Iron homeostasis is essential for maintaining the physiological requirement for iron while preventing iron overload. Multicopper ferroxidases regulate the oxidation of Fe(II) to Fe(III), circumventing the generation of harmful hydroxyl-free radicals. Ceruloplasmin is the major multicopper ferroxidase in blood; however, hephaestin, a membrane-bound ceruloplasmin homolog, has been implicated in the export of iron from duodenal enterocytes into blood. These ferroxidases supply transferrin, the iron-carrier protein in plasma, with Fe(III). Transferrin circulates through blood and delivers iron to cells via the transferrin receptor pathway. Due to the insoluble and reactive nature of free Fe(III), the oxidation of Fe(II) upon exiting the duodenal enterocyte may require an interaction between the ferroxidase and transferrin. In Chapter 3, the putative interaction of transferrin with ceruloplasmin and a soluble form of recombinant hephaestin was investigated. Utilizing native polyacrylamide gel electrophoresis, covalent cross-linking and surface plasmon resonance, a stable interaction between the two proteins was not detected. The lack of interaction between hephaestin and transferrin prompted the investigation into the localization of hephaestin in the human small intestine. Hephaestin has been reported to have both intracellular and extracellular locations in murine tissue. In the Appendix, the location of hephaestin in human tissue was investigated using a novel polyclonal antibody. Hephaestin was localized to the basolateral membrane and an intracellular location of the enterocyte, as well as a novel location in the myenteric plexus of the duodenum. The delivery of iron to cells via the transferrin receptor pathway is well established; however, little is known about the interaction of transferrin with the transferrin receptor at the molecular level. In Chapters 4 and 5, surface plasmon resonance was employed to further characterize the binding event between transferrin and the transferrin receptor. It was found that mutations affecting iron release in transferrin did not impact receptor binding. However, when N-lobe residues predicted to form contacts with the transferrin receptor were targeted, significant changes in the transferrin receptor binding kinetics and affinity were observed.

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The study objectives were to measure the concentrations of albumin, prealbumin and transferrin in amniotic fluid (AF), and to establish if these concentrations were associated with infant birth weight (BW). At St Mary's Hospital (Montreal, Quebec), 294 AF samples were collected from mothers undergoing routine amniocentesis (12-19 weeks gestation). Exclusion criteria included subjects having gestational diabetes, multiple births or fetal genetic abnormalities. AF samples were analyzed by capillary electrophoresis (CE) at 190 nm. Analysis of variance and multiple linear regressions were performed. AF prealbumin could not be detected by CE. However, ANCOVA showed that transferrin was different among BW categories. Multiple regressions showed the parameter estimates for transferrin and albumin were negative, but neither was associated with BW in our study population. In contrast, transferrin was negatively associated with BW in our LBW infants. Our study shows that 2nd trimester AF transferrin may emerge as a biomarker for poor in-utero growth.

Iron homeostasis is essential for maintaining the physiological requirement for iron while preventing iron overload. Multicopper ferroxidases regulate the oxidation of Fe(II) to Fe(III), circumventing the generation of harmful hydroxyl-free radicals. Ceruloplasmin is the major multicopper ferroxidase in blood; however, hephaestin, a membrane-bound ceruloplasmin homolog, has been implicated in the export of iron from duodenal enterocytes into blood. These ferroxidases supply transferrin, the iron-carrier protein in plasma, with Fe(III). Transferrin circulates through blood and delivers iron to cells via the transferrin receptor pathway. Due to the insoluble and reactive nature of free Fe(III), the oxidation of Fe(II) upon exiting the duodenal enterocyte may require an interaction between the ferroxidase and transferrin. In Chapter 3, the putative interaction of transferrin with ceruloplasmin and a soluble form of recombinant hephaestin was investigated. Utilizing native polyacrylamide gel electrophoresis, covalent cross-linking and surface plasmon resonance, a stable interaction between the two proteins was not detected. The lack of interaction between hephaestin and transferrin prompted the investigation into the localization of hephaestin in the human small intestine. Hephaestin has been reported to have both intracellular and extracellular locations in murine tissue. In the Appendix, the location of hephaestin in human tissue was investigated using a novel polyclonal antibody. Hephaestin was localized to the basolateral membrane and an intracellular location of the enterocyte, as well as a novel location in the myenteric plexus of the duodenum. The delivery of iron to cells via the transferrin receptor pathway is well established; however, little is known about the interaction of transferrin with the transferrin receptor at the molecular level. In Chapters 4 and 5, surface plasmon resonance was employed to further characterize the binding event between transferrin and the transferrin receptor. It was found that mutations affecting iron release in transferrin did not impact receptor binding. However, when N-lobe residues predicted to form contacts with the transferrin receptor were targeted, significant changes in the transferrin receptor binding kinetics and affinity were observed.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph.D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Microbiology & Immunology. Includes bibliographical references.