(Taken from application) The broad objective of the proposed research is to demonstrate the importance of specific intestinal bacteria in the metabolism of xenobiotics. The principle hypothesis which is to be tested is: intestinal microbes possess a cytochrome P450 system, which may be a new family of cytochromes P450, and this system is used to metabolize various xenobiotics present in the intestine.
The specific aims are (1) to use the Polymerase Chain Reaction (PCR) technique to amplify the constant heme binding region of the cytochrome P450 gene from intestinal microorganisms, (2) to clone and sequence the gene encoding for cytochrome P450 in intestinal microbes and (3) to purify and characterize cytochrome P450 from specific intestinal microorganisms. The proposed studies will screen specific intestinal strains as well as mixed cultures from human feces for the presence of the cytochrome P450 gene. Molecular biology techniques including PCR, Southern Blot, nucleotide and amino acid sequencing will be used to detect and identify the cytochrome P450 gene. Biochemical techniques such as the difference spectrum will be employed to detect functional cytochrome P450 and its ability to bind substrate. The proposed research will demonstrate the presence of cytochromes P450, which is associated with xenobiotic metabolism, in intestinal bacteria. These studies will also determine if new and unique isoforms of cytochrome P450 exist, which will either validate or contradict the current modeling studies. Finally, analysis of the specific enzymatic activity of the newly isolated cytochrome P450 will allow testing of various xenobiotics to which humans are exposed on a daily basis, and determine what effects these metabolites will have on the surrounding gastrointestinal environment and other parts of the body.
| Stamm, W E; Norrby, S R (2001) Urinary tract infections: disease panorama and challenges. J Infect Dis 183 Suppl 1:S1-4 |
