Natural products have been serving as rich resources of potential therapeutic agents. Many of the presently known natural products were isolated based on cytotoxicity. There are, however, a number of organic molecules that are devoid of strong cytotoxicity. DNA microarray technology can expand our ability to uncover novel compounds that have been 'invisible' to the conventional screening methods. Our long-range goal is to establish various assay methodologies for the discovery of novel therapeutic agents. The objective of this application, which is the first step in pursuit of that goal, is to determine how DNA microarray can be implemented into the isolation of natural products. The central hypothesis of this application is that transcription profiling can detect compounds that are invisible to cytotoxicity assay. This hypothesis was formulated based on the fact that not every gene is involved in the cell proliferation. The rationale for the proposed research is that, once the methodology is established, the approach is expected to facilitate the discovery of novel compounds. The method can be applied even to natural resources that have already been screened by other screening methods. In our approach, DNA microarray is employed for initial screening of crude mixtures. Once differentially expressed genes are obtained, the isolation of active compounds can be guided by more focused and less expensive methods, such as reverse-transcriptase polymerase chain reaction (RT-PCR). In order to test the central hypothesis, the following two specific aims will be pursued: 1. Identify clusters of genes differentially expressed in endothelial cells by Keishi-bukuryo-gan, a Japanese Kampo medicine formulation for conditions associated with blood stasis. 2. Determine the differences between RT-PCR and cytotoxicity-guided purification. The innovative feature of the proposed work is that the approach is based on a unique combination of specialized chemical and biological techniques, namely, microscale purification/ derivatization of natural products and transcriptome profiling using DNA microarray. It is our expectation that the current study will found the basis for an effective application of genomic tools for natural products isolation. The results from this pilot study will be significant, because the methods coming out of this study will open up new avenues for the discovery of previously unnoticed natural products.
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