the transporters along the midgut in whole-mounts of An. gambiae larvae using in situ hybridization with transcript-specific dioxygenin-RNA probes. The apical/basal (polar) integration of the transporters and H v V-ATPases will be determined by confocal microscopy of immuno-labeling preparations. Aim 3 seeks to determine electrochemical motive forces across basal/apical membranes and phosphorylation potentials in specified midgut cells using capillary electrophoresis and ion selective microelectrodes. Trans-membrane voltages will be measured with microelectrodes in midgut of intact and semi-intact An. gambiae larvae. Aim 4 examines the mechanism, efficiency, and role of electrochemical coupling between H + V-ATPase and identified transporters in midgut alkalinization using non-invasive self-referencing ion-selective microelectrodes (SERIS LIX) and time-lapse photography of a lumen alkalinization profile in vivo. With this proposed study, integration of the molecular and electrochemical mechanism of a specific physiological process will be defined for the first time, which is crucial not only for understanding midgut alkalinization in disease vector mosquitoes but other types of transporting epithelia as well. Since the work is to be done on a malaria vector, it has health relevance because the larval midgut is the target for Bti, TMOF, and other larvicides and is an apt model for the plasmodium interaction with the adult-female midgut.

描述（由申请人提供）：利用完整的基因组以及幼虫中肠的形态简单性和细胞可及性。冈比亚，我们将创建一个综合的生理模型，该模型整合上皮转运的分子，细胞和电化学机制。特别是，我们希望了解流明碱化的综合生理学，这对于幼虫消化和养分摄取至关重要。我们假设碱化和养分的吸收取决于旋转的和纵向离子梯度，这些梯度是由特异性的空间分布和幼虫中gut中二级酸/基础转运蛋白之间的特定空间分布和电化学相互作用所产生的。我们已经确定了AN中的八个基因。冈比亚基因组，编码从An的推​​定酸/碱转运蛋白和设计的组织特异性cDNA文库。冈比亚幼虫，大大提高了克隆效率。我们还开发了独特的纳米级分析方法，使我们能够通过亚细胞分辨率分析上皮组织。 为了探索蚊子中肠的综合生理学和功能基因组学，我们将完成四个特定目标。 AIM 1使用初步的生物信息学数据来克隆酸/碱基转运蛋白。冈比亚幼虫；基因产物将通过对爪蟾卵母细胞中转录表达的电化学分析来评估。 AIM 2以An的整个成绩将转运蛋白定位在Midgut。 gambiae幼虫使用原位杂交与转录本特异性二氧蛋白-RNA探针。转运蛋白和H V-V-ATPase的顶部/基底（极）整合将通过免疫标记制剂的共聚焦显微镜确定。 AIM 3旨在使用毛细管电泳和离子选择性微电极确定指定的中肠细胞中基底/顶膜和磷酸化电位的电化学动力。跨膜电压将用完整和半完整的Midgut中的微电极测量。冈比亚幼虫。 AIM 4使用非侵入性自我引用的离子微电极（Seris lix）和Vivo中Lumen Alkalinization photogramy摄影，研究了H + V-ATPase与Midgut碱化中鉴定的转运蛋白之间电化学耦合的机制，效率和作用。通过这项提出的研究，将首次定义特定生理过程的分子和电化学机制的整合，这不仅对于理解疾病媒介蚊子中的中肠碱化至关重要，而且对其他类型的运输上皮植物至关重要。由于该工作要在疟疾载体上进行，因此它具有健康相关性，因为幼虫中肠是BTI，TMOF和其他幼虫的靶标，并且是与成人女性中肠质量相互作用的适当模型。