Free Radic Research Articles

Estrogen Receptor α Enhances the Rate of Oxidative DNA Damage by Targeting an Equine Estrogen Catechol Metabolite to the Nucleus

Exposure to estrogens increases the risk of breast and endometrial cancer. It is proposed that the estrogen receptor (ER) may contribute to estrogen carcinogenesis by transduction of the hormonal signal and as a "Trojan horse" concentrating genotoxic estrogen metabolites in the nucleus to complex with DNA, enhancing DNA damage. 4-Hydroxyequilenin (4-OHEN), the major catechol metabolite of equine estrogens present in estrogen replacement formulations, autoxidizes to a redox-cycling quinone that has been shown to cause DNA damage. 4-OHEN was found to be an estrogen of nanomolar potency in cell culture using a luciferase reporter assay and, using a chromatin immunoprecipitation assay, was found to activate ERalpha binding to estrogen-responsive genes in MCF-7 cells. DNA damage was measured in cells by comparing ERalpha(+) versus ERalpha(-) cells and 4-OHEN versus menadione, a reactive oxygen species (ROS)-generating, but non-estrogenic, quinone. 4-OHEN selectively induced DNA damage in ERalpha(+) cells, whereas menadione-induced damage was not dependent on cellular ER status. The rate of 4-OHEN-induced DNA damage was significantly enhanced in ERalpha(+) cells, whereas ER status had no effect on the rate of menadione-induced damage. Imaging of ROS induced by 4-OHEN showed accumulation selective for the nucleus of ERalpha(+) cells within 5 min, whereas in ERalpha(-) or menadione-treated cells, no selectivity was observed. These data support ERalpha acting as a Trojan horse concentrating 4-OHEN in the nucleus to accelerate the rate of ROS generation and thereby amplify DNA damage. The Trojan horse mechanism may be of general importance beyond estrogen genotoxins.

Endothelin-1 Inhibits Thick Ascending Limb Transport via Akt-stimulated Nitric Oxide Production

Endothelin-1 inhibits sodium reabsorption in the thick ascending limb (THAL) via stimulation of nitric oxide (NO) production. The mechanism whereby endothelin-1 stimulates THAL NO is unknown. We hypothesized that endothelin-1 stimulates THAL NO production by activating phosphatidylinositol 3-kinase (PI3K), stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport. We measured 1) NO production by fluorescence microscopy using DAF2-DA, 2) Akt activity using a fluorescence resonance energy transfer-based Akt reporter, 3) phosphorylated NOS3 and Akt by Western blotting, and 4) NKCC2 activity by fluorescence microscopy. In isolated THAL, endothelin-1 (1 nmol/liter) increased NO production from 0.23 +/- 0.24 to 2.81 +/- 0.32 fluorescence units/min (p < 0.001; n = 5) but failed to stimulate NO production in THALs isolated from NOS3-/- mice. Wortmannin (150 nmol/liter), a PI3K inhibitor, reduced endothelin-1-stimulated NO by 83% (0.49 +/- 0.13 versus 3.31 +/- 0.49 fluorescence units/min for endothelin-1 alone; p < 0.006; n = 5). Endothelin-1 stimulated Akt activity by 0.16 +/- 0.02 arbitrary units as measured by fluorescence resonance energy transfer (p < 0.001; n = 5) and increased phosphorylation of Akt at Ser473 by 56 +/- 11% (p < 0.002; n = 7). Dominant-negative Akt blocked endothelin-1-induced NO by 60 +/- 8% (p < 0.001 versus control; n = 6), and an Akt inhibitor had a similar effect. Endothelin-1 increased phosphorylation of NOS3 at Ser1177 by 89 +/- 24% (p < 0.01; n = 7) but had no effect on Ser633. Endothelin-1 inhibited NKCC2 activity, an effect that was blocked by dominant-negative Akt and NOS inhibition. We conclude that endothelin-1 stimulates THAL NO production by activating PI3K, stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport.

Increased Enzymatic O-GlcNAcylation of Mitochondrial Proteins Impairs Mitochondrial Function in Cardiac Myocytes Exposed to High Glucose

Increased nuclear protein O-linked beta-N-acetylglucosamine glycosylation (O-GlcNAcylation) mediated by high glucose treatment or the hyperglycemia of diabetes mellitus contributes to cardiac myocyte dysfunction. However, whether mitochondrial proteins in cardiac myocytes are also submitted to O-GlcNAcylation or excessive O-GlcNAcylation alters mitochondrial function is unknown. In this study, we determined if mitochondrial proteins are O-GlcNAcylated and explored if increased O-GlcNAcylation is linked to high glucose-induced mitochondrial dysfunction in neonatal rat cardiomyocytes. By immunoprecipitation, we found that several mitochondrial proteins, which are members of complexes of the respiratory chain, like subunit NDUFA9 of complex I, subunits core 1 and core 2 of complex III, and the mitochondrial DNA-encoded subunit I of complex IV (COX I) are O-GlcNAcylated. By mass spectrometry, we identified that serine 156 on NDUFA9 is O-GlcNAcylated. High glucose treatment (30 mm glucose) increases mitochondrial protein O-GlcNAcylation, including those of COX I and NDUFA9 which are reduced by expression of O-GlcNAcase (GCA). Increased mitochondrial O-GlcNAcylation is associated with impaired activity of complex I, III, and IV in addition to lower mitochondrial calcium and cellular ATP content. When the excessive O-GlcNAc modification is reduced by GCA expression, mitochondrial function improves; the activity of complex I, III, and IV increases to normal and mitochondrial calcium and cellular ATP content are returned to control levels. From these results we conclude that specific mitochondrial proteins of cardiac myocytes are O-GlcNAcylated and that exposure to high glucose increases mitochondrial protein O-GlcNAcylation, which in turn contributes to impaired mitochondrial function.

Cysteine S-Nitrosylation Protects Protein-tyrosine Phosphatase 1B against Oxidation-induced Permanent Inactivation

Protein S-nitrosylation mediated by cellular nitric oxide (NO) plays a primary role in executing biological functions in cGMP-independent NO signaling. Although S-nitrosylation appears similar to Cys oxidation induced by reactive oxygen species, the molecular mechanism and biological consequence remain unclear. We investigated the structural process of S-nitrosylation of protein-tyrosine phosphatase 1B (PTP1B). We treated PTP1B with various NO donors, including S-nitrosothiol reagents and compound-releasing NO radicals, to produce site-specific Cys S-nitrosylation identified using advanced mass spectrometry (MS) techniques. Quantitative MS showed that the active site Cys-215 was the primary residue susceptible to S-nitrosylation. The crystal structure of NO donor-reacted PTP1B at 2.6 A resolution revealed that the S-NO state at Cys-215 had no discernible irreversibly oxidized forms, whereas other Cys residues remained in their free thiol states. We further demonstrated that S-nitrosylation of the Cys-215 residue protected PTP1B from subsequent H(2)O(2)-induced irreversible oxidation. Increasing the level of cellular NO by pretreating cells with an NO donor or by activating ectopically expressed NO synthase inhibited reactive oxygen species-induced irreversible oxidation of endogenous PTP1B. These findings suggest that S-nitrosylation might prevent PTPs from permanent inactivation caused by oxidative stress.

RETRACTED: Expression of the longevity proteins by both red and white wines and their cardioprotective components, resveratrol, tyrosol, and hydroxytyrosol

Resveratrol increases longevity through SirT1, which is activated with NAD(+) supplied by an anti-aging enzyme PBEF. SirT1 interacts with an anti-aging transcription factor, FoxO1, which is negatively regulated by Akt. Since white wine could have similar health benefits as red wine, we determined if white wine and its cardioprotective components possess anti-aging properties by feeding rats with these compounds. The hearts expressed SirT, FoxO, and PBEF in the order of white wine>resveratrol>tyrosol>hydroxytyrosol>red wine, while cardioprotection shown by reduction of infarct size and cardiomyocyte apoptosis followed a different pattern: resveratrol>red wine>hydroxytyrosol>white wine>tyrosol, suggesting the existence of different signaling mechanisms for the induction of longevity and survival.

Synergistic induction of heme oxygenase-1 by the components of the antioxidant supplement Protandim

Protandim is an antioxidant supplement that consists of five ingredients, namely, ashwagandha, bacopa extract, green tea extract, silymarin, and curcumin, each with known therapeutic properties. Protandim was formulated with the objective of combining multiple phytochemicals at low nontoxic doses to gain synergy among them. A recent clinical study demonstrated the in vivo antioxidant effects of Protandim (S.K. Nelson et al., 2006, Free Radic. Biol. Med. 40, 341–347). The objective of the present study was to determine if the components of Protandim induce heme oxygenase-1 (HO-1) in a synergistic manner in cultured MIN6 cells, a mouse β-cell line, and in SK-N-MC cells, a human neuroblastoma cell line. When the components of Protandim were tested alone at low doses, curcumin showed minimal induction, whereas the others were unable to induce the HO-1 promoter, assayed by transient transfection. All components together, however, produced a strongly synergistic induction of around three- to ninefold in a dose-dependent manner, greatly exceeding the sum of the parts. Similar findings were obtained for the expression of HO-1 at the mRNA and protein levels. Protandim-mediated HO-1 induction involved the presence of ARE sites in the HO-1 promoter and nuclear translocalization of the transcription factor Nrf2, which binds to ARE sites. The involvement of multiple signaling pathways, including PI3-kinase/Akt, p38MAPK, and PKCδ, in HO-1 induction seems to be the probable mechanism of synergy between the components of Protandim. There were significant increases in the levels of total glutathione in Protandim-treated cells. These findings suggest that the use of a combination of phytochemicals may be an efficient method for the induction of antioxidant enzymes.

Overexpression of Glucose-6-phosphate Dehydrogenase Extends the Life Span of Drosophila melanogaster

The redox state of tissues tends to become progressively more prooxidizing during the aging process. The hypothesis tested in this study was that enhancement of reductive capacity by overexpression of glucose-6-phosphate dehydrogenase (G6PD), a key enzyme for NADPH biosynthesis, could protect against oxidative stress and extend the life span of transgenic Drosophila melanogaster. Overexpression of G6PD was achieved by combining a UAS-G6PD responder transgene at one of four independent loci with either a broad expression (armadillo-GAL4, Tubulin-GAL4, C23-GAL4, and da-GAL4) or a neuronal driver (D42-GAL4 and Appl-GAL4). The mean life spans of G6PD overexpressor flies were extended, in comparison with driver and responder controls, as follows: armadillo-GAL4 (up to 38%), Tubulin-GAL4 (up to 29%), C23-GAL4 (up to 27%), da-GAL4 (up to 24%), D42-GAL4 (up to 18%), and Appl-GAL4 (up to 16%). The G6PD enzymatic activity was increased, as were the levels of NADPH, NADH, and the GSH/GSSG ratio. Resistance to experimental oxidative stress was enhanced. Furthermore, metabolic rates and fertility were essentially the same in G6PD overexpressors and control flies. Collectively, the results demonstrate that enhancement of the NADPH biosynthetic capability can extend the life span of a relatively long-lived strain of flies, which supports the oxidative stress hypothesis of aging.

Enzymatic Mechanism Controls Redox-mediated Protein-DNA Interactions at the Replication Origin of Kinetoplast DNA Minicircles

Kinetoplast DNA (kDNA) is the mitochondrial DNA of trypanosomatids. Its major components are several thousand topologically interlocked DNA minicircles. Their replication origins are recognized by universal minicircle sequence-binding protein (UMSBP), a CCHC-type zinc finger protein, which has been implicated with minicircle replication initiation and kDNA segregation. Interactions of UMSBP with origin sequences in vitro have been found to be affected by the protein's redox state. Reduction of UMSBP activates its binding to the origin, whereas UMSBP oxidation impairs this activity. The role of redox in the regulation of UMSBP in vivo was studied here in synchronized cell cultures, monitoring both UMSBP origin binding activity and its redox state, throughout the trypanosomatid cell cycle. These studies indicated that UMSBP activity is regulated in vivo through the cell cycle dependent control of the protein's redox state. The hypothesis that UMSBP's redox state is controlled by an enzymatic mechanism, which mediates its direct reduction and oxidation, was challenged in a multienzyme reaction, reconstituted with pure enzymes of the trypanosomal major redox-regulating pathway. Coupling in vitro of this reaction with a UMSBP origin-binding reaction revealed the regulation of UMSBP activity through the opposing effects of tryparedoxin and tryparedoxin peroxidase. In the course of this reaction, tryparedoxin peroxidase directly oxidizes UMSBP, revealing a novel regulatory mechanism for the activation of an origin-binding protein, based on enzyme-mediated reversible modulation of the protein's redox state. This mode of regulation may represent a regulatory mechanism, functioning as an enzyme-mediated, redox-based biological switch.

Ferritin Contains Less Iron (59Fe) in Cells When the Protein Pores Are Unfolded by Mutation

Ferric minerals in ferritins are protected from cytoplasmic reductants and Fe2+ release by the protein nanocage until iron need is signaled. Deletion of ferritin genes is lethal; two critical ferritin functions are concentrating iron and oxidant protection (consuming cytoplasmic iron and oxygen in the mineral). In solution, opening/closing (gating) of eight ferritin protein pores controls reactions between external reductant and the ferritin mineral; pore gating is altered by mutation, low heat, and physiological urea (1 mm) and monitored by CD spectroscopy, protein crystallography, and Fe2+ release rates. To study the effects of a ferritin pore gating mutation in living cells, we cloned/expressed human ferritin H and H L138P, homologous to the frog open pore model that was unexpressable in human cells. Human ferritin H L138P behaved like the open pore ferritin model in vitro as follows: (i) normal protein cage assembly and mineralization, (ii) increased iron release (t1/2) decreased 17-fold), and (iii) decreased alpha-helix (8%). Overexpression (> 4-fold), in HeLa cells, showed for ferritin H L138P equal protein expression and total cell 59Fe but increased chelatable iron, 16%, p < 0.01 (59Fe in the deferoxamine-containing medium), and decreased 59Fe in ferritin, 28%, p < 0.01, compared with wild type. The coincidence of decreased 59Fe in open pore ferritin with increased chelatable 59Fe in cells expressing the ferritin open pore mutation suggests that ferritin pore gating influences to the amount of iron (59Fe) in ferritin in vivo.

Differential Activation of ERK and Rac Mediates the Proliferative and Anti-proliferative Effects of Hyaluronan and CD44

Hyaluronan, a widely distributed component of the extracellular matrix, exists in a high molecular weight (native) form and lower molecular weight form (HMW- and LMW-HA, respectively). These different forms of hyaluronan bind to CD44 but elicit distinct effects on cellular function. A striking example is the opposing effects of HMW- and LMW-HA on the proliferation of vascular smooth muscle cells; the binding of HMW-HA to CD44 inhibits cell cycle progression, whereas the binding of LMW-HA to CD44 stimulates cell cycle progression. We now report that cyclin D1 is the primary target of LMW-HA in human vascular smooth muscle cells, as it is for HMW-HA, and that the opposing cell cycle effects of these CD44 ligands result from differential regulation of signaling pathways to cyclin D1. HMW-HA binding to CD44 selectively inhibits the GTP loading of Rac and Rac-dependent signaling to the cyclin D1 gene, whereas LMW-HA binding to CD44 selectively stimulates ERK activation and ERK-dependent cyclin D1 gene expression. These data describe a novel mechanism of growth control in which a ligand-receptor system generates opposing effects on mitogenesis by differentially regulating signaling pathways to a common cell cycle target. They also emphasize how a seemingly subtle change in matrix composition can have a profound effect on cell proliferation.

Antiinflammatory Actions of HDL

Atherosclerosis is an inflammatory disorder characterized by the accumulation of macrophages and T-lymphocytes in the arterial intima. The macrophages are derived from blood monocytes that adhere to, and transmigrate across, an activated or injured endothelial surface. The firm adhesion of monocytes to the endothelium requires expression of integrins such as CD11/CD18 on the monocyte surface and endothelial expression of the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin. Monocytes that are firmly tethered to the endothelium transmigrate into the subendothelial space in a process that is dependent on endothelial expression of chemokines such as monocyte chemotactic protein-1 (MCP-1).1 See accompanying article on page 2071 After entering the artery wall, monocytes differentiate into macrophages in processes that are mediated by macrophage colony stimulating factor (CSF) and granulocyte-macrophage CSF.2 Macrophages express a range of scavenger receptors that bind and internalize modified LDL in an unregulated manner. This leads to a progressive accumulation of lipids in the macrophages and the formation of foam cells: the hallmark cells of atherosclerotic lesions. The results of numerous epidemiological studies have established that …

Physical and Functional Interaction between Human Oxidized Base-specific DNA Glycosylase NEIL1 and Flap Endonuclease 1

The S phase-specific activation of NEIL1 and not of the other DNA glycosylases responsible for repairing oxidatively damaged bases in mammalian genomes and the activation of NEIL1 by proliferating cell nuclear antigen (PCNA) suggested preferential action by NEIL1 in oxidized base repair during DNA replication. Here we show that NEIL1 interacts with flap endonuclease 1 (FEN-1), an essential component of the DNA replication. FEN-1 is present in the NEIL1 immunocomplex isolated from human cell extracts, and the two proteins colocalize in the nucleus. FEN-1 stimulates the activity of NEIL1 in vitro in excising 5-hydroxyuracil from duplex, bubble, forked, and single-stranded DNA substrates by up to 5-fold. The disordered region near the C terminus of NEIL1, which is dispensable for activity, is necessary and sufficient for high affinity binding to FEN-1 (K(D) approximately = 0.2 microm). The interacting interface of FEN-1 is localized in its disordered C-terminal region uniquely present in mammalian orthologs. Fine structure mapping identified several Lys and Arg residues in this region that form salt bridges with Asp and Glu residues in NEIL1. NEIL1 was previously shown to initiate single nucleotide excision repair, which does not require FEN-1 or PCNA. The present study shows that NEIL1 could also participate in strand displacement repair synthesis (long patch repair (LP-BER)) mediated by FEN-1 and stimulated by PCNA. Interaction between NEIL1 and FEN-1 is essential for efficient NEIL1-initiated LP-BER. These studies strongly implicate NEIL1 in a distinct subpathway of LP-BER in replicating genomes.

Fibroblast senescence

Fibroblast senescence

Phosphate Is Essential for Inhibition of the Mitochondrial Permeability Transition Pore by Cyclosporin A and by Cyclophilin D Ablation

Energized mouse liver mitochondria displayed the same calcium retention capacity (a sensitive measure of the propensity of the permeability transition pore (PTP) to open) irrespective of whether phosphate, arsenate, or vanadate was the permeating anion. Unexpectedly, however, phosphate was specifically required for PTP desensitization by cyclosporin A (CsA) or by genetic inactivation of cyclophilin D (CyP-D). Indeed, when phosphate was replaced by arsenate, vanadate, or bicarbonate, the inhibitory effects of CsA and of CyP-D ablation on the PTP disappeared. After loading with the same amount of Ca(2+) in the presence of arsenate or vanadate but in the absence of phosphate, the sensitivity of the PTP to a variety of inducers was identical in mitochondria from wild-type mice, CyP-D-null mice, and wild-type mice treated with CsA. These findings call for a reassessment of conclusions on the role of the PTP in cell death that are based on the effects of CsA or of CyP-D ablation.

Long Patch Base Excision Repair in Mammalian Mitochondrial Genomes

The mitochondrial genome is highly susceptible to damage by reactive oxygen species (ROS) generated endogenously as a byproduct of respiration. ROS-induced DNA lesions, including oxidized bases, abasic (AP) sites, and oxidized AP sites, cause DNA strand breaks and are repaired via the base excision repair (BER) pathway in both the nucleus and mitochondria. Repair of damaged bases and AP sites involving 1-nucleotide incorporation, named single nucleotide (SN)-BER, was observed with mitochondrial and nuclear extracts. During SN-BER, the 5'-phosphodeoxyribose (dRP) moiety, generated by AP-endonuclease (APE1), is removed by the lyase activity of DNA polymerase gamma (pol gamma) and polymerase beta in the mitochondria and nucleus, respectively. However, the repair of oxidized deoxyribose fragments at the 5' terminus after strand break would require 5'-exo/endonuclease activity that is provided by the flap endonuclease (FEN-1) in the nucleus, resulting in multinucleotide repair patch (long patch (LP)-BER). Here we show the presence of a 5'-exo/endonuclease in the mitochondrial extracts of mouse and human cells that is involved in the repair of a lyase-resistant AP site analog via multinucleotide incorporation, upstream and downstream to the lesion site. We conclude that LP-BER also occurs in the mitochondria requiring the 5'-exo/endonuclease and pol gamma with 3'-exonuclease activity. Although a FEN-1 antibody cross-reacting species was detected in the mitochondria, it was absent in the LP-BER-proficient APE1 immunocomplex isolated from the mitochondrial extract that contains APE1, pol gamma, and DNA ligase 3. The LP-BER activity was marginally affected in FEN-1-depleted mitochondrial extracts, further supporting the involvement of an unidentified 5'-exo/endonuclease in mitochondrial LP-BER.

Inhibitory Action of NoxA1 on Dual Oxidase Activity in Airway Cells

Imbalance between pro- and antioxidant mechanisms in the lungs can compromise pulmonary functions, including blood oxygenation, host defense, and maintenance of an anti-inflammatory environment. Thus, tight regulatory control of reactive oxygen species is critical for proper lung function. Increasing evidence supports a role for the NADPH oxidase dual oxidase (Duox) as an important source for regulated H2O2 production in the respiratory tract epithelium. In this study Duox expression, function, and regulation were investigated in a fully differentiated, mucociliary airway epithelium model. Duox-mediated H2O2 generation was dependent on calcium flux, which was required for dissociation of the NADPH oxidase regulatory protein Noxa1 from plasma membrane-bound Duox. A functional Duox1-based oxidase was reconstituted in model cell lines to permit mutational analysis of Noxa1 and Duox1. Although the activation domain of Noxa1 was not required for Duox function, mutation of a proline-rich domain in the Duox C terminus, a potential interaction motif for the Noxa1 Src homology domain 3, caused up-regulation of basal and stimulated H2O2 production. Similarly, knockdown of Noxa1 in airway cells increased basal H2O2 generation. Our data indicate a novel, inhibitory function for Noxa1 in Duox regulation. This represents a new paradigm for control of NADPH oxidase activity, where second messenger-promoted conformational change of the Nox structure promotes oxidase activation by relieving constraint induced by regulatory components.

Nitrosylation of ISG15 Prevents the Disulfide Bond-mediated Dimerization of ISG15 and Contributes to Effective ISGylation

The expression of the ubiquitin-like molecule ISG15 (UCRP) and protein modification by ISG15 (ISGylation) are strongly activated by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. Inducible nitric-oxide synthase (iNOS) is induced by the similar stimuli as ISG15 and enhances the production of nitric oxide (NO), a pleiotropic free radical with antipathogen activity. Here, we report that cysteine residues (Cys-76 and -143 in mouse, Cys-78 in human) of ISG15 can be modified by NO, and the NO modification of ISG15 decreases the dimerization of ISG15. The mutation of the cysteine residue of ISG15 to serine improves total ISGylation. The NO synthase inhibitor S-ethylisothiourea reduces endogenous ISGylation. Furthermore, ectopic expression of iNOS enhanced total ISGylation. Together, these results suggest that nitrosylation of ISG15 enhances target protein ISGylation. This is the first report of a relationship between ISGylation and nitrosylation.

HDL: Close to Our Memories?

The last decade has witnessed an explosion in studies of the role of lipoproteins in brain function. Neurons require a continuous supply of lipids for membrane synthesis and acetylcholine production. Indeed, the brain is a site of intense lipid turnover—even though the central nervous system (CNS) accounts for only 2.1% of body weight, it contains 23% of total body cholesterol.1 Lipid metabolism in the brain is tightly controlled locally, as plasma lipoproteins are shielded from the brain by the blood-brain barrier. Although neuronal cells are capable of de novo synthesis of a wide spectrum of molecular species of lipids, they rely heavily on exogenous sources and readily bind and internalize lipoproteins of the extracellular fluid.2 Equally, neurons need to dispose of excess lipids; lipoprotein-mediated lipid transport is therefore bidirectional and includes cellular efflux of cholesterol.3 See accompanying article on page 1556 Human cerebrospinal fluid (CSF) primarily contains spherical lipoproteins of approximately 10 to 12 nm in diameter with hydrated density in the range 1.063 to 1.25 g/mL, thereby resembling HDL in human plasma.3,4⇓ Lipid concentrations in CSF are however much lower (eg, 300- to 400-fold for total cholesterol and phospholipids) as compared to the plasma compartment.4 Apolipoproteins (apo) E and A-I are the major apolipoproteins in human CSF (typical concentration range: 0.1 to 0.4 mg/dL3,4⇓), with apoA-II, A-IV, J, D, C-II, C-III, C-IV, and H equally present.3,4⇓ Importantly, CSF lipoproteins carry amyloid-β (Aβ), a 39- to 43-aa peptide produced in neuronal cells, which is the major component of senile amyloid plaques.5 The metabolism of CSF lipoproteins remains poorly characterized but seems to be distinct from that of plasma lipoproteins. ApoE-rich HDL are synthesized locally in CNS and secreted by astrocytes as discoidal complexes enriched in free cholesterol.6 High …

Bioactivation of Nitroglycerin by Purified Mitochondrial and Cytosolic Aldehyde Dehydrogenases

Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or is also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and, like 1,2-GDN formation, were sensitive to ALDH inhibitors. GTN caused biphasic sGC activation with apparent EC50 values of 42 ± 2.9 and 3.1 ± 0.4 μm in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. These data may explain the coupling of ALDH2-catalyzed GTN metabolism to sGC activation in vascular smooth muscle.

Hepatocyte-specific c-Met Deletion Disrupts Redox Homeostasis and Sensitizes to Fas-mediated Apoptosis

The hepatocyte growth factor and its receptor c-Met direct a pleiotropic signal transduction pathway that controls cell survival. We previously demonstrated that mice lacking c-Met (Met-KO) in hepatocytes were hypersensitive to Fas-induced liver injury. In this study, we used primary hepatocytes isolated from Met-KO and control (Cre-Ctrl) mice to address more directly the protective effects of c-Met signaling. Loss of c-Met function increased sensitivity to Fas-mediated apoptosis. Hepatocyte growth factor suppressed apoptosis in Cre-Ctrl but not Met-KO hepatocytes concurrently with up-regulation of NF-kappaB and major antiapoptotic proteins Bcl-2 and Bcl-xL. Intriguingly, Met-KO hepatocytes exhibited intrinsic activation of NF-kappaBas well as Bcl-2 and Bcl-xL. Furthermore, unchallenged Met-KO cells displayed oxidative stress as evidenced by overproduction of reactive oxygen species, which was associated with greater NADPH and Rac1 activities, was blocked by the known NADPH oxidase inhibitors, and was paralleled by increased lipid peroxidation and reduced glutathione (GSH) content. N-Acetylcysteine, an antioxidant and GSH precursor, significantly reduced Jo2-induced cell death. Conversely, the GSH-depleting agent buthionine sulfoximine completely abolished the protective effects of N-acetylcysteine in Met-KO hepatocytes. In conclusion, genetic inactivation of c-Met in mouse hepatocytes caused defects in redox regulation, which may account for the increased sensitivity to Fas-induced apoptosis and adaptive up-regulation of NF-kappaB survival signaling. These data provide evidence that intact c-Met signaling is a critical factor in the protection against excessive generation of endogenous reactive oxygen species.