Pharmacology Of Neurotoxins

我们正在验证这一假设，即伴随着干细胞表型的发育有特定的蛋白质组变化。研究干细胞的分化过程可以阐明正常人脑发育过程中发生的复杂事件。大鼠骨髓基质细胞(BMSCs)是一种具有自我更新功能的多能干细胞，具有广阔的应用前景。在培养条件下，BMSCs可以被诱导为表达神经元标志蛋白的神经元表型。神经元表型表达与终末分化平行，使细胞分裂停止。这些研究的目的是描述具有表型变化特征的蛋白质组变化。基质细胞中的蛋白质将使用二维凝胶和质谱学技术进行表征。 我们正在寻求确定神经精神疾病的蛋白质生物标记物，例如儿童患者感染链球菌后的强迫症。免疫亲和策略的组合将被用于从患者血清中分离蛋白质。来自这些蛋白质的多肽的质谱图将被比较，以确定那些患者状态和疾病特征的特征。 我们已经开始使用串联亲和纯化(TAP)策略来阐明酵母中DNA复制复合体的必要成分。我们产生了针对POL12和Tdp1酵母基因的TAP标签。POL12基因TAP复合体将确认与已知复合体相关的蛋白质相互作用，而Tdp1复合体将定义与(Tyr-DNA磷酸二酯酶基因)密切相关的蛋白质。基于从这些亲和纯化中获得的蛋白质鉴定，我们将针对与DNA复制和Tdp1复合体相关的其他酵母基因来确认它们之间的关联。然后，与酵母中的那些基因同源的哺乳动物基因将成为靶点，这些复合体将通过质谱学得到充分的表征。这一策略将是迭代的，每个目标基因都提供了关于合成体、Tyr-DNA磷酸二酯酶的作用以及功能相互作用网络中组成蛋白的鉴定的额外确认。

We are testing the hypothesis that there are specific proteome changes accompanying stem cell phenotype development. Investigating the differentiation process of stem cells could elucidate the complex events that occur during normal human brain development. Rat bone marrow stromal cells (BMSCs) are multipotent stem cells that are self-renewing with broad potential. When grown in culture, BMSCs can be induced to a neuronal phenotype expressing neuronal marker proteins. Neuronal phenotype expression parallels terminal differentiation, halting cell division. The objective of these studies is to profile proteome changes characteristic of the phenotypic changes. Proteins from the stromal cells will be characterized using two-dimensional gel and mass spectrometric techniques. We are seeking to identify protein biomarkers of neuropsychiatric disorders, such as the obsessive compulsive syndrome that follows streptococcal infections in pediatric patients. A combination of immunoaffinity strategies will be used to separate proteins from patient sera. The mass spectra of peptides from these proteins will be compared to identify those characteristic of patient state and the diesease trait. We have started to use a tandem affinity purification (TAP) strategy to elucidate the required components of the DNA replication complex in yeast. We generated TAP tags targeting the POL12 and TDP1 yeast genes. The POL12 gene TAP complex will confirm the protein interactions associated with a known complex and the TDP1 complex will define proteins tightly associated with (tyr-DNA phosphodiesterase gene). Based upon protein identifications that are made from these affinity purifications, we will target additional yeast genes associated with DNA replication and the TDP1 complex to confirm the associations. Mammalian genes orthologous to those in yeast will then be targeted, and the complexes will be fully characterized both by mass spectrometry. This strategy will be iterative, with each targeted gene providing additional confirmation regarding the synthesome, the role of the tyr-DNA phosphodiesterase and the identification of the component proteins in a functional interaction network.