title: Modulation of proline-rich akt substrate survival signaling pathways by oxidative stress in mouse brains after transient focal cerebral ischemia.
abstract: OBJECTIVE
  A proline-rich Akt substrate (PRAS) contributes to the regulation of apoptosis after a variety of cell death stimuli, as well as in an in vivo transient focal cerebral ischemia (tFCI) model. We reported previously that overexpression of copper/zinc-superoxide dismutase (SOD1) reduces apoptotic cell death after tFCI. Our present study was designed to clarify the relationship between the PRAS signaling pathway and oxidative stress in the regulation of apoptosis after tFCI.
  
  
  METHODS
  We used a tFCI model with SOD1 transgenic mice and wild-type littermates to examine the expression of phosphorylated PRAS (pPRAS) by Western blotting and immunohistochemistry and the interaction of pPRAS with phosphorylated Akt (pPRAS/pAkt) or the 14-3-3 protein (pPRAS/14-3-3) by coimmunoprecipitation. Direct oxidation of the carbonyl groups, an indication of oxidative injury to total and individual proteins caused by tFCI, was examined using a 2,4-dinitrophenylhydrazone reaction assay.
  
  
  RESULTS
  Expression of pPRAS, pPRAS/pAkt, and pPRAS/14-3-3 decreased 2 hours after tFCI. Oxidized hydroethidine did not colocalize with expression of pPRAS. Individual oxidized carbonyls in pPRAS remarkably increased 2 hours after tFCI but were significantly reduced by SOD1 2 hours after tFCI. Expression of pPRAS, pPRAS/pAkt, and pPRAS/14-3-3 was promoted by SOD1 during the same time course.
  
  
  CONCLUSIONS
  These results suggest that overexpression of SOD1 may affect the PRAS pathway after tFCI by reducing the direct oxidative reaction to pPRAS after reperfusion injury.
authors.affiliation: Department of Neurosurgery, Program in Neurosciences, Stanford University School of Medicine, California, USA.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Saito
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hayashi
authors.forename: Shuzo
authors.initials: S
authors.lastname: Okuno
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Nishi
authors.forename: Pak H
authors.initials: PH
authors.lastname: Chan
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D001921:Brain / Q000473:pathology*; D002545:Brain Ischemia / Q000473:pathology*; D006579:Heterozygote; D006835:Hydrazones / Q000737:chemistry; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D020287:In Situ Nick-End Labeling; D002546:Ischemic Attack, Transient / Q000473:pathology; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000502:physiology*; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction; D013482:Superoxide Dismutase / Q000378:metabolism; D013997:Time Factors
chemical_list: D048948:14-3-3 Proteins; C001072:2,4-dinitrophenylhydrazone; C484523:AKT1S1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D006835:Hydrazones; D010750:Phosphoproteins; C508191:proline-rich Akt substrate, 40 kDa protein, mouse; D011392:Proline; D013482:Superoxide Dismutase; D051057:Proto-Oncogene Proteins c-akt; D010100:Oxygen