NADH:Cytochrome b5 Reductase (cb5r) catalyzes the reduction of the ferric iron (Fe3+) atom of the heme cofactor found within cytochrome b5 (cb5) by the reduction of the FAD cofactor of cb5r from reducing equivalents of the physiological electron donor, reduced nicotinamide adenine dinucleotide (NADH). Cb5r is characterized by the presence of two domains necessary for proper enzyme function: a flavin-binding domain and a pyridine nucleotide-binding domain. Within these domains are highly conserved "motifs" necessary for the correct binding and orientation of both the NADH coenzyme and the FAD cofactor. To address the importance of these conserved motifs, site-directed mutagenesis was utilized to generate a series of variants of residues located within the motifs to allow for the full characterizations. Second, naturally occurring recessive congenital methemoglobinemia (RCM) mutants found in proximity to these highly conserved motifs were analyzed utilizing site-directed mutagenesis. In addition, a canine variant of the cb5r soluble domain was cloned, generated and characterized and compared with the WT rat domain. The canine construct showed a high degree of sequence homology to that of the corresponding human and rat sequences. Characterization of the canine variant indicated that it possessed comparable functional characteristics to the rat variant. Investigation of the pyrophosphate-associating residues, Y112 and Q210, indicated that each played a role in the proper association and anchoring of NADH to the enzyme. The RCM type I mutants, T116S and E212K, caused a moderate decrease in efficiency of the enzyme. The presence of both mutations interact synergistically to generate a more substantially decreased function Analysis of the "180GtGitP185" NADH-binding motif and the preceding residue G179 revealed that these residues are vital in enabling proper NADH association. The residues of this motif were shown to be important in determining nucleotide specificity and properly positioning the NADH and flavin cofactor for efficient electron transfer. RCM variants A178T and A178V were shown to decrease catalytic efficiency or protein stability respectively, leading to disease phenotype. Analysis of the NADH-binding motif "273CGxxxM278" indicated that this motif facilitates electron transfer from substrate to cofactor and is important in release of NAD+ from the enzyme after electron transfer.

NADH:Cytochrome b5 Reductase (cb5r) catalyzes the two electron reduction of the iron center of the heme cofactor found within cytochrome b5 (cb5) utilizing reducing equivalents of the nicotinamide adenine dinucleotide (NADH) coenzyme. Cb5r is characterized by two domains necessary for proper enzyme function: a flavin-binding domain and a pyridine nucleotide-binding domain. Within these domains are highly conserved "motifs" necessary for the proper binding and orientation of both the NADH coenzyme and the FAD cofactor. To address the importance of these conserved motifs site-directed mutagenesis was utilized to generate a series of variants upon residues found within the motifs to allow for the full characterizations. Second, naturally occurring recessive congenital methemoglobinemia (RCM) mutants that are found within or in close proximity to these highly conserved motifs were analyzed utilizing site-directed mutagenesis. The flavin-binding motif "91RxYSTxxSN97" was characterized by the generation of variants T94H, T94G, T94P, P95I, V96S, and S97N. In addition to this, the naturally occurring double mutant P92H/E255- was fully characterized to establish a role of the P92 residue giving rise to RCM. The role of the "124GRxxST127" was determined by the introduction of a positive charge, charge reversal, and conserved amino acid mutations through site-directed mutagenesis of the G124, K125, and M126 residues. Based on the data presented here, each of the residues of the GRxxST motif are directly involved in maintaining the proper binding and orientation of the cb5r flavin prosthetic group. Analysis of the NADH-binding motif "273CGxxx-M278" was accomplished through the characterization of the type II RCM variant M272- and the type I RCM variant P275L. This demonstrates that the deletion of the M272 residue causes a frame shift leading to the inability of the NADH substrate to bind. The introduction of the P275L variant showed that substrate affinity was diminished, yet turnover was comparable to wild-type cytochrome b5 reductase, indicating that although P275 is required for proper substrate binding it is not essential for overall catalytic function. Finally, analysis of the naturally occurring double mutant G75S/V252M provided the first insight into a methemoglobinemia variant that possessed mutations in both the FAD-binding and NADH-binding domains.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

A research program was developed to investigate basic and applied aspects of toxicity, both lethal and sublethal, of hemoxidants, particularly nitrite, on fish, non-fish aquatic vertebrates, and crayfish. The major objectives of this research were to determine A) acute and sublethal toxicity of nitrite to selected aquatic organisms: 1. aquatic salamander larvae, Ambystoma texanum, 2. swamp crayfish, Procambarus simulans, 3. bluegill, Lepomis macrochirus, 4. bullfrog, tadpoles, Rana catesbiana, 5. channel catfish, Ictalurus punctatus, B) the influence of environmental chloride on acute and sublethal exposures to hemoxidants: 1. on acute nitrite toxicity to salamander larvae, crayfish, and bluegill, 2. on nitrite-induced methemoglobinemia in bullfrog tadpoles, Rana catesbian, C) the effect of environmental hydrogen ion concentrations (pH) on acute nitrite toxicity 1. to the crayfish, Procambarus simulans, 2. to the bluegill, Lepomis macrochirus, D) the effect of temperature in sublethal exposures to nitrite 1. methemoglobin formation in channel catfish exposed at different acclimation temperatures, 2. recovery from methemoglobinemia at different acclimation temperatures, E) the effect of the fish anesthetic TMS-222 on nitrite-induced methemoglobinemia in channel catfish 1. supression of nitrite-induced methemoglobinemia, 2. dose-response curve for TMS-222 induced methemoglobinemia, and F) if a methemoglobin reductase system is present in channel catfish.

Orientador : Felix Guillermo Reyes Reyes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-04T02:35:55Z (GMT). No. of bitstreams: 1 Elias_EledeMartins_M.pdf: 3405812 bytes, checksum: 457a65d1aaa744e4deed5d27239bf848 (MD5) Previous issue date: 2005 / Mestrado / Ciência de Alimentos / Mestre em Ciência de Alimentos

We describe a patient who developed hemolysis and methemoglobinemia due to rasburicase (RBU) and was found to have glucose-6-phosphate dehydrogenase (G6PD) deficiency. This is a rare clinical scenario that provides valuable insight into complex diagnosis and management of these life-threatening complications. A 59-year-old male presented to the VA Medical Center with 11 days of epigastric abdominal pain radiating to the back and flank, associated with bloating and lower extremity edema. He was transferred to our facility for percutaneous nephrostomy tube (PCN) placement for renal dysfunction (creatinine 5.5). Computed tomography (CT) scan of the abdomen and pelvis revealed 17 cm retroperitoneal mass engulfing major vessels, involving right renal hilum and ureter with moderate-severe right hydronephrosis. CT guided biopsy of the mass showed intermediate-large malignant cells that were CD20, CD23, BCL2 and BCL6 positive; CD10 and MUM1 negative. Fluorescent in-situ hybridization resulted after discharge to reveal no MYC rearrangement, confirming a diagnosis of diffuse large B-cell lymphoma. Positron-emission tomography CT revealed extensive retroperitoneal lymphadenopathy with pelvic extension into internal and external iliac vessels, encasing aorta, inferior vena cava and anteriorly displacing pancreas and bowel and contiguous involvement of the right kidney with hypermetabolic activity. Moderate right sided pleural effusion was also seen. Creatinine improved with PCN. Uric acid was 10.3 with lactate dehydrogenase (LDH) 953. The patient received RBU for tumor lysis syndrome (TLS). However, pulse oximetry showed an oxygen saturation of 70-80%, though the patient had only mild dyspnea. CT pulmonary embolism (CTPE) showed segmental PE. Therapeutic lovenox was initiated. He underwent thoracentesis with symptomatic improvement but continued to desaturate on pulse oximetry. Arterial blood gas on 100% oxygen via non-rebreather revealed methemoglobin of 4.5% without hypoxemia. LDH worsened to 1668 with low haptoglobin and direct hyperbilirubinemia, suggestive of hemolysis. G6PD was deficient at 0.8 U/g. Treatment was conservative with cautious use of red cell transfusions and supplemental oxygen. Due to hyperbilirubinemia, chemotherapy was started with dose-adjusted etoposide, prednisone, oncovin, cyclophosphamide, and rituximab - with adriamycin withheld upfront. As hemolysis improved the patient received dose-reduced adriamycin. RBU is a recombinant urate oxidase used in managing TLS. It converts uric acid to allantoin, producing hydrogen peroxide, which oxidizes hemoglobin to methemoglobin. G6PD deficiency decreases cellular ability to reduce glutathione and thus detoxify hydrogen peroxide. This causes life-threatening methemoglobinemia and hemolysis. Methylene blue is contraindicated due to the risk of worsening hemolysis in G6PD deficiency. Methemoglobinemia is typically treated in such cases with exchange transfusion or hyperbaric oxygen therapy.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 197 pages. Includes vita. Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references (leaves 188-197). Also available online.