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Jeffrey M. Vinocur * St. Christopher’s Hospital for Children Research Day, June 8, 2001

Mechanism of Protein Tyrosine Phosphorylation During Hypoxia in the Cerebral Cortex of Newborn Piglets. Jeffrey M. Vinocur * St. Christopher’s Hospital for Children Research Day, June 8, 2001. * coauthored with Anita C. Truttmann, Syed H. Haider,

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Jeffrey M. Vinocur * St. Christopher’s Hospital for Children Research Day, June 8, 2001

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  1. Mechanism of Protein Tyrosine Phosphorylation During Hypoxia in the Cerebral Cortex of Newborn Piglets Jeffrey M. Vinocur* St. Christopher’s Hospital for Children Research Day, June 8, 2001 * coauthored with Anita C. Truttmann, Syed H. Haider, Om P. Mishra, and Maria Delivoria-Papadopoulos

  2. Background • Hypoxic brain injury • Apoptosis • Phosphorylation

  3. Hypothesis Protein tyrosine phosphatase activity is decreased during hypoxia in cortical neuronal cell membranes of newborn piglets.

  4. Experimental Protocol Anesthesia: Halothane 4%, Nitrous oxide, Tubocurarine (0.3 mg/kg), Fentanyl (50 ug/kg), Lidocaine 1%

  5. Methods (1):Preparation of P2 Membranes • Brain tissue was homogenized in a sucrose buffer. • Homogenate was centrifuged at 1,000 g for 10 minutes, and subsequent supernatant at 40,000 g for 1 hour. • Pellet was resuspended in HEPES buffer and assayed for protein content (Lowry et al., 1951).

  6. Methods (2):ATP and PCr Determination • Hypoxia can be documented by measuring a decrease in high-energy phosphates. • Cerebral cortical tissue concentrations of ATP and PCr (phosphocreatine) were determined biochemically using the method of Lamprecht et al., 1974.

  7. phosphatase dephosphorylation p-nitrophenol (pNP) colorless yellow max absorbance at 405 nm ε = 20,000 cm-1 M-1 Methods (3):Protein Tyrosine Phosphatase Assay p-nitrophenyl phosphate (pNPP)

  8. Using bpV(phen), a specific inhibitor of protein tyrosine phosphatases, we can find PTP activity as the difference between total and inhibited activities. Methods (3):Protein Tyrosine Phosphatase Assay

  9. Data:Documentation of Hypoxia * p < 0.001 vs. normoxia Mean ± SD

  10. Data:Protein Tyrosine Phosphatase Activity 525.20 ± 64.24 * 138.75 ± 35.79 * p < 0.001 vs. normoxia Mean ± SD

  11. Summary Hypoxia results in significant decrease in protein tyrosine phosphatase activity in cortical cell membranes of newborn piglets.

  12. Speculation • The decreased protein tyrosine phosphatase activity may be due to free radicals. • Bcl-2 phosphorylation may prevent deactivation of Bax, leading to caspase-mediated apoptosis.

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