Biomedical subjects

Jay L Zweier

Publications and source records attributed to Jay L Zweier.

At least 37 records · Page 2Linked to original sources

PubMed2006

Hydroxylation of D-phenylalanine as a novel approach to detect hydroxyl radicals: application to cardiac pathophysiology.

OBJECTIVE: Research in the pathophysiology of ischemia/reperfusion or redox signaling is hindered by lack of simple methodology to measure short-lived oxygen radicals. In the presence of hydroxyl radical ((*)OH), d-phenylalanine (d-Phe) yields para-, meta- and ortho-tyrosine. We have previously demonstrated that d-Phe can accurately detect (*)OH formation in chemical, enzymatic and cellular systems by simple HPLC methodology [Anal Biochem 290:138;2001]. In the present study, we tested whether d-Phe hydroxylation can be used to detect (*)OH formation in intact organs. METHODS: Rat hearts were perfused with buffer containing 5 mM d-Phe and subjected to 30 min of total global ischemia at 37 degrees C followed by 45 min of reperfusion. Quantitative analysis of the three hydroxytyrosine isomers was achieved by HPLC-based electrochemical detection of cardiac venous effluent, with the analytical cells operating in the oxidative mode. The detection limit of this assay was <10 fmol. RESULTS: Under baseline conditions, hydroxytyrosine release from the heart was very low ( congruent with0.8 nmol/min/g). However, a prominent tyrosine burst occurred immediately upon post-ischemic reflow. In cardiac effluent collected 40 s into reperfusion, the hydroxytyrosine concentration was more than 40 times greater than at baseline; hydroxytyrosine concentration then progressively declined, to return to pre-ischemic values by 5 min of reperfusion. In parallel experiments, formation of hydroxytyrosines was markedly reduced in hearts reperfused in the presence of the (*)OH scavenger mannitol. Inclusion of 5 mm d-Phe in the perfusion medium altered neither basal cardiac function nor coronary vascular tone, but it enhanced recovery of myocardial function during post-ischemic reperfusion, consistent with direct reaction with (*)OH. CONCLUSION: Our results demonstrate that d-Phe is a sensitive method for detection of (*)OH generation in the heart. Since d-Phe is not a substrate for endogenous enzymes, it can be exploited as a reliable method to measure (*)OH formation under a variety of pathophysiological conditions.

Animals↗

PubMed2006

Direct and indirect roles of cytochrome b in the mediation of superoxide generation and NO catabolism by mitochondrial succinate-cytochrome c reductase.

Mitochondrial superoxide (O2*-) production is an important mediator of oxidative cellular injury. Succinate-cytochrome c reductase (SCR) of the electron transport chain has been implicated as an essential part of the mediation of O2*- generation and an alternative target of nitric oxide (NO) in the regulation of mitochondrial respiration. The Q cycle mechanism plays a central role in controlling both events. In the present work, O2*- generation by SCR was measured with the EPR spin-trapping technique using DEPMPO (5-diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide) as the spin trap. In the presence of succinate, O2*- generation from SCR was detected as the spin adduct DEPMPO/*OOH. Inhibitors of the Q(o*-) site only marginally reduced (20-30%) this O2*- production, suggesting a secondary role of Q(o*-) in the mediation of O2*- generation. Addition of cyanide significantly decreased (approximately 70%) O2*- production, indicating the involvement of the heme component. UV-visible spectral analysis revealed that oxidation of ferrocytochrome b was accompanied by cytochrome c(1) reduction, and the reaction was mediated by the formation of an O2*- intermediate, indicating a direct role for cytochrome b in O2*- generation. In the presence of NO, DEPMPO/*OOH production was progressively diminished, implying that NO interacted with SCR or trapped the O2*-. The consumption of NO by SCR was investigated by electrochemical detection using an NO electrode. In the presence of succinate, SCR-mediated NO consumption was observed and inhibited by the addition of superoxide dismutase, suggesting the involvement of O2*-. Under the conditions of argon saturation, the NO consumption rate was not enhanced by succinate, suggesting a direct role for O2*- in the mediation of NO consumption. In the presence of succinate, oxidation of the ferrocytochrome b moiety of SCR was accelerated by the addition of NO, and was inhibited by argon saturation, indicating an indirect role for cytochrome b in the mediation of NO consumption.

Animals↗

PubMed2006

Characterization of the mechanism of cytochrome P450 reductase-cytochrome P450-mediated nitric oxide and nitrosothiol generation from organic nitrates.

Mammalian cytochrome P450 reductase (CPR) and cytochrome P450 (CP) play important roles in organic nitrate bioactivation; however, the mechanism by which they convert organic nitrate to NO remains unknown. Questions remain regarding the initial precursor of NO that serves to link organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of CPR-CP-mediated organic nitrate bioactivation, EPR, chemiluminescence NO analyzer, NO electrode, and immunoassay studies were performed. With rat hepatic microsomes or purified CPR, the presence of NADPH triggered organic nitrate reduction to NO2(-). The CPR flavin site inhibitor diphenyleneiodonium inhibited this NO2(-) generation, whereas the CP inhibitor clotrimazole did not. However, clotrimazole greatly inhibited NO2(-)-dependent NO generation. Therefore, CPR catalyzes organic nitrate reduction, producing nitrite, whereas CP can mediate further nitrite reduction to NO. Nitrite-dependent NO generation contributed <10% of the CPR-CP-mediated NO generation from organic nitrates; thus, NO2(-) is not the main precursor of NO. CPR-CP-mediated NO generation was largely thiol-dependent. Studies suggested that organic nitrite (R-O-NO) was produced from organic nitrate reduction by CPR. Further reaction of organic nitrite with free or microsome-associated thiols led to NO or nitrosothiol generation and thus stimulated the activation of sGC. Thus, organic nitrite is the initial product in the process of CRP-CP-mediated organic nitrate activation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.

Animals↗

PubMed2006

The role of oxidants and free radicals in reperfusion injury.

While timely reperfusion of acute ischemic myocardium is essential for myocardial salvage, reperfusion results in a unique form of myocardial damage. Functional alterations occur, including depressed contractile function and decreased coronary flow as well as altered vascular reactivity. Both myocardial stunning and infarction are seen. Over the last two decades, it has become increasingly clear that oxidant and oxygen radical formation is greatly increased in the post-ischemic heart and serves as a critical central mechanism of post-ischemic injury. This oxidant formation is generated through a series of interacting pathways in cardiac myocytes and endothelial cells and triggers subsequent leukocyte chemotaxis and inflammation. Nitric oxide (NO) production and NO levels are also greatly increased in ischemic and post-ischemic myocardium, and this occurs through NO synthase (NOS)-dependent NO formation and NOS-independent nitrite reduction. Recently, it has been shown that the pathways of oxygen radical and NO generation interact and can modulate each other. Under conditions of oxidant stress, NOS can switch from NO to oxygen radical generation. Under ischemic conditions, xanthine oxidase can reduce nitrite to generate NO. NO and peroxynitrite can inhibit pathways of oxygen radical generation, and, in turn, oxidants can inhibit NO synthesis from NOS. Ischemic preconditioning markedly decreases NO and oxidant generation, and this appears to be an important mechanism contributing to preconditioning-induced myocardial protection.

Chemotaxis, Leukocyte↗

PubMed2006

Reversal to cisplatin sensitivity in recurrent human ovarian cancer cells by NCX-4016, a nitro derivative of aspirin.

Ovarian cancer is a gynecological malignancy that is commonly treated by cytoreductive surgery followed by cisplatin treatment. However, the cisplatin treatment, although successful initially, is not effective in the treatment of the recurrent disease that invariably surfaces within a few months of the initial treatment. The refractory behavior is attributed to the increased levels of cellular thiols apparently caused by the cisplatin treatment. This observation prompted us to choose a cytotoxic drug whose activity is potentiated by cellular thiols with enhanced specificity toward the thiol-rich cisplatin-resistant cells. We used NCX-4016 [2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester], a derivative of aspirin containing a nitro group that releases nitric oxide in a sustained fashion for several hours in cells and in vivo, and we studied its cytotoxic efficacy against human ovarian cancer cells (HOCCs). Cisplatin-sensitive and cisplatin-resistant (CR) HOCCs were treated with 100 microM NCX-4016 for 6 h, and/or 0.5 microg/ml cisplatin for 1 h and assayed for clonogenecity. NCX-4016 significantly reduced the surviving fractions of cisplatin-sensitive (63 +/- 6%) and CR (70 +/- 10%) HOCCs. NCX-4016 also caused a 50% reduction in the levels of cellular glutathione in CR HOCCs. Treatment of cells with NCX-4016 followed by cisplatin showed a significantly greater extent of toxicity when compared with treatment of cells with NCX-4016 or cisplatin alone. In conclusion, this study showed that NCX-4016 is a potential inhibitor of the proliferation of CR HOCCs and thus might specifically kill cisplatin-refractory cancer cells in patients with recurrent ovarian cancer.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2006

Optimization of data acquisition for EPR imaging.

In electron paramagnetic resonance imaging (EPRI), long data acquisition time is one of the major problems limiting successful and useful biological EPRI experiments. Depending on the configuration (spatial distribution of paramagnetic species), information embedded in some objects can be characterized using a smaller number of projections, while others may require significantly larger number of projections to generate similar results. In order to optimize the acquisition process, it is therefore important to acquire a different number of projections for different objects. In this paper, a prediction scheme is demonstrated that can determine the number of projections required to achieve a preset reconstruction quality for a given object. After acquiring first few projections, corresponding partially filled k-space is analyzed. The complexity of data (to interpolate) in k-space is quantified and used to predict the number of required projections. All the projections are acquired using a mean-square difference-based adaptive acquisition technique that is also demonstrated in this work. The purpose of this non-uniform acquisition is to reduce redundancy in the acquired data which in turn decreases the number of projections required for the given object. It is also demonstrated that the performance of non-uniform acquisition is content dependant, and for certain configurations it may not be as effective as uniform acquisition in preserving signal from low intensity regions. The prediction scheme along with the non-uniform acquisition is tested using computer simulations, imaging of experimental phantoms, and in vivo imaging. Results indicate that the proposed method may save up to 50% of acquisition time. The techniques in this manuscript are described for 2D spatial imaging but can be extended to 3D imaging.

Algorithms↗

PubMed2006

Vitamin C-induced activation of phospholipase D in lung microvascular endothelial cells: regulation by MAP kinases.

Our earlier studies have shown that vitamin C at pharmacological doses (mM) induces loss of redox-dependent viability in bovine lung microvascular endothelial cells (BLMVECs) that is mediated by oxidative stress. Therefore, here, we investigated the vitamin C-induced activation of the lipid signaling enzyme, phospholipase D (PLD) in BLMVECs. Monolayer cultures of BLMVECs were treated with vitamin C (0-10 mM) for different time periods (0-2 h) and the activity of PLD was determined. Vitamin C induced activation of PLD in BLMVECs in a time- and dose-dependent fashion that was significantly attenuated by antioxidants, p38 mitogen-activated protein kinase (p38 MAPK)-specific inhibitor (SB203580), extracellular signal-regulated protein kinase (ERK)-specific inhibitor (PD98059), and transient transfection of cells with dominant-negative (DN)-p38 MAPK and DN-ERK1/ERK2. Vitamin C also induced phosphorylation and enhanced the activities of p38 MAPK and ERK in BLMVECs in a time-dependent fashion. It was also evident that vitamin C induced translocation of PLD(1) and PLD(2), association of p38 MAPK and ERK with PLD(1) and PLD(2), threonine phosphorylation of PLD(1) and PLD(2) and SB203580- and PD98059-inhibitable threonine phosphorylation of PLD(1) in BLMVECs. Transient transfection of BLMVECs with DN-p38 MAPK and DN-ERK1/ERK2 resulted in marked attenuation of vitamin C-induced phosphorylation of threonine in PLD(1) and PLD(2). We, for the first time, showed that vitamin C at pharmacological doses, activated PLD in the lung microvascular ECs through oxidative stress and MAPK activation.

Animals↗

PubMed2006

In vivo proton electron double resonance imaging of the distribution and clearance of nitroxide radicals in mice.

Proton electron double resonance imaging (PEDRI) is an emerging technique that utilizes the Overhauser effect to enable in vivo and in vitro imaging of free radicals in biological systems. Nitroxide spin probes enable measurement of tissue redox state based on their reduction to diamagnetic hydroxylamines. PEDRI instrumentation at 0.02 T was applied to assess the ability to image the in vivo distribution, clearance, and metabolism of nitroxide radicals in living mice. Using phantoms of 2,2,5,5-tetramethyl-3-carboxylpyrrolidine-N-oxyl (PCA) in normal saline the dependence of the enhancement on RF power and spin probe concentration was determined. Enhancements of up to -23 were obtained in phantoms with 2 mM levels. Maximum enhancement of -7 was observed in vivo. Coronal images of nitroxide-infused mice enabled visualization of the kinetics of spin probe uptake and clearance in different organs including the great vessels, heart, lungs, kidneys, and bladder with an in-plane spatial resolution of 0.6 mm. PEDRI of living mice was also performed using 3-carbamoyl-proxyl and 2,2,6,6-tetramethyl-4-oxopiperidine-N-oxyl to compare the different rate of clearance and metabolism among different nitroxide probes. PCA, due to its intravascular compartmentalization, provided the sharpest contrast for the vascular system and highest enhancement values in the PEDRI images among the three nitroxides.

Animals↗

PubMed2006

Automated on-the-fly detection and correction procedure for EPR imaging data acquisition.

Fast and reliable data acquisition is a major requirement for successful and useful biological electron paramagnetic resonance imaging (EPRI) experiments. Even a technologically advanced and professionally supervised EPRI system can exhibit instabilities initiated by perturbations such as animal motion, microphonics, and temperature changes. As a result, part of an acquired data set may become corrupted with excessive noise and distortions, which in turn may degrade the quality of the reconstructed image. In this work an automated scheme to monitor the system performance and stability over the course of an experiment is demonstrated. This method ensures that the quality of the acquired data is maintained during the experiment. For this purpose, four parameters including noise content and integration of each acquired projection are quantified and measured against those of the zero-gradient (ZG) projection, which is set as a quality benchmark. Projections with parameter values that differ substantially from the expected values are identified as damaged and consequently are reacquired. Therefore, the proposed technique not only effectively monitors the quality of acquisition, it also saves a substantial amount of acquisition time because it eliminates the necessity of repeating the entire experiment in cases in which only a small fraction of the data are corrupted.

Algorithms↗

PubMed2006

Modified Alderman-Grant resonator with high-power stability for proton electron double resonance imaging.

Proton-electron double resonance imaging (PEDRI) requires a dual EPR/NMR resonator. In order to saturate electron spins, long high-power RF EPR pulses are needed. Dissipated power causes resonator and sample heating, resulting in instability of the resonator frequency and coupling, which in turn causes a reduction in sensitivity. To overcome this problem, we designed, built, and tested a modified Alderman-Grant (MAG) resonator for in vivo and in vitro applications. We improved the temperature stability by using fused quartz in critical structural components of the resonator and employing a design that minimized moving parts. All of the components of the resonator and matching circuit, including coupling capacitors, were rigidly positioned on the surface of the quartz tube. Electrical coupling circuitry between the feeding cable and resonator was employed to provide independent coupling and tuning adjustments, which improved the ease and speed of operation. The equivalent schematic of the matching circuit is presented along with calculations of critical parameters. The resonator exhibits no frequency shift with 20 W of continuous power irradiation, and can handle intermittent power of 60 W for up to 3 min without detuning, enabling stable and reliable data acquisition for PEDRI of biological samples.

Computer-Aided Design↗

PubMed2006

Real-time calculation and visualization of spectra in field-cycled dynamic nuclear polarization spectroscopy.

Field-cycled dynamic nuclear polarization (FC-DNP), which is based on the Overhauser effect, provides a new way to perform in vivo measurements of free radicals in biological systems. Since it measures the alterations of the nuclear magnetic resonance (NMR) signal in the presence of paramagnetic molecules, a customized program is usually needed in FC-DNP experiments to extract spectral information from the acquired NMR signals. While this program can be designed to calculate the spectrum after all the NMR signals are collected, the batch-processing mode inevitably causes delay and is not convenient for in vivo applications. In this paper, we report the development of a real-time DNP spectrum calculation and visualization program, called RT_DNP, for FC-DNP experiments. A dynamic data exchange (DDE) client was implemented to enable real-time receipt of the system information and the NMR signals from a commercial NMR console. The received NMR signals and experimental parameters were then used to calculate the DNP spectrum during the data acquisition. The real-time DNP spectrum calculation and visualization program was tested in experiments. A seamless integration of the program into a commercial NMR console has been achieved.

Computer Systems↗

PubMed2006

4-Hydroxy-2-nonenal upregulates endogenous antioxidants and phase 2 enzymes in rat H9c2 myocardiac cells: protection against overt oxidative and electrophilic injury.

This study was undertaken to determine if 4-hydroxy-2-nonenal (HNE) could upregulate antioxidants and phase 2 enzymes in rat H9c2 myocardiac cells, and if the upregulated defenses led to cytoprotection against oxidative and electrophilic injury. Incubation of H9c2 cells with HNE at noncytotoxic concentrations resulted in significant induction of cellular catalase, glutathione (GSH), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase 1 (NQO1), as determined by enzyme activity and/or protein expression. HNE treatment caused increased mRNA expression of catalase, gamma-glutamylcysteine ligase, GST-A1, and NQO1. Pretreatment of H9c2 cells with HNE led to significant protection against cytotoxicity induced by reactive oxygen and nitrogen species. HNE-pretreated cells also exhibited increased resistance to injury elicited by subsequent cytotoxic concentrations of HNE. Taken together, this study demonstrates that several antioxidants and phase 2 enzymes in H9c2 cells are upregulated by HNE and that the increased defenses afford protection against overt oxidative and electrophilic cardiac cell injury.

Aldehydes↗

PubMed2006

Antioxidants and phase 2 enzymes in cardiomyocytes: Chemical inducibility and chemoprotection against oxidant and simulated ischemia-reperfusion injury.

The increasing recognition of the role for oxidative stress in cardiac disorders has led to extensive investigation on the protection by exogenous antioxidants against oxidative cardiac injury. On the other hand, another strategy for protecting against oxidative cardiac injury may be through upregulation of the endogenous antioxidants and phase 2 enzymes in the myocardium by chemical inducers. However, our current understanding of the chemical inducibility of cardiac cellular antioxidants and phase 2 enzymes is very limited. In this study, using rat cardiac H9c2 cells we have characterized the concentration- and time-dependent induction of cellular antioxidants and phase 2 enzymes by 3H-1,2-dithiole-3-thione (D3T), and the resultant chemoprotective effects on oxidative cardiac cell injury. Incubation of H9c2 cells with D3T resulted in a marked concentration- and time-dependent induction of a number of cellular antioxidants and phase 2 enzymes, including catalase, reduced glutathione (GSH), GSH peroxidase, glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase-1 (NQO1). D3T treatment of H9c2 cells also caused an increase in mRNA expression of catalase, gamma-glutamylcysteine ligase catalytic subunit, GR, GSTA1, M1 and P1, and NQO1. Moreover, both mRNA and protein expression of Nrf2 were induced in D3T-treated cells. D3T pretreatment led to a marked protection against H9c2 cell injury elicited by various oxidants and simulated ischemia-reperfusion. D3T pretreatment also resulted in decreased intracellular accumulation of reactive oxygen in H9c2 cells after exposure to the oxidants as well as simulated ischemia-reperfusion. This study demonstrates that a series of endogenous antioxidants and phase 2 enzymes in H9c2 cells can be induced by D3T in a concentration- and time-dependent fashion, and that the D3T-upregulated cellular defenses are accompanied by a markedly increased resistance to oxidative cardiac cell injury.

Animals↗

PubMed2005

The synthesis and aqueous superoxide anion scavenging of water-dispersible lutein esters.

Xanthophyll carotenoids of the C40 series, which includes commercially important compounds such as lutein, zeaxanthin, and astaxanthin, have poor aqueous solubility in the native state. Hawaii Biotech, Inc. (HBI) and others have shown that the aqueous dispersibility of derivatized carotenoids can be increased by varying the chemical structure of the esterified moieties. In the current study, the published series of novel, highly water-dispersible C40 carotenoid derivatives has been extended to include (3R,3'R,6'R)-lutein (beta,epsilon-carotene-3,3'-diol) derivatives. Two novel derivatives were synthesized by esterification with inorganic phosphate and succinic acid, respectively, and subsequently converted to the sodium salts. Red-orange, clear, aqueous suspensions were obtained after addition of these novel derivatives to USP-purified water. Aqueous dispersibility of the disuccinate sodium salt of lutein was 2.85 mg/mL; the diphosphate salt demonstrated a >10-fold increase in dispersibility at 29.27 mg/mL. As reported previously, these aqueous suspensions were obtained without the addition of heat, detergents, co-solvents, or other additives. The direct aqueous superoxide scavenging abilities of these novel derivatives were subsequently evaluated by electron paramagnetic resonance (EPR) spectroscopy in a well-characterized in vitro isolated human neutrophil assay. The novel derivatives were nearly identical aqueous-phase scavengers, demonstrating dose-dependent suppression of the superoxide anion signal (as detected by spin-trap adducts of DEPMPO) in the millimolar range. These lutein-based soft drugs will likely find utility in those commercial and clinical applications for which aqueous-phase singlet oxygen quenching and direct radical scavenging may be required.

Anions↗

PubMed2005

Heat shock-induced attenuation of hydroxyl radical generation and mitochondrial aconitase activity in cardiac H9c2 cells.

A mild heat shock (hyperthermia) protects cells from apoptotic and necrotic deaths by inducing overexpression of various heat shock proteins (Hsps). These proteins, in combination with the activation of the nitric oxide synthase (NOS) enzyme, play important roles in the protection of the myocardium against a variety of diseases. In the present work we report that the generation of potent reactive oxygen species (ROS), namely *OH in cardiac H9c2 cells, is attenuated by heat shock treatment (2 h at 42 degrees C). Western blot analyses showed that heat shock treatment induced overexpression of Hsp70, Hsp60, and Hsp25. The observed *OH was found to be derived from the superoxide (O(2)(-)*) generated by the mitochondria. Whereas the manganese superoxide dismutase (MnSOD) activity was increased in the heat-shocked cells, the mitochondrial aconitase activity was reduced. The mechanism of O(2)(-)* conversion into *OH in mitochondria is proposed as follows. The O(2)(-)* leaked from the electron transport chain, oxidatively damages the mitochondrial aconitase, releasing a free Fe(2+). The aconitase-released Fe(2+) combines with H(2)O(2) to generate *OH via a Fenton reaction and the oxidized Fe(3+) recombines with the inactivated enzyme after being reduced to Fe(2+) by other cellular reductants, turning it over to be active. However, in heat-shocked cells, because of higher MnSOD activity, the excess H(2)O(2) causes irreversible damage to the mitochondrial aconitase enzyme, thus inhibiting its activity. In conclusion, we propose that attenuation of *OH generation after heat shock treatment might play an important role in reducing the myocardial ischemic injury, observed in heat shock-treated animals.

Aconitate Hydratase↗

PubMed2005

Superoxide generation from mitochondrial NADH dehydrogenase induces self-inactivation with specific protein radical formation.

Mitochondrial superoxide (O(2)(.)) production is an important mediator of oxidative cellular injury. While NADH dehydrogenase (NDH) is a critical site of this O(2)(.) production; its mechanism of O(2)(.) generation is not known. Therefore, the catalytic function of NDH in the mediation of O(2)(.) generation was investigated by EPR spin-trapping. In the presence of NADH, O(2)(.) generation from NDH was observed and was inhibited by diphenyleneiodinium chloride (DPI), indicating involvement of the FMN-binding site of NDH. Addition of FMN increased O(2)(.) production. Destruction of the cysteine ligands of iron-sulfur clusters decreased O(2)(.) generation, suggesting a secondary role of this site. This inhibitory effect was reversed by addition of FMN. However, FMN addition could not reverse the inhibition of NDH by either DPI or heat denaturation, demonstrating involvement of both FMN and its FMN-binding protein moiety in the catalysis of O(2)(.) generation. O(2)(.) production by NDH also induced self-inactivation. Immunospin-trapping with anti-DMPO antibody and subsequent mass spectrometry was used to define the sites of oxidative damage of NDH. A DMPO adduct was detected on the 51-kDa subunit and was O(2)(.)-dependent. Alkylation of the cysteine residues of NDH significantly inhibited NDH-DMPO spin adduct formation, indicating involvement of protein thiyl radicals. LC/MS/MS analysis of a tryptic digest of the 51-kDa polypeptide revealed that cysteine (Cys(206)) and tyrosine (Tyr(177)) were specific sites of NDH-derived protein radical formation. Thus, two domains of the 51-kDa subunit, Gly(200)-Ala-Gly-Ala-Tyr-Ile-Cys(206)-Gly-Glu-Glu-Thr-Ala-Leu-Ile-Glu-Ser-Ile-Glu-Gly-Lys(219) and Ala(176)-Tyr(177)-Glu-Ala-Gly-Leu-Ile-Gly-Lys(184), were demonstrated to be susceptible to oxidative attack, and their oxidative modification results in decreased electron transfer activity.

Amino Acid Sequence↗

PubMed2005

Nitric oxide release from the unimolecular decomposition of the superoxide radical anion adduct of cyclic nitrones in aqueous medium.

Nitrones such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO) have become the spin-traps of choice for the detection of transient radical species in chemical and biological systems using electron paramagnetic resonance (EPR) spectroscopy. The mechanism of decomposition of the superoxide radical anion (O2(.-)) adducts of DMPO, DEPMPO and EMPO in aqueous solutions was investigated. Our findings suggest that nitric oxide (NO) was formed during the decomposition of the O2(.-) adduct as detected by EPR spin trapping using Fe(II)N-methyl-d-glucamine dithiocarbamate (MGD). Nitric oxide release was observed from the O2(.-) adduct formed from hypoxanthine-xanthine oxidase, PMA-activated human neutrophils, and DMSO solution of KO2. Nitric oxide formation was not observed from the independently generated hydroxyl radical adduct. Formation of nitric oxide was also indirectly detected as nitrite (NO2(.-)) utilizing the Griess assay. Nitrite concentration increases with increasing O2(.-) concentration at constant DMPO concentration, while NO2(.-) formation is suppressed at anaerobic conditions. Moreover, large excess of DMPO also inhibits NO2(.-) formation which can be attributed to the oxidation of DMPO to hydroxamic acid nitroxide (DMPO-X) by nitrogen dioxide (NO2), a precursor to NO2(.-). Product analysis was also conducted to further elucidate the mechanism of adduct decay using gas chromatography-mass spectrometry (GC-MS) technique.

Anions↗

PubMed2005

Involvement of phospholipid, biomembrane integrity, and NO peroxidase activity in the NO catabolism by cytochrome c oxidase.

The physiological regulation of mitochondrial respiration by NO has been reported to result from the reversible binding of NO to the two-electron reduced binuclear center (Fe(2+)(a3)-Cu(1+)(B)) of cytochrome c oxidase (CcO). Although the role of CcO and its derived catalytic intermediates in the catabolism of NO has been documented, little has been established for the enzyme in its fully oxidized state (Fe(3+)(a3)-Cu(2+)(B)). We report: (1) CcO, in its fully oxidized state, represents the major component of the mitochondrial electron transport chain for NO consumption as controlled by the binding of NO to its binuclear center. Phospholipid enhances NO consumption by fully oxidized CcO, whereas the consumption of NO is slowed down by membrane structure and membrane potential when CcO is embedded in the phospholipid bilayer. (2) In the presence of H(2)O(2), CcO was shown to serve as a mitochondria-derived NO peroxidase. A CcO-derived protein radical intermediate was induced and involved in the modulation of NO catabolism.

Animals↗