Jun Du, M.Sc. Chemistry, 2005

Anti-apoptotic and antioxidant defenses in the freeze tolerant wood frog, Rana sylvatica.

Abstract: Multiple biochemical adaptations support natural freeze tolerance by wood frogs, Rana sylvatica. The present research explored the role of anti-apoptotic and antioxidant defenses in organ survival of freeze/thaw stresses using PCR and Western blotting to analyze the expression of selected genes and proteins. The STAT family of transcription factors mediate both pro- and anti-apoptotic gene responses. Elevated amounts of phosphorylated (active) Stat5 (Tyr694) and/or phospho-Stat3 (Ser727) in selected frog organs during freeze/thaw suggest activation of anti-apoptotic defenses to help organs recover from metabolic insults caused by freezing. However, levels of phospho-Stat1 (Tyr701), a pro-apoptotic signal, also rose in kidney and muscle during thawing. Increased amounts of anti-apoptotic proteins including Bcl-2 and phospho- Bcl-2 (Ser70) in liver and skeletal muscle and Bcl-xL (and phospho-Bcl-2) in kidney could help counteract freeze-induced apoptotic signals that were evidenced by higher levels of Bad protein (liver, muscle, kidney) and phospho-Bad (Ser112) (kidney) and enhanced DNA laddering. Antioxidant defense via glutathione S-transferase (GST) was evaluated by analyzing the expression of GST isozymes. GST Pi protein rose in four organs during freeze/thaw and GST Pi mRNA was freeze up-regulated in liver. GST M1/2, M5, A3 and A5 were freeze- or thaw- responsive in selected organs. Freeze-induced changes in the transcription factors, Nrf2 and MafG, and elevated MafG in the nucleus suggest that these regulate the freeze up-regulation of antioxidant enzymes by targeting the antioxidant response element of genes. Both anti-apoptotic and antioxidant defenses are important aspects of natural freezing survival.