N-acylation of phosphatidylethanolamine (PE) with free fatty acids catalyzed by N-acyl phosphatidylethanolamine (NAPE) synthase was reported in cotyledons of 24-h-old cotton seedlings. Here I report subcellular localization of this enzyme. Differential centrifugation, sucrose density gradient fractionation,aqueous two-phase partitioning and electron microscopy techniques were utilized to elucidate subcellular site(s) of NAPE synthase. Marker enzymes were used to locate organelles in subcellular fractions. Differential centrifugation indicated that NAPE synthase is present in more than one organelle and it is a membrane bound enzyme. Sucrose density gradient fractionations indicated that NAPE synthase is present in membranes derived from endoplasmic reticulum (ER),Golgi and possibly plasma membrane (PM) but not mitochondria, glyoxysomes or plastids. Aqueous two-phase partitioning experiments with cotton and spinach tissues supported these results but Goigi appeared to be the major site of NAPE synthesis. Electron microscopy of subcellular fractions was used to examine isolated fractions to provide visual confirmation of our biochemical results. Collectively, these results indicate that NAPE is synthesized in plant ER, Golgi and possibly PM.

An in vitro model of Rapid Acceleration Impact (RAI) Injury was used to study the effects of multiple impact (220 g/impact, 3-5 sec intervals) trauma on cultures of murine CNS cells. Investigations with spinal cord cultures showed that 1) multiple impacts delivered tangential to the plane of cell growth caused neuronal death (12% after 3 impacts to 46% after 10 impacts); 2) multiple impacts delivered normal to the plane of cell growth were much less effective (8% dead after 10 impacts); 3) most neuronal death occurred within 15 minutes after injury 4) morphological changes observed included increased nuclear prominence and somal swelling; and 5) pretreatment with ketamine (0.1mM) reduced cell death from 51 to 14% and reduced somal swelling. Identical studies performed on cortical cultures revealed minimal differences between the two tissues in their response to multiple tangential impacts.