Showing papers by "Balaraman Kalyanaraman published in 2005"
TL;DR: Analysis of the fluorescence characteristics of ethidium (E(+)) and 2-OH-E(+) strongly suggests that the currently available fluorescence methodology is not suitable for quantitating intracellular O(2)(.-).
Abstract: Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide (). By using mass spectral and NMR techniques, the chemical structure of this product was determined as 2-hydroxyethidium (2-OH-E+). By using an authentic standard, we developed an HPLC approach to detect and quantitate the reaction product of HE and formed in bovine aortic endothelial cells after treatment with menadione or antimycin A to induce intracellular reactive oxygen species. Concomitantly, we used a spin trap, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), to detect and identify the structure of reactive oxygen species formed. BMPO trapped the that formed extracellularly and was detected as the BMPO-OH adduct during use of the EPR technique. BMPO, being cell-permeable, inhibited the intracellular formation of 2-OH-E+. However, the intracellular BMPO spin adduct was not detected. The definitive characterization of the reaction product of with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of . Analysis of the fluorescence characteristics of ethidium (E+) and 2-OH-E+ strongly suggests that the currently available fluorescence methodology is not suitable for quantitating intracellular . We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable reactive oxygen species for detecting intracellular .
541 citations
TL;DR: It is concluded that selective uptake of Mito-CP into the mitochondria is responsible for inhibiting peroxide-mediated Tf-Fe uptake and apoptosis and restoration of the proteasomal function.
191 citations
TL;DR: It is concluded that mitochondria-derived ROS and calcium play a key role in stimulating DOX-induced 'intrinsic and extrinsic forms' of apoptosis in cardiac cells with Fas L expression via the NFAT signalling mechanism.
Abstract: Doxorubicin (DOX), a widely used antitumour drug, causes dose-dependent cardiotoxicity. Cardiac mitochondria represent a critical target organelle of toxicity during DOX chemotherapy. Proposed mechanisms include generation of ROS (reactive oxygen species) and disturbances in mitochondrial calcium homoeostasis. In the present study, we probed the mechanistic link between mitochondrial ROS and calcium in the embryonic rat heart-derived H9c2 cell line and in adult rat cardiomyocytes. The results show that DOX stimulates calcium/calcineurin-dependent activation of the transcription factor NFAT (nuclear factor of activated T-lymphocytes). Pre-treatment of cells with an intracellular calcium chelator abrogated DOX-induced nuclear NFAT translocation, Fas L (Fas ligand) expression and caspase activation, as did pre-treatment of cells with a mitochondria-targeted antioxidant, Mito-Q (a mitochondria-targeted antioxidant consisting of a mixture of mitoquinol and mitoquinone), or with adenoviral-over-expressed antioxidant enzymes. Treatment with GPx-1 (glutathione peroxidase 1), MnSOD (manganese superoxide dismutase) or a peptide inhibitor of NFAT also inhibited DOX-induced nuclear NFAT translocation. Pre-treatment of cells with a Fas L neutralizing antibody abrogated DOX-induced caspase-8- and -3-like activities during the initial stages of apoptosis. We conclude that mitochondria-derived ROS and calcium play a key role in stimulating DOX-induced ‘intrinsic and extrinsic forms’ of apoptosis in cardiac cells with Fas L expression via the NFAT signalling mechanism. Implications of ROS- and calcium-dependent NFAT signalling in DOX-induced apoptosis are discussed.
173 citations
TL;DR: The reaction between HE and the Fremy's salt should provide a facile route for the synthesis of 2-OH-E+, a diagnostic marker product of the HE/O2.- reaction.
92 citations
TL;DR: BMPO and DEPMPO adducts were more stable, considering the stability of their maximal signal, than DMPO adduct in the tested cellular systems, and it was observed that O2*- spin adductions were reduced to their corresponding hydroxylAdducts in the cellular system.
87 citations
TL;DR: It is concluded that a rapid intramolecular electron transfer reaction between the tyrosyl radical and the Cys residue impedes tyrosine nitration and induces corresponding thiyl radical and nitrosocysteine product.
71 citations
TL;DR: Western blot analysis demonstrated that induction of biopterin synthesis in HAECs and ARCM by LPS does not entail augmentation of constitutive GTPCH-I protein levels, suggesting that allosteric regulation of GTP CH-I activity in the cardiovascular system maybe an important mechanism regulating BH4 levels through GFRP signaling.
46 citations
TL;DR: The mechanisms of reaction defined herein allow to rationalize the biochemistry of peroxynitrite based on direct and free radical-mediated processes and contribute to the understanding of the antioxidant actions of LA and DHLA.
41 citations
TL;DR: The mechanism of dephosphorylation and activation of death-associated protein kinase (DAPK) and the role of lysosome in neuroblastoma cells treated with mitochondrial toxins, such as MPP+ and rotenone is investigated.
41 citations
TL;DR: nNOS overexpression in SH-SY5Y cells greatly enhanced proteasome activity and mitigated MPP+-induced apoptosis, and implications of BH4 depletion in dopaminergic cells and sepiapterin supplementation to augment the striatal nNOS activity in the pathogenesis mechanism and treatment of Parkinson disease are discussed.
32 citations
TL;DR: Chemical cross-linking revealed that Mn2+ influences the stoichiometry of interaction between the CD-MPR and lysosomal enzymes by increasing the oligomeric state of the receptor from dimer to higher order oligomers, and Asp-103 has been identified as the key residue which mediates the effects of divalent cations on the binding properties of the CD
Abstract: The 46 kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) plays an essential role in the biogenesis of lysosomes by diverting newly synthesized mannose 6-phosphate (Man-6-P)-containing lysosomal enzymes from the secretory pathway to acidified endosomes. Previous crystallographic studies of the CD-MPR have identified 11 amino acids within its carbohydrate binding pocket. These residues were evaluated quantitatively by assaying the binding affinity of mutant receptors containing a single amino acid substitution toward a lysosomal enzyme. The results show that substitution of Gln-66, Arg-111, Glu-133, or Tyr-143 results in a >800-fold decrease in affinity, demonstrating these four amino acids are essential for carbohydrate recognition by the CD-MPR. Solution binding and surface plasmon resonance analyses demonstrated that the presence of Mn2+ enhanced the affinity of the CD-MPR for a lysosomal enzyme by 2- to 4-fold and increased the stoichiometry of the interaction between a heterogeneous population of a lysosomal enzyme and the receptor by approximately 3-fold. In contrast, substitution of Asp-103 results in a protein that no longer exhibits enhanced binding affinities or altered stoichiometry in the presence of cations, and electron spin resonance demonstrated that the D103S mutant exhibits a 6-fold lower affinity for Mn2+ than the wild-type receptor (Kd = 3.7 6 1.4 mM versus 0.6 6 0.1 mM). Chemical cross-linking revealed that Mn2+ influences the stoichiometry of interaction between the CD-MPR and lysosomal enzymes by increasing the oligomeric state of the receptor from dimer to higher order oligomers. Taken together, these studies provide the molecular basis for high affinity carbohydrate recognition by the CD-MPR. Furthermore, Asp-103 has been identified as the key residue which mediates the effects of divalent cations on the binding properties of the CD-MPR.
TL;DR: It is concluded that the thiol oxidase activity of SOD1 stimulates carbonate anion radical (CO3*-) formation in the presence of HCO3- and that the CO3*- formed in the S OD1/Cys/ H CO3- system is responsible for oxidation and hydroxylation reactions.
Abstract: Here, we investigated the effect of bicarbonate anion (HCO3-) on the peroxidase activity stimulated by the thiol oxidase activity of copper, zinc superoxide dismutase (SOD1) using electron spin res...
01 Jan 2005
TL;DR: This chapter discusses the key role of cofactors such as tetrahydrobiopterin and protein-protein interactions in superoxide release from endothelial NOS and the pathophysiological effects of NOS-mediated superoxide generation in cardiovascular system.
Abstract: Application of the loop gap resonator technology and improvement in spin trapping methodologies have enormously increased our understanding of the mechanisms of superoxide formation from nitric oxide synthases. In this chapter we discuss the key role of cofactors such as tetrahydrobiopterin and protein-protein interactions in superoxide release from endothelial NOS. The pathophysiological effects of NOS-mediated superoxide generation in cardiovascular system are discussed.
TL;DR: While Cr(V) was inactive toward NO, spin trapping experiments with 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide and oxygen consumption measurements showed that Cr( V) reacts with superoxide by a one-electron-transfer mechanism to generate oxygen and Cr(IV).
Abstract: Chromium(VI) is a recognized toxicant whose effects have been linked to its reduction to lower oxidation states. Although Cr(VI) is reduced by several systems, it is anticipated that its reduction by nitric oxide synthase (NOS) could have significant effects in endothelial and brain cells that express high constitutive levels of the enzyme. This possibility was examined by electron paramagnetic resonance that showed the formation of a stable Cr(V) species from NOS/Cr(VI). The formation of Cr(V) was calcium/calmodulin-independent indicating that Cr(VI) to Cr(V) reduction occurs at the flavin-containing domain of NOS. Accordingly, Cr(VI) reduction by the reductase domain of NOS and the chimera protein cytochrome-P450-reductase+tail-nNOS also generated Cr(V). Activation of tetrahydrobiopterin (BH(4))-free NOS with calcium/calmodulin diminished Cr(V) steady-state levels while increasing superoxide formation. Since SOD restored Cr(V) to control levels, this result was taken as evidence for a reaction between Cr(V) and superoxide. Supplementation of NOS with BH(4) cofactor not only failed to increase Cr(V) yields but generated superoxide and hydroxyl radical. Since the holoenzyme does not generate superoxide, this reaction indicated that Cr(V) mediates the oxidation of BH(4)-bound to the enzyme. In the presence of L-arginine, however, Cr(VI) neither enhances superoxide release nor inhibits NO formation from fully active NOS. This suggests that L-arginine protects BH(4) from Cr(V)-mediated oxidation. While Cr(V) was inactive toward NO, spin trapping experiments with 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide and oxygen consumption measurements showed that Cr(V) reacts with superoxide by a one-electron-transfer mechanism to generate oxygen and Cr(IV). Thus, reduction of Cr(VI) to Cr(V) by NOS occurs in resting and fully active states. It is likely that the reaction between Cr(V) and superoxide influences the cytotoxic mechanisms of Cr(VI) in cells.
01 Jan 2005
TL;DR: EPR has been employed in attempts to understand the basis of specific pathophysiologies in which free radicals have a postulated role, including pulmonary free radical damage, free radicals and sickle cell disease, and the potential role of oxidative stress in the induction of cancer.
Abstract: EPR has been employed in attempts to understand the basis of specific pathophysiologies in which free radicals have a postulated role. Examples described include pulmonary free radical damage, free radicals and sickle cell disease, free radicals in amyotrophic lateral sclerosis, melanin and free radicals and the potential role of oxidative stress in the induction of cancer. The final section of the chapter describes the use of NMR as the spectroscopic measure of spin-trapped radicals, after they have reacted further to form diamagnetic species.
TL;DR: The interrelationship between oxidative stress, iron-signaling, and nitric oxide-dependent proteasomal function provides a rational mechanism that connects both oxidative and nitrative modifications.
Abstract: In this chapter, oxidant‐induced transferrin receptor‐mediated iron‐signaling and apoptosis are described in endothelial and neuronal cells exposed to oxidants. The role of nitric oxide in the regulation of iron homeostasis and oxidant‐induced apoptosis is described. The interrelationship between oxidative stress, iron‐signaling, and nitric oxide‐dependent proteasomal function provides a rational mechanism that connects both oxidative and nitrative modifications.
TL;DR: In this chapter, the upregulation of the proteasomal enzymes in response to nitric oxide in endothelial cells is discussed and the role of NO-dependent proteolytic signaling mechanism is important to the overall understanding of the redox signaling in oxidative endothelial cell injury.
Abstract: In this chapter, the upregulation of the proteasomal enzymes in response to nitric oxide (NO) in endothelial cells is discussed. The cytoprotective effects of NO are discussed in relation to iron homeostasis, oxidative stress, and the cellular proteasomal function. The role of NO-dependent proteolytic signaling mechanism is important to the overall understanding of the redox signaling in oxidative endothelial cell injury.