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Victor Darley-Usmar

Publications and source records attributed to Victor Darley-Usmar.

15 recordsLinked to original sources
PubMed2006

S-adenosylmethionine prevents chronic alcohol-induced mitochondrial dysfunction in the rat liver.

An early event that occurs in response to alcohol consumption is mitochondrial dysfunction, which is evident in changes to the mitochondrial proteome, respiration defects, and mitochondrial DNA (mtDNA) damage. S-adenosylmethionine (SAM) has emerged as a potential therapeutic for treating alcoholic liver disease through mechanisms that appear to involve decreases in oxidative stress and proinflammatory cytokine production as well as the alleviation of steatosis. Because mitochondria are a source of reactive oxygen/nitrogen species and a target for oxidative damage, we tested the hypothesis that SAM treatment during alcohol exposure preserves organelle function. Mitochondria were isolated from livers of rats fed control and ethanol diets with and without SAM for 5 wk. Alcohol feeding caused a significant decrease in state 3 respiration and the respiratory control ratio, whereas SAM administration prevented these alcohol-mediated defects and preserved hepatic SAM levels. SAM treatment prevented alcohol-associated increases in mitochondrial superoxide production, mtDNA damage, and inducible nitric oxide synthase induction, without a significant lessening of steatosis. Accompanying these indexes of oxidant damage, SAM prevented alcohol-mediated losses in cytochrome c oxidase subunits as shown using blue native PAGE proteomics and immunoblot analysis, which resulted in partial preservation of complex IV activity. SAM treatment attenuated the upregulation of the mitochondrial stress chaperone prohibitin. Although SAM supplementation did not alleviate steatosis by itself, SAM prevented several key alcohol-mediated defects to the mitochondria genome and proteome that contribute to the bioenergetic defect in the liver after alcohol consumption. These findings reveal new molecular targets through which SAM may work to alleviate one critical component of alcohol-induced liver injury: mitochondria dysfunction.

Animals↗
PubMed2005

Evidence of cardiovascular protection by moderate alcohol: role of nitric oxide.

Epidemiological evidence indicates that moderate alcohol consumption reduces the incidence of heart disease. Endothelial nitric oxide synthase (eNOS) is a key regulator of vascular homeostasis and myocardial functions through the controlled production of nitric oxide (*NO). These studies were conducted to determine if the apparent alcohol-associated cardioprotection is mediated, in part, through modulation of the eNOS protein and activity in the cardiovascular system. Rats were fed alcohol and eNOS protein and *NO production were evaluated at the end of 8 weeks. Myocardial and vascular function was assessed ex vivo in a subset of animals. Moderate alcohol improved postischemic myocardial systolic and diastolic function and attenuated the postischemic reduction in coronary vascular resistance. Moderate alcohol also enhanced maximum vascular relaxation by 26 +/- 0.2% and increased plasma *NO production concomitant with a greater than 2.5-fold increase in eNOS protein. Higher levels of alcohol impaired maximum vascular relaxation by 22 +/- 0.1%. These results suggest that moderate alcohol improves postischemic myocardial functions and increases *NO production by vascular endothelium. An increase in *NO may explain, at least in part, the cardioprotective benefits of moderate alcohol consumption.

Animals↗
PubMed2005

Mitochondrial proteomics in free radical research.

The importance of mitochondrial dysfunction in numerous diseases has long been appreciated. The impact of oxidative and nitrosative stress on mitochondrial function is complex; however, recent progress in the field using proteomics technologies has begun to shed light on the molecular defects responsible for mitochondrial and cellular dysfunction. This review focuses on the state-of-the-art technologies being used and current research endeavors in the field of mitochondrial proteomics with emphasis on those advancements being made in the field of free radical biology to identify the importance of alterations to the mitochondrial proteome in the development of disease.

Alcohols↗
PubMed2005

RSA 2004: combined basic research satellite symposium - session three: alcohol and mitochondrial metabolism: at the crossroads of life and death.

This article summarizes the proceedings of the RSA 2004 Combined Basic Research Satellite Meeting convened at the Westin Bayshore Resort and Marina, Vancouver, CA. One of the sessions "Alcohol and mitochondrial metabolism: At the crossroads of life and death" featured five speakers and was chaired by Drs. Jan Hoek and Sam Zakhari. The presentations were 1) INTRODUCTION: Alcohol and cellular energy metabolism by Jan Hoek, 2) Ethanol-dependent dysfunction of mitochondrial energy metabolism: the role of NO by Victor Darley-Usmar, 3) Ethanol and apoptosis in the heart by Gyorgy Hajnoczky, 4) Alcohol and mitochondrial biogenesis in development by Thomas Knudsen, and 5) Alcohol, mitochondrial function and cardiac preconditioning by Daria Mochly-Rosen.

Animals↗
PubMed2004

Modification of the mitochondrial proteome in response to the stress of ethanol-dependent hepatotoxicity.

Mitochondria are particularly susceptible to increased formation of reactive oxygen and nitrogen species in the cell that can occur in response to pathological and xenobiotic stimuli. Proteomics can give insights into both mechanism of pathology and adaptation to stress. Herein we report the use of proteomics to evaluate alterations in the levels of mitochondrial proteins following chronic ethanol exposure in an animal model. Forty-three proteins showed differential expression, 13 increased and 30 decreased, as a consequence of chronic ethanol. Of these proteins, 25 were not previously known to be affected by chronic ethanol emphasizing the power of proteomic approaches in revealing global responses to stress. Both nuclear and mitochondrially encoded gene products of the oxidative phosphorylation complexes in mitochondria from ethanol-fed rats were decreased suggesting an assembly defect in this integrated metabolic pathway. Moreover mtDNA damage was increased by ethanol demonstrating that the effects of ethanol consumption extend beyond the proteome to encompass mtDNA. Taken together, we have demonstrated that chronic ethanol consumption extends to a modification of the mitochondrial proteome far broader than realized previously. These data also suggest that the response of mitochondria to stress may not involve non-discriminate changes in the proteome but is restricted to those metabolic pathways that have a direct role in a specific pathology.

Alcohol Drinking↗
PubMed2004

Modification of Cytochrome c by 4-hydroxy- 2-nonenal: evidence for histidine, lysine, and arginine-aldehyde adducts.

4-Hydroxy-2-nonenal (4HNE), a major secondary product of lipid peroxidation, has been associated with a number of disease states involving oxidative stress. Despite the recognized importance of post-translational modification of proteins by products such as 4HNE, little is known of the modification of cytochrome c by this reagent and its analysis by mass spectrometry. The purpose of this study was to investigate the chemical interaction of 4HNE and cytochrome c, a protein essential to cellular respiration, under in vitro conditions. Isoelectric focusing of native and 4HNE-modified cytochrome c using immobilized pH gradient (IpG) strips showed a decrease in the pI of the 4HNE-modified protein suggesting modification of charged amino acids. Reaction of 4HNE with cytochrome c resulted in increases in molecular weight consistent with the addition of four 4HNE residues as determined by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS). Samples of both native and 4HNE-modified cytochrome c were enzymatically digested and subjected to peptide mass fingerprinting using MALDI-TOF MS. Analysis of these samples using LC-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) provided sequence information that was used to determine specific residues to which the aldehyde adducted. Taken together, the data indicated that H33, K87, and R38 were modified by 4HNE. Mapping these results onto the X-ray crystal structure of native cytochrome c suggest that 4HNE adduction to cytochrome c could have significant effects on tertiary structure, electron transport, and ultimately, mitochondrial dysfunction.

Aldehydes↗
PubMed2003

Oxidative modification of hepatic mitochondria protein thiols: effect of chronic alcohol consumption.

Redox modification of mitochondrial proteins is thought to play a key role in regulating cellular function, although direct evidence to support this hypothesis is limited. Using an in vivo model of mitochondrial redox stress, ethanol hepatotoxicity, the modification of mitochondrial protein thiols was examined using a proteomics approach. Specific labeling of reduced thiols in the mitochondrion from the livers of control and ethanol-fed rats was achieved by using the thiol reactive compound (4-iodobutyl)triphenylphosphonium (IBTP). This molecule selectively accumulates in the organelle and can be used to identify thiol-containing proteins. Mitochondrial proteins that have been modified are identified by decreased labeling with IBTP using two-dimensional SDS-PAGE followed by immunoblotting with an antibody directed against the triphenylphosphonium moiety of the IBTP molecule. Analyses of these data showed a significant decrease in IBTP labeling of thiols present in specific mitochondria matrix proteins from ethanol-fed rats compared with their corresponding controls. These proteins were identified as the low-K(m) aldehyde dehydrogenase and glucose-regulated protein 78. The decrease in IBTP labeling in aldehyde dehydrogenase was accompanied by a decrease in specific activity of the enzyme. These data demonstrate that mitochondrial protein thiol modification is associated with chronic alcohol intake and might contribute to the pathophysiology associated with hepatic injury. Taken together, we have developed a protocol to chemically tag and select thiol-modified proteins that will greatly enhance efforts to establish posttranslational redox modification of mitochondrial protein in in vivo models of oxidative or nitrosative stress.

Aldehyde Dehydrogenase↗
PubMed2003

Control of mitochondrial respiration by NO*, effects of low oxygen and respiratory state.

Nitric oxide (NO.) inhibits mitochondrial respiration by binding to the binuclear heme a3/CuB center in cytochrome c oxidase. However, the significance of this reaction at physiological O2 levels (5-10 microM) and the effects of respiratory state are unknown. In this study mitochondrial respiration, absorption spectra, [O2], and [NO.] were measured simultaneously at physiological O2 levels with constant O2 delivery, to model in vivo respiratory dynamics. Under these conditions NO. inhibited mitochondrial respiration with an IC50 of 0.14 +/- 0.01 microm in state 3 versus 0.31 +/- 0.04 microM in state 4. Spectral data indicate that the higher sensitivity of state 3 respiration to NO. is due to greater control over respiration by an NO.-dependent spectral species in the respiratory chain in this state. These results are discussed in the context of regulation of respiration by NO. in vivo and its implications for the control of vessel-parenchymal O2 gradients.

Electron Transport↗
PubMed2003

Polyphenols, inflammatory response, and cancer prevention: chlorination of isoflavones by human neutrophils.

An important aspect of the risk of cancer is the involvement of the inflammatory response. Currently, antiinflammatory agents are used in chemopreventive strategies. For example, aspirin is recommended for the prevention of colon cancer as well as breast and other cancers. The inflammatory response involves the production of cytokines and proinflammatory oxidants such as hypochlorous acid (HOCl) and peroxynitrite (ONO2-) produced by neutrophils and macrophages, respectively. These oxidants react with phenolic tyrosine residues on proteins to form chloro- and nitrotyrosine. Diets rich in polyphenols (green tea catechins, soy isoflavones) have also been shown to be chemopreventive. The aromatic nature of polyphenols makes them potential targets of HOCl and ONO2-. These reactions may create novel pharmacophores at the site of inflammation. Previous studies in the neutrophil-like cell line, differentiated HL-60 cells, demonstrated the formation of chlorinated and nitrated isoflavones. In this study we have examined whether similar reactions occur in freshly isolated human neutrophils. After induction of a respiratory burst with a phorbol ester, isoflavones and their metabolites were identified by liquid chromatography-tandem mass spectrometry and then quantitatively measured by LC-mass spectrometry using multiple-reaction ion monitoring. The data obtained indicate that both chlorinated and nitrated genistein are formed by human neutrophils. The extent of chlorination of genistein was markedly increased by the phorbol ester whereas the low level of nitration of genistein was constitutive and unaffected. These data imply a potential role for modified forms of genistein that would be produced in the inflammatory environment in and around a tumor.

Anticarcinogenic Agents↗
PubMed2002

Mitochondria, nitric oxide, and cardiovascular dysfunction.

Cardiovascular diseases encompass a wide spectrum of abnormalities with diverse etiologies. The molecular mechanisms underlying these disorders include a variety of responses such as changes in nitric oxide- (NO) dependent cell signaling and increased apoptosis. An interesting aspect that has received little or no attention is the role mitochondria may play in the vascular changes that occur in both atherosclerosis and hypertension. With the changing perspective of the organelle from simply a role in metabolism to a contributor to signal transduction pathways, the role of mitochondria in cells with relatively low energy demands such as the endothelium has become important to understand. In this context, the definition of the NO-cytochrome c oxidase signaling pathway and the influence this has on cytochrome c release is particularly important in understanding apoptotic mechanisms involving the mitochondrion. This review examines the role of compromised mitochondrial function in a variety of vascular pathologies and the modulation of these effects by NO. The interaction of NO with the various mitochondrial respiratory complexes and the role NO plays in modulating mitochondrial-mediated apoptosis in these systems will be discussed.

Animals↗
PubMed2002

Mitochondrial function in response to cardiac ischemia-reperfusion after oral treatment with quercetin.

Polyphenolic compounds present in red wines, such as the flavonol quercetin, are thought capable of cardioprotection through mechanisms not yet clearly defined. It has been established that mitochondria play a critical role in myocardial recovery from ischemia-reperfusion (I-R) damage, and in vitro experiments indicate that quercetin can exert a variety of direct effects on mitochondrial function. The effects of quercetin at concentrations typically found in 1-2 glasses of red wine on cardiac I-R and mitochondrial function in vivo are not known. Quercetin was administered to rats (0.033 mg/kg per day by gavage for 4 d). Isolated Langendorff perfused hearts were subjected to I-R, and cardiac functional parameters determined both before and after I-R. Mitochondria were isolated from post-I-R hearts and their function assessed. Compared to an untreated control group, quercetin treatment significantly decreased the impairment of cardiac function following I-R. This protective effect was associated with improved mitochondrial function after I-R. These results indicate that oral low dose quercetin is cardioprotective, possibly via a mechanism involving protection of mitochondrial function during I-R.

Administration, Oral↗
PubMed2002

Inhibition of mitochondrial protein synthesis results in increased endothelial cell susceptibility to nitric oxide-induced apoptosis.

Mutations in mitochondrial DNA, affecting the activity of respiratory complexes, have been implicated in many chronic degenerative diseases. Mitochondrial proteins coded for by both the mitochondrial and nuclear genes are known to have important signaling roles in apoptosis. However, the impact of the inhibition of mitochondrial protein synthesis on apoptosis is largely unknown. This inhibition is particularly important in NO-dependent cytotoxicity, which is believed to have a significant mitochondrial component and depend on other factors such as glycolysis. In this study we have examined whether the inhibition of mitochondrial protein synthesis by chloramphenicol increases the susceptibility of endothelial cells to undergo NO-dependent apoptosis in glucose-free media. Bovine aortic endothelial cells were treated with chloramphenicol, which resulted in a decreased ratio of mitochondrial complex IV to cytochrome c and increased oxidant production in the cell. Inhibition of mitochondrial protein synthesis was associated with a greater susceptibility of the cells to apoptosis induced by NO in glucose-free medium.

Adenosine Triphosphate↗
PubMed2002

Specific modification of mitochondrial protein thiols in response to oxidative stress: a proteomics approach.

Mitochondria play a central role in redox-linked processes in the cell through mechanisms that are thought to involve modification of specific protein thiols, but this has proved difficult to assess. In particular, specific labeling and quantitation of mitochondrial protein cysteine residues have not been achieved due to the lack of reagents available that can be applied to the intact organelle or cell. To overcome these problems we have used a combination of mitochondrial proteomics and targeted labeling of mitochondrial thiols using a novel compound, (4-iodobutyl)triphenylphosphonium (IBTP). This lipophilic cation is accumulated by mitochondria and yields stable thioether adducts in a thiol-specific reaction. The selective uptake into mitochondria, due to the large membrane potential across the inner membrane, and the high pH of the matrix results in specific labeling of mitochondrial protein thiols by IBTP. Individual mitochondrial proteins that changed thiol redox state following oxidative stress could then be identified by their decreased reaction with IBTP and isolated by two-dimensional electrophoresis. We demonstrate the selectivity of IBTP labeling and use it to show that glutathione oxidation and exposure to an S-nitrosothiol or to peroxynitrite cause extensive redox changes to mitochondrial thiol proteins. In conjunction with blue native gel electrophoresis, we used IBTP labeling to demonstrate that thiols are exposed on the matrix faces of respiratory Complexes I, II, and IV. This novel approach enables measurement of the thiol redox state of individual mitochondrial proteins during oxidative stress and cell death. In addition the methodology has the potential to identify novel redox-dependent modulation of mitochondrial proteins.

Animals↗
PubMed2002

Nitric oxide and cGMP-dependent protein kinase regulation of glucose-mediated thrombospondin 1-dependent transforming growth factor-beta activation in mesangial cells.

Excessive transforming growth factor-beta (TGF-beta) activity in hyperglycemia contributes to the development of diabetic nephropathy. Glucose stimulation of TGF-beta activity and matrix synthesis are dependent on autocrine thrombospondin 1 (TSP1) to convert latent TGF-beta to its biologically active form. The mechanisms by which glucose regulates TSP1 are not known. High glucose inhibits nitric oxide (NO) bioavailability and decreased NO increases TGF-beta activity and extracellular matrix accumulation. Yet, the impact of NO signaling on TSP1 activation of TGF-beta is unknown. We tested the role of NO signaling in the regulation of TSP1 expression and TSP1-dependent TGF-beta activity in rat mesangial cells exposed to high glucose. On exposure to 30 mm glucose, NO accumulation in the conditioned media and intracellular cGMP levels were significantly decreased. The addition of an NO donor prevented the glucose-dependent increase in TSP1 mRNA, protein, and TGF-beta bioactivity. The effects of the NO donor were blocked by ODQ (a soluble guanylate cyclase inhibitor) or Rp-8-pCPT-cGMPS (an inhibitor of cGMP-dependent protein kinase). These effects of high glucose were also reversed by the nitric-oxide synthase cofactor tetrahyrobiopterin (BH(4)). These results show that high glucose mediates increases in TSP1 expression and TSP1-dependent TGF-beta bioactivity through down-modulation of NO-cGMP-dependent protein kinase signaling.

Animals↗