Antimicrobial Proteins Secreted by the Epididymis

描述（由申请人提供）：该提案侧重于附睾中天然存在的抗菌蛋白的开发、调节和发现，重点是防御素和cathelicidins。天然抗菌蛋白是先天免疫系统的一部分，它们可能保护生殖道免受病原微生物的入侵，从而有助于预防附睾炎等疾病。第一个目的是在附睾腔液中鉴定新的抗微生物附睾蛋白，并测试它们的生物活性。生殖器官产生多种抗菌物质，其中一些天然抗生素可能仍有待鉴定。将使用逐步方法检测附睾不同区域腔液中的抗菌活性，随后鉴定、表征和分析抗菌蛋白的特异性。第二个目的是确定在大鼠附睾发育过程中所选择的防御素和cathelicidin的mRNA和蛋白质的表达模式。这些研究将确定表达的时间起始，量化发育过程中表达水平的变化，并将表达定位于特定的上皮细胞类型。第三个目的是通过测试雄激素、腔液、暴露于细菌产物和阻塞对所选基因表达的影响来确定调节附睾中抗菌蛋白表达的因素。方法包括通过凝胶覆盖法和其他细菌学测定、北方和西方分析、真实的时间PCR、原位杂交和免疫组织化学、2-D凝胶电泳、通过质谱的微测序和分子生物学的标准方法检测抗微生物蛋白。一组已知存在于大鼠附睾中的抗微生物蛋白将在目标2和3中进行研究：大鼠β防御素1和2（RBD-1和RBD-2），Bin 1b，cathelicidin（rCRAMP）和防御素样分子E-3。拟议的研究将增加我们对男性生殖系统中先天抗菌蛋白的了解，它们也为发现和表征新的抗生素制剂提供了机会。

DESCRIPTION (provided by applicant): This proposal focuses on the development, regulation and discovery of naturally occurring antimicrobial proteins in the epididymis, highlighting the defensins and cathelicidins. Natural antimicrobial proteins are part of the innate immune system, and they likely protect the reproductive tract from invasion by pathogenic microbes thus helping to prevent diseases such as epididymitis. The first aim is to identify novel antimicrobial epididymal proteins in epididymal luminal fluid and to test their biological activities. The reproductive organs produce a variety of antimicrobial substances and it is likely that some of these natural antibiotics remain to be characterized. A step-wise approach will be used to test for antibacterial activity in luminal fluid from different regions of the epididymis, and subsequently to identify, characterize, and analyze the specificities of antimicrobial proteins. The second aim is to determine the pattern of expression of mRNA and protein for selected defensins and cathelicidin during development of the rat epididymis. These studies will determine the temporal onset of expression, quantify changes in expression levels during development, and localize expression to specific epithelial cell types. The third aim is to determine factors regulating expression of antimicrobial proteins in the epididymis by testing the effects of androgens, luminal fluid, exposure to bacterial products, and obstruction on expression of selected genes. Methods include detection of antimicrobial proteins by a gel overlay method and other bacteriologic assays, northern and western analyses, real time PCR, in situ hybridization, and immunohistochemistry, 2-D gel electrophoresis, microsequencing by mass spectrometry, and standard approaches of molecular biology. A set of antimicrobial proteins known to be present in the rat epididymis will be studied in aims 2 & 3: rat beta defensins 1 and 2 (RBD-1 and RBD-2), Bin1b, a cathelicidin (rCRAMP), and the defensin-like molecule E-3. The proposed studies will increase our knowledge of innate antimicrobial proteins in the male reproductive system, and they also present the opportunity for discovery and characterization of new antibiotic agents.