Acetylation is essential for nuclear heme oxygenase-1-enhanced tumor growth and invasiveness.

Overexpression of heme oxygenase-1 (HO-1), an endoplasmic reticulum-anchored enzyme, is observed in many cancers. HO-1 nuclear translocation has been shown to correlate with progression of several cancers. We recently reported that HO-1 is susceptible to intramembrane proteolysis and translocates to the nucleus to promote cancer growth and invasiveness without depending on its enzymatic activity. In the present study, we show that the HO-1 lacking C-terminal transmembrane segment (t-HO-1) was susceptible to acetylation by p300 and CREB-binding protein (CBP) histone acetyltransferase in the nucleus. Mass spectrometry analysis of HO-1 isolated from human embryonic kidney cells 293T (HEK293T) cells overexpressing CBP and t-HO-1 revealed two acetylation sites located at K243 and K256. Mutation of both lysine residues to arginine (R) abolished t-HO-1-enhanced tumor cell growth, migration and invasion. However, mutation of the lysine residues to glutamine (Q), a mimic of acetylated lysine, had no significant effect on t-HO-1-mediated tumorigenicity. Mechanistic studies demonstrated that transcriptional factor JunD interacted with wild-type (WT) t-HO-1 and mutant carrying K243/256Q but not K243/256 R mutation. Moreover, JunD-induced AP-1 transcriptional activity was significantly enhanced by coexpression with WT and acetylation-mimic but not acetylation-defective t-HO-1. Consistent with the in vitro observations, the implication of K243/256 acetylation in t-HO-1-enhanced tumorigenicity was also demonstrated in xenograft models. Immunohistochemistry performed with a specific antibody against acetyl-HO-1 showed the positive acetyl-HO-1 nuclear staining in human lung cancer tissues but not in the corresponding non-tumor tissues, supporting its clinical significance. Collectively, our findings highlight the importance of nuclear HO-1 post-translational modification in the induction of cancer progression.

Signal peptide peptidase-mediated nuclear localization of heme oxygenase-1 promotes cancer cell proliferation and invasion independent of its enzymatic activity.

Heme oxygenase-1 (HO-1) is a heme-degrading enzyme anchored in the endoplasmic reticulum by a carboxyl-terminal transmembrane segment (TMS). HO-1 is highly expressed in various cancers and its nuclear localization is associated with the progression of some cancers. Nevertheless, the mechanism underlying HO-1 nuclear translocation and its pathological significance remain elusive. Here we show that the signal peptide peptidase (SPP) catalyzes the intramembrane cleavage of HO-1. Coexpression of HO-1 with wild-type SPP, but not a dominant-negative SPP, promoted the nuclear localization of HO-1 in cells. Mass spectrometry analysis of cytosolic HO-1 isolated from HeLa cells overexpressing HO-1 and SPP revealed two adjacent intramembrane cleavage sites located after S275 and F276 within the TMS. Mutations of S275F276 to A275L276 significantly hindered SPP-mediated HO-1 cleavage and nuclear localization. Nuclear HO-1 was detected in A549 and DU145 cancer cell lines expressing high levels of endogenous HO-1 and SPP. SPP knockdown or inhibition significantly reduced nuclear HO-1 localization in A549 and DU145 cells. The positive nuclear HO-1 stain was also evident in lung cancer tissues expressing high levels of HO-1 and SPP. Overexpression of a truncated HO-1 (t-HO-1) lacking the TMS in HeLa and H1299 cells promoted cell proliferation and migration/invasion. The effect of t-HO-1 was not affected by a mutation in the catalytic site. However, blockade of t-HO-1 nuclear localization abolished t-HO-1-mediated effect. The tumorigenic effect of t-HO-1 was also demonstrated in the mouse model. These findings disclose that SPP-mediated intramembrane cleavage of HO-1 promotes HO-1 nuclear localization and cancer progression independent of HO-1 enzymatic activity.

p53-Mediated transactivation of LIMK2b links actin dynamics to cell cycle checkpoint control.

The p53 tumor suppressor protein is widely known for its role as a sequence-specific transcription factor that regulates the expression of stress response genes. Here, we report the identification of LIMK2, which encodes a kinase that regulates actin dynamics through phosphorylation of cofilin, as a p53 target upregulated by DNA damage. Interestingly, the splice variant LIMK2b, but not LIMK2a, was induced in a p53-dependent manner through an intronic consensus p53-binding site. Depletion of LIMK2b leads to early exit of G2/M arrest after DNA damage, whereas its overexpression prolongs the arrest. These responses are recapitulated by ectopic expression of the active cofilin S3A mutant and the inactive cofilin S3D mutant, respectively, suggesting that LIMK2b may modulate G2/M arrest through cofilin phosphorylation. Furthermore, in support of its potential role as a tumor suppressor, LIMK2b was downregulated in esophageal and thyroid cancers, as well as in a number of established cancer cell lines, and its expression suppresses cancer cell migration. Taken together, our results unveil a novel pathway whereby LIMK2b, acting downstream of p53, ensures proper execution of checkpoint arrest by modulating the dynamics of actin polymerization.

Identification and macrophage-activating activity of glycolipids released from intracellular Mycobacterium bovis BCG.

Intracellular mycobacteria release cell wall glycolipids into the endosomal network of infected macrophages. Here, we characterize the glycolipids of Mycobacterium bovis BCG (BCG) that are released into murine bone marrow-derived macrophages (BMMØ). Intracellularly released mycobacterial lipids were harvested from BMMØ that had been infected with 14C-labelled BCG. Released BCG lipids were resolved by thin-layer chromatography, and they migrated similarly to phosphatidylinositol dimannosides (PIM2), mono- and diphosphatidylglycerol, phosphatidylethanolamine, trehalose mono- and dimycolates and the phenolic glycolipid, mycoside B. Culture-derived BCG lipids that co-migrated with the intracellularly released lipids were purified and identified by electrospray ionization mass spectrometry. When delivered on polystyrene microspheres, fluorescently tagged BCG lipids were also released into the BMMØ, in a manner similar to release from viable or heat-killed BCG bacilli. To determine whether the released lipids elicited macrophage responses, BCG lipid-coated microspheres were delivered to interferon gamma-primed macrophages (BMMØ or thioglycollate-elicited peritoneal macrophages), and reactive nitrogen intermediates as well as tumour necrosis factor-alpha and monocyte chemoattractant protein-1 production were induced. When fractionated BCG lipids were delivered on the microspheres, PIM2 species reproduced the macrophage-activating activity of total BCG lipids. These results demonstrate that intracellular mycobacteria release a heterogeneous mix of lipids, some of which elicit the production of proinflammatory cytokines from macrophages that could potentially contribute to the granulomatous response in tuberculous diseases.

Studies of phospholipid metabolism, proliferation, and secretion of stably transfected insulinoma cells that overexpress group VIA phospholipase A2.

A cytosolic 84 kDa Group VIA phospholipase A2 (iPLA2beta) that does not require Ca2+ for catalysis was cloned from Chinese hamster ovary (CHO) cells, murine P388D1 cells, pancreatic islet beta-cells, and other sources. Proposed iPLA2beta functions include participation in phosphatidylcholine (PC) homeostasis by degrading excess PC generated in CHO cells that overexpress CTP:phosphocholine cytidylyltransferase (CT), which catalyzes the rate-limiting step in PC biosynthesis; participation in biosynthesis of arachidonate-containing PC species in P388D1 cells by generating lysophosphatidylcholine (LPC) acceptors for arachidonate incorporation; and participation in signaling events in insulin secretion from islet beta-cells. To further examine iPLA2beta functions in beta-cells, we prepared stably transfected INS-1 insulinoma cell lines that overexpress iPLA2beta activity eightfold compared to parental INS-1 cells or to INS-1 cells transfected with an empty retroviral vector that did not contain iPLA2beta cDNA. The iPLA2beta-overexpressing cells exhibit a twofold increase in CT activity compared to parental cells but little change in rates of [3H]choline incorporation into or disappearance from PC. Electrospray ionization (ESI) tandem mass spectrometric measurements indicate that iPLA2beta-overexpressing cells have 1.5-fold higher LPC levels than parental INS-1 cells but do not exhibit increased rates of [3H]arachidonate incorporation into phospholipids, and incorporation is unaffected by a bromoenol lactone (BEL) suicide substrate inhibitor of iPLA2beta. The rate of appearance of arachidonate-containing phosphatidylethanolamine species visualized by ESI mass spectrometry is also similar in iPLA2beta-overexpressing and parental INS-1 cells incubated with supplemental arachidonic acid, and this process is unaffected by BEL. Compared to parental INS-1 cells, iPLA2beta-overexpressing cells proliferate more rapidly and exhibit amplified insulin secretory responses to a protein kinase C-activating phorbol ester, glucose, and a cAMP analog. These findings suggest that iPLA2beta plays a signaling role in beta-cells that differs from housekeeping functions in PC biosynthesis and degradation in P388D1 and CHO cells.

Reactive chlorinating species produced by myeloperoxidase target the vinyl ether bond of plasmalogens: identification of 2-chlorohexadecanal.

Plasmalogens contain a vinyl ether bond linking the sn-1 aliphatic chain to the glycerol backbone of this predominant phospholipid molecular subclass, which is found in many mammalian tissues. The present study demonstrates that the vinyl ether bond of plasmalogens is a molecular target of the reactive chlorinating species produced by myeloperoxidase. Analysis by thin layer chromatography revealed that reactive chlorinating species produced by myeloperoxidase target the vinyl ether bond of the plasmalogen, lysoplasmenylcholine (1-O-hexadec-1'-enyl-sn-glycero-3-phosphorylcholine), resulting in the production of a neutral lipid. Capillary gas chromatographic analyses demonstrated that the neutral lipid generated from lysoplasmenylcholine was neither hexadecanal nor did it contain masked hexadecanal (i.e. the vinyl ether) because the dimethyl acetal of hexadecanal produced by acid methanolysis derivatization was no longer present. Electrospray ionization mass spectrometry of the myeloperoxidase-generated neutral lipid product was consistent with the production of a 16-carbon fatty aldehyde containing one chlorine atom. Furthermore, proton NMR analysis indicated that this neutral lipid product was a 2-chloro-fatty aldehyde. Additional structural analysis of this neutral lipid by gas chromatography-mass spectrometry of the underivatized product as well as its pentafluorobenzyl oxime-derivative product was consistent with the neutral lipid being 2-chlorohexadecanal. The reactive chlorinating species, hypochlorous acid and chlorine gas, both attacked the vinyl ether bond of lysoplasmenylcholine resulting in the production of 2-chlorohexadecanal. The production of 2-chlorohexadecanal was dependent on the presence of the plasmalogen masked aldehyde (i.e. the vinyl ether) in the substrate because the free fatty aldehyde, hexadecanal, was not converted to 2-chlorohexadecanal by the reactive chlorinating species generated by myeloperoxidase. Taken together, the present studies demonstrate for the first time the targeting of the vinyl ether bond of plasmalogens by the reactive chlorinating species produced by myeloperoxidase resulting in the production of novel chlorinated fatty aldehydes.

A novel mouse model of lipotoxic cardiomyopathy.

Inherited and acquired cardiomyopathies are associated with marked intracellular lipid accumulation in the heart. To test the hypothesis that mismatch between myocardial fatty acid uptake and utilization leads to the accumulation of cardiotoxic lipid species, and to establish a mouse model of metabolic cardiomyopathy, we generated transgenic mouse lines that overexpress long-chain acyl-CoA synthetase in the heart (MHC-ACS). This protein plays an important role in vectorial fatty acid transport across the plasma membrane. MHC-ACS mice demonstrate cardiac-restricted expression of the transgene and marked cardiac myocyte triglyceride accumulation. Lipid accumulation is associated with initial cardiac hypertrophy, followed by the development of left-ventricular dysfunction and premature death. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and cytochrome c release in transgenic hearts suggest that cardiac myocyte death occurs, in part, by lipid-induced programmed cell death. Taken together, our data demonstrate that fatty acid uptake/utilization mismatch in the heart leads to accumulation of lipid species toxic to cardiac myocytes. This novel mouse model will provide insight into the role of perturbations in myocardial lipid metabolism in the pathogenesis of inherited and acquired forms of heart failure.

Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2beta ) indicate a signaling rather than a housekeeping role for iPLA2beta.

A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.

Reactive nitrogen oxygen species metabolize N-acetylbenzidine.

A close association has been reported for certain types of cancers influenced by aromatic amines and infection/inflammation. Reactive nitric oxygen species (RNOS), components of the inflammatory response, are bactericidal and tumoricidal, and contribute to the deleterious effects attributed to inflammation on normal tissues. This study assessed the possible transformation of the aromatic amine N-acetylbenzidine (ABZ) by RNOS. RNOS were generated by various conditions to react with ABZ, and samples were evaluated by HPLC. Conditions which generate nitrogen dioxide radical (NO(2)(-) + myeloperoxidase + H(2)O(2), ONOO(-), and NO(2)(-) + HOCl) produced primarily a single new product termed 3'-nitro-ABZ. The myeloperoxidase-catalyzed reaction with 0.3 mM NO(2)(-) was completely inhibited by 1 mM cyanide, and not effected by 100 mM chloride with or without 1 mM taurine. In contrast, conditions which generate N(2)O(3), such as spermine NONOate, did not produce 3'-nitro-ABZ, but rather two compounds termed 4'-OH-AABP and AABP. (1)H NMR and mass spectrometry identified 3'-nitro-ABZ as 3'-nitro-N-acetylbenzidine, 4'-OH-AABP as 4'-OH-4-acetylaminobiphenyl, and AABP as 4-acetylaminobiphenyl. Human polymorphonuclear neutrophils incubated with [(3)H]ABZ and stimulated with beta-phorbol 12-myristate 13-acetate produced 3'-nitro-ABZ in the presence of NO(2)(-) (0.1-1 mM). Neutrophil 3'-nitro-ABZ formation was verified by mass spectrometry and was consistent with myeloperoxidase oxidation of NO(2)(-). The results demonstrate that ABZ forms unique products in the presence of nitrosating and nitrating RNOS, which could influence the carcinogenic process and serve as biomarkers for these reactive species.

Methemoglobin oxidation of N-acetylbenzidine to form a sulfinamide.

Aromatic amine sulfinamide adducts of hemoglobin are biomarkers of exposure and evidence for cytochrome P-450 N-hydroxylation. The possible peroxidatic formation of an N-acetylbenzidine (ABZ) sulfinamide adduct by methemoglobin was examined. Following addition of H2O2, 0.06 mM [3H]ABZ was metabolized by methemoglobin. With 0.3 mM glutathione, a new peak was observed, ABZ-SG, representing 17% of the total radioactivity. N'-Hydroxy-N-acetylbenzidine and 4'-nitro-4-acetylaminobiphenyl were not detected. Optimal ABZ-SG formation was observed with 3 uM methemoglobin, 0.1 to 0.3 mM glutathione, and pH 5.5. Higher concentrations of glutathione were inhibitory. Without glutathione, an H2O2-to-ABZ molar ratio of 1:1 resulted in complete metabolism of ABZ. This ratio increased to greater than 2:1 with 0.3 mM glutathione. Nearly complete inhibition of ABZ-SG formation by cyanide (10 mM), ascorbic acid (0.1 mM), 5,5-dimethyl-1-pyrroline N-oxide (50 mM), thiourea (1 mM), and azide (0.3 mM), and the lack of inhibition by mannitol (50 mM) and superoxide dismutase (2 microg) is consistent with a methemoglobin-mediated peroxidatic reaction, which does not involve hydroxyl radical or superoxide. ABZ-SG was identified by electrospray ionization/mass spectrometry as N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide. Conjugate was hydrolyzed by 0.1 N HCl and NaOH, was relatively stable at pH 5.5 and 7.4, and was susceptible to gamma-glutamyltranspeptidase treatment. Formation of an ABZ sulfinamide conjugate with hemoglobin was demonstrated. The results demonstrate that methemoglobin can catalyze the peroxidatic formation of an ABZ sulfinamide adduct, perhaps by a diimine monocation intermediate.

Structural determination of glycosphingolipids as lithiated adducts by electrospray ionization mass spectrometry using low-energy collisional-activated dissociation on a triple stage quadrupole instrument.

Structural characterization of glycosphingolipids as their lithiated adducts using low-energy collisional-activated dissociation (CAD) tandem mass spectrometry with electrospray ionization (ESI) is described. The tandem mass spectra contain abundant fragment ions reflecting the long chain base (LCB), fatty acid, and the sugar constituent of the molecule and permit unequivocal identification of cerebrosides, di-, trihexosyl ceramides and globosides. The major fragmentation pathways arise from loss of the sugar moiety to yield a lithiated ceramide ion, which undergoes further fragmentation to form multiple fragment ions that confirm the structures of the fatty acid and LCB. The mechanisms for the ion formation and the possible configuration of the fragment ions, resulting from CAD of the lithiated molecular ions ([M + Li]+) of monoglycosylceramides are proposed. The mechanisms were supported by CAD and source CAD tandem mass spectra of various cerebrosides and of their analogous molecules prepared by H-D exchange. Constant neutral loss and precursor ion scannings to identify galactosylceramides with sphingosine or sphinganine LCB subclasses, and with specific N-2-hydroxyl fatty acid subclass in mixtures are also demonstrated.

Elevated levels of protein-bound p-hydroxyphenylacetaldehyde, an amino-acid-derived aldehyde generated by myeloperoxidase, are present in human fatty streaks, intermediate lesions and advanced atherosclerotic lesions.

Reactive aldehydes might have a pivotal role in the pathogenesis of atherosclerosis by covalently modifying low-density lipoprotein (LDL). However, the identities of the aldehyde adducts that form on LDL in vivo are not yet clearly established. We previously demonstrated that the haem protein myeloperoxidase oxidizes proteins in the human artery wall. We also have shown that p-hydroxyphenylacetaldehyde (pHA), the aldehyde that forms when myeloperoxidase oxidizes L-tyrosine, covalently modifies the N(epsilon)-lysine residues of proteins. The resulting Schiff base can be quantified as N(epsilon)-[2-(p-hydroxyphenyl)ethyl]lysine (pHA-lysine) after reduction with NaCNBH(3). Here we demonstrate that pHA-lysine is a marker for LDL that has been modified by myeloperoxidase, and that water-soluble, but not lipid-soluble, antioxidants inhibit the modification of LDL protein. To determine whether myeloperoxidase-generated aldehydes might modify LDL in vivo, we used a combination of isotope-dilution GC-MS to quantify pHA-lysine in aortic tissues at various stages of lesion evolution. We also analysed LDL isolated from atherosclerotic aortic tissue. Comparison of normal and atherosclerotic aortic tissue demonstrated a significant elevation (more than 10-fold) of the reduced Schiff base adduct in fatty streaks, intermediate lesions and advanced lesions compared with normal aortic tissue. Moreover, the level of pHA-lysine in LDL recovered from atherosclerotic aortic intima was 200-fold that in plasma LDL of healthy donors. These results indicate that pHA-lysine, a specific covalent modification of LDL, is generated in human atherosclerotic vascular tissue. They also raise the possibility that reactive aldehydes generated by myeloperoxidase have a role in converting LDL into an atherogenic lipoprotein.

Characterization of phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate by electrospray ionization tandem mass spectrometry: a mechanistic study.

Structural characterization of phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PI-4P), and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) by collisionally activated dissociation (CAD) tandem mass spectrometry with electrospray ionization is described. In negative ion mode, the major fragmentation pathways under low energy CAD for PI arise from neutral loss of free fatty acid substituents ([M - H - RxCO2H]-) and neutral loss of the corresponding ketenes ([M - H - R'xCH=C=O]-), followed by consecutive loss of the inositol head group. The intensities of the ions arising from neutral loss of the sn-2 substituent as a free fatty acid ([M - H - R2CO2H]-) or as a ketene ([M - H - R'2CH=C=O] ) are greater than those of ions reflecting corresponding losses of the sn-1 substutient. This is consistent with our recent finding that ions reflecting those losses arise from charge-driven processes that occur preferentially at the sn-2 position. These features permit assignment of the position of the fatty acid substituents on the glycerol backbone. Nucleophilic attack of the anionic phosphate onto the C-1 or the C-2 of the glycerol to which the fatty acids attached expels sn-1 (R1CO2-) or sn-2 (R2CO2-) carboxylate anion, respectively. This pathway is sterically more favorable at sn-2 than at sn-1. However, further dissociations of [M - H - RxCO2H - inositol] , [M - H - RxCO2H]-, and [M - H - RxCH=C=O]- precursor ions also yield RxCO2- ions, whose abundance are affected by the collision energy applied. Therefore, relative intensities of the RxCO2- ions in the spectrum do not reflect their positions on the glycerol backbone and determination of their regiospecificities based on their ion intensities is not reliable. The spectra also contain specific ions at m/z 315, 279, 259, 241, and 223, reflecting the inositol head group. The last three ions are also observed in the tandem spectra of the [M - H]- ions of phosphatidylinositol monophosphate (PI-P) and phosphatidylinositol bisphosphate (PI-P2), in addition to the ions at m/z 321 and 303, reflecting the doubly phosphorylated inositol ions. The PI-P2 also contains unique ions at m/z 401 and 383 that reflect the triply phosphorylated inositol ions. The [M - H]- ions of PI-P and PI-P2 undergo fragmentation pathways similar to that of PI upon CAD. However, the doubly charged ([M - 2H]2-) molecular ions undergo fragmentation pathways that are typical of the [M - H]- ions of glycerophosphoethanolamine, which are basic. These results suggest that the further deprotonated gaseous [M - 2H]2 ions of PI-P and PI-P2 are basic precursors.

Charge-remote and charge-driven fragmentation processes in diacyl glycerophosphoethanolamine upon low-energy collisional activation: a mechanistic proposal.

A mechanistic study of diacyl glycerophosphoethanolamine fragmentation under low energy collision-activated dissociation with electrospray ionization tandem mass spectrometry is reported. The fragmentation pathways leading to the formation of carboxylate anions (RxCO2-) (x = 1, 2) and the formation of the ions representing neutral loss of ketene ([M - H - Rx'CH=C=O]-) are charge-driven processes, which are governed by the gas-phase basicity and the steric configuration of the molecules. The fragmentation pathway for the formation of the [M - H - RxCO2H]- ions, reflecting neutral loss of fatty acid, is a charge-remote process, which involves the participation of the hydrogens at C-1 and C-2 of the glycerol, resulting in [M - H - R2CO2H]- > [M - H - R1CO2H]-. The preferential formations of R2CO2- > R1CO2-, and of [M - H - R2'CH=C=O]- > [M - H - R1'CH=C=O]- are attributed to the findings that charge-driven processes are sterically more favorable at sn-2. The observation of the abundance of [M - H - Rx'CH=C=O]- > [M - H - RxCO2H]- is attributed to the fact that the [M - H]- ions of GPE are basic precursor ions, which undergo preferential loss of ketene than loss of acid. The major pathway for the formation of RxCO2- ions arises from the nucleophilic attack of the anionic charge site of the phosphate on the C-1 or C-2 of the glycerol to render a charge transfer. The sterically more favorable attack on the C-2 than C-2 of the glycerol results in the abundance of R2CO2- > R1CO2-. These features of tandem spectra readily identify and locate the fatty acid substituents of GPE in the glycerol backbone.

Electrospray ionization mass spectrometry analyses of nuclear membrane phospholipid loss after reperfusion of ischemic myocardium.

The role of nuclear membrane phospholipids as targets of phospholipases resulting in the generation of nuclear signaling messengers has received attention. In the present study, we have exploited the utility of electrospray ionization mass spectrometry to determine the phospholipid content of nuclei isolated from perfused hearts. Rat heart nuclei contained choline glycerophospholipids composed of palmitoyl and stearoyl residues at the sn-1 position with oleoyl, linoleoyl, and arachidonoyl residues at the sn-2 position. Diacyl molecular species were the predominant molecular subclass in the choline glycerophospholipids, with the balance of the molecular species being plasmalogens. In the ethanolamine glycerophospholipid pool from rat heart nuclei approximately 50% of the molecular species were plasmalogens, which were enriched with arachidonic acid at the sn-2 position. A 50% loss of myocytic nuclear choline and ethanolamine glycerophospholipids was observed in hearts rendered globally ischemic for 15 min followed by 90 min of reperfusion in comparisons with the content of these phospholipids in control perfused hearts. The loss of nuclear choline and ethanolamine glycerophospholipids during reperfusion of ischemic myocardium was partially reversed by the calcium-independent phospholipase A(2) (iPLA(2)) inhibitor bromoenol lactone (BEL), suggesting that the loss of nuclear phospholipids during ischemia/reperfusion is mediated, in part, by iPLA(2). Western blot analyses of isolated nuclei from ischemic hearts demonstrated that iPLA(2) is translocated to the nucleus after myocardial ischemia. Taken toghether, these studies have demonstrated that nuclear phospholipid mass decreases after myocardial ischemia by a mechanism that involves, at least in part, phospholipolysis mediated by iPLA2.

Electrospray ionization mass spectrometric analyses of changes in tissue phospholipid molecular species during the evolution of hyperlipidemia and hyperglycemia in Zucker diabetic fatty rats.

The Zucker diabetic fatty (ZDF) rat is a genetic model of type II diabetes mellitus in which males homozygous for nonfunctional leptin receptors (fa/fa) develop obesity, hyperlipidemia, and hyperglycemia, but rats homozygous for normal receptors (+/+) remain lean and normoglycemic. Insulin resistance develops in young fa/fa rats and is followed by evolution of an insulin secretory defect that triggers hyperglycemia. Because insulin secretion and insulin sensitivity are affected by membrane phospholipid fatty acid composition, we have determined whether metabolic abnormalities in fa/fa rats are associated with changes in tissue phospholipids. Electrospray ionization mass spectrometric analyses of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) molecular species from tissues of prediabetic (6 wk of age) and overtly diabetic (12 wk) fa/fa rats and from +/+ rats of the same ages indicate that arachidonate-containing species from heart, aorta, and liver of prediabetic fa/fa rats made a smaller contribution to GPC total ion current than was the case for +/+ rats. There was a correspondingly larger contribution from species with sn-2 oleate or linoleate substituents in fa/fa heart and aorta. The relative contributions of arachidonate-containing GPC species increased in these tissues as fa/fa rats aged and were equal to or greater than those for +/+ rats by 12 wk. For heart and aorta, relative contributions from GPE species with sn-2 arachidonate or docosahexaenoate substituents to the total ion current increased and those from species with sn-2 oleate or linoleate substituents fell as fa/fa rats aged, but these tissue lipid profiles changed little with age in +/+ rats. GPC and GPE profiles for brain, kidney, sciatic nerve, and red blood cells were similar among fa/fa and +/+ rats at 6 and 12 wk of age, and pancreatic islets from fa/fa and +/+ rats exhibited similar GPC and GPE profiles at 12 wk of age. Under-representation of arachidonate-containing GPC and GPE species in some fa/fa rat tissues at 6 wk could contribute to insulin resistance, but depletion of islet arachidonate-containing GPC and GPE species is unlikely to explain the evolution of the insulin secretory defect that is well-developed by 12 wk of age.

Nitrating reactive nitric oxygen species transform acetaminophen to 3-nitroacetaminophen.

Nitrating reactive nitric oxygen species (RNOS) elicit many of the deleterious effects of the inflammatory response. Their high reactivity and short half-life make RNOS analysis difficult. Reaction of acetaminophen (APAP) with RNOS generated by various conditions was evaluated by HPLC. When [(14)C]APAP was incubated at pH 7.4, the same new product (3NAP) was produced by at least three separate pathways represented by the following conditions: myeloperoxidase oxidation of NO(2)(-), NO(2)Cl, and ONOO(-) or Sin-1. Diethylamine NONO and spermine NONO did not convert APAP to 3NAP. 3NAP was stable at pH 5, 7.4, or 9, and at pH 7.4 with ONOO(-), spermine NONO, Sin-1, or H(2)O(2). HOCl transformed 3NAP, which was prevented by APAP, ascorbic acid, taurine, or NO(2)(-). ONOO(-)-derived 3NAP was identified by (1)H NMR as 3-nitroacetaminophen or 3-nitro-N-acetyl-p-aminophenol, and the product mass was verified by EI/ESI mass spectrometry. Human polymorphonuclear neutrophils incubated with [(14)C]APAP and stimulated with beta-phorbol 12-myristate 13-acetate produced 3NAP in the presence of NO(2)(-). Neutrophil 3NAP formation was verified by mass spectrometry and was consistent with myeloperoxidase oxidation of NO(2)(-). Spermine NONO supported 3NAP formation by stimulated cells in the absence of NO(2)(-). Results demonstrate that 3NAP is a product of nitrating RNOS generated by at least three separate pathways and may be a biomarker for nitrating mediators of inflammation.

Charge-driven fragmentation processes in diacyl glycerophosphatidic acids upon low-energy collisional activation. A mechanistic proposal.

A mechanistic study of diacyl glycerophosphatidic acid (GPA) under low energy collisionally activated decomposition (CAD) with electrospray ionization tandem mass spectrometry is reported. The fragmentation pathways leading to the formation of carboxylate anions [RxCO2-], (x = 1, 2) and the formation of the ions representing neutral loss of fatty acid ([M-H-RxCO2H] ) and neutral loss of ketene ([M-H-R'xCH-C=O] ) (Rx=R'xCH2) are charge-driven processes that are governed by the gas-phase basicity and the steric configuration of the molecules. The preferential formation of the ions of [M-H-R2CO2H]- > [M-H-R1CO2H]- and [M-H-R'2CH=C=O]- > [M-H-R'1CH=C=O]- are attributed to the fact that loss of fatty acid and loss of ketene are sterically more favorable at sn-2. While the observation of the abundance of [M-H-RxCO2H]- > [M-H-R'xCH=C=O]- is attributed to the acidity of the gas phase ion of GPA, which undergoes a more facile neutral loss of acid than loss of ketene. The major pathway leading to the formation of RxCO2- ion under low energy CAD arises from further fragmentation of the [M-H-RxCO2H]- ions by neutral loss of 136, resulting in an abundance of R1CO2- > R2CO2-. The differential formation of the carboxylate anions permits accurate assignment of the regiospecificity of the fatty acid substituents of GPA molecules by tandem mass spectrometry.

N-Acetylbenzidine-DNA adduct formation by phorbol 12-myristate-stimulated human polymorphonuclear neutrophils.

N'-(3'-Monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is the major adduct in exfoliated urothelial cells and in peripheral white blood cells of workers exposed to benzidine. This study was designed to assess the metabolic pathways leading to dGp-ABZ formation in human peripheral white blood cells. [(3)H]-N-Acetylbenzidine (ABZ) transformation was assessed using myeloperoxidase (MPO), hypochlorous acid (HOCl), and human peripheral white blood cells in the absence and presence of DNA or dGp. MPO metabolism required H(2)O(2), but not NaCl. While transformation by HOCl was completely inhibited by 10 mM taurine, the level of metabolism of ABZ by MPO was only reduced 56%. Transformation by either MPO or HOCl was inhibited by 100 mM DMPO, 1 mM glutathione, and 1 mM ascorbic acid. Glutathione formed a new product with MPO, but not with HOCl. Previously identified oxidation products of ABZ, N'-hydroxy-N-acetylbenzidine or 4'-nitro-4-acetylaminobiphenyl, were not detected. With DNA or dGp present, a new product was observed that corresponded to synthetic dGp-ABZ in its HPLC elution profile, in nuclease P(1) hydrolysis to dG-ABZ, and in (32)P-postlabeling analysis. The HOCl-derived adduct was identified by electrospray ionization mass spectrometry, with collision-activated dissociation, as dGp-ABZ. Metabolism of [(3)H]ABZ by peripheral blood cells was stimulated about 3-fold with 30 ng/mL beta-phorbol 12-myristate 13-acetate (PMA). Using (32)P-postlabeling, dGp-ABZ was detected only in the presence of PMA and its level was increased more than 300-fold if either 0.7 mg/mL DNA or dGp was present. Indomethacin (0.1 mM) did not alter adduct formation. With dGp, dGp-ABZ formation could be detected with as little as 0.12 x 10(6) neutrophils. Using specific chromatographic and enzymatic techniques, neutrophil-derived dGp-ABZ was identical to the synthetic standard. Thus, these results are consistent with human polymorphonuclear neutrophils forming dGp-ABZ by a peroxidatic mechanism involving MPO.