The activity of superoxide dismutase (SOD) was determined in three kiwifruit (Actinidia) species including A. deliciosa, A. chinensis, and A. arguta. Among the species tested, the highest SOD activity was found in crude extracts prepared from fruit tissues of A. deliciosa. The highest enzyme activity was localized in seed, followed by locules, core and outer pericarp (OP). SOD activity in crude extract of whole fruit remained stable for at least one month when stored at -20℃. The effect of synthetic protease inhibitors (PI) on SOD activity was investigated. Supplementing crude kiwifruit extracts with PI improved SOD activity in freshly prepared extracts, and in extracts stored at 4℃, but had no effect on those stored at -20℃. Among the PI used, iodoacetamide (an inhibitor of cysteine proteases, for example, actinidin which is a principal protease found in kiwifruit) and PMSF (an inhibitor of serine proteases), had the most and least influence on SOD activity in crude kiwifruit extracts, respectively. There was a significant increase in SOD activity in kiwifruit (that were relatively firm) when the fruits were stored at low temperature (4℃). An increase in SOD activity was also correlated with a decrease in fruit firmness. Staining fruit tissues with nitroblue tetrazolium (NBT) provided evidence for stress-induced superoxide generation in kiwifruit tissues. Taken together, the changes in SOD activity and the capacity for stress-inducible superoxide production in post-harvest kiwifruit suggest that SOD might play a fundamental role in the storage life/ripening of kiwifruit.

The activity of superoxide dismutase (SOD) was determined in three kiwifruit (Actinidia) species including A. deliciosa, A. chinensis, and A. arguta. Among the species tested, the highest SOD activity was found in crude extracts prepared from fruit tissues of A. deliciosa. The highest enzyme activity was localized in seed, followed by locules, core and outer pericarp (OP). SOD activity in crude extract of whole fruit remained stable for at least one month when stored at -20℃. The effect of synthetic protease inhibitors (PI) on SOD activity was investigated. Supplementing crude kiwifruit extracts with PI improved SOD activity in freshly prepared extracts, and in extracts stored at 4℃, but had no effect on those stored at -20℃. Among the PI used, iodoacetamide (an inhibitor of cysteine proteases, for example, actinidin which is a principal protease found in kiwifruit) and PMSF (an inhibitor of serine proteases), had the most and least influence on SOD activity in crude kiwifruit extracts, respectively. There was a significant increase in SOD activity in kiwifruit (that were relatively firm) when the fruits were stored at low temperature (4℃). An increase in SOD activity was also correlated with a decrease in fruit firmness. Staining fruit tissues with nitroblue tetrazolium (NBT) provided evidence for stress-induced superoxide generation in kiwifruit tissues. Taken together, the changes in SOD activity and the capacity for stress-inducible superoxide production in post-harvest kiwifruit suggest that SOD might play a fundamental role in the storage life/ripening of kiwifruit.

Indiana University-Purdue University Indianapolis (IUPUI) / Flavocytochrome b558, the catalytic core of the phagocytic NADPH oxidase, mediates the transfer of electrons from NADPH to molecular oxygen to generate superoxide for host defense. Flavocytochrome b is a membrane heterodimer consisting of a large subunit gp91phox (NOX2) and a smaller subunit, p22phox. Localization of flavocytochrome b to the phagosome is essential for microbial killing, yet the subcellular distribution of flavocytochrome b in macrophages and how it is incorporated into macrophage phagosomes is not well characterized. In neutrophils, flavocytochrome b localizes primarily to specific granules that are rapidly mobilized to the phagosome upon stimulation. In contrast to neutrophils, macrophages do not contain specific granules, and trafficking of membrane proteins to the phagosome is more dynamic, involving fission and fusion events with endosomal compartments. We hypothesized that in macrophages, flavocytochrome b localizes to both plasma membrane and endosomal compartments that deliver flavocytochrome b to the phagosome. We generated fluorescently tagged versions of both p22phox and gp91phox, and rigorously verified their functionality in Chinese Hamster Ovary cells. Localization of flavocytochrome b was then examined in both RAW 264.7 murine macrophages and primary murine bone marrow derived macrophages (BMDM) in the presence and absence of interferon gamma (IFNg). We found that in “resting” macrophages, flavocytochrome b localizes primarily to the Rab11-positive endosome recycling compartment that recycles to the plasma membrane. In addition, phagocytosis assays showed flavocytochrome b is incorporated into the phagocytic cup and colocalized with Rab11 at the base of the cup, suggesting Rab11-positive endosomes may be involved in trafficking of flavocytochrome b between intracellular membranes and forming or nascent phagosomes. However, in IFNg activated macrophages, flavocytochrome b was localized predominantly in the plasma membrane, with little present in endosomal compartments. This shift in flavocytochrome b distribution occurred following sustained exposure to IFNg and correlated with increased flavocytochrome b protein expression and increased extracellular production of superoxide. Taken together, our results suggest the IFNg-induced redistribution of flavocytochrome b may be important for enhancing the production of superoxide at the cell surface and may be a potential new mechanism by which IFNg enhances antimicrobial activity in macrophages.

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Thesis--Wisconsin. / Vita. Includes bibliographical references.

Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 92-101). Also available via the World Wide Web.

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 127-134). Also available on the World Wide Web.

published_or_final_version / Anatomy / Master / Master of Medical Sciences

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No description available.