Xanthine oxidase (XO) is an important source of reactive metabolites of oxygen. There is evidence that XO may be up-regulated in the liver during sepsis and that it may play a role in pathophysiologic liver-lung interactions in ADDS. Marked differences in XO activity between species and in different organs has impeded clarification of the role of XO in humans with ADDS by obscuring the applicability of animal studies to man. We have studied the regulation of the gene encoding XO in the rat. We have found promotor sequences that may modulate both liver specific transcriptional regulation during sepsis. We propose to extend these observations to study the regulation of XDH/XO in the sheep model of LPS- induced ADDS and to the human gene. Our hypothesis is: In patients with gram negative sepsis, increased transcription of XO and release of XO from liver contribute to the pathogenesis of ADDS. The gene(s) encoding XDH/XO activity may be expressed in an organ or cell-specific manner in the liver, an may be up-regulated during sepsis.
Our specific aims are: 1: To study hepatic XO expression and release after LPS in vivo in the sheep model. We will clone the sheep XDH/XO cDNA expressed in the sheep liver, determine whether LPS in vivo induces increased transcription of the XDH/XO gene and determine if the XO is released from the liver. 2: To characterize mechanisms of organ and cell-specific expression of XDH- XO in the liver in response to LPS in sheep. We will use in situ hybridization to determine which cells in the liver express XDH both at baseline and as regulated during sepsis. Then we will clone, and sequence the sheep XDH promotor. We will identify cis-acting elements in the sheep promotor involved in sepsis-induced transcription in the sheep liver, and analyze the trans-acting factors involved in liver specific expression. 3. To clone the regulatory elements of the structural human XDH/XO gene(s). Little is known about the structure of the human gene(s), about the mechanism(s) of its tissue specific regulation, or about its altered regulation during sepsis. We will clone the human XDH/XO regulatory regions and establish the areas of presumed protein-DNA binding for the human gene. We anticipate that these studies will shed light on role of hepatic XO in ADDS, and the mechanisms(s) by which it is regulated in sepsis.
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