The overall goal of the proposed project is to investigate the role of thiols, particularly glutathione (GSH) and cysteine, in affecting the extent of tissue injury caused by renal ischemia and reperfusion. Although extensively studied for the past two decades, ischemic injury to the kidney constitutes the most common cause of acute renal failure in hospitalized patients and causes significant morbidity and mortality. The overall mortality of patients suffering from acute renal failure is approximately 50%, a statistic that has remained unchanged for the past decade. The involvement of glutathione (GSH) in renal ischemia will be investigated since the metabolism of this intracellular thiol serves as an important defense mechanism to protect cells from oxidative stress by quenching free radical chain reactions. Recent studies have clearly indicated that at least one component of ischemic injury is due to free radical-mediated lipid peroxidation. Using an in vivo model of renal ischemia, we demonstrated that renal levels of GSH are depleted rapidly during ischemia. This loss of GSH was observed to closely parallel an accumulation of cysteine in the kidney, which was subsequently lost from the tissue upon resumption of blood flow. In order to assess the significance of the loss of GSH on the functional recovery of the kidney, methods were developed to after the level of renal GSH. Surprisingly, prior elevation of GSH with the monoethylester of GSH led to a greater degree of ischemic injury. Proposed experiments will test the hypothesis that the elevated GSH levels cause a recruitment and activation of granulocytes into the postischemic tissue that subsequently caused the enhanced tissue damage. Since cysteine is a considerably more reactive thiol that is GSH, additional experiments will test the hypothesis that the oxidation of cysteine, which accumulates during ischemia, leads to generation of oxygen free radicals during the initial period of blood reflow. Other experiments will evaluate if cysteine or GSH is utilized during this period to oxidize critical sulfhydral groups present in enzymes. Antibodies against xanthine oxidase will be utilized to assess if this enzyme, known to be generated by thiolation of the dehydrogenase form, is produced in the kidney during ischemia and/or blood reflow. R01A20279 In the fluorescent pseudomonads an independent 3-gene cluster is responsible for producing tryptophan synthase. In addition to the 2-gene operon encoding the enzyme's alpha and beta polypeptides, a divergently transcribed activator gene (trpI) belonging to a new family of bacterial regulators mediates induction of the enzyme by its substrate, indoleglycerol phosphate. We will examine the mechanism of this activation through: purification of the trpI gene product to homogeneity; identification of its DNA-binding domain by evolutionary comparison, directed mutagenesis and partial proteolysis; identification of its chromosomal recognition site by evolutionary comparison, deletion analysis and both spontaneous and directed mutagenesis; development of a filter-binding assay to complement the available footprinting and gel retardation assays; making fusions to place other genes (trpEGDC and xylE) under trpI control. In addition we will investigate the generality of the recently described """"""""tunnel"""""""" connecting the two active sites in the three- dimensional structure of Salmonella typhimurium tryptophan synthase by examining its relationship to an unusual class of """"""""repairable"""""""" trpB mutants studied earlier. We will: clone and sequence most of the existing mutants (using the polymerase chain reaction) to establish the nature and location of the amino acid substitution; overproduce selected mutant proteins by use of a high-expression vector; characterize the extent of """"""""repairability"""""""" by the trpA gene product and ammonium ions; correlate the enzymatic defect with the ability of normal and borohydride-reduced beta 2 subunits to bind indole.
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| Yelamarty, R V; Miller, B A; Scaduto Jr, R C et al. (1990) Three-dimensional intracellular calcium gradients in single human burst-forming units-erythroid-derived erythroblasts induced by erythropoietin. J Clin Invest 85:1799-809 |
| Slusser, S O; Grotyohann, L W; Martin, L F et al. (1990) Glutathione catabolism by the ischemic rat kidney. Am J Physiol 258:F1546-53 |
