Search tips
Search criteria

Results 1-10 (10)

Clipboard (0)

Select a Filter Below

Year of Publication
Document Types
1.  Humans with Atherosclerosis have Impaired ABCA1 Cholesterol Efflux and Enhanced HDL Oxidation by Myeloperoxidase 
Circulation research  2014;114(11):1733-1742.
The efflux capacity of HDL with cultured macrophages associates strongly and negatively with CAD status, indicating that impaired sterol efflux capacity might be a marker—and perhaps mediator—of atherosclerotic burden. However, the mechanisms that contribute to impaired sterol efflux capacity remain poorly understood.
To determine the relationship between myeloperoxidase-mediated oxidative damage to apoA-I, the major HDL protein, and the ability of HDL to remove cellular cholesterol by the ABCA1 pathway.
Methods and Results
We quantified both site-specific oxidation of apoA-I and HDL’s ABCA1 cholesterol efflux capacity in control subjects and subjects with stable coronary artery disease or acute coronary syndrome. The CAD and ACS subjects had higher levels of chlorinated tyrosine-192 and oxidized methionine-148 than the control subjects. In contrast, plasma levels of MPO did not differ between the groups. HDL from the CAD and ACS subjects was less able to accept cholesterol from cells expressing ABCA1 than HDL from control subjects. Levels of chlorinated tyrosine and oxidized methionine associated inversely with ABCA1 efflux capacity and positively with atherosclerotic disease status. These differences remained significant after adjusting for HDL-cholesterol levels.
Our observations indicate that MPO may contribute to the generation of dysfunctional HDL with impaired ABCA1 efflux capacity in humans with atherosclerosis. Quantification of chlorotyrosine and oxidized methionine in circulating HDL might be useful indicators of the risk of cardiovascular disease that are independent of HDL-cholesterol.
PMCID: PMC4076170  PMID: 24647144
Acute coronary syndrome; cardiovascular diseases; 3-chlorotyrosine; mass spectrometry; myeloperoxidase; selected reaction monitoring
2.  Independent roles of methionine sulfoxide reductase A in mitochondrial ATP synthesis and as antioxidant in retinal pigment epithelial cells 
Free radical biology & medicine  2013;65:10.1016/j.freeradbiomed.2013.10.006.
The antioxidant enzyme methionine sulfoxide reductase A (MsrA) is highly expressed in the retinal pigment epithelium (RPE), a support tissue for neighboring photoreceptors. MsrA protein levels correlate with sensitivity of RPE in culture to experimental oxidative stress. To investigate whether and how MsrA affects RPE functionality regardless of oxidative stress, we tested the effects of acute silencing or overexpression of MsrA on the phagocytosis of photoreceptor outer segment fragments (POS), a demanding, daily function of the RPE that is essential for vision. Endogenous MsrA localized to mitochondria and cytosol of rat RPE in culture. RPE cells manipulated to express higher or lower levels of MsrA than control cells showed no signs of cell death but increased or decreased, respectively, POS binding as well as engulfment. These effects of altered MsrA protein concentration on phagocytosis were independent of the levels of oxidative stress. However, altering MsrA expression had no effect on phagocytosis when mitochondrial respiration was inhibited. Furthermore, ATP content directly correlated with MsrA protein levels in RPE cells that used mitochondrial oxidative phosphorylation for ATP synthesis but not in RPE cells that relied on glycolysis alone. Overexpressing MsrA was sufficient to increase specifically the activity of complex-IV of the respiratory chain, while activity of complex-II and mitochondrial content were unaffected. Thus, MsrA likely enhances ATP synthesis by increasing complex-IV activity. Such contribution of MsrA to energy metabolism is independent of its function in protection from elevated oxidative stress but contributes to routine but vital photoreceptor support by RPE cells.
PMCID: PMC3859712  PMID: 24120970
mitochondria; MsrA; oxidative stress; phagocytosis; respiratory chain; retinal pigment epithelium
3.  Biomarkers of Oxidative Stress Study V: Ozone exposure of rats and its effect on lipids, proteins and DNA in plasma and urine 
Ozone exposure effect on free radical-catalyzed oxidation products of lipids, proteins and DNA in the plasma and urine of rats was studied as a continuation of the international Biomarker of Oxidative Stress Study (BOSS) sponsored by NIEHS/NIH. The goal was to identify a biomarker for ozone-induced oxidative stress and to assess whether inconsistent results often reported in the literature might be due to the limitations of the available methods for measuring the various types of oxidative products. The time and dose-dependent effects of ozone exposure on rat plasma lipid hydroperoxides, malondialdehyde, F2-isoprostanes, protein carbonyls, methionine oxidation, tyrosine- and phenylalanine oxidation products, as well as urinary malondialdehyde and F2-isoprostanes were investigated with various techniques. The criterion used to recognize a marker in the model of ozone exposure was that a significant effect could be identified and measured in a biological fluid seen at both doses at more than one time point. No statistically significant differences between the experimental and control groups at either ozone dose and time point studied could be identified in this study. Tissue samples were not included. Despite all the work accomplished in the BOSS study of ozone, no available product of oxidation in biological fluid has yet met the required criteria of being a biomarker. The current negative findings as a consequence of ozone exposure are of great importance, because they document that in complex systems, as the present in vivo experiment, the assays used may not provide meaningful data of ozone oxidation, especially in human studies.
PMCID: PMC3968235  PMID: 23608465
biomarkers; oxidative stress; ozone exposure; rats
4.  Mycobacterium tuberculosis expresses methionine sulfoxide reductases A and B that protect from killing by nitrite and hypochlorite 
Molecular microbiology  2009;71(3):583-593.
Methionine sulfoxide reductases (Msr’s) reduce methionine sulfoxide to methionine and protect bacteria against reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Many organisms express both MsrA, active against methionine-(S)-sulfoxide, and MsrB, active against methionine-(R)-sulfoxide. Mycobacterium tuberculosis (Mtb) expresses MsrA, which protects ΔmsrA-E. coli from ROI and RNI (St. John et al., 2001). However, the function of MsrA in Mtb has not been defined, and it is unknown whether Mtb expresses MsrB. We identified MsrB as the protein encoded by Rv2674 in Mtb and confirmed the distinct stereospecificities of recombinant Mtb MsrA and MsrB. We generated strains of Mtb deficient in MsrA, MsrB or both and complemented the mutants. Lysates of singly deficient strains displayed half as much Msr activity as wild type against N-acetyl methionine sulfoxide. However, in contrast to other bacteria, single mutants were no more vulnerable than wild type to killing by ROI/RNI. Only Mtb lacking both MsrA and MsrB was more readily killed by nitrite or hypochlorite. Thus, MsrA and MsrB contribute to the enzymatic defenses of Mtb against ROI and RNI.
PMCID: PMC3697012  PMID: 19040639
5.  A High-Throughput Screening Compatible Assay for Activators and Inhibitors of Methionine Sulfoxide Reductase A 
The methionine sulfoxide reductase (Msr) system has been shown to play an important role in protecting cells against oxidative damage. This family of enzymes can repair damage to proteins resulting from the oxidation of methionine residues to methionine sulfoxide, caused by reactive oxygen species. Previous genetic studies in animals have shown that increased levels of methionine sulfoxide reductase enzyme A (MsrA), an important member of the Msr family, can protect cells against oxidative damage and increase life span. A high-throughput screening (HTS) compatible assay has been developed to search for both activators and inhibitors of MsrA. The assay involves a coupled reaction in which the oxidation of NADPH is measured by either spectrophotometric or fluorometric analysis. Previous studies had shown that MsrA has a broad substrate specificity and can reduce a variety of methyl sulfoxide compounds, including dimethylsulfoxide (DMSO). Since the chemicals in the screening library are dissolved in DMSO, which would compete with any of the standard substrates used for the determination of MsrA activity, an assay has been developed that uses the DMSO that is the solvent for the compounds in the library as the substrate for the MsrA enzyme. A specific activator of MsrA could have important therapeutic value for diseases that involve oxidative damage, especially age-related diseases, whereas a specific inhibitor of MsrA would have value for a variety of research studies.
PMCID: PMC2978062  PMID: 20515413
6.  The biological activity of FasL in human and mouse lungs is determined by the structure of its stalk region 
The Journal of Clinical Investigation  2011;121(3):1174-1190.
Acute lung injury (ALI) is a life-threatening condition in critically ill patients. Injury to the alveolar epithelium is a critical event in ALI, and accumulating evidence suggests that it is linked to proapoptotic Fas/FasL signals. Active soluble FasL (sFasL) is detectable in the bronchoalveolar lavage (BAL) fluid of patients with ALI, but the mechanisms controlling its bioactivity are unclear. We therefore investigated how the structure of sFasL influences cellular activation in human and mouse lungs and the role of oxidants and proteases in modifying sFasL activity. The sFasL in BAL fluid from patients with ALI was bioactive and present in high molecular weight multimers and aggregates. Oxidants generated from neutrophil myeloperoxidase in BAL fluid promoted aggregation of sFasL in vitro and in vivo. Oxidation increased the biological activity of sFasL at low concentrations but degraded sFasL at high concentrations. The amino-terminal extracellular stalk region of human sFasL was required to induce lung injury in mice, and proteolytic cleavage of the stalk region by MMP-7 reduced the bioactivity of sFasL in human cells in vitro. The sFasL recovered from the lungs of patients with ALI contained both oxidized methionine residues and the stalk region. These data provide what we believe to be new insights into the structural determinants of sFasL bioactivity in the lungs of patients with ALI.
PMCID: PMC3049393  PMID: 21285513
7.  Methionine Sulfoxide Reductases B1, B2, and B3 Are Present in the Human Lens and Confer Oxidative Stress Resistance to Lens Cells 
Methionine-sulfoxide reductases are unique, in that their ability to repair oxidized proteins and MsrA, which reduces S-methionine sulfoxide, can protect lens cells against oxidative stress damage. To date, the roles of MsrB1, -B2 and -B3 which reduce R-methionine sulfoxide have not been established for any mammalian system. The present study was undertaken to identify those MsrBs expressed by the lens and to evaluate the enzyme activities, expression patterns, and abilities of the identified genes to defend lens cells against oxidative stress damage.
Enzyme activities were determined with bovine lens extracts. The identities and spatial expression patterns of MsrB1, -B2, and -B3 transcripts were examined by RT-PCR in human lens and 21 other tissues. Oxidative stress resistance was measured using short interfering (si)RNA–mediated gene-silencing in conjunction with exposure to tert-butyl hydroperoxide (tBHP) and MTS viability measurements in SRA04/01 human lens epithelial cells.
Forty percent of the Msr enzyme activity present in the lens was MsrB, whereas the remaining enzyme activity was MsrA. MsrB1 (selenoprotein R, localized in the cytosol and nucleus), MsrB2 (CBS-1, localized in the mitochondria), and MsrB3 (localized in the endoplasmic reticulum and mitochondria) were all expressed by the lens. These genes exhibit asymmetric expression patterns between different human tissues and different lens sublocations, including lens fibers. All three genes are required for lens cell viability, and their silencing in lens cells results in increased oxidative-stress–induced cell death.
The present data suggest important roles for both MsrA and -Bs in lens cell viability and oxidative stress protection. The differential tissue distribution and lens expression patterns of these genes, coupled with increased oxidative-stress–induced cell death on their deletion provides evidence that they are important for lens cell function, resistance to oxidative stress, and, potentially, cataractogenesis.
PMCID: PMC1351357  PMID: 15914630
8.  I-PLA2 Activation during Apoptosis Promotes the Exposure of Membrane Lysophosphatidylcholine Leading to Binding by Natural Immunoglobulin M Antibodies and Complement Activation 
Deficiency of serum immunoglobulin (Ig)M is associated with the development of a lupus-like disease in mice. Recent studies suggest that classical complement components facilitate the clearance of apoptotic cells and that failure to do so predisposes mice to lupus. Since IgM is a potent activator of the classical complement pathway, we examined IgM binding to dying cells. IgM, but not IgG, bound to apoptotic T cells through the Fab′ portion of the antibody. Exposure of apoptotic cell membranes to phospholipase (PL) A2 increased, whereas PLD reduced, IgM binding and complement activation. Absorption studies combined with direct plate binding assays, revealed that IgM antibodies failed to bind to phosphatidyl lipids, but did recognize lysophosphatidylcholine and the phosphorylcholine head group. Both iPLA2 and cPLA2 are activated during apoptosis. Since inhibition of iPLA2, but not cPLA2, attenuated IgM binding to apoptotic cells, these results strongly suggest that the endogenous calcium independent PLA2, iPLA2, is involved in the hydrolysis of plasma membrane phospholipids and exposure of the epitope(s) recognized by IgM. We propose that recognition of dying cells by natural IgM antibodies is, in part, responsible for complement activation on dying cells leading to their safe clearance.
PMCID: PMC2194002  PMID: 12208880
IgM; apoptosis; complement; macrophages; autoimmunity
9.  Oxidative Regulation of Large Conductance Calcium-Activated Potassium Channels 
The Journal of General Physiology  2001;117(3):253-274.
Reactive oxygen/nitrogen species are readily generated in vivo, playing roles in many physiological and pathological conditions, such as Alzheimer's disease and Parkinson's disease, by oxidatively modifying various proteins. Previous studies indicate that large conductance Ca2+-activated K+ channels (BKCa or Slo) are subject to redox regulation. However, conflicting results exist whether oxidation increases or decreases the channel activity. We used chloramine-T, which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation in the cloned human Slo (hSlo) channel expressed in mammalian cells. In the virtual absence of Ca2+, the oxidant shifted the steady-state macroscopic conductance to a more negative direction and slowed deactivation. The results obtained suggest that oxidation enhances specific voltage-dependent opening transitions and slows the rate-limiting closing transition. Enhancement of the hSlo activity was partially reversed by the enzyme peptide methionine sulfoxide reductase, suggesting that the upregulation is mediated by methionine oxidation. In contrast, hydrogen peroxide and cysteine-specific reagents, DTNB, MTSEA, and PCMB, decreased the channel activity. Chloramine-T was much less effective when concurrently applied with the K+ channel blocker TEA, which is consistent with the possibility that the target methionine lies within the channel pore. Regulation of the Slo channel by methionine oxidation may represent an important link between cellular electrical excitability and metabolism.
PMCID: PMC2225619  PMID: 11222629
chloramine-T; methionine; methionine sulfoxide; methionine sulfoxide reductase; cysteine
10.  C-Reactive Protein Binds to Apoptotic Cells, Protects the Cells from Assembly of the Terminal Complement Components, and Sustains an Antiinflammatory Innate Immune Response 
The Journal of Experimental Medicine  2000;192(9):1353-1364.
C-reactive protein (CRP) is a serum protein that is massively induced as part of the innate immune response to infection and tissue injury. As CRP has been detected in damaged tissues and is known to activate complement, we assessed whether apoptotic lymphocytes bound CRP and determined the effect of binding on innate immunity. CRP bound to apoptotic cells in a Ca2+-dependent manner and augmented the classical pathway of complement activation but protected the cells from assembly of the terminal complement components. Furthermore, CRP enhanced opsonization and phagocytosis of apoptotic cells by macrophages associated with the expression of the antiinflammatory cytokine transforming growth factor β. The antiinflammatory effects of CRP required C1q and factor H and were not effective once cells had become necrotic. These observations demonstrate that CRP and the classical complement components act in concert to promote noninflammatory clearance of apoptotic cells and may help to explain how deficiencies of the classical pathway and certain pentraxins lead to impaired handling of apoptotic cells and increased necrosis with the likelihood of immune response to self.
PMCID: PMC2193350  PMID: 11067883
apoptosis; C-reactive protein; complement; macrophages; autoimmunity

Results 1-10 (10)