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）：利用按蚊幼虫中肠的完整基因组以及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，整合上皮运输的分子、细胞和电化学机制。特别是，我们希望了解管腔碱化的综合生理学，这对于幼虫的消化和营养吸收至关重要。我们假设碱化和营养吸收取决于跨上皮和纵向离子梯度，这是由幼虫中肠中初级 H + V-ATP 酶和次级酸/碱转运蛋白之间的特定空间分布和电化学相互作用产生的。我们已经在 An 中鉴定出八个基因。冈比亚基因组，编码假定的酸/碱转运蛋白，并设计了来自冈比亚安的组织特异性 cDNA 文库。冈比亚幼虫，这极大地提高了克隆效率。我们还开发了独特的纳米级分析方法，使我们能够以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，我们将完成四个具体目标。目标 1 使用初步生物信息学数据从 An 中克隆酸/碱转运蛋白。冈比亚幼虫；将通过爪蟾卵母细胞转录表达的电化学分析来评估基因产物。目标 2 将转运蛋白定位在 An 整个中肠中。冈比亚幼虫使用转录特异性双加氧配基 RNA 探针进行原位杂交。转运蛋白和 H v V-ATP 酶的顶端/基底（极性）整合将通过免疫标记制剂的共聚焦显微镜确定。目标 3 寻求使用毛细管电泳和离子选择性微电极确定特定中肠细胞中跨基底/顶膜的电化学动力和磷酸化电位。将使用完整和半完整 An 中肠中的微电极测量跨膜电压。冈比亚幼虫。目标 4 使用非侵入性自参考离子选择性微电极 (SERIS LIX) 和管腔碱化曲线的延时摄影，检查 H + V-ATP 酶和已识别的转运蛋白在中肠碱化中的电化学耦合的机制、效率和作用体内。通过这项拟议的研究，将首次定义特定生理过程的分子和电化学机制的整合，这不仅对于了解病媒蚊子的中肠碱化至关重要，而且对于其他类型的运输上皮细胞也至关重要。由于这项工作是在疟疾载体上进行的，因此它具有健康相关性，因为幼虫中肠是 Bti、TMOF 和其他杀幼虫剂的目标，并且是疟原虫与成年雌性中肠相互作用的合适模型。