Acquisition of a flash chromatography and HPLC preparative system

项目摘要/摘要 父母资助的目标是开发有效的唾液酸酶抑制剂。唾液酸酶(也称为 神经氨酸酶)是负责清除唾液酸(SIA)残基(脱氢)的糖苷酶。 从糖偶联物的葡聚糖部分。通过分析，唾液酸酶能够调节功能。 和稳定性，并参与生理和病理 小路。先前和我们最近的研究表明，溶酶体Neu1唾液酸酶可以重新定位到细胞中 巨噬细胞表面的内毒素刺激，导致TLR4受体缺失，导致 TLR4的激活和随后促炎细胞因子的产生。TLR4的调节失调 内毒素的激活会导致慢性和急性炎症性疾病，这些疾病往往会导致危险 像败血症这样的疾病，仍然缺乏特定的药物治疗。唾液酸酶抑制剂是有用的工具 用于研究唾液酸酶的功能及其生物途径的相关机制。更重要的是， 有效的唾液酸酶抑制剂可以作为药物来调节由肺炎引起的病理通路 唾液酸酶，如TLR4激活失调。我们最近的研究表明，目前可用的PAN 唾液酸酶抑制剂和微生物唾液酸酶抑制剂不能有效地抑制哺乳动物唾液酸酶。 已有几种哺乳动物唾液酸酶抑制剂的报道。然而，目前的唾液酸酶抑制剂设计 通常侧重于活性部位结合，而忽略了活性酶的亚细胞定位， 因此，它们在体内的有效性较低，甚至可能是有毒的，因为它们会影响体内的其他唾液酸酶。 这些细胞。因此，本应用的目的是开发Neu1的位置特异性抑制剂 唾液酸酶，并确定Neu1唾液酸酶在内毒素/TLR4信号通路中的作用，这将是 通过三个特定的目的完成：(1)Neu1唾液酸酶的表达和细胞表面的重新定位 巨噬细胞对内毒素的刺激；(2)开发溶酶体靶向Neu1唾液酸酶抑制剂 调节内毒素/TLR4信号通路的缺失；(3)开发细胞表面靶向Neu1唾液酸酶 有效调节内毒素/TLR4信号通路失活的抑制剂。本研究具有创新性。 因为它使用了一种新的方法，克服了目前在(A)分析唾液酸酶表达方面的限制 和重定位以及(B)抑制亚细胞位置的唾液酸酶。拟议中的项目意义重大 因为它将(I)揭示对内毒素/TLR4信号通路至关重要的特定信号转导和(Ii) 开发新的唾液酸酶抑制剂以有效调节脂多糖/TLR4信号通路中的脱氢作用。 最后，这项提案将加强克利夫兰州立大学的研究和教育基础设施 大学，让本科生学习广泛的实验技术、数据 在现代科学调查中使用的分析和陈述技巧。

Project Summary/Abstract The goal of the parent grant is to develop effective sialidase inhibitors. Sialidases (also called neuraminidases) are glycosidases responsible for the removal of sialic acid (Sia) residues (desialylation) from glycan portions of glycocojugates. By desialylation, sialidases are able to modulate the functionality and stability of the Sia-containing molecules and are involved in both physiological and pathological pathways. Previous and our recent study indicate that lysosomal Neu1 sialidase could relocate to the cell surface of macrophages upon LPS stimulation, where it causes desialylation of TLR4 receptor, leading to TLR4 activation and subsequent production of pro-inflammatory cytokines. Dysregulation of TLR4 activation by LPS is responsible for chronic and acute inflammatory disorders that often causes dangerous disease like sepsis that still lacks specific pharmacological treatment. Sialidase inhibitors are useful tools for studying sialidase function and related mechanisms of the biological pathways. More importantly, effective sialidase inhibitors can be used as drugs to regulate the pathological pathways caused by sialidase, such as dysregulated TLR4 activation. Our recent study indicates that currently available pan sialidase inhibitor and microbial sialidase inhibitors could not inhibit mammalian sialidase effectively. Several mammalian sialidase inhibitors have been reported. However, current sialidase inhibitor design has usually focused on active-site binding, neglecting the subcellular localization of the active enzyme, therefore, they are less effective in vivo or may be even toxic as they will affect other sialidases inside of the cells. Therefore, the objective of this application is to develop location-specific inhibitor for Neu1 sialidase and define the Neu1 sialidase’s involvement in LPS/TLR4 signaling pathway, which will be accomplished by three specific aims: (1) Profile Neu1 sialidase expression and cell surface relocation in macrophages upon LPS stimulation; (2) Develop lysosome-targeting Neu1 sialidase inhibitors for effective regulating desialylation in LPS/TLR4 signaling pathway; (3) Develop cell surface-targeting Neu1 sialidase inhibitors for effective regulating desialylation in LPS/TLR4 signaling pathway. This study is innovative because it uses a novel approach that overcomes the current limitations in (a) profiling sialidase expression and relocation and (b) inhibiting sialidase at subcellular location. The proposed project is significant because it will (i) uncover specific desialylation that is critical to the LPS/TLR4 signal pathway and (ii) develop novel sialidase inhibitors for effective regulation of desialylation in LPS/TLR4 signaling pathway. Finally, this proposal will enhance the infrastructure of research and education at Cleveland State University, allowing undergraduate students to learn a broad spectrum of experimental techniques, data analysis and presentation skills used in modern scientific investigations.