Peroxynitrite, a highly oxidative molecule, plays a role in neuronal cell death following traumatic brain injury (TBI). A peptide comprised of the HIV-1 tat transduction domain fused to nine cysteine residues (Tat-9c) was previously designed to act as an exogenous target for nitrosylation by peroxynitrite. The present study’s aim was to explore the efficacy of Tat-9c in maintaining neurological function following TBI. Rats treated with Tat-9c exhibited significant improvement in performance compared to controls 24 hrs following TBI in the Beam-Walk task but not in the Rota-Rod task. Injured animals, given the drug, show a recovery as indicated by similar performance on the Morris Water Maze task compared to sham controls. These findings suggest Tat-9c may constitute a potential therapy for improving motor and cognitive function following TBI.

Peroxynitrite, a highly oxidative molecule, plays a role in neuronal cell death following traumatic brain injury (TBI). A peptide comprised of the HIV-1 tat transduction domain fused to nine cysteine residues (Tat-9c) was previously designed to act as an exogenous target for nitrosylation by peroxynitrite. The present study’s aim was to explore the efficacy of Tat-9c in maintaining neurological function following TBI. Rats treated with Tat-9c exhibited significant improvement in performance compared to controls 24 hrs following TBI in the Beam-Walk task but not in the Rota-Rod task. Injured animals, given the drug, show a recovery as indicated by similar performance on the Morris Water Maze task compared to sham controls. These findings suggest Tat-9c may constitute a potential therapy for improving motor and cognitive function following TBI.

Peroxynitrite, a highly oxidative molecule, plays a role in neuronal cell death following traumatic brain injury (TBI). A peptide comprised of the HIV-1 tat transduction domain fused to nine cysteine residues (Tat-9c) was previously designed to act as an exogenous target for nitrosylation by peroxynitrite. The present study’s aim was to explore the efficacy of Tat-9c in maintaining neurological function following TBI. Rats treated with Tat-9c exhibited significant improvement in performance compared to controls 24 hrs following TBI in the Beam-Walk task but not in the Rota-Rod task. Injured animals, given the drug, show a recovery as indicated by similar performance on the Morris Water Maze task compared to sham controls. These findings suggest Tat-9c may constitute a potential therapy for improving motor and cognitive function following TBI.

Peroxynitrite, a highly oxidative molecule, plays a role in neuronal cell death following traumatic brain injury (TBI). A peptide comprised of the HIV-1 tat transduction domain fused to nine cysteine residues (Tat-9c) was previously designed to act as an exogenous target for nitrosylation by peroxynitrite. The present study’s aim was to explore the efficacy of Tat-9c in maintaining neurological function following TBI. Rats treated with Tat-9c exhibited significant improvement in performance compared to controls 24 hrs following TBI in the Beam-Walk task but not in the Rota-Rod task. Injured animals, given the drug, show a recovery as indicated by similar performance on the Morris Water Maze task compared to sham controls. These findings suggest Tat-9c may constitute a potential therapy for improving motor and cognitive function following TBI.

Thesis (Ph. D.)--University of Chicago, Department of Neurobiology, Pharmacology, and Physiology, 2001. / Includes bibliographical references. Also available on the Internet.

No description available.

Monomethylmercury (CH3Hg+ and its complexes; hereafter referred to as MeHg) in the intracellular environment is known to be predominantly bonded to thiol-containing biomolecules but the identities of these target biomolecules remain unknown. Some evidence suggests that binding with glutathione acts as a detoxification mechanism for MeHg, while binding with L-cysteine permits MeHg transport across the blood–brain barrier resulting in neurotoxicity. However, the occurrence of these complexes in biological tissues has not been confirmed analytically, and little is known about their kinetic stability. In this thesis, methylmercury L-cysteinate (CH3HgCys) and methylmercury L-glutathionate (CH3HgGlu) were synthesized and structurally characterized by proton nuclear magnetic resonance (1H NMR), electrospray ionization mass spectrometry (ESI-MS), and X-ray crystallography. A new analytical method was developed combining high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICPMS). The method was capable of separating and analyzing CH3HgCys and CH3HgGlu complexes, as well as CH3HgX and inorganic HgX (X = H2O, OH-, or Cl-), with detection limits at the sub-micromolar levels. Using a new enzymatic hydrolysis method to isolate MeHg species in biological tissues, the HPLC-ICPMS method was successfully applied for the determination of MeHg speciation in the muscle tissue of dogfish (Squalus acanthius). These results provide the first analytical evidence for the presence and dominance of CH3HgCys in fish muscle. The analytical method was also used to study the kinetic stability of CH3HgCys and CH3HgGlu under a range of environmental and intracellular conditions. In general, CH3HgGlu was more stable than CH3HgCys under light exposure or darkness. The stability of both compounds decreases dramatically with increasing ionic strength (I). Half-life of CH3HgCys decreases from 34.1 h (I = 0.01 M) to 5.9 h (I = 0. 5 M) and the half-life of CH3HgGlu decreases from 259 h (I = 0.01 M) to 35.9 h (I = 0. 5 M). Suggesting major differences in their cycling in freshwater (I < 0.01M), seawater (I ≈ 0.7M) and body fluids (I ≈ 0.16 M). The analytical technique and the findings from this thesis research provide a new analytical framework for the study of MeHg speciation in natural waters, and the metallomics of MeHg in biological systems.

Monomethylmercury (CH3Hg+ and its complexes; hereafter referred to as MeHg) in the intracellular environment is known to be predominantly bonded to thiol-containing biomolecules but the identities of these target biomolecules remain unknown. Some evidence suggests that binding with glutathione acts as a detoxification mechanism for MeHg, while binding with L-cysteine permits MeHg transport across the blood–brain barrier resulting in neurotoxicity. However, the occurrence of these complexes in biological tissues has not been confirmed analytically, and little is known about their kinetic stability. In this thesis, methylmercury L-cysteinate (CH3HgCys) and methylmercury L-glutathionate (CH3HgGlu) were synthesized and structurally characterized by proton nuclear magnetic resonance (1H NMR), electrospray ionization mass spectrometry (ESI-MS), and X-ray crystallography. A new analytical method was developed combining high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICPMS). The method was capable of separating and analyzing CH3HgCys and CH3HgGlu complexes, as well as CH3HgX and inorganic HgX (X = H2O, OH-, or Cl-), with detection limits at the sub-micromolar levels. Using a new enzymatic hydrolysis method to isolate MeHg species in biological tissues, the HPLC-ICPMS method was successfully applied for the determination of MeHg speciation in the muscle tissue of dogfish (Squalus acanthius). These results provide the first analytical evidence for the presence and dominance of CH3HgCys in fish muscle. The analytical method was also used to study the kinetic stability of CH3HgCys and CH3HgGlu under a range of environmental and intracellular conditions. In general, CH3HgGlu was more stable than CH3HgCys under light exposure or darkness. The stability of both compounds decreases dramatically with increasing ionic strength (I). Half-life of CH3HgCys decreases from 34.1 h (I = 0.01 M) to 5.9 h (I = 0. 5 M) and the half-life of CH3HgGlu decreases from 259 h (I = 0.01 M) to 35.9 h (I = 0. 5 M). Suggesting major differences in their cycling in freshwater (I < 0.01M), seawater (I ≈ 0.7M) and body fluids (I ≈ 0.16 M). The analytical technique and the findings from this thesis research provide a new analytical framework for the study of MeHg speciation in natural waters, and the metallomics of MeHg in biological systems.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitous membrane protein that exchanges one intracellular H+ for an extracellular Na+, thereby regulating cell pH and volume. NHE1 catalytic activity is mediated by a transmembrane (TM) domain with 12 transmembrane segments. We performed cysteine scanning mutagenesis on TMVI (Asn227–Ile249) of NHE1. Each residue of TMVI was mutated into a cysteine in the background of a cysteineless NHE1 protein. MTSET and MTSES are sulfhydryl reactive membrane impermeable compounds able to react with accessible cysteines. Asp238Cys, Pro239Cys, and Glu247Cys expressed inactive NHE1. Asn227Cys, Ile233Cys, and Leu243Cys were strongly inhibited by MTSET, suggesting their pore lining properties. More mutations were introduced to characterize critical residues in TMVI. The Glu248Gln and Leu243Ala mutants were more susceptible to limited proteolytic attack by trypsin suggesting an altered conformation. The results suggest that Glu248 and Leu243 are important in protein structure, stability, and folding.

Proteases are enzymes that cleave protein amide bonds. Proteases are involved in a myriad of biological processes and are considered good targets for drug design. The proteases described herein are cysteine proteases, which utilize a cysteine residue thiol to attack the amide carbonyl, leading to amide bond cleavage. Irreversible inhibitors of cysteine proteases react with the active site cysteine, forming a covalent bond and rendering the enzyme inactive. The first project involved the design and synthesis of aza-peptide epoxide inhibitors for calpain, a clan CA, ubiquitous, calcium-activated human enzyme involved in neurodegeneration. These inhibitors proved to be poor inactivators of calpain, demonstrating that the aza-peptide epoxide is a warhead specific to clan CD cysteine proteases (caspases, gingipains). Subsequently, a known epoxide inhibitor of calpain was optimized to create a more potent inhibitor. Several of these inhibitors were more potent than the parent, and all were demonstrated to inhibit calpain in a breast cancer cell line which was treated with paclitaxel to spike calpain activity. The second project involved the design and solid phase synthesis of aza-peptide Michael acceptor caspases inhibitors. The two goals of this project were to develop a solid phase method for synthesis of inhibitors that are tedious to synthesize in solution phase, and to use a variety of amino acid residues to determine the optimal interactions in the P3? position for various caspases. The synthesis was successful, and the optimal P3? residues were determined. The third project involved the kinetic evaluation of aza-peptide epoxide and Michael acceptor inhibitor designed for the gingipains. Gingipains K and R are virulence factors in the pathology of Porphyromonas gingivalis involved in gingivitis and periodontal disease. These inhibitors proved to be extremely potent inactivators of gingipains, with some of the highest rates of inhibition measured in the Powers laboratory. Gingipain K preferred larger, aromatic moieties in the P1? position, while gingipain R preferred the Michael acceptor inhibitors, with the P1? substituent having less of an impact on potency.