A series of plasmids encoding various Klebsiella pneumoniae nif (nitrogen fixation) genes were constructed to determine which were required to produce active iron (Fe) protein in Escherichia coli, a species which does not normally fix nitrogen. The greatest success was achieved with binary plasmid systems that produced nifA regulatory protein under the control of a tac promoter on one plasmid, which then induced synthesis of nifH and nifM proteins from their native promoter sites on a second plasmid. nifH protein, the monomeric subunit of Fe protein, produced in the presence of nifM constituted nearly 10% of the whole cell protein and exhibited the corresponding amount of C2H2-reducing activity in nitrogenase assays conducted in vitro. nifH protein formed in the absence of nifM constituted 4.7% of the whole cell protein and exhibited no detectable activity in assays of whole cell extracts. The plasmid-encoded Fe protein was purified to homogeneity and was found to be indistinguishable from that isolated from derepressed wild type K. pneumoniae, having a similar specific activity, approximately 4 Fe/dimer of 68 kDa, and similar epr features. Although these experiments do not exclude the participation of other E. coli gene products in the maturation of nifH protein, they limit the nif-specific genes required for active Fe protein production to nifA, nifH, and nifM. Since nifA is thought to be the required activator protein involved in nif operon transcription, the simplest explanation for these observations is that nifH codes for the peptide of the Fe protein, while nifM acts to convert this nifH peptide to the functioning Fe protein of nitrogenase. In the absence of nifM, only an inactive nifH polypeptide is produced.

A protein analog of a purple copper center has been constructed from a recombinant blue copper protein (Pseudomonas aeruginosa azurin) by replacing the loop containing the three ligands to the blue copper center with the corresponding loop of the CuA center in cytochrome c oxidase (COX) from Paracoccus denitrificans. The electronic absorption in the UV and visible region (UV-vis) and electron paramagnetic resonance (EPR) spectra of this analog are remarkably similar to those of the native CuA center in COX from Paracoccus denitrificans. The above spectra can be obtained upon addition of a mixture of Cu2+ and Cu+. Addition of Cu2+ only results in a UV-vis spectrum consisting of absorptions from both a purple copper center and a blue copper center. This spectrum can be converted to the spectrum of a pure purple copper by a prolonged incubation in the air, or by addition of excess ascorbate. The azurin mutant reported here is an example of an engineered purple copper center with the A480/A530 ratio greater than 1 and with no detectable hyperfines, similar to those of the CuA sites in COX of bovine heart and of Paracoccus denitrificans.

The glycolytic enzyme phosphoglycerate mutase exists in two evolutionarily unrelated forms. Vertebrates have only the 2,3-bisphosphoglycerate-dependent enzyme (dPGM), whilst higher plants have only the cofactor-independent enzyme (iPGM). Certain eubacteria possess genes encoding both enzymes, and their respective metabolic roles and activities are unclear. We have over-expressed, purified and characterised the two PGMs of Escherichia coli. Both are expressed at high levels, but dPGM has a 10-fold higher specific activity than iPGM. Differential inhibition by vanadate was observed. The presence of an integral manganese ion in iPGM was confirmed by EPR spectroscopy.

Addition of the Lewis acid Zn(2+) to (TBP(8)Cz)Mn(V)(O) induces valence tautomerization, resulting in the formation of [(TBP(8)Cz(+•))Mn(IV)(O)-Zn(2+)]. This new species was characterized by UV-vis, EPR, the Evans method, and (1)H NMR and supported by DFT calculations. Removal of Zn(2+) quantitatively restores the starting material. Electron-transfer and hydrogen-atom-transfer reactions are strongly influenced by the presence of Zn(2+).

1. The EPR spectrum at 15 degrees K of soybean lipoxygenase-1 in borate buffer pH 9.0 has been studied in relation to the presence of substrate (linoleic acid), product (13-L-hydroperoxylinoleic acid) and oxygen. 2. The addition of 13-L-hydroperoxylinoleic acid to lipoxygenase-1 at pH 9.0 gives rise to the appearance of EPR lines at g equals 7.5, 6.2, 5.9 and 2.0, and an increased signal at g equals 4.3. 3. In view of the effect of the end product on both the kinetic lag period of the aerobic reaction and the fluorescence of the enzyme, it is concluded that 13-L-hydroperoxylinoleic acid is required for the activation of soybean lipoxygenase-1. Thus it is proposed that the enzyme with iron in the ferric state is the active species. 4. A reaction scheme is presented in which the enzyme alternatingly exists in the ferric and ferrous states for both the aerobic and anaerobic reaction.

Stopped-flow absorption and freeze-quench electron paramagnetic resonance (EPR) and Mössbauer spectroscopies have been used to obtain evidence for the intermediacy of a (mu-1,2-peroxo)diiron(III/III) complex on the pathway to the tyrosyl radical and (mu-oxo)diiron(III/III) cluster during assembly of the essential cofactor in the R2 subunit of ribonucleotide reductase from mouse. The complex accumulates to approximately 0.4 equiv in the first few milliseconds of the reaction and decays concomitantly with accumulation of the previously detected diiron(III/IV) cluster, X, which generates the tyrosyl radical and product (mu-oxo)diiron(III/III) cluster. Kinetic complexities in the reaction suggest the existence of an anti-cooperative interaction of the monomers of the R2 homodimer in Fe(II) binding and perhaps O2 activation. The detection of the (mu-1,2-peroxo)diiron(III/III) complex, which has spectroscopic properties similar to those of complexes previously characterized in the reactions of soluble methane monooxygenase, stearoyl acyl carrier protein Delta9 desaturase, and variants of Escherichia coli R2 with the iron ligand substitution, D84E, provides support for the hypothesis that the reactions of the diiron-carboxylate oxidases and oxygenases commence with the formation of this common intermediate.

The Bacillus subtilis oxalate decarboxylase (EC ), YvrK, converts oxalate to formate and CO(2). YvrK and the related hypothetical proteins YoaN and YxaG from B. subtilis have been successfully overexpressed in Escherichia coli. Recombinant YvrK and YoaN were found to be soluble enzymes with oxalate decarboxylase activity only when expressed in the presence of manganese salts. No enzyme activity has yet been detected for YxaG, which was expressed as a soluble protein without the requirement for manganese salts. YvrK and YoaN were found to catalyze minor side reactions: oxalate oxidation to produce H(2)O(2); and oxalate-dependent, H(2)O(2)-independent dye oxidations. The oxalate decarboxylase activity of purified YvrK was O(2)-dependent. YvrK was found to contain between 0.86 and 1.14 atoms of manganese/subunit. EPR spectroscopy showed that the metal ion was predominantly but not exclusively in the Mn(II) oxidation state. The hyperfine coupling constant (A = 9.5 millitesla) of the main g = 2 signal was consistent with oxygen and nitrogen ligands with hexacoordinate geometry. The structure of YvrK was modeled on the basis of homology with oxalate oxidase, canavalin, and phaseolin, and its hexameric oligomerization was predicted by analogy with proglycinin and homogentisate 1,2-dioxygenase. Although YvrK possesses two potential active sites, only one could be fully occupied by manganese. The possibility that the C-terminal domain active site has no manganese bound and is buried in an intersubunit interface within the hexameric enzyme is discussed. A mechanism for oxalate decarboxylation is proposed, in which both Mn(II) and O(2) are cofactors that act together as a two-electron sink during catalysis.

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the assignment of the low field g value at 2.18 consistent with the seven line pattern observed at 9.31 GHz, 10 K. S-band spectra at 20 K are better resolved than the X-band spectra recorded at 10 K. The features observed at 2.4, 3.4, 9.31 and 35 GHz are explained by a mixed-valence [Cu(1.5)..Cu(1.5)] S = 1/2 species with the unpaired electron delocalized between two equivalent Cu nuclei. The resemblance of the N2OR S-band spectra to the spectra for the EPR-detectable Cu of cytochrome c oxidase suggests that the S-band spectrum for cytochrome c oxidase measured below 30 K may also contain hyperfine splittings from two approximately equivalent Cu nuclei.

Lysine 2,3-aminomutase catalyzes the interconversion of l-alpha-lysine and l-beta-lysine. The enzyme contains an iron-sulfur cluster with unusual properties, and it requires pyridoxal-5'-phosphate (PLP) and S-adenosylmethionine (AdoMet) for activity. The reaction proceeds by a substrate radical rearrangement mechanism, in which the external aldimine formed between PLP and lysine is initially converted into a lysyl-radical intermediate by hydrogen abstraction from C3. The present research concerns the mechanism by which a hydrogen-abstracting species is generated at the active site of lysine 2,3-aminomutase. Earlier tritium tracer experiments have implicated the 5'-deoxyadenosyl moiety of AdoMet in this process. AdoMet is here shown to interact with the iron-sulfur cluster at the active site of Clostridial lysine 2,3-aminomutase. Reduction of the iron-sulfur cluster from its EPR-silent form [4Fe-4S]2+ to the fully reduced form [4Fe-4S]1+ requires the presence of either AdoMet or S-adenosylhomocysteine (SAH) and a strong reducing agent such as dithionite or deazariboflavin and light. The reduced forms are provisionally designated E-[4Fe-4S]1+/AdoMet and E-[4Fe-4S]1+/SAH, and they display similar low-temperature EPR spectra centered at gav = 1.91. The reduced form E-[4Fe-4S]1+/AdoMet is fully active in the absence of any added reducing agent, whereas the form E-[4Fe-4S]1+/SAH is not active. It is postulated that the active form E-[4Fe-4S]1+/AdoMet is in equilibrium with a low concentration of a radical-initiating form that contains the 5'-deoxyadenosyl radical. Initiation of the radical rearrangement mechanism is postulated to take place by action of the 5'-deoxyadenosyl radical in abstracting a hydrogen atom from carbon-3 of lysine, which is bound as its external aldiminine with PLP. This process accounts for the results of tritium tracer experiments, it explains the radical rearrangement mechanism, and it rationalizes the roles of AdoMet and the [4Fe-4S] cluster in the reaction.

The purpose of this study was to examine the efficacy of a chemotherapeutic drug, doxorubicin (DOX), loaded in pH-sensitive micelles poly(l-histidine) (M(n):5K)-b-PEG (M(n):5K) micelles. The micelles were designed to target the acidic extracellular pH of solid tumors. Studies of pH-dependent cytotoxicity, growth rate of the tumor, pharmacokinetics and biodistribution were conducted. In vitro DOX uptake upon A2780 cells by incubating the cells in a pH 6.8 complete medium at a concentration of 20 microg DOX/ml in the micelle formulation was more than five times that of pH 7.4 condition for initial 20 min. In vivo pharmacokinetic data showed that AUC (area under concentration curve) and half life time (t(1/2)) (plasma half life) of DOX in the pH sensitive micelles increased about 5.8- and 5.2-fold of free DOX in phosphate buffered saline (PBS), respectively. It appeared that DOX in the pH-sensitive micelles preferentially accumulated in the tumor site. The distributions at 12 h post injection in other organs including liver, kidney, spleen, lung and heart were not significantly different from those of DOX in PBS at a 6 mg DOX/kg dose. The in vivo test of anti-tumor activity was performed with human ovarian carcinoma A2780 which was subcutaneously xenografted in female nu/nu athymic mice. The pH-sensitive micelle formulation significantly retarded tumor growth rate without serious body weight loss. The triggered drug release by the reduced tumor pH is believed to be a major mechanism of the observed efficacy after passive accumulation of the micelles by EPR effect. This may have resulted in a local high dose of drug in the tested solid tumor.

Several model tumour systems are now known to display increased vascular permeability compared with normal tissues, permitting their selective targeting using macromolecular drugs. Preliminary clinical observations suggest that this pathology may be reflected in at least some types of human cancer, and this may have important implications in facilitating macromolecular drug treatments, including antibody targeting and delivery of DNA for gene therapy. The enhanced permeability of tumour vasculature is thought to be regulated by tumour-secreted growth factors, with vascular permeability facor (VPF), also known as vascular endothelial growth factor (VEGF), emerging as a particularly likely candidate. VPF/VEGF is known to be an important regulator of tumour-angiogenesis in vivo, and it exerts its endothelium-specific effects via its receptors KDR/Flk-1 and Flt-1 on the endothelial cell membrane. Although the precise mechanism of VEGF's permeabilising action is not yet understood, it is likely to contribute to the enhanced permeability and retention (EPR) effect in tumours which is thought to underlie the anticancer activity of macromolecular drugs.

Oxidative stress has been suggested to contribute to neuronal apoptosis associated with Alzheimer's disease (AD). Copper may participate in oxidative stress through redox-cycling between its +2 and +1 oxidation states to generate reactive oxygen species (ROS). In vitro, copper binds to the amyloid-beta peptide of AD, and in vivo, copper is associated with amyloid plaques characteristic of AD. As a result, the AbetaCu(I) complex may be a critical reactant involved in ROS associated with AD etiology. To characterize the AbetaCu(I) complex, we have pursued X-ray absorption (XAS) and electron paramagnetic resonance (EPR) spectroscopy of AbetaCu(II) and AbetaCu(I) (produced by ascorbate reduction of AbetaCu(II)). The AbetaCu(II) complex Cu K-edge XAS spectrum is indicative of a square-planar Cu(II) center with mixed N/O ligation. Multiple scattering analysis of the extended X-ray absorption fine structure (EXAFS) data for AbetaCu(II) indicates that two of the ligands are imidazole groups of histidine ligands, indicating a (N(Im))(2)(N/O)(2) Cu(II) ligation sphere for AbetaCu(II). After reduction of the AbetaCu(II) complex with ascorbate, the edge region decreases in energy by approximately 4 eV. The X-ray absorption near-edge spectrum region of AbetaCu(I) displays an intense pre-edge feature at 8984.1(2) eV. EXAFS data fitting yielded a two-coordinate geometry, with two imidazole ligands coordinated to Cu(I) at 1.877(2) A in a linear geometry. Ascorbate reduction of AbetaCu(II) under inert atmosphere and subsequent air oxidation of AbetaCu(I) to regenerate AbetaCu(II) was monitored by low-temperature EPR spectroscopy. Slow reappearance of the AbetaCu(II) EPR signal indicates that O(2) oxidation of the AbetaCu(I) complex is kinetically sluggish and Abeta damage is occurring following reoxidation of AbetaCu(I) by O(2). Together, these results lead us to hypothesize that Cu(I) is ligated by His13 and His14 in a linear coordination environment in Alphabeta, that Abeta may be playing a neuroprotective role, and that metal-mediated oxidative damage of Abeta occurs over multiple redox cycles.

The organometallic H cluster at the active site of [FeFe]-hydrogenase consists of a 2Fe subcluster coordinated by cyanide, carbon monoxide, and a nonprotein dithiolate bridged to a [4Fe-4S] cluster via a cysteinate ligand. Biosynthesis of this cluster requires three accessory proteins, two of which (HydE and HydG) are radical S-adenosylmethionine enzymes. The third, HydF, is a GTPase. We present here spectroscopic and kinetic studies of HydF that afford fundamental new insights into the mechanism of H-cluster assembly. Electron paramagnetic spectroscopy reveals that HydF binds both [4Fe-4S] and [2Fe-2S] clusters; however, when HydF is expressed in the presence of HydE and HydG (HydF(EG)), only the [4Fe-4S] cluster is observed by EPR. Insight into the fate of the [2Fe-2S] cluster harbored by HydF is provided by FTIR, which shows the presence of carbon monoxide and cyanide ligands in HydF(EG). The thorough kinetic characterization of the GTPase activity of HydF shows that activity can be gated by monovalent cations and further suggests that GTPase activity is associated with synthesis of the 2Fe subcluster precursor on HydF, rather than with transfer of the assembled precursor to hydrogenase. Interestingly, we show that whereas the GTPase activity is independent of the presence of the FeS clusters on HydF, GTP perturbs the EPR spectra of the clusters, suggesting communication between the GTP- and cluster-binding sites. Together, the results indicate that the 2Fe subcluster of the H cluster is synthesized on HydF from a [2Fe-2S] cluster framework in a process requiring HydE, HydG, and GTP.

BACKGROUND

The proximal femur is the most common site of surgery for bone metastases, and stabilization may be achieved through intramedullary fixation (IMN) or endoprosthetic reconstruction (EPR). Intramedullary devices are less expensive, less invasive, and may yield improved function over endoprostheses. However, it is unclear which, if either, has any advantages.

OBJECTIVE

We determined whether function, complications, and survivorship differed between the two approaches.

METHODS

We retrospectively reviewed 158 patients with 159 proximal femur metastatic lesions treated with surgical stabilization. Forty-six were stabilized with IMN and 113 were treated with EPR. The minimum followup was 0.25 months (mean, 16 months; median, 17 months; range, 0.25-86 months).

RESULTS

The mean Musculoskeletal Tumor Society score was 24 of 30 (80%) after IMN and 21 of 30 (70%) after EPR. There were 12 complications (26%) in the IMN group, including 10 nonunions, six of which went on to mechanical failure. There were complications in 20 of 113 (18%) of the EPR group, which consisted of 10 dislocations (9%) and 10 infections (9%). There were no mechanical failures with EPR. Both implants remained functional for the limited lifespan of these patients in each group at all time intervals. EPRs were associated with increased implant longevity compared with IMNs (100% versus 85% 5-year survival, respectively) and a decreased rate of mechanical failure (0% versus 11%, respectively) when compared with the intramedullary devices.

CONCLUSIONS

Patients with metastatic disease to the proximal femur may live for long periods of time, and these patients may undergo stabilization with either IMN or EPR with comparable functional scores and the implant survivorship exceeding patient survivorship at all time intervals. Endoprostheses demonstrate a lower mechanical failure rate and a higher rate of implant survivorship without mechanical failure than IMN devices.

METHODS

Level III, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

An important clue to the mechanism for O(2) tolerance of certain [NiFe]-hydrogenases is the conserved presence of a modified environment around the iron-sulfur cluster that is proximal to the active site. The O(2)-tolerant enzymes contain two cysteines, located at opposite ends of this cluster, which are glycines in their O(2)-sensitive counterparts. The strong correlation highlights special importance for electron-transfer activity in the protection mechanism used to combat O(2). Site-directed mutagenesis has been carried out on Escherichia coli hydrogenase-1 to substitute these cysteines (C19 and C120) individually and collectively for glycines, and the effects of each replacement have been determined using protein film electrochemistry and electron paramagnetic resonance (EPR) spectroscopy. The "split" iron-sulfur cluster EPR signal thus far observed when oxygen-tolerant [NiFe]-hydrogenases are subjected to oxidizing potentials is found not to provide any simple, reliable correlation with oxygen tolerance. Oxygen tolerance is largely conferred by a single cysteine (C19), replacement of which by glycine removes the ability to function even in 1% O(2).

We have used chemical protein synthesis and advanced physical methods to probe dynamics-function correlations for the HIV-1 protease, an enzyme that has received considerable attention as a target for the treatment of AIDS. Chemical synthesis was used to prepare a series of unique analogues of the HIV-1 protease in which the flexibility of the "flap" structures (residues 37-61 in each monomer of the homodimeric protein molecule) was systematically varied. These analogue enzymes were further studied by X-ray crystallography, NMR relaxation, and pulse-EPR methods, in conjunction with molecular dynamics simulations. We show that conformational isomerization in the flaps is correlated with structural reorganization of residues in the active site, and that it is preorganization of the active site that is a rate-limiting factor in catalysis.

A growing body of evidence indicates that phytooxylipins play important roles in plant defense responses. However, many enzymes involved in the biosynthesis of these metabolites are still elusive. We have purified one of these enzymes, the peroxygenase (PXG), from oat microsomes and lipid droplets. It is an integral membrane protein requiring detergent for its solubilization. Proteinase K digestion showed that PXG is probably deeply buried in lipid droplets or microsomes with only about 2 kDa at the C-terminal region accessible to proteolytic digestion. Sequencing of the N terminus of the purified protein showed that PXG had no sequence similarity with either a peroxidase or a cytochrome P450 but, rather, with caleosins, i.e. calcium-binding proteins. In agreement with this finding, we demonstrated that recombinant thale cress and rice caleosins, expressed in yeast, catalyze hydroperoxide-dependent mono-oxygenation reactions that are characteristic of PXG. Calcium was also found to be crucial for peroxygenase activity, whereas phosphorylation of the protein had no impact on catalysis. Site-directed mutagenesis studies revealed that PXG catalytic activity is dependent on two highly conserved histidines, the 9 GHz EPR spectrum being consistent with a high spin pentacoordinated ferric heme.

The mechanism of cobalt(II)-porphyrin-mediated cyclopropanation of olefins with diazoesters was studied. The first step--reaction of cobalt(II)-porphyrin with ethyl diazoacetate (EDA)--was examined using EPR and ESI-MS techniques. EDA reacts with cobalt(II)-porphyrin to form a 1:1 Co(por)(CHCOOEt) adduct that exists as two isomers: the 'bridging carbene' C' in which the 'carbene' is bound to the metal and the pyrrolic nitrogen of the porphyrin that has a d(7) configuration on the metal, and the 'terminal carbene' C in which the 'carbene' behaves as a redox noninnocent ligand having a d(6) cobalt center and the unpaired electron residing on the 'carbene' carbon atom. The subsequent reactivities of the thus formed 'cobalt carbene radical' with propene, styrene, and methyl acrylate were studied using DFT calculations. The calculations suggest that the formation of the carbene is the rate-limiting step for the unfunctionalized Co(II)(por) and that the cyclopropane ring formation proceeds via a stepwise radical process: Radical addition of the 'carbene radical' C to the C=C double bonds of the olefins results in formation of the gamma-alkyl radical intermediates D. Species D then easily collapse in almost barrierless ring-closure reactions (TS3) to form the cyclopropanes. This radical mechanism readily explains the high activity of Co(II)(por) species in the cyclopropanation of electron-deficient olefins such as methyl acrylate.

The copper content of recombinant CotA laccase from Bacillus subtilis produced by Escherichia coli cells is shown to be strongly dependent on the presence of copper and oxygen in the culture media. In copper-supplemented media, a switch from aerobic to microaerobic conditions leads to the synthesis of a recombinant holoenzyme, while the maintenance of aerobic conditions results in the synthesis of a copper-depleted population of proteins. Strikingly, cells grown under microaerobic conditions accumulate up to 80-fold more copper than aerobically grown cells. In vitro copper incorporation into apoenzymes was monitored by optical and electron paramagnetic resonance (EPR) spectroscopy. This analysis reveals that copper incorporation into CotA laccase is a sequential process, with the type 1 copper center being the first to be reconstituted, followed by the type 2 and the type 3 copper centers. The copper reconstitution of holoCotA derivatives depleted in vitro with EDTA results in the complete recovery of the native conformation as monitored by spectroscopic, kinetic and thermal stability analysis. However, the reconstitution of copper to apo forms produced in cultures under aerobic and copper-deficient conditions resulted in incomplete recovery of biochemical properties of the holoenzyme. EPR and resonance Raman data indicate that, presumably, folding in the presence of copper is indispensable for the correct structure of the trinuclear copper-containing site.

Many small molecules, including bioactive molecules and approved drugs, spontaneously form colloidal aggregates in aqueous solution at micromolar concentrations. Though it is widely accepted that aggregation leads to artifacts in screens for ligands of soluble proteins, the effects of colloid formation in cell-based assays have not been studied. Here, seven anticancer drugs and one diagnostic reagent were found to form colloids in both biochemical buffer and in cell culture media. In cell-based assays, the antiproliferative activities of three of the drugs were substantially reduced when in colloidal form as compared to monomeric form; a new formulation method ensured the presence of drug colloids versus drug monomers in solution. We also found that Evans Blue, a dye classically used to measure vascular permeability and to demonstrate the "enhanced permeability and retention (EPR) effect" in solid tumors, forms colloids that adsorb albumin, as opposed to older literature that suggested the reverse.

Delivery of nanoparticle drugs to tumors relies heavily on the enhanced permeability and retention (EPR) effect. While many consider the effect to be equally effective on all tumors, it varies drastically among the tumors' origins, stages, and organs, owing much to differences in vessel leakiness. Suboptimal EPR effect represents a major problem in the translation of nanomedicine to the clinic. In the present study, we introduce a photodynamic therapy (PDT)-based EPR enhancement technology. The method uses RGD-modified ferritin (RFRT) as "smart" carriers that site-specifically deliver (1)O2 to the tumor endothelium. The photodynamic stimulus can cause permeabilized tumor vessels that facilitate extravasation of nanoparticles at the sites. The method has proven to be safe, selective, and effective. Increased tumor uptake was observed with a wide range of nanoparticles by as much as 20.08-fold. It is expected that the methodology can find wide applications in the area of nanomedicine.

BACKGROUND

Diesel exhaust particulate (DEP) is a key arbiter of the adverse cardiovascular effects of air pollution.

OBJECTIVE

We assessed the in vitro effects of DEP on vascular function, nitric oxide (NO) availability, and the generation of oxygen-centered free radicals.

METHODS

We assessed the direct vascular effects of DEP (10-100 microg/mL) in isolated rat aortic rings using myography. We investigated NO scavenging and oxygen-centered free radical generation using an NO electrode and electron paramagnetic resonance (EPR) with the Tempone-H (1-hydroxyl-2,2,6,6-tetramethyl-4-oxo-piperidine) spin trap, respectively.

RESULTS

Acetylcholine-induced relaxation was attenuated by DEP (maximum relaxation reduced from 91 +/- 4% to 49 +/- 6% with 100 microg/mL DEP; p < 0.001) but was restored by superoxide dismutase (SOD; maximum relaxation, 73 +/- 6%; p < 0.001). DEP caused a modest inhibition of relaxation to NO donor drugs, an effect that could be reversed by SOD (p < 0.01). At 10 microg/mL, DEP did not affect verapamil-induced relaxation (p = 0.73), but at 100 microg/mL DEP inhibited relaxation (p < 0.001) by a mechanism independent of SOD. NO concentrations generated by 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO; 10 microM) were reduced by DEP (100 microg/mL; from 5.2 +/- 0.4 to 3.3 +/- 0.4 microM; p = 0.002). Free radical generation was increased by DEP (10 microg/mL; 9-fold increase in EPR spectra; p = 0.004) in a manner that could be attenuated by SOD (p = 0.015).

CONCLUSIONS

DEP caused oxidative stress through the generation of oxygen-centered free radicals that reduced the bioavailability of endothelium-derived NO without prior interaction with the lung or vascular tissue. These findings provide a mechanism for the adverse cardiovascular effects of particulate air pollution.

Studies in experimental traumatic brain injury (TBI) suggest both deleterious and protective effects of inducible nitric oxide synthase (iNOS). Early after injury, iNOS may be detrimental via formation of peroxynitrite and iNOS inhibitors are protective. In contrast, we reported impaired long-term functional outcome after TBI in iNOS knockout (ko) versus wild-type (wt) mice. To elucidate potential neuroprotective and neurotoxic mechanisms for iNOS, we studied nitric oxide formation by electron paramagnetic resonance (EPR) spectroscopy using diethyldithiocarbamate-iron (DETC-Fe) as a spin trap and markers of nitrosative (S-nitrosothiol (RSNO, Fluorescent assay); nitrotyrosine (3NT, ELISA)) and oxidative stress (ascorbate, HPLC) at 72 h after controlled cortical impact (CCI) in iNOS ko and wt and in uninjured iNOS ko and wt mice. 3NT immunostaining with macrophage and myeloperoxidase (MPO) dual labeling was also assessed in brain sections. Brain DETC-Fe-NO low-temperature EPR signal intensity was approximately 2-fold greater in wt versus iNOS ko at 72 h after CCI. Ascorbate levels decreased in injured hemisphere in wt and iNOS ko versus uninjured -this decrease was more pronounced in iNOS ko. In wt mice, RSNO and 3NT levels were increased after CCI versus uninjured (50% and 400%, respectively, P < 0.05). RSNO levels were not increased in iNOS ko after CCI. Nitrotyrosine levels increased after CCI in wt and ko versus respective uninjured -this increase was more pronounced in wt (2.34 +/- 0.95 versus 1.27 +/- 0.49 pmol/mg protein, P < 0.05). Increased 3NT immunoreactivity was detected in wt versus iNOS ko at 72 h after CCI, and colocalized with macrophage marker and MPO. Our data support a role for iNOS-derived NO as an endogenous antioxidant after CCI. iNOS also contributes protein nitrosylation and nitration. Colocalization of 3NT with macrophages and MPO suggests generation of nitrating agents by macrophages and/or phagocytosis of nitrated proteins.