Antimicrobial Proteins Secreted by the Epididymis

描述（由申请人提供）：本提案侧重于附睾中天然存在的抗菌蛋白的开发、调控和发现，重点是防御素和抗菌素。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菌附睾蛋白并检测其生物活性。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。分步方法将用于测试附睾不同区域腔液中的抗菌活性，并随后识别、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选定的防御素和抗菌肽mRNA和蛋白的表达模式。这些研究将确定表达的时间开始，量化发育过程中表达水平的变化，并将表达定位到特定的上皮细胞类型。第三个目的是通过测试雄激素、管腔液、暴露于细菌产物和阻塞对选定基因表达的影响，确定调节附睾抗菌蛋白表达的因素。方法包括通过凝胶覆盖法检测抗菌蛋白和其他细菌学分析，northern和western分析，实时PCR，原位杂交，免疫组织化学，2d凝胶电泳，质谱微测序和分子生物学标准方法。目标2和目标3将研究一组已知存在于大鼠附睾中的抗菌蛋白：大鼠β -防御素1和2 (RBD-1和RBD-2)，抗菌素Bin1b （rCRAMP）和防御素样分子E-3。所提出的研究将增加我们对男性生殖系统中先天抗菌蛋白的认识，并为发现和表征新的抗生素提供机会。

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.