Chlorite dismutase has been purified from the chlorate-metabolizing bacterium Ideonella dechloratans. The purified enzyme is tetrameric, with a relative molecular mass of 25,000 for the subunit, and contains about 0.6 heme/subunit as isolated. Its catalytic properties are similar, but not identical, to those found for a similar enzyme purified earlier from the bacterium GR-1. The heme group in Ideonella chlorite dismutase is readily reduced by dithionite, in contrast to the GR-1 enzyme, and redox titration gave a value of -21 mV for the midpoint potential at pH 7. The heme group has been characterized by optical and EPR spectroscopy. It is high-spin ferric at neutral pH, with spectroscopic properties similar to those found for cytochrome c peroxidase. In the alkaline pH range, a low-spin compound is formed. A 22-residue N-terminal amino acid sequence has been determined and no homologue has been found in the protein sequence databases.

We report the generation and characterization of a new high-spin iron(IV)-oxo complex supported by a trigonal nonheme pyrrolide platform. Oxygen-atom transfer to [(tpa(Mes))Fe(II)](-) (tpa(Ar) = tris(5-arylpyrrol-2-ylmethyl)amine) in acetonitrile solution affords the Fe(III)-alkoxide product [(tpa(Mes2MesO))Fe(III)](-) resulting from intramolecular C-H oxidation with no observable ferryl intermediates. In contrast, treatment of the phenyl derivative [(tpa(Ph))Fe(II)](-) with trimethylamine N-oxide in acetonitrile solution produces the iron(IV)-oxo complex [(tpa(Ph))Fe(IV)(O)](-) that has been characterized by a suite of techniques, including mass spectrometry as well as UV-vis, FTIR, Mössbauer, XAS, and parallel-mode EPR spectroscopies. Mass spectral, FTIR, and optical absorption studies provide signatures for the iron-oxo chromophore, and Mössbauer and XAS measurements establish the presence of an Fe(IV) center. Moreover, the Fe(IV)-oxo species gives parallel-mode EPR features indicative of a high-spin, S = 2 system. Preliminary reactivity studies show that the high-spin ferryl tpa(Ph) complex is capable of mediating intermolecular C-H oxidation as well as oxygen-atom transfer chemistry.

Traumatic fractures place a substantial burden on health-care systems worldwide. Although detailed information about incidence, distribution, and risk factors for traumatic fractures is vital for planning and prevention, in China, national data are unavailable. We aimed to do an up-to-date national survey on the population-weighted incidence of traumatic fractures in China.

The China National Fracture Study (CNFS) was a retrospective epidemiological study that recruited a nationally representative sample from eight provinces, 24 urban cities, and 24 rural counties in China using stratified random sampling and the probability proportional to size method. All eligible household members who had lived in their current residence for 6 months or longer were personally interviewed by trained research teams about traumatic fractures of the trunk, arms, or legs (not including the skull, sternum, and ribs) that had occurred in 2014. Telephone surveys were used for participants who were non-contactable after repeated visits. Fracture cases were verified by clinical records, medical history, and radiographs by orthopaedic surgeons and radiologists. We estimated incidence rates for traumatic fractures for the overall population and for subgroups by age and sex, as well as by demographic factors such as ethnic origin, occupation, geographical region, and residency category. We also studied potential associations between fractures and various factors of interest, such as age, ethnic origin, education, smoking, alcohol drinking, sleep time per day, and history of previous fracture. Data were weighted during statistical analysis to ascertain the national incidence rate. This study is registered with the Chinese Clinical Trial Registry, number ChiCTR-EPR-15005878.

Between Jan 19, 2015, and May 16, 2015, 535 836 individuals were selected and invited to participate in the study. Questionnaires from 23 649 (4%) individuals were excluded due to missing items, insufficient responses, or logical errors. Following exclusions, 512 187 (96%) individuals participated in the CNFS, consisting of 259 649 (51%) boys and men and 252 538 (49%) girls and women. Of these individuals, 1763 individuals had experienced traumatic fractures during 2014 (n=1833). The population-weighted incidence rate of traumatic fractures of the trunk, arms, or legs was 3·21 (95% CI 2·83-3·59) per 1000 population in 2014 (3·65, 3·12-4·18 in men and 2·75, 2·46-3·04 in women). For all ages, sleeping less than 7 h per day was identified as a risk factor for traumatic fractures. We identified previous fracture history as a risk factor for adults aged 15 years and older. Alcohol consumption incurred a risk effect for men aged 15 years and older and women aged 15-64 years.

Our results provide detailed information about fracture incidence, distribution, and risk factors, which can now be used as an up-to-date clinical evidence base for national health-care planning and preventive efforts in China and elsewhere. Specific public health policies that focus on decreasing alcohol consumption, prohibiting drunk driving, promoting smoking cessation, and encouraging individuals to obtain sufficient sleep and maintain a healthy bodyweight should be urgently implemented to help reduce the risk of traumatic fractures.

The Hebei Province Medical Science Special Major Projects Research Fund.

The N-methyl-N-nitroso-urea streptozotocin is an antibiotic with diabetogenic, carcinogenic and antitumor activity thought to act via alkylation of DNA and proteins. Evidence points to a release of bioactive nitric oxide (NO) from streptozotocin as an additional cytotoxic activity of this drug. Here we show by EPR spectroscopy, that NO is not generated during spontaneous decay of streptozotocin but that its metabolization in rat hepatocytes and pancreatic islet cells yields NO. This NO formation is not due to a NO synthase (NOS) activity since NO formation in hepatocytes in the presence of streptozotocin is not blocked by the NOS inhibitor NG-methyl-L-arginine. By iNOS-specific RT-PCR no positive signal for specific mRNA presence was obtained in streptozotocin-treated cells, proving that iNOS activity was not induced during cell isolation procedures and did not account for the NO release. Furthermore, early DNA-strand breaks induced either by SZ or by the NO donor nitroprusside were both significantly reduced in the presence of an intracellular NO scavenger. In contrast, DNA damage found after incubation with the purely alkylating agent methylmethanesulfonate was not inhibited by the NO trap. These results prove that intracellular formation of NO occurs during degradation of SZ within cells. This NO appears to contribute significantly to streptozotocin-induced cytotoxicity.

Pretreatment of rat hepatocytes with low-dose nitrogen oxide (addition of SNAP in vitro or induction of nitric oxide synthase in vitro or in vivo) imparts resistance to killing and decrease in aconitase and mitochondrial electron transfer from a second exposure to a higher dose of SNAP. Induction of this resistance is prevented by cycloheximide, indicating upregulation of protective protein(s). Ferritin levels are increased as are non-heme iron-NO EPR signals. Tin-protoporphyrin (SnPP) prevents protection, suggesting involvement of hsp32 (heme oxygenase) and/or guanylyl cyclase (GC). Cross-resistance to H2O2 killing is also observed, which is also prevented by cycloheximide and SnPP. Thus, hepatocytes possess inducible protective mechanisms against nitrogen oxide and reactive oxygen toxicity.

The biosynthesis of chloramphenicol requires a beta-hydroxylation tailoring reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the nonribosomal peptide synthetase CmlP. EPR, Mössbauer, and optical spectroscopies reveal that CmlA contains an oxo-bridged dinuclear iron cluster, a metal center not previously associated with nonribosomal peptide synthetase chemistry. Single-turnover kinetic studies indicate that CmlA is functional in the diferrous state and that its substrate is L-PAPA covalently bound to CmlP. Analytical studies show that the product is hydroxylated L-PAPA and that O(2) is the oxygen source, demonstrating a monooxygenase reaction. The gene sequence of CmlA shows that it utilizes a lactamase fold, suggesting that the diiron cluster is in a protein environment not previously known to effect monooxygenase reactions. Notably, CmlA homologs are widely distributed in natural product biosynthetic pathways, including a variety of pharmaceutically important beta-hydroxylated antibiotics and cytostatics.

Copolymer of styrene-maleic acid (SMA) was used to construct micelles containing doxorubicin by means of a hydrophobic interaction between the styrene moiety of SMA and doxorubicin (Dox). The micelles obtained (SMA-Dox) showed a high solubility in water and a constant doxorubicin release rate of about 3-4%/day in vitro. The SMA-Dox micelle preparation was less (36-70%) cytotoxic to the SW480 human colon cancer cell line in vitro compared with free doxorubicin. In vivo assay of SMA-Dox in ddY mice bearing S-180 tumor revealed a potent anticancer effect with no remarkable toxicity up to a dose of 100 mg/kg of free doxorubicin equivalent. The drug concentration in tumor after administration of SMA-Dox was 13 times higher than that after the free drug. This result can be attributed to the enhanced permeability and retention (EPR) effect of macromolecular drugs observed in solid tumors. Complete blood counts and cardiac histology showed no serious side effects for intravenous (i.v.) doses of the micellar formulation as high as 100 mg/kg doxorubicin equivalent in mice. These data indicate that i.v. administration of SMA-Dox micellar formulation can enhance the therapeutic effect of doxorubicin while reducing greatly cardiac and bone marrow toxicity, which should allow safe use of high doses of this agent.

OBJECTIVE

Hemoglobin (Hb) becomes toxic when released from the erythrocyte. The acute phase protein haptoglobin (Hp) binds avidly to Hb and decreases oxidative damage to Hb itself and to the surrounding proteins and lipids. However, the molecular mechanism underpinning Hp protection is to date unclear. The aim of this study was to use electron paramagnetic resonance (EPR) spectroscopy, stopped flow optical spectrophotometry, and site-directed mutagenesis to explore the mechanism and specifically the role of specific tyrosine residues in this protection.

RESULTS

Following peroxide challenge Hb produces reactive oxidative intermediates in the form of ferryl heme and globin free radicals. Hp binding increases the steady state level of ferryl formation during Hb-catalyzed lipid peroxidation, while at the same time dramatically inhibiting the overall reaction rate. This enhanced ferryl stability is also seen in the absence of lipids and in the presence of external reductants. Hp binding is not accompanied by a decrease in the pK of ferryl protonation; the protonated ferryl species still forms, but is intrinsically less reactive. Ferryl stabilization is accompanied by a significant increase in the concentration of the peroxide-induced tyrosine free radical. EPR spectral parameters and mutagenesis studies suggest that this radical is located on tyrosine 145, the penultimate C-terminal amino acid on the beta Hb subunit.

METHODS

Hp binding decreases both the ferryl iron and free radical reactivity of Hb.

CONCLUSIONS

Hp protects against Hb-induced damage in the vasculature, not by preventing the primary reactivity of heme oxidants, but by rendering the resultant protein products less damaging.

Certain short peptides, which are able to translocate across cell membranes with a low lytic activity, can be useful as carriers (vectors) for hydrophilic molecules. We have studied three such cell penetrating peptides: pAntp ('penetratin'), pIsl and transportan. pAntp and pIsl originate from the third helix of homeodomain proteins (Antennapedia and Isl-1, respectively). Transportan is a synthetic chimera (galanin and mastoparan). The peptides in the presence of various phospholipid vesicles (neutral and charged) and SDS micelles have been characterized by spectroscopic methods (fluorescence, EPR and CD). The dynamics of pAntp were monitored using an N-terminal spin label. In aqueous solution, the CD spectra of the three peptides show secondary structures dominated by random coil. With phospholipid vesicles, neutral as well as negatively charged, transportan gives up to 60% alpha-helix. pAntp and pIsl bind significantly only to negatively charged vesicles with an induction of around 60% beta-sheet-like secondary structure. With all three peptides, SDS micelles stabilize a high degree of alpha-helical structure. We conclude that the exact nature of any secondary structure induced by the membrane model systems is not directly correlated with the common transport property of these translocating peptides.

Although nitric oxide synthase (NOS) is widely considered as the major source of NO in biological cells and tissues, direct evidence demonstrating NO formation from the purified enzyme has been lacking. It was recently reported that NOS does not synthesize NO, but rather generates nitroxyl anion (NO-) that is subsequently converted to NO by superoxide dismutase (SOD). To determine if NOS synthesizes NO, electron paramagnetic resonance (EPR) spectroscopy was applied to directly measure NO formation from purified neuronal NOS. In the presence of the NO trap Fe2+-N-methyl-D-glucamine dithiocarbamate, NO gives rise to characteristic EPR signals with g = 2.04 and aN = 12.7 G, whereas NO- is undetectable. In the presence of L-arginine (L-Arg) and cofactors, NOS generated prominent NO signals. This NO generation did not require SOD, and it was blocked by the specific NOS inhibitor N-nitro-L-arginine methyl ester. Isotope-labeling experiments with L-[15N]Arg further demonstrated that NOS-catalyzed NO arose from the guanidino nitrogen of L-Arg. Measurement of the time course of NO formation demonstrated that it paralleled that of L-citrulline. The conditions used in the prior study were shown to result in potent superoxide generation, and this may explain the failure to measure NO formation in the absence of SOD. These experiments provide unequivocal evidence that NOS does directly synthesize NO from L-Arg.

NADH:quinone oxidoreductase (complex I) plays a pivotal role in cellular energy production. It employs a series of redox cofactors to couple electron transfer to the generation of a proton-motive force across the inner mitochondrial or bacterial cytoplasmic membrane. Complex I contains a noncovalently bound flavin mononucleotide at the active site for NADH oxidation and eight or nine iron-sulfur clusters to transfer electrons between the flavin and a quinone-binding site. Understanding the mechanism of complex I requires the properties of these clusters to be defined, both individually and as an ensemble. Most functional information on the clusters has been gained from EPR spectroscopy, but some clusters are not observed by EPR and attributing the observed signals to the structurally defined clusters is difficult. The current consensus picture relies on correlating the spectra from overexpressed subunits (containing one to four clusters) with those from intact complexes I. Here, we analyze spectra from the overexpressed NuoG subunit from Escherichia coli complex I and compare them with spectra from the intact enzyme. Consequently, we propose that EPR signals N4 and N5 have been misassigned: signal N4 is from NuoI (not NuoG) and signal N5 is from the conserved cysteine-ligated [4Fe-4S] cluster in NuoG (not from the cluster with a histidine ligand). The consequences of reassigning the EPR signals and their associated functional information on the free energy profile for electron transfer through complex I are discussed.

To investigate the involvement of a hemoglobin radical in the human oxyhemoglobin (oxyHb) or metHb/H2O2 system, we have used a new approach called "immuno-spin trapping," which combines the specificity and sensitivity of both spin trapping and antigen:antibody interactions. Previously, a novel rabbit polyclonal anti-DMPO nitrone adduct antiserum, which specifically recognizes protein radical-derived nitrone adducts, was developed and validated in our laboratory. In the present study, the formation of nitrone adducts on hemoglobin was shown to depend on the oxidation state of the iron heme, the concentrations of H2O2 and DMPO, and time as determined by enzyme-linked immunosorbent assay (ELISA) and by Western blotting. The presence of reduced glutathione or L-ascorbate significantly decreased the level of nitrone adducts on metHb in a dose-dependent manner. To confirm the ELISA results, Western blotting analysis showed that only the complete system (oxy- or metHb/DMPO/H2O2) generates epitopes recognized by the antiserum. The specific modification of tyrosine residues on metHb by iodination nearly abolished antibody binding, while the thiylation of cysteine residues caused a small but reproducible decrease in the amount of nitrone adducts. These findings strongly suggest that tyrosine residues are the site of formation of the immunochemically detectable hemoglobin radical-derived nitrone adducts. In addition, we were able to demonstrate the presence of hemoglobin radical-derived nitrone adducts inside red blood cells exposed to H2O2 and DMPO. In conclusion, our new approach showed several advantages over EPR spin trapping with the anti-DMPO nitrone adduct antiserum by demonstrating the formation of tyrosyl radical-derived nitrone adduct(s) in human oxyHb/metHb at much lower concentrations than was possible with EPR and detecting radicals inside RBC exposed to H2O2.

ATP-driven pumping of a variety of drugs out of cells by the human P-glycoprotein poses a serious problem to medical therapy. High level heterologous expression of human P-glycoprotein, in the yeast Saccharomyces cerevisiae, has facilitated biophysical studies in purified proteoliposome preparations. Membrane permeability of transported drugs and consequent lack of an experimentally defined drug position have made resolution of the transport mechanism difficult by classical techniques. To overcome these obstacles we devised a novel EPR spin-labeled verapamil for use as a transport substrate. Spin-labeled verapamil was an excellent transport substrate with apparent turnover number, K(m) and K(i) values of 5.8 s(-1), 4 microm, and 210 microm, respectively, at pH 7.4 and 37 degrees C. The apparent affinities were approximately 10-fold higher than for unlabeled verapamil. Spin-labeled verapamil stimulated ATPase activity approximately 5-fold, was relatively hydrophilic, and had a very low flip-flop rate, making it an ideal transport substrate. The K(m) for MgATP activation of transport was 0.8 mm. By measuring the mobility of spin-labeled verapamil during transport experiments, we were able to resolve the location of the drug in proteoliposome suspensions. Steady state gradients of spin-labeled verapamil within the range of K(i)/K(m) ratios were observed.

To efficiently deliver therapeutics into cancer cells, a number of strategies have been recently investigated. The toxicity associated with the administration of chemotherapeutic drugs due to their random interactions throughout the body necessitates the development of drug-encapsulating nanopreparations that significantly mask, or reduce, the toxic side effects of the drugs. In addition to reduced side effects associated with drug encapsulation, nanocarriers preferentially accumulate in tumors as a result of its abnormally leaky vasculature via the Enhanced Permeability and Retention (EPR) effect. However, simple passive nanocarrier delivery to the tumor site is unlikely to be enough to elicit a maximum therapeutic response as the drug-loaded carriers must reach the intracellular target sites. Therefore, efficient translocation of the nanocarrier through the cell membrane is necessary for cytosolic delivery of the cargo. However, crossing the cell membrane barrier and reaching cytosol might still not be enough for achieving maximum therapeutic benefit, which necessitates the delivery of drugs directly to intracellular targets, such as bringing pro-apoptotic drugs to mitochondria, nucleic acid therapeutics to nuclei, and lysosomal enzymes to defective lysosomes. In this review, we discuss the strategies developed for tumor targeting, cytosolic delivery via cell membrane translocation, and finally organelle-specific targeting, which may be applied for developing highly efficacious, truly multifunctional, cancer-targeted nanopreparations.

The use of spin echoes to obtain spectroscopic EPR images (spectral-spatial images) at 250 MHz is described. The advantages of spin echoes-larger signals than the free induction decay, better phase characteristics for Fourier transformation, and decay shapes undistorted by instrumental dead time-are clearly shown. An advantage is gained from using a crossed loop resonator that isolates the 250-W pump power by greater than 50 dB from the observer arm preamplifiers. The echo decay rates can be used to determine the oxygen content in solutions containing 1 mM trityl concentrations. Two- and three-dimensional images of oxygen concentration are presented.

Voltage-gated sodium channels have essential roles in electrical signalling. Prokaryotic sodium channels are tetramers consisting of transmembrane (TM) voltage-sensing and pore domains, and a cytoplasmic carboxy-terminal domain. Previous crystal structures of bacterial sodium channels revealed the nature of their TM domains but not their C-terminal domains (CTDs). Here, using electron paramagnetic resonance (EPR) spectroscopy combined with molecular dynamics, we show that the CTD of the NavMs channel from Magnetococcus marinus includes a flexible region linking the TM domains to a four-helix coiled-coil bundle. A 2.9 Å resolution crystal structure of the NavMs pore indicates the position of the CTD, which is consistent with the EPR-derived structure. Functional analyses demonstrate that the coiled-coil domain couples inactivation with channel opening, and is enabled by negatively charged residues in the linker region. A mechanism for gating is proposed based on the structure, whereby splaying of the bottom of the pore is possible without requiring unravelling of the coiled-coil.

Allergic conjunctivitis (AC) is a common allergic eye disease characterized by clinical symptoms such as itchiness, conjunctival congestion, elevated Ag-specific IgE, mast cell activation, and local eosinophil infiltration. In this study we established a murine model for Ag-induced AC to understand the pathogenesis of the disease. Cell transfer experiments indicated that AC can be divided into early and late phase responses (EPR and LPR). EPR was associated with IgE responses, leading to itchiness, whereas LPR was characterized by local eosinophil infiltration. Both EPR and LPR were significantly inhibited in STAT6-deficient mice, and adoptive transfer of Th2 cells reconstituted LPR. Furthermore, SOCS3 was highly expressed at the disease site, and T cell-specific expression of SOCS3 deteriorated clinical and pathological features of AC, indicating that Th2-mediated SOCS3 expression controls the development and persistence of AC. Reduction of the expression level in SOCS3 heterozygous mice or inhibition of function in dominant-negative SOCS3 transgenic mice clearly reduced the severity of AC. In contrast, constitutive expression of SOCS5, a specific inhibitor of IL-4 signaling, resulted in reduced eosinophil infiltration. These results suggest that negative regulation of the Th2-mediated response by dominant-negative SOCS3 and SOCS5 could be a target for therapeutic intervention in allergic disease.

Nanoparticles are efficient to safely deliver therapeutic and imaging contrast agents to tumors for cancer diagnostic and therapy, if they can escape the reticuloendothelial system (RES) and accumulate in tumors either passively due to the enhanced permeability and retention (EPR) effect or actively via a specific ligand. The main hallmark of nanoparticles is their large surface areas, which, depending of their chemical compositions, surface coatings, electric charges, sizes and shapes, will generate complex, extremely dynamic and continuous interactions and exchanges between the nanoparticles and the different molecules present in the blood. Special attention will be paid to explain how the nanoparticles were improved step by step in order to adapt our increasing knowledge on their biophysics. In particular, we will discuss the influence of PEGylation, the difficulties to generate actively targeted particles and finally the actual trends in the manufacturing of "third-generation" smart particles.

The heart of the oxygen-evolving complex (OEC) of photosystem II is a Mn4OxCa cluster that cycles through five different oxidation states (S0 to S4) during the light-driven water-splitting reaction cycle. In this study we interpret the recently obtained 55Mn hyperfine coupling constants of the S0 and S2 states of the OEC [Kulik et al. J. Am. Chem. Soc. 2005, 127, 2392-2393] on the basis of Y-shaped spin-coupling schemes with up to four nonzero exchange coupling constants, J. This analysis rules out the presence of one or more Mn(II) ions in S0 in methanol (3%) containing samples and thereby establishes that the oxidation states of the manganese ions in S0 and S2 are, at 4 K, Mn4(III, III, III, IV) and Mn4(III, IV, IV, IV), respectively. By applying a "structure filter" that is based on the recently reported single-crystal EXAFS data on the Mn4OxCa cluster [Yano et al. Science 2006, 314, 821-825] we (i) show that this new structural model is fully consistent with EPR and 55Mn-ENDOR data, (ii) assign the Mn oxidation states to the individual Mn ions, and (iii) propose that the known shortening of one 2.85 A Mn-Mn distance in S0 to 2.75 A in S1 [Robblee et al. J. Am. Chem. Soc. 2002, 124, 7459-7471] corresponds to a deprotonation of a mu-hydroxo bridge between MnA and MnB, i.e., between the outer Mn and its neighboring Mn of the mu3-oxo bridged moiety of the cluster. We summarize our results in a molecular model for the S0 ->> S1 and S1 ->> S2 transitions.

The development of traditional tumor-targeted drug delivery systems based on EPR effect and receptor-mediated endocytosis is very challenging probably because of the biological complexity of tumors as well as the limitations in the design of the functional nano-sized delivery systems. Recently, multistage drug delivery systems (Ms-DDS) triggered by various specific tumor microenvironment stimuli have emerged for tumor therapy and imaging. In response to the differences in the physiological blood circulation, tumor microenvironment, and intracellular environment, Ms-DDS can change their physicochemical properties (such as size, hydrophobicity, or zeta potential) to achieve deeper tumor penetration, enhanced cellular uptake, timely drug release, as well as effective endosomal escape. Based on these mechanisms, Ms-DDS could deliver maximum quantity of drugs to the therapeutic targets including tumor tissues, cells, and subcellular organelles and eventually exhibit the highest therapeutic efficacy. In this review, we expatiate on various responsive modes triggered by the tumor microenvironment stimuli, introduce recent advances in multistage nanoparticle systems, especially the multi-stimuli responsive delivery systems, and discuss their functions, effects, and prospects.

Pirfenidone (Pf), a new broad-spectrum anti-fibrotic agent, is known to offer protection against lung fibrosis in vivo in laboratory animals, and against mitogenesis and collagen formation by human lung fibroblasts in vitro. Because reactive oxygen species are thought to be involved in these events, we investigated the mechanism(s) by which Pf ameliorates oxidative stress and its effects on NADPH-dependent lipid peroxidation. Pf has been shown to cause inhibit NADPH-dependent lipid peroxidation in sheep liver microsomes in a dose-dependent manner. The concentration of Pf required to cause 50% inhibition of lipid peroxidation was approximately 6 mM. Pf was found to be ineffective as a superoxide radical scavenger. Pf was also ineffective in decomposing H2O2 and chelating iron. In deoxyribose degradation assays, Pf was a potent scavenger of hydroxyl radicals with a rate constant of 5.4 x 10(9) M(-1) sec(-1). EPR spectroscopy in combination with spin trapping techniques, using a Fenton type reaction and DMPO as a spin-trapping agent, Pf scavenged hydroxyl radicals in a dose-dependent manner. The concentration of Pf required to inhibit 50% signal height was approximately 2.5 mM. Because iron was used in the Fenton reaction, the ability of Pf in chelating iron was verified in a fluorescent competitive assay using calcein as the fluorescent probe. Pf up to 10 mM concentration was ineffective in chelating either Fe2+ or Fe3+ in this system. We propose that Pf exerts its beneficial effects, at least in part, through its ability to scavenge toxic hydroxyl radicals.

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1(fl/fl)) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1(fl/fl)Tie2cre). Macrophages from Gch1(fl/fl)Tie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1(fl/fl)Tie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1(fl/fl)Tie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1(fl/fl)Tie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1(fl/fl)Tie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1(fl/fl)Tie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation.

One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy and small-angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein's hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.

The aqueous-membrane partitioning of alamethicin, a voltage-gated channel-forming peptide, was measured as a function of the membrane spontaneous curvature. EPR spectroscopy was used to measure the partitioning of the peptide in lipid compositions formed from dioleoylphosphatidylcholine (DOPC) and varied percentages of dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylethanolamine-N-methyl (DOPE-Me), or dioleoylphosphatidylethanolamine-N,N-dimethyl (DOPE-Me2). When the mole fraction of DOPE in mixtures of DOPC/DOPE is increased the binding of alamethicin decreases, and the increase in binding free energy is found to be linearly dependent upon the mole fraction of DOPE in the mixture. Addition of DOPE-Me or DOPE-Me2 also increases the binding free energy, except that the effect is reduced relative to that of DOPE. The free-energy increase per mole fraction of DOPE was found to be 1400 cal/mol, whereas for DOPE-Me and DOPE-Me2 the free-energy changes were 980 and 630 cal/mol, respectively. When the free-energy changes for alamethicin binding are compared with the previously determined spontaneous curvatures for mixtures of DOPC/DOPE and DOPC/DOPE-Me, the free energy of binding is found to be linearly dependent upon the spontaneous curvature of the bilayer lipids. The effects of membrane lipid unsaturation on the partitioning of alamethicin were also measured and are qualitatively consistent with this conclusion. The sensitivity to spontaneous curvature and the cooperativity that is seen in the binding curves for alamethicin are postulated to be a result of a localized thinning of the bilayer promoted by this peptide.