MOLECULAR STUDY OF INSECT VECTOR ANTIHEMOSTATIC FACTORS

在从脊椎动物取食的过程中，吸血昆虫遇到 它们的宿主的止血反应的组成部分，特别是 血管收缩和血小板栓的形成。因此，委员会认为， 食血昆虫，唾液成分中含有血小板 抗聚集活性（腺苷三磷酸双磷酸酶），许多产生唾液 血管扩张剂这些成分也可能影响寄生虫的过程 通过改变在进食部位的宿主免疫应答来建立。 尽管它们很重要，但对它们的分子生物学知之甚少。 这些物质。 我建议在分子水平上比较腺苷三磷酸双磷酸酶和血管舒张 在两种昆虫中独立进化出的吸血剂， 是重要的疾病媒介：黄热病蚊子埃及伊蚊 和锥蝽Rhodnius prolixus。具体而言，该项目 涉及： (1)分离和测序前速激肽原的cDNA克隆， 被加工以释放Ae中的血管舒张性速激肽。埃及人。 (2)三种NO结合血红素的cDNA克隆的分离和测序 蛋白质，在A.冗长 (3)分离并测序Rhodnius腺苷三磷酸双磷酸酶的cDNA克隆， 与伊蚊腺苷三磷酸双磷酸酶的比较; (4)使用这些克隆作为探针来研究腺苷三磷酸双磷酸酶的调节， 两种昆虫的血管扩张剂; (5)使用血红素蛋白克隆产生重组野生型， 位点特异性突变蛋白，以研究结构/功能 可逆NO结合的关系和生物物理学;以及 (6)分离和测序基因组克隆，包括Rhodnius 血红素蛋白基因，研究基因组织和调控这一 功能独特的蛋白质组。 最终，这项研究将允许更详细地了解 与早期阶段相关的生化和药理学事件 吸血昆虫传播寄生虫的证据比较两 已经独立进化出吸血的物种， 这可能是多种病媒昆虫共有的。

In the course of feeding from vertebrates, blood feeding insects encounter components of their host's hemostatic responses, especially vasoconstriction and formation of a platelet plug. Consequently, hematophagous insects have salivary components with platelet antiaggregating activity (apyrase), and many produce salivary vasodilators. These components may also influence the course of parasite establishment by modifying the host immune response at the feeding site. Despite their importance, little is known of the molecular biology of these substances. I propose to compare, at the molecular level, apyrase and vasodilatory agents in two insects that have independently evolved blood feeding, and are significant disease vectors: the yellow fever mosquito Aedes aegypti and the triatomine bug Rhodnius prolixus. Specifically, the project involves: (1) isolating and sequencing cDNA clones for the preprotachykinin, which is processed to release vasodilatory tachykinins in Ae. aegypti. (2) isolating and sequencing cDNA clones for three NO.binding heme proteins, which function as vasodilators in A. prolixus. (3) isolating and sequencing cDNA clones for the Rhodnius apyrase, for comparison with the Aedes apyrase; (4) using these clones as probes to study regulation of the apyrase and vasodilators in both insects; (5) using the heme protein clones to produce recombinant wild-type and site-specific mutant proteins, to study the structure/function relationships and biophysics of reversible NO binding; and (6) isolating and sequencing genomic clones which include the Rhodnius heme protein genes, to study gene organization and regulation of this functionally unique group of proteins. Ultimately, this research will permit a more detailed knowledge of the biochemical and pharmacological events associated with the earliest stages of parasite transmission by blood-feeding insects. Comparison of two species, which have independently evolved blood feeding, will point to congruencies which may be common to a variety of vector insects.