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The understanding of the mechanism, oxidant(s) involved and how and what protein radicals are produced during the reaction of wild-type SOD1 (Cu,Zn-superoxide dismutase) with H2O2 and their fate is incomplete, but a better understanding of the role of this reaction is needed. We have used immuno-spin trapping and MS analysis to study the protein oxidations driven by human (h) and bovine (b) SOD1 when reacting with H2O2 using HSA (human serum albumin) and mBH (mouse brain homogenate) as target models. In order to gain mechanistic information about this reaction, we considered both copper- and CO3•− (carbonate radical anion)-initiated protein oxidation. We chose experimental conditions that clearly separated SOD1-driven oxidation via CO3•− from that initiated by copper released from the SOD1 active site. In the absence of (bi)carbonate, site-specific radical-mediated fragmentation is produced by SOD1 active-site copper. In the presence of (bi)carbonate and DTPA (diethylenetriaminepenta-acetic acid) (to suppress copper chemistry), CO3•− produced distinct radical sites in both SOD1 and HSA, which caused protein aggregation without causing protein fragmentation. The CO3•− produced by the reaction of hSOD1 with H2O2 also produced distinctive DMPO (5,5-dimethylpyrroline-N-oxide) nitrone adduct-positive protein bands in the mBH. Finally, we propose a biochemical mechanism to explain CO3•− production from CO2, enhanced protein radical formation and protection by (bi)carbonate against H2O2-induced fragmentation of the SOD1 active site. Our present study is important for establishing experimental conditions for studying the molecular mechanism and targets of oxidation during the reverse reaction of SOD1 with H2O2; these results are the first step in analysing the critical targets of SOD1-driven oxidation during pathological processes such as neuroinflammation.
- carbonate radical anion
- immuno-spin trapping
- nitrone adduct
- peroxymonocarbonate
- protein radical
- superoxide dismutase
Much of the mechanistic biochemical evidence regarding SOD1 (Cu,Zn-superoxide dismutase)-driven pathogenic oxidative mechanisms has been obtained by studying the reaction between bovine or human SOD1 and H2O2 [1–4]. The reaction of SOD1 and H2O2 may be particularly important in sites of inflammation, particularly in peroxisomes, which are known to have high local concentrations of H2O2 and SOD1, and where the degradation of damaged proteins occurs [5]. The study of radical pathways induced by H2O2/SOD1-driven peroxidation may be important to the understanding of the molecular mechanisms of tissue damage during neuroinflammation, as occurs, for example, in fALS (familial amyotrophic lateral sclerosis).
Mechanistically, the reaction of SOD1 with H2O2 proceeds in several sequential steps (Reactions 1 and 2) that end with the formation of an enzyme-bound oxidant [i.e. (CuO)+↔(Cu-OH•)2+↔Cu3+] at the enzyme active site [6,7]. This species promotes oxidation of one or more histidine residues at the SOD1 active site [8,9], partial copper release [10,11], enzyme inactivation [6] and SOD1 fragmentation at its active site [10,12].(Reaction 1) (Reaction 2)
The copper-bound oxidant at the enzyme active site (Reactions 1 and 2) is generally proposed to oxidize (bi)carbonate to the diffusible carbonate radical anion (CO3•−) [7,13–16]. CO2 has been postulated to be the chemical entity in (bi)carbonate buffer that reacts with the strong oxidant at the SOD1 active site to give CO3•− [14]; however, the chemical mechanism of this reaction remains controversial [7,17–19]. Much of our current knowledge of SOD1-driven oxidation has been gained by studying the oxidation of chemical compounds (e.g. NADPH [20], dichlorofluorescein [11], peroxidase substrates [12,21], ethanol, spin-trap compounds [21,22], formate and azide [13]), the loss of superoxide dismutase activity, or the activity or the structure of protein or nucleic acid targets [23]. Because of its diffusibility and powerful oxidative nature, CO3•− may form distinct radicals in SOD1 itself or tissue proteins, which may be trapped with DMPO (5,5-dimethylpyrroline-N-oxide) to form stable nitrone adducts for further characterization [12,24]. However, in cells and tissues, it is difficult to study the formation of protein radicals using ESR with or without spin trapping because of their rapid decay and the severe overlap of their spectra.
We have developed a new biochemical tool with which to study protein [25–27] and DNA [24,28] radicals called immuno-spin trapping [26,27] that helps to identify protein radicals induced by SOD1-driven oxidation in cells and tissues. Immuno-spin trapping [29] involves the trapping of protein radicals with the spin trap DMPO in situ and in real time and the further detection of the protein radical–DMPO nitrone adducts with an anti-DMPO serum [25] by using heterogeneous immunoassays and also by MS [30]. In principle, this technology allows the simultaneous detection of more than one protein radical at the same time and in the same reaction system, as they are formed during tissue oxidative damage [24,29].
We have previously shown that (bi)carbonate, but not DTPA (diethylenetriaminepenta-acetic acid), protects SOD1 against H2O2-induced fragmentation at its active site and that, in the presence of DTPA, (bi)carbonate is required in order to observe SOD1 nitrone adducts with immuno-spin trapping [12]. In the present study, we have used immuno-spin trapping and MS to understand the mechanism of protein radical formation induced by the bovine and human SOD1/HSA (human serum albumin) or mBH (mouse brain homogenate)/H2O2 system. To accomplish this goal, we chose experimental conditions that would separate the two major radical pathways of protein modification by H2O2-induced SOD1-driven oxidation: copper (both active site and released)- and CO3•−-triggered radical reactions. Furthermore, we have analysed the way in which these two different initiators of radical reactions contribute to oxidizing target proteins and SOD1 itself using HSA and mBH as models. In the present study, we also propose a novel mechanism to explain how (bi)carbonate blocks the fragmentation of the SOD1 active site by the enzyme-bound oxidant (Reaction 2).
bSOD1 (bovine SOD1) (from bovine erythrocytes) and beef liver catalase were purchased from Roche Applied Science. Sodium bicarbonate (99.7–100.3% purity) was purchased from Alfa Aesar. The spin-trap DMPO was purchased from Alexis Biochemicals, purified twice by vacuum sublimation at room temperature (15–25 °C), and stored under an argon atmosphere at −80 °C until use. The DMPO concentration was measured at 228 nm, assuming a molar absorption coefficient of 7800 M−1·cm−1. Reagent-grade 30% H2O2 was obtained from Fisher Scientific. The H2O2 concentration was verified using UV–visible absorption at 240 nm (ε240=43.6 M−1·cm−1). Recombinant hSOD1 (human SOD1) (expressed in Escherichia coli) was from BioVision. Erythrocytic hSOD1 and all other reagents were purchased from Sigma Chemical Co. Buffers were treated with Chelex® 100 ion-exchange resin (Bio-Rad Laboratories) to remove transition metals usually found as contaminants. The pH of the buffer solutions containing bicarbonate was adjusted to 7.4 by bubbling with a 19:1 N2/CO2 gas mixture.
SOD1, HSA and mBH preparation
Our bSOD1 and hSOD1 (from erythrocytes and recombinant) solutions were prepared and determined to be free from detectable unbound copper as described previously [31]. SOD1 concentration was determined by measuring the absorbance at 258 nm (ε258=10.3 mM−1·cm−1) at pH 7.4 [32]. HSA solutions were prepared as described previously [31,33]. Our HSA preparations were free from detectable copper contamination, and the free sulfhydryl/albumin ratio was similar to that reported in [31,33]. The HSA concentration was determined by using the BCA (bicinchoninic acid) assay (Pierce) or assuming its UV–visible molar absorption coefficient of ε280=35700 M−1·cm−1. Male C576J/BL mice (25–30 g of body weight) were killed following institutionally approved protocols, and brains were collected in ice-cold 10 mM PB [sodium phosphate buffer (pH 7.4)], washed in the same buffer and homogenized (1 g/ml). The homogenate was centrifuged at 11700 g and the supernatant was dialysed (3 kDa cut-off) against 10 mM PB. The protein concentration in the dialysed mBH was determined using the BCA assay.
Chemical reactions
Typically, the reactions of 15 μM SOD1, 7.5 μM (0.5 mg/ml) HSA or 0.5 mg/ml mBH and 0.1 mM H2O2 were carried out in the presence of 100 mM DMPO in 100 mM Chelex®-bound BB [sodium (bi)carbonate buffer (pH 7.4)], or 100 mM Chelex®-bound PB, or in PB containing a physiological concentration of (bi)carbonate (i.e. ∼25 mM) and with or without 0.1 mM DTPA. Solutions were incubated at 37 °C for 1 h, and the reaction was stopped by adding 10 IU of catalase to eliminate excess H2O2. The pH of the reaction mixtures after the reactions was completed was between 7.3 and 7.6.
Measurement of H2O2-induced bathocuproine-assisted reduction of released Cu2+
The H2O2-induced Cu2+ release from bSOD1 or hSOD1 and its reduction to Cu+ was monitored using the specific Cu+ chelator BCDS (bathocuproine disulfonic acid) [34]. Cu+ binds to BCDS and produces a complex [Cu+(BCDS)2]3− that exhibits a characteristic absorbance maximum at 480 nm, with ε480=12540 M−1·cm−1.
Measurement of SOD1 activity
bSOD1 and hSOD1 activity was measured using the ferricytochrome c reduction assay [11,32]. For controls, the ratio of ferricytochrome c reduction was measured in samples containing 15 μM active or heat-inactivated SOD1 (incubation for 40 min at 75 °C) [32].
Anti-DMPO serum
A rabbit antiserum against the nitrone form of DMPO was obtained in our laboratory [25] and was used to develop immuno-spin trapping [26,27,29]; this antiserum has been used successfully to detect protein [27,29] and DNA [24,28] radicals. The anti-DMPO serum is commercially available from Alexis Biochemicals, Cayman Chemicals, AbCam, Chemicon International and Oxford Biomedical Research.
Immuno-spin trapping assays
DMPO–protein radical-derived nitrone adducts were determined using a standard ELISA and Western blot as described previously [12,29]. Briefly, the reaction mixture was separated by reducing SDS/PAGE [4–12% NuPAGE Novex Bis-Tris gels (Invitrogen); 1.2 μg of protein/lane]. After the separation of protein, gels were stained using Coomassie Blue, or the proteins were blotted to a nitrocellulose membrane, and the nitrone adducts were detected by Western blotting. Briefly, immunocomplexes were detected by exposing the membrane to NBT (Nitro Blue Tetrazolium)/BCIP (5-bromo-4-chloroindol-3-yl phosphate) One Step reagent from Pierce for 15 min or, when indicated, by enhanced chemiluminescence using a CDP-Star II (Roche Molecular Biochemicals)/Nitro Block II (Tropix) system. Where indicated, a MagicMark™ XP Western Protein Standard (Invitrogen) was used as a molecular-mass marker that glows after Western blot development [24].
Measurement of H2O2-induced SOD1-driven oxidation of guaiacol
The peroxidative activity of bSOD1 or hSOD1/H2O2 with or without (bi)carbonate or DTPA was measured using guaiacol as a substrate. The reaction was initiated by adding 5 μM SOD1 to 100 mM Chelex®-bound buffers (pH 7.4), containing 0.5 mM guaiacol and 1 mM H2O2 (final volume 1 ml). Oxidation of guaiacol to tetraguaiacol was measured spectrophotometrically in a Beckman DU® 640 spectrometer at 470 nm and at room temperature for 15 min. A molar absorption coefficient of 26.6 mM−1·cm−1 was used to quantify tetraguaiacol formation.
Mass spectrometric identifications from gel bands
In-gel tryptic digestion
The protein bands were manually excised from the gel, cut into small pieces and transferred into a 96-well microtitre plate. Gel pieces were subjected to automatic tryptic digestion using an Investigator™ Progest protein digestion station (Genomic Solutions). Briefly, gel bands were sequentially washed twice with 25 mM ammonium bicarbonate buffer (pH 7) and acetonitrile, dehydrated, rehydrated with 25 μl of the enzyme solution and digested at 37 °C for 8 h. The enzyme solution used was sequencing-grade modified trypsin (Promega Corporation) at a concentration of 0.01 mg/ml in 25 mM ammonium bicarbonate buffer (pH 7). Resulting tryptic peptides were extracted from the gel, freeze-dried and stored at −80 °C. Before mass spectrometric analysis, the peptides were reconstituted in 40 μl of a 97:3 solution of water/acetonitrile (containing 0.1% formic acid).
ESI (eletrospray ionization) MS
For the nano-LC (liquid chromatography)–ESI–MS/MS (tandem MS) analyses, we used an Agilent XCT Ultra ion trap (Agilent Technologies) equipped with an HPLC-Chip Cube MS interface and an Agilent 1100 nano-LC system. Injections of 30 μl of the peptide digests were made on to a 40 nl enrichment column followed by a 43 mm×75 μm analytical column packed with Zorbax 300SB C18 particles. Peptides were separated and eluted using a linear gradient of 3–50% acetonitrile (containing 0.1% formic acid) over 40 min, followed by a linear gradient of 50–95% acetonitrile over 7 min at a flow rate of 500 nl/min. The ion-trap mass spectrometer was operated in the positive-ion mode, standard enhanced mode using the following settings: capillary voltage, −2150 V; mass range, 300–1500; ICC smart target (number of ions in the trap before scan out), 100000 or 200 ms of accumulation; and MS/MS fragmentation amplitude, 1.0 V. During the LC–MS/MS analyses, automated data-dependent acquisition software was employed with the six most abundant ions (threshold requirement of 10000 counts) from each spectrum selected for MS/MS analysis.
Following the analyses, the MS/MS data were extracted and analysed using Spectrum Mill MS Proteomics software (Agilent Technologies). To generate peak lists, the raw data files were processed using the Data Extractor function with the following parameters: deconvoluted ions of 300–6000 Da and a retention time of 10–60 min. MS scans with the same precursor m/z were merged based on a ±1.4 m/z window and a ±15 s retention time window. Using the extracted data, searches were performed against the NCBI non-redundant protein database using the MS/MS search function. Parameters used for the searches included: precursor mass tolerance, ±1.5 Da; product mass tolerance, ±1 Da; enzyme specificity, trypsin, with maximum two missed cleavage sites; variable modifications, oxidized methionine and N-terminal pyroglutamic acid; and at least two unique peptides matched peak intensity, 80%; species, mouse. Proteins with a summed MS/MS search score of 30 or greater were considered for validation. At this scoring threshold, the false-positive rate was essentially 0% as determined by searching against a reversed sequence database. All MS/MS sequence assignments used for protein identifications were validated manually.
Parallel SOD1-centred radical and enzyme inactivation induced by H2O2
We observed H2O2-induced bSOD1 and erythrocytic hSOD1 inactivation in BB (Figure 1A). In agreement with our previous work [12] and that of other authors [11,18], we did not find any effect on H2O2-induced SOD1 inactivation when the (bi)carbonate concentration was 25 mM or less in PB with DTPA.
When (bi)carbonate was added to a bSOD1/H2O2 or hSOD1/H2O2 system in fresh argon-bubbled (to purge carbon dioxide) PB containing the copper chelator DTPA, the amount of H2O2-induced bSOD1-centred radicals observed as monomers and dimers increased, as seen by Western blotting (Figure 1B). The hSOD1 enzyme produced similar results (results not shown). Although bSOD1-centred radicals were detected at the physiological (bi)carbonate concentration of 25 mM, higher (bi)carbonate concentrations led to more nitrone adduct formation, demonstrating that the (bi)carbonate-dependent mechanism was not saturated under physiological conditions. We could not find any H2O2-induced SOD1 nitrone adducts in argon-purged freshly prepared Chelex®-bound PB containing 0.1 mM DTPA in a closed system, but we could detect them by Western blotting when PB was equilibrated with air at room temperature for 15 min, presumably due to the absorption of CO2 (results not shown). In PB with 0.1 mM DTPA, the SOD1 nitrone adduct (SOD1-self-oxidized) formation was totally dependent on (bi)carbonate. It increased with incubation time (Figure 1C) and was completely prevented by cyanide (results not shown), suggesting that (bi)carbonate and copper redox cycling at the enzyme active site is essential for producing SOD1 nitrone adducts.
In Figures 1(A)–1(C), the copper chelator DTPA was added to eliminate the contribution of H2O2-induced free copper-mediated SOD1 oxidation; however, it is known that CO3•− reacts relatively rapidly with DTPA (rate constant ∼1.7×107 M−1·s−1 [35]), and thus the concentration of DTPA was carefully adjusted to avoid underestimating CO3•−-triggered oxidations. Indeed, we observed that concentrations of DTPA higher than 0.5 mM inhibited CO3•−-mediated guaiacol oxidation (Figure 1D), suggesting that high concentrations of DTPA scavenge CO3•−. Accordingly, in subsequent experiments, we used 0.1 mM DTPA, which is over 3-fold higher than the copper content of the SOD1 solutions used in our experiments.
Albumin protects bSOD1 and hSOD1 activity by acting as an alternative target for radical modification
In order to investigate the potential for the induction of SOD1-driven oxidation in surrounding proteins, we used HSA as a model target. We observed that HSA blocked H2O2-induced SOD1 self-inactivation in BB, but in PB it had no effect (Figure 2A). This suggested that HSA protects SOD1 activity by scavenging the diffusible CO3•− that enhances H2O2-induced SOD1 inactivation. When we separated the reaction mixture using SDS/PAGE and stained the gels with Coomassie Blue, we observed that both proteins were extensively fragmented when the reaction was carried out in PB (Figure 2B, left-hand panel). DTPA protected HSA against fragmentation, but did not affect SOD1 fragmentation at its active site, as assessed by the ∼5 and ∼10 kDa SOD1 fragments [10,12] (Figure 2B, left-hand panel). However, when we performed the reaction in BB with DTPA, the active site of SOD1 was protected against site-specific fragmentation (Figure 2B, right panel; [12]), but extensive fragmentation at other sites of both SOD1 and HSA still occurs until DTPA is added. Although active-site fragmentation is not occurring, copper must still be released, presumably as a result of histidine oxidation (Table 1). This released copper must be causing protein fragmentation through Fenton-type chemistry. Similar results were obtained when erythrocytic bSOD1 was replaced by hSOD1 (results not shown).
As modulated by the copper chelator DTPA and (bi)carbonate, two structural consequences of free radical chemistry can be delineated in the formation of H2O2-induced SOD1-driven protein radicals: fragmentation and aggregation. Fragmentation other than at the active site appears to be mediated by copper released from the active site of SOD1. In the absence of DTPA and (bi)carbonate, we saw evidence of both pathways, as protein nitrone adducts (both fragments and intact proteins) increased with the concentration of H2O2 (Figure 2C, left-hand panel). At the same H2O2 concentration, the number of radical sites was higher when the reaction was carried out in 100 mM BB rather than in 100 mM PB (Figure 2C, middle panel). But in BB with 0.1 mM DTPA, we observed distinct protein nitrone adducts of monomers and aggregates from both proteins, but not of fragments (Figure 2C, right-hand panel). These results suggest that DTPA inhibits the Fenton-like chemistry triggered by copper released from the SOD1, which results in protein fragmentation. Once the released copper rebinds to specific residues on SOD1 and HSA, in the presence of excess H2O2, these residues can also act as additional sites for CO3•− generation (see below). It is noteworthy that this amplifying copper-dependent radical chemistry occurs at protein copper-binding sites other than the active site of SOD1.
SOD1-driven copper-dependent radical damage
We further examined SOD1-driven copper-triggered radical chemistry in proteins by reacting SOD1 and HSA with H2O2 in PB without DTPA (Figure 3). Under these conditions, HSA and SOD1 itself were targets of copper-mediated site-specific fragmentation (Figure 3A, left-hand panel). H2O2-induced fragmentation of SOD1 at its active site was evident by its ∼5 and ∼10 kDa fragments [10,12] and was not prevented by DTPA, although DTPA did strongly inhibit nitrone adduct formation of the protein (Figure 3A, right-hand panel). This result suggests that the formation of H2O2-induced copper-bound oxidant at the SOD1 active site precedes release of copper. Released copper would then be re-bound to HSA and other sites in SOD1 itself to react with excess H2O2 and induce free-radical-mediated site-specific fragmentation. To test this hypothesis, we measured H2O2-induced copper release from the bSOD1 active site by BCDS-assisted reduction of Cu2+ to Cu+ as the formation of its red [Cu+(BCDS)2]3− complex (Figure 3B). Depending on the concentration of H2O2, 15 min of reaction was enough to release a significant amount of copper, an amount that would be sufficient to produce protein radicals, as we found previously by immuno-spin trapping in the HSA/Cu2+/H2O2 system [31]. After 2 h, 5 mM H2O2 released approx. one-half of the available copper at the active site.
To study the effect of phosphate, (bi)carbonate and DTPA on oxidation of substrates too bulky to access the SOD1 active site, we examined the oxidation of guaiacol to tetraguaiacol (Figure 4A). We did not observe guaiacol oxidation when SOD1 was allowed to react with H2O2 in argon-purged (to eliminate CO2) PB, with or without DTPA. The strongest oxidation of guaiacol was observed when the reaction was carried out in 100 mM BB; this oxidation was inhibited by almost 30% with 0.1 mM DTPA and by over 50% with 100 mM PB. Phosphate may compete with (bi)carbonate for copper binding and also for access to the positively charged channel to the SOD1 active site. We obtained similar results when bSOD1 was replaced with hSOD1 (results not shown). Other peroxidase substrates such as ABTS [2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] and NADPH were used with similar results (results not shown). We also observed that H2O2-induced loss of copper from the SOD1 active site was enhanced by (bi)carbonate and inhibited by phosphate (Figure 4B). Presumably, the oxidation of histidine residues at the SOD1 active site by CO3•− is responsible for this copper release, which occurs without SOD1 active-site fragmentation.
With or without DTPA, (bi)carbonate was observed to enhance the production of H2O2-induced SOD1 and HSA nitrone adducts (Figure 4C). However, levels of protein radicals were higher in the absence of DTPA than in its presence. Products of copper-mediated site-specific modification of SOD1 and HSA were observed when the reaction was carried out without DTPA, but they disappeared when DTPA was included (compare smears in Figure 4C left- and right-hand panels).
Radical chemistry induced by SOD1-driven CO3•− oxidations
Thus (bi)carbonate enhances H2O2-induced loss of copper from the enzyme active site, presumably by oxidizing active-site histidine residues, and this copper then re-binds to other SOD1 sites or to target proteins such as HSA. The re-bound copper acts as additional sites for peroxymonocarbonate reduction to CO3•− [31] (see also Figure 5), thus preventing site-specific fragmentation. Accordingly, in the following experiments, we used a physiological concentration of (bi)carbonate (i.e. ∼25 mM) in 100 mM PB with 0.1 mM DTPA in order to specifically study CO3•−-triggered free radicals in SOD1 itself and HSA (Figure 6A). Under these conditions, SOD1 and HSA radicals were observed as their monomer and dimer and required the addition of all components (Figure 6A, right-hand panel). This result confirms the proposal of Bonini at al. [4] that the CO3•− formed at the active site of SOD1 can diffuse out the channel to react with BSA, forming diverse radicals. CO3•−-triggered protein radicals increased with incubation time (results not shown). The oxidation profile of HSA (Figure 6A) was independent of whether bSOD1 or hSOD1 was used, establishing that the tryptophan radical formed from hSOD1 has little role in HSA oxidation (see Figure 7). In addition, SOD1-driven CO3•−-triggered oxidation of HSA, but not of SOD1 itself, was partially prevented by adding guaiacol to the reaction mixture (Figure 6B), with concomitant oxidation to tetraguaiacol (results not shown). These results suggest that, by scavenging CO3•−, guaiacol protected HSA against SOD1-driven oxidation.
Gaining further insights into the species oxidized by the (bi)carbonate-dependent species formed at the active site of SOD1, we observed that CA (carbonic anhydrase/dehydratase) enhanced H2O2-induced SOD1 and HSA nitrone adducts and was also a target for its own catalysed reactions, producing CA nitrone adducts (Figure 6C). Figure 7(A) shows a protein staining and a Western blot of hSOD1, isolated from human erythrocytes and allowed to react with H2O2 and DMPO in the absence of copper chemistry. Figure 7(A), middle panel, also shows the (bi)carbonate-dependent formation of a protein radical in the enzyme. The right-hand panel in Figure 7(A) shows that the formation of hSOD1 and HSA-centred radicals are dependent on (bi)carbonate and H2O2. Control experiments and dose-dependent effect of (bi)carbonate on hSOD1-driven protein radical production are shown in Figure 7(B). Human SOD1 isolated from transformed E. coli produced results similar to those of the human and bovine erythrocytic enzymes (not shown). Once again, our results show the importance of a (bi)carbonate-dependent and diffusible species capable of generating DMPO-trappable radical sites in SOD1 itself and in any other protein in the reaction microenvironment when SOD1 reacts with H2O2 in the presence of DTPA.
Protein radical formed by the hSOD1-driven CO3•−-mediated oxidations in mBH
We have devised experimental conditions to study protein oxidation driven by the bSOD1 or hSOD1/H2O2 systems that are important to understanding the mechanism of possible protein modifications in vivo. Here we used the SOD1/H2O2/DTPA/(bi)carbonate system to generate CO3•− without copper-derived OH•-like-induced oxidations. As a target protein mixture for CO3•−, we used mBH (Figure 8). We partially characterized the corresponding Coomassie Blue-stained gel bands using LC–ESI–MS/MS analyses. The Coomassie Blue-stained protein bands (corresponding to the anti-DMPO bands observed by Western blotting) were excised from the gel, digested and then subjected to LC–MS/MS analyses for protein identification.
Following LC–MS/MS analyses, the raw data were extracted and searched against the NCBI non-redundant database (species: mouse only) (Table 2). Only those proteins with a summed MS/MS score greater than 30 and at least two distinct tryptic peptides are listed. The immuno-spin trapping analysis detected six distinct protein bands. As shown in Table 2, many proteins were identified in each band of the Western blot. One or more of the identified proteins in each band may be a target of CO3•−-triggered modification, leading to formation of protein radicals that are trapped by DMPO. These protein radicals can be assigned to mitochondrial proteins, heat-shock proteins and cell cytoskeleton proteins (Table 2). The above results exhibit the utility of the combined approaches of immuno-spin trapping and MS to detect potential targets for oxidation by CO3•− in a complex system such as mBH.
The study of the mechanism of protein radical formation by the wild-type SOD1/H2O2 system is important to the understanding of tissue damage in neuroinflammatory pathologies such as fALS. In the present study, we investigated the H2O2-induced wild-type bSOD1- and hSOD1-driven production of protein radicals in SOD1 itself and the model protein targets HSA and mBH under experimental conditions that clearly separated copper- from CO3•−-initiated radical chemistry (Figure 5).
In some studies of the SOD1 and H2O2 reaction, the role of copper- and CO3•−-triggered oxidations is difficult to distinguish (Figure 5). In the absence of copper chelators, Bonini et al. [4] were the first to detect BSA radicals formed by CO3•−-triggered radical chemistry following the H2O2-induced peroxidative activity of SOD1 in the presence of (bi)carbonate; however, SOD1 radicals were not detected [4,19]. According to our results, this system would involve the reduction of peroxymonocarbonate to CO3•− by copper-bound oxidants located at the SOD1 active site and at many other sites in both SOD1 and BSA. In the absence of DTPA, the SOD1/HSA/H2O2 reaction in BB produced greater oxidative damage, mainly to side-chain residues (Table 1), than that in PB, as demonstrated by increases in DMPO-trappable SOD1 radicals and SOD1 inactivation (Figures 1A and 1B). In the absence of DTPA, reduction of peroxymonocarbonate and formation of CO3•− will occur at many sites outside the enzyme active site where copper is re-bound. In agreement with these observations, we have found previously that (bi)carbonate protected against site-specific fragmentation, but enhanced side-chain radical formation in HSA [31].
DTPA prevents the re-binding of copper to SOD1 or HSA, but, as previously reported, does not prevent oxidative damage to SOD1 at the enzyme active site [12]. In addition, the oxidants formed at the SOD1 active site (Reactions 1 and 2) are generated in systems whether or not they contain DTPA, owing to the fact that this chelating agent does not have access to the SOD1 active site [8,13]. This can explain the failure of HSA, guaiacol or ABTS, even at high concentrations, to protect against SOD1's active-site fragmentation (∼10 and ∼5 kDa fragments) and to completely block SOD1 inactivation.
The copper-bound oxidant (an OH•-like species) at the SOD1 active site may well inactivate SOD1 at a different site from that inactivated by CO3•−. CO3•− is the species responsible for the oxidation of peroxidase substrates [11,13,21] and for oxidation of a key tryptophan residue and formation of a Trp–Trp cross-linkage in human SOD1 [36]. (Bi)carbonate was required for DMPO-trappable protein radical formation and caused protein aggregation of SOD1, possibly due to the formation of side-chain radicals (Table 1). In any case, in our results, we have found very little difference between hSOD1 (recombinant or erythrocytic) and bSOD1, although bSOD1 does not contain any tryptophan (compare Figures 1B and 7A).
Previously, we have shown that CA enhances the (bi)carbonate-enhanced, H2O2-induced, copper-catalysed oxidation of a peroxidase substrate, suggesting the involvement of CO2 in the formation of CO3•− [31]. Indeed, in the present study (Figure 6C), we have shown that when CA is included in the SOD1/H2O2/HSA/DTPA system with physiological concentrations of (bi)carbonate, it produces a dramatic concentration-dependent increase in (bi)carbonate-enhanced, H2O2-induced, SOD1-driven protein radicals. Our results indicate that, under our experimental conditions, peroxidase substrates, such as guaiacol, ABTS, NADPH, and proteins protect SOD1 against inactivation by 0.1 mM H2O2 in 100 mM (bi)carbonate with 0.1 mM DTPA because they scavenge CO3•−. Our results also suggest that the diffusible CO3•− can oxidize critical residues for SOD1 activity or for the transport of the superoxide radical anion through the enzyme cationic channel. In fact, at non-physiological concentrations above 25 mM, (bi)carbonate increases the rate of inactivation of SOD1 (Figure 1A) [17]. The peroxidase substrate guaiacol competes with HSA for CO3•−, thus guaiacol acts as an alternative target to HSA with a consequent decrease in HSA-centred radical formation (Figure 6B), but does not completely prevent SOD1 inactivation (results not shown). This could be related to the proximity of SOD1 to CO3•− as it is formed [15].
In order to explain the pathways of CO3•−-initiated radical chemistry and the apparent discrepancy between protein radicals and dismutase activity, we propose a novel mechanism involving CO2 based on the enhancing effect of CA on protein radical formation (Figure 6C). Indeed, peroxymonocarbonate, which has been proposed to be an important physiological oxidant [4], oxidizes biological targets by a two-electron mechanism, and its oxidizing power is enhanced when reduced to CO3•− by catalysis with metal centres [4,31,37] (Figure 5). For the formation of CO3•−, we propose the formation of a free peroxymonocarbonate anion intermediate (HOOCO2−, see Figure 5); CO2 and hydrogen peroxide (OOH−/H2O2) are in equilibrium with HOOCO2− with a Keq=0.33 M−1 (Reaction 3) [19,38–40]. The addition of the deprotonized water (−OH) to CO2 is catalysed by CA. Analogously, we make the novel proposal that addition of deprotonized hydrogen peroxide (OOH−) to CO2 is likewise catalysed by CA: (Reaction 3)
Accordingly, the equilibrium shown in Reaction 3 probably proceeds via the intermediacy of CO2, where HOOCO2− is essentially a CO2 adduct of the OOH− [38]. The reduction of HOOCO2− by H2O2-induced Cu+-SOD1 (Reaction 1) forms CO3•− directly, without any need for the formation of the high-energy bound oxidant at the enzyme's active site (Reaction 2). It has been suggested recently that the equilibrium constant for Reaction 3 is increased in the presence of biological targets such as proteins, lipids and CA mimetics, which may alter the equilibrium of gaseous CO2/dissolved CO2/HCO3− [19]. Our proposal of a direct role for CA in peroxymonocarbonate anion formation (Reaction 3) is supported by the fact that the established mechanism of CA for the formation of (bi)carbonate (Reaction 4) [41] is very similar for peroxymonocarbonate [38], except that the base of hydrogen peroxide (OOH−) nucleophilically attacks CO2 instead of the base of water (OH−) where the Zn2+ of CA catalyses the deprotonation of HOOH and H2O respectively: (Reaction 4)
The ease of diffusion of peroxymonocarbonate anion through the anion channel of SOD1 and the substantially lower oxidation potential of CO3•− (E° ∼1.78 V) relative to that of the strong oxidant at the active site (E° ∼2.31 V) should be reflected in a much more kinetically favoured formation of protein radicals and would explain the protection afforded by (bi)carbonate against the fragmentation at the SOD1 active site induced by H2O2, which probably is initiated by the abstraction of the hydrogen of the amide bond [42] linking two amino acids near the active-site copper of SOD1. In addition, CO3•− diffuses and produces additional DMPO-trappable radical sites in the side chains of SOD1 itself and HSA (Table 1), but it does not produce protein fragmentation [31]. Our results suggest that CO3•− can produce protein radicals or oxidize proteins at side chains [43], but it cannot induce backbone cleavages and further protein fragmentation as the copper-bound oxidant does [12,31]. The enhanced inhibition of the enzyme by (bi)carbonate may be the consequence of the oxidation of residues by the diffusible CO3•−, which may be important for driving superoxide radical anion through the SOD1 anion channel to its active site.
Our model of hSOD1-driven CO3•−-initiated protein oxidation is supported by our finding that the SOD1/H2O2/DTPA/(bi)carbonate system, in which Cu+ reduces peroxymonocarbonate to form CO3•−, oxidizes a set of well-distinguished bands of proteins in mBH (Figure 8). Immuno-spin trapping in combination with MS technology [30,44] has allowed the identification of potential targets of CO3•−-mediated oxidations in mBH. The identification of the protein radicals and their subcellular location using murine models of fALS [45] may be important to our understanding of the molecular mechanisms of fALS.
We have established experimental conditions that separate copper- from CO3•−-initiated protein oxidations in the SOD1/H2O2 system, which may help us to develop an experimental model to characterize biological targets in fALS. The identification of these proteins may be a starting point for investigations aimed at identifying critical proteins in neurons overexpressing the hSOD1 mutant proteins that are targets for oxidation and thus may have a critical role in this neuroinflammatory disease.
This research was supported by the Intramural Research Program of the National Institute of Environmental Health Sciences of the National Institutes of Health. D.C.R. acknowledges support from the National Institute of Environmental Health Sciences [grant number R00ES015415] and the start-up funds from the Presbyterian Health Foundation.
We thank Ms Mary Mason and Dr Ann Motten for helping in the pre-submission editing of this manuscript.
Abbreviations: ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); BB, sodium (bi)carbonate buffer (pH 7.4); BCA, bicinchoninic acid; BCDS, bathocuproine disulfonic acid; CA, carbonic anhydrase; DMPO, 5,5-dimethylpyrroline-N-oxide; DTPA, diethylenetriaminepenta-acetic acid; ESI, electrospray ionization; fALS, familial amyotrophic lateral sclerosis; HSA, human serum albumin; LC, liquid chromatography; mBH, mouse brain homogenate; MS/MS, tandem MS; PB, sodium phosphate buffer (pH 7.4); SOD1, Cu,Zn-superoxide dismutase; bSOD1, bovine SOD1; hSOD1, human SOD1
- © The Authors Journal compilation © 2009 Biochemical Society


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Tickets available by Friday for Sarah Palin
Gates will open at 6 a.m. Saturday at Sims Park in New Port Richey, and John McCain's running mate Sarah Palin will speak by 9 a.m., said Bill Bunting, Pasco County GOP chairman.
At least two GOP offices will have tickets by Friday: Hudson and New Port Richey (details after the jump).
Palin is also scheduled to be in Virginia and North Carolina later on Saturday to finish the "Road to Victory" tour.
-- David DeCamp, Times staff writer
New Port Richey
The Palm Center; 5509 Grand Blvd. (at Gulf Blvd.); 727-815-7671
7414 SR 52; near Zimmerman/across from the Bank of America; 727-863-5400

subset: commoncrawl


text: Additional Programs for Individuals with Brain Injury who are Medicaid Eligible Centennial Care Centennial Care is the new name of the New Mexico Medicaid program. Centennial Care will begin January 1, 2014 and services will be provided by four managed care organizations (MCOs). These services include physical health, behavioral health, long-term care and community benefits. Most people who are currently enrolled in a New Mexico Medicaid program are eligible for Centennial Care. Eligibility for all Medicaid programs requires that individuals meet certain federal guidelines. These include citizenship, residency and income requirements. To apply for Centennial Care coverage, go to Apply for Benefits


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Worst Monday: Balloting open
Finally, a Monday with some meat! Twelve games—plenty of opportunities for heroes or (in our case) goats.
Are you gazing this morning at a double-digit deficit in the standings for the week? March out the offenders!
Entering's a snap:
1. Send an email to
2. Put Worst Monday in the subject line along with your Monday point total.
3. Attach a screen shot of your roster and their points scored for Monday. (You can paste the screen shot in a Word document and attach that.) We need the screen shot—don't spell out the tallies in the email.
4. Add brief biographical material.
We'll sift through the entries & give the lowest score on Wednesday. Each weekly winner gets a year of Heater Magazine. The winner with the lowest score for the season gets a free copy of the 2010 Graphical Player, coming out in December.
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Welcome to the Muppet Central Forum!
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When you outgrew Sesame Street...
Discussion in 'Classic Sesame Street' started by Philo and Gunge, Jan 1, 2004.
Page 3 of 5
Page 3 of 5


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Notes to Self:
1/ Discovering who I am, how I think and make 2/ Constructing success 3/ Unpacking big goals. 4/ One step at a time. 5/ Being myself 6/ Due 4/24/14: "Look Book" for my Fashion Accessory + Wearable Collection. 7/ I can do this. 8/ This school is completely worth the tuition.
— Madisen Sowers, Junior, Product Design, MAD ABOUT IT


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CONTEST: Pro-Marijuana People, Time to Win Big
It's simple, really. Below is a list of 3 Myth/Fact combinations about weed. One of them is totally bogus. The other two are completely legit.
The 10th person to correctly say which one is bogus (posted to the wall of our Facebook page) wins a copy of Marijuana Is Safer: So Why Are We Driving People to Drink? by Steve Fox, Paul Armentano, and Mason Tvert.
The contest begins RIGHT NOW! Whenever you're reading this.
1. MYTH: Today's marijuana is significantly stronger and thus more dangerous than the marijuana of the past.
FACT: The potency of today's cannabis is only slightly higher, on average, than the pot of twenty or thirty years ago. Marijuana's increased potency, however, is not associated with increased health risks.
2. MYTH: Smoking pot with your children actually increases the likelihood they'll get a high paying job.
FACT: This is up for debate. It has been noted to increase forms of filial bonds, on the other hand.
3. MYTH: Using marijuana will inevitably lead to the use of "harder" drugs like cocaine and heroin.
FACT: The overwhelming majority of marijuana users never try another illicit substance.


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So on Christmas I got a pair of Vibrams Bikila LS's, and have been wearing them around for a week to break them in before I took to working out with them. Today was the test! I ran my weekly sprint(on the treadmill because its kinda nasty outside), and proceeded to lift weights(an upper body day though). I will have to say that I am super impressed with how it felt to run in those. After a few minutes of warmup and about 10 minutes of alternating sprint/walking I had no shin splints, sore joints, or anything...and actually was bounding with energy. I am sold! Not just on the Vibrams, but TPB in general!
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text:
CSB Reports Second Quarter Financial Results
Trudy K. Everhart, president and CEO of Community State Bank of Orbisonia and Orbisonia Community Bancorp, Inc. has announced unaudited financial results as of June 30, 2017.
Due to safety concerns for our customers and employees, effective September 5, 2017, our Waterfall branch will become a drive-thru only facility. Along with the drive-thru service, we will continue to have our night deposit and ATM available.
We apologize for any inconvenience that this may cause; however, we feel that this is the best solution at this time. We value you as a customer and look forward to serving you for years to come. For any questions, please contact us at 866-874-5552.
CSB On The Go Mobile Banking App
CSB would like to introduce our new Mobile Banking App called CSB On The Go. It offers a convenient, fast, easy, and secure way to access your accounts on the go from your mobile device. The CSB On The Go Mobile Banking App allows our customers to check balances, view transactions, receive text alerts, and much more.
Learn More About Our Mobile Banking App
Standard message and data rates may apply.
CSB Mobile Banking App now includes Online Deposit Anywhere!! After downloading and logging into the CSB On The Go Mobile Banking App, CSB Online Deposit Anywhere will be available.
CSB Online Deposit Anywhere
Community State Bank of Orbisonia Deposit Anywhere Mobile Deposit Service allows you to make deposits to a Consumer Checking or Savings account that may be used in connection with the Deposit Anywhere Mobile Deposit Service, and the functions within our Mobile Banking Service application (Mobile App) to scan checks and to transmit the images of the checks and associated data to us. In order to use the Service, our Mobile Banking Service application (Mobile App) must be installed on your Wireless Device.
Customer Security Alert: Robotic Phishing Phone Call
Please be aware of a fraud scheme that is attempting to deceive banking card customers of their card information. Fraudsters are using an automated system to call claiming to be representing Barkley Card Fraud Detection and pressing "1" to be connected to a "detection specialist". Immediately the call is asking for card information including card number, expiration date, and PIN number. If you receive a call like this please disconnect the call immediately. These calls are being received on both land line and mobile phone numbers.
While not new, telephone scams continue to cheat unsuspecting consumers. Whether being concealed as a computer company, fraud detection agency, or bank, this brand of fraudster usually requests consumers to share their sensitive personal information, such as social security numbers or payment card account details.
The most important thing to remember in these situations is to never give card, banking, or personal information to someone who calls you. While your financial institution may call to report suspicious activity or to confirm that you made a particular transaction, you will not be asked to provide your account information. If this happens to you, dial the number on the back of your payment card to verify the call. Never use the telephone number the caller provides.
Scammers continue to call, because they sometimes succeed. If you are suspicious, end the call immediately. Better to be safe than scammed. If you have received this or a similar type of call and have provided the caller with any form of personal information please contact our Electronic Banking Department at (814) 447-5552.
Customer Alert: Fraudulent Customer Solicitation
Customer Alert: Community State Bank of Orbisonia does not contact customers about card activation. We have received reports of a suspicious call regarding card activation status or stating your card has been deactivated. This is a fraudulent call, if you have any questions about this type of call or the potential calls you may receive about your ATM or Debit Mastercard please call your local branch office or our electronic banking department at (800) 626-2130.


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Flamengo 11 X 0 Friburguense - Gols - Campeonato Carioca Sub-20 2012
Frauches Mattheus - 3 Recife Nixon Adryan - 2 Rafinha e Fernandinho - 2 fizeram os gols do jogo.
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I had a rooted S4 with Gummy ROM instaled.
This week the game Puzzle & Dragons launched a patch, and it stopped working on my cel phone.
When I changed the Gummy ROM to a Google Edition one, without the cool stuff of a tweak box, it started working again.
I'm able to install Xposed Framework and the game still works, but every time I install a module / tweak box (all from this list http://forum.xda-developers.com/show....php?t=2327541), do the steps and reboot the phone, the game stops working.
I also tried to reinstall the game after installing the tweak box.
I'd like to know what can be messing with the game.
I really miss the advanced power-off menu between other things, but I don't want to stop playing this game right now.
Also, I don't know if this is happening to another game / app / etc


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PULASKI — A Grammy Award-nominated singer, poet and storyteller will bring the Martin Hall Auditorium stage alive with the richness of the South when Minton Sparks performs at Martin Methodist College on Tuesday, Feb. 11
PULASKI — A Grammy Award-nominated singer, poet and storyteller will bring the Martin Hall Auditorium stage alive with the richness of the South when Minton Sparks performs at Martin Methodist College on Tuesday, Feb. 11
The event, which begins at 7 p.m., is part of the college's 2013-14 Della Clayton Lee Cultural Arts Series. Admission is $7 per person, with all students (any age, any school) and all members of the MMC Alumni Association admitted free of charge.
To reserve seats, call (931) 363-9882.
It's hard to envision singular performances by a poet and short story author, a character actor and a songwriter being offered on a single bill. That is, until one Minton Sparks takes the stage. For she is all three — a lean, literate livewire in a flower print church dress who balances writerly, theatrical and musical gifts as easily as she balances the prop pocketbook on her slender wrist.
As Nashville-based musician Marshall Chapman once wrote: "Imagine, if you will, Flannery O'Connor and the ghost of Hank Williams having an affair that results in the birth of an illegitimate child — I have seen Minton Sparks. And if she's not the ghost child of the woman who wrote 'Wise Blood' and the man who sang 'I'll Never Get Out of This World Alive,' then cotton doesn't grow in a cotton field."
Minton Sparks fuses music, poetry and her intoxicating gift for storytelling to paint word pictures of the rural South that put you square in the middle of the people and the places she knows like the back of her hand.
A wildly original poet, performance artist, novelist, teacher and essayist, she was born in a Tennessee college town and raised among her Southern family in and around Arkansas. She earned degrees from the University of the South and Vanderbilt University. Her appearances range from the prestigious Jonesborough National Storytelling Festival all the way to the American Songbook Series at Lincoln Center in New York City.
Minton's books, "Desperate Ransom" and "White Lightning," and her writing have received wide acclaim from NPR's Weekend "All Things Considered" and BBC's Bob Harris Show. Her performances have enthralled audiences across the United States and Europe. Her DVD, "Open Casket" and three CDs, which feature blues sensation Keb Mo', the legendary Waylon Jenningsand the internationally acclaimed Irish songstress Maura O'Connell, are sought after worldwide. She has appeared on stage with Ben Folds, John Prine, Punch Brothers, Jacob Dylan, Rosanne Cash and country legend Pam Tillis.
"I'm not sure we've ever had a performer quite like Minton Sparks as part of the Della Clayton Lee series," said Grant Vosburgh, the college's director of communications and coordinator of the cultural arts series. "My first introduction to her was watching one of her video clips, and I have since watched it over and over. She is at once captivating and entertaining. I found myself laughing out loud at her brilliant insights of the South that I certainly know, all the while nodding in affirmation that she had captured it exactly right.
"As a bonus, she will conduct a storytelling workshop from 9-11 a.m. Wednesday, Feb. 12, in the Gault Fine Arts Center recital hall. There is no admission charge to the workshop, and the public is invited," Vosburgh said.
For more information about the concert or the workshop call (931) 363-9882.


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JK Roses
Bespoke balloon and floral decorations at great prices!
Many special offers available!
At JK Roses we specialise in creating floral and balloon decorations for our customers for events both large and small.
Whether it be for a party, a wedding or to brighten up an office or restaurant, JK Roses can provide superb quality and amazing visual impact for competitive prices.
We are more than happy to visit customers across North London, Bedfordshire, Hertfordshire and Berkshire from our base in the Welwyn Hatfield area of Hertfordshire
Please feel free to give us a call to discuss your ideas for decoration for your events and we will be happy to give you a competitive price.


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(LOS ANGELES) — Michael Lohan collapsed during an Alcoholics Anonymous meeting on Tuesday and was transported to a hospital, reports RadarOnline.com.
Lindsay Lohan's father was taken by ambulance to Palm Beach Gardens Hospital, where doctors determined that he had a blood clot in his lung. A source told the site that Lohan underwent a CAT scan and an MRI, and he was given medication to dissolve the clot.
The source added, "Michael's still waiting on the test results and we're not sure when he'll be released from the hospital."
Lohan has been in Florida attending a 120-day court-appointed rehab and domestic violence intervention program. The rehab stint stems from a domestic violence case involving ex Kate Major.
The 51-year-old has visited the emergency room several times while in the Sunshine State. Lohan had surgery to unclog his artery and repair blockage in his heart in early December. Shortly after that, he was rushed to the hospital with a staph infection.
Copyright 2011 ABC News Radio
Adam Forsgren, EastIdahoNews.com columnist
Adam Forsgren, EastIdahoNews.com columnist
Brian Stelter, CNN


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What's Hot Today
SONS OF ANARCHY "Dorylus" Season 4 Episode 3 airs on Tuesday September 20 at 10pm on FX. Episode Synopsis : SAMCRO encounters an unlikely foe when a piece of their business goes missing. Read More... http://www.daemonstv.com/2011/09/20/sons-of-anarchy-dorylus-season-4-episode-3/?utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+daemonstv+%28Daemon%27s+TV%29&utm_content=Google+Reader
On the latest episode of Sons of Anarchy , Clay and Jax pushed for the club to step in to the dangerous world of running drugs for the Mexican cartel. What impact will this have on next week's installment? Titled "Dorylus," it will focus on the campaign by President and Vice President to convince other SAMCRO members to vote their way, while the following promo teases that Gemma learns of this plan and tries to talk Clay out of it. Look for her to also confront Tara about Maureen's letters. Elsewhere, according to the official FX synopsis, Kozik is "challenged to a game of basketball." Really, that's what it says. http://www.tvfanatic.com/2011/09/sons-of-anarchy-sneak-preview-a-major-confrontation/


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Al Capp had numerous assistants at various times throughout the run of Li'l Abner. They are not easily identifiable. The story and art credits listed as Al Capp should more accurately be ascribed to Al Capp Studios. Until everyone who contributed to the art and story can be identified, story and art credits will read Al Capp.
Distributed by American News Company.
The cover is a composite of figures from Al Capp's model sheets. The Wolf Gal figure looks like it doesn't belong in the scene. The television is probably new art by Mel Lazarus.
Located on the left side of the page between pages 3 and 4 of "Lonesome Polecat Goes to School."
On the first page, copyright 1949 by Toby Press, Inc.


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Do you get bored with your curls?
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Oregano (formerly babywavy)
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Join Date: Dec 2004
Posts: 10,336
Friday, January 12, 2007 at 03:13PM
I do this all the time, but then I regret cutting it b/c I can never get it as long as I want! I always break down and chop it, and then I'm pissed at myself a week later!
But I'm the queen of dying my hair - something else I need to break myself of. I hate the upkeep, and having roots, and my hair is so dry and porous that the dye just ruins it.
But to answer your original question, yes I get bored with my curls all the time. Right now I've been blowing it out all the time. My pregnancy seems to be pulling out the curl, and it's annoying that it falls out 20 minutes later, so I just gave up. Plus, my hair is short right now, and I feel like the curls make me look so young. And not in a good way. I feel like I look cutesy, Shirley Temple young.
My goal is to grow it out one length in hopes of pulling out the curl and getting more of a wave, that would be easier to maintain. I'm so SICK of having this producty coif that can't blow around, and I can't run my fingers through.
~ the artist formerly known as babywavy ~
Please excuse any typos. For the time being, we are blaming it on my computer.


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When Mr. Earl Grey Becomes a Lady2012-04-16 3 18 Share Tweet
Developing 35mm Earl Grey 100 ISO pushed to Lady Grey 400 ISO is possible: just process "Earl" as a "Lady".
Everything was ok to test my Yashica electron 35 mm bought for 20€ along the seaside market in Marina di Pisa.
It was Christmas time, family and friends were with me. It was a usual Italian-southern-sunny days falling in the middle of winter. Perfect in and out conditions to load an Earl Grey 100.
Yet…too much southern-food, too much worldwide-drink maybe, since I pushed the film to 400 ISO by mistake. There was no way to find any suggestions for developing times for Earl Grey pushed to 400. I checked on web looking for tips. Even Lomo-friends appear not to have ever fallen in such an experience.
Then, I thought that the easiest is the simplest (but maybe not the best): let's try to develop Mr. Earl as a Lady. Indeed, I used the same developing times as for 400 ISO Lady Grey: Rodinal 09 one shot 1+50 10 minutes (20°) (as seen on Lomography Lady Grey)
And here are the results:
Lomography's Earl Grey is an exquisite black and white 35mm ISO 100 film that will surely give your shots an extra dose of style and class. Whether you're taking landscapes or portraits, you will get jaw-dropping results with Earl Grey super-fine grain and wide tonal range. See our selection of Lomography films here.
written by tsaramaso on 2012-04-16 #gear #tutorials #bw #35mm #tipster #development #film-processing #lady-grey #early-grey #pushing-100-to-400


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My dog Abby and I just love this place, the staff is amazing and the place is first class. Everything is clean and all the dog's look happy because they are. I would recomend Barkinglot to any dog lover looking for safe and friendly place for their beloved pet.
Albert R
Thank you, thank you Toni, Geter and staff for the great week you gave our beloved pups, Abbey and Murfy! It was such a load off our minds to know what good care and attention they were getting. We will recommend you highly every chance we get, and will definitely be coming back! -
Don and Brenda Kimball
We just picked up our dog, Cricket, from a week long stay at The Barking Lot and I cannot say enough good things about the facility and and the caring people who work there. From the moment we walked through the door to the moment we packed up her things at the end of her stay, our dog was given the best care. While we were away, we were able to watch her play with the other dogs and interact with the staff via several webcams. We were most impressed watching the staff engage the dogs-they played with and interacted with all of the dogs. They really treated them as if they were their own. There are two outside areas -one for larger dogs and another for smaller pooches. There are also two indoor areas. The facility itself is top-notch- in addition to pools outside they also have a misting tent. When we picked up Cricket this afternoon, she had been given a bath. We travel quite often and we will be bringing our dog back to the barking lot without a doubt!
Caroline K.
Coppell, Texas


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We like to think we are in control of our lives, of what we feel and what we think. But scientists are now discovering this is often an illusion.
Surprising scientific experiments are revealing that what you think you'll do and what you will actually do can be very different.
Your unconscious mind is often calling the shots, influencing the decisions you make, from who you fall in love with to what you eat!
A recent study demonstrated the mind's tendency to favour positive information over negative, the latter being tacitly edited out. So, when we say things like, "we just have to be positive and keep going" we may be expressing a deeper truth about how the mind has evolved to persuade us that the future will be better than the past.
Research has unveiled that the human mind is programmed for positivity, and it is this deeply ingrained positivity that has allowed us to survive and thrive against other species over thousands of years.
This innate ability to expect the best regardless of circumstances has allowed us to take risks as a species that have ultimately led us to conquer the world.
It seems that even considering pros and cons, possible obstacles, likely challenges, fearful outcomes… our "go for it" tendencies have led us to massively and quickly evolve as a species.
However, when it comes to health and weight loss, our overly optimistic views may be contributing to making us the fattest and unhealthiest species to walk this planet.
Studies show that it's this positivity that keeps smokers smoking, even though they are fully aware it will kill them. It's the same positivity that leads us to choose to eat foods our bodies are not designed for, knowing the negative consequences.
This same optimism leads us to eat cake when we know perfectly well it will sabotage our weight loss efforts. Fully aware of the pros and cons, we think "ah sure, it'll be fine" and tuck into the piece of cake.
Scientists have shown that most of our decisions, actions, emotions and behaviour depend on the 95% of brain activity that is beyond our conscious awareness, which means that at least 95 % of our life is ruled by the programming in our unconscious mind.
As soon as we forget to be conscious, the unconscious is back in charge again. It seems the term "unconscious eating" is more a rule than an exception, and it's because of this that we continue to sabotage our own health and weight loss efforts.
But, does this mean we have no say or control over our health or our size?
Thankfully, studies in Neuroscience have shown that Neuro-linguistic Programming, Hypnosis, and Meditation can help re-wire the brain structure by creating new neural pathways, reprogramming the unconscious mind and building new more useful patterns of thinking that will lead to better decisions when it comes to cake, or anything else for that matter.
Like this post? Feel free to post any questions or comments in the Facebook comment box below.


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AMD Implements Cost Cutting Workforce Reduction, Carrell Killebrew is Outby Anand Lal Shimpi on November 4, 2011 11:10 AM EST
I had a feeling I was going to have to write a post like this after I published that Rick Bergman left AMD. Those of you who remember the RV770 and RV870 stories may remember the name Carrell Killebrew, one of many AMDers who was instrumental in the complete turnaround of their GPU strategy. If you flip back through the RV770 story you'll remember that there was one AMD executive who supported Carrell's strategy of a small(er)-die RV770. The relevant excerpt from the RV770 story is below:
There were many individuals at ATI that were responsible for the RV770 we know today getting green lighted. ATI's Rick Bergman was willing to put himself and his career on the line, because if this didn't work, he'd be one to blame. Carrell recalled a story where Rick Bergman and others were at a table discussing RV770; Rick turned to Matt Skynner and asked him if he thought they could really do it, if they could make RV770 a smaller-than-NVIDIA GPU and still be successful, if it was possible to create a halo in the Performance segment. Matt apparently pondered the question, turned to Rick and said "I think we can". Carrell felt that ATI might not have gone down that path if it weren't for Matt Skynner's support and Rick Bergman making sure that the project was executed as well as it ended up being.
With Bergman gone, I was afraid Carrell would follow. Unfortunately, it's a lot worse. Not only is Carrell Killebrew gone, but around 10% of AMD's workforce join him in yesterday's announced layoffs. Many of the PR people we work with at AMD are now gone, as well as higher ups like Carrell Killebrew and Patrick Moorhead.
I'd like to wish all of the AMDers the best of luck going forward. The beauty of the tech industry is there are always options. Hopefully we'll see some of these folks appear at Apple, Intel, NVIDIA and Qualcomm.
Source: Icrontic
19 Comments
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Beenthere - Friday, November 04, 2011 - linkBest wishes to those who are departing AMD. It's never easy being booted. In life when one door closes, often another opportunity presents itself.
Let's hope Read knows what he is doing and can get AMD sorted out. He can't do it alone so there will be new people coming into AMD as well. AMD has some good products now and more in the pipeline. They need to execute better at GF and TSMC for AMD to move forward in big strides. Reply
FATCamaro - Friday, November 04, 2011 - linkI can't believe you would go up on stage with jeans that show your unit.... Reply
ezekiel68 - Friday, November 04, 2011 - linkFunny, I hadn't noticed. I guess it all depends on what you're looking for. Reply
tipoo - Friday, November 04, 2011 - linkIts a shame they sold all their mobile technology a few years back, what they could really do with right now is a good ARM SoC. I think they should still be able to make one though, ARM will licence out a core to anyone who pays, and AMD has both CPU and graphics building expertise. Few sectors are growing as rapidly as ARM SoC's. Its clear that continuing to compete with Intel directly will be difficult for them and they will have to fight tooth and nail for every bit of profits, I think they could build a formidiable ARM SoC though, if only they started three years ago. That's one of the reasons their last CEO got the boot, right? I hope they still do it though, better late then never, and Windows 8 on ARM should be pretty big. Reply
Penti - Saturday, November 05, 2011 - linkTheir technology is successful at Qualcomm and Broadcom at least. That is actually better then something failing in house. That's more then you can say about nVidias tech that has been marginalized by being tied to their own products now rather then an addition/companion to another SoC/platform. Their survival in that field is dependent on that it makes it in the tablet market, but it got tough competition from TI, Samsung, Qualcomm, Freescale and other now. GF already does ARM SoC's for mayor vendors and semi-companies there really are no reason for AMD to cook a standard ARM IP macro or soft SoC. GF already has in-house developed ARM IP tech of their own. But they got other clients. Competition would be far grater in this field though also. Windows 8 tablets should pretty much exclusively be x86 I guess. Reply
marc1000 - Friday, November 04, 2011 - linkbut the last AMD cpu i bought was Athlon64, and it now seems that my last GPU will be the Radeon5770 that is still in my system.
i guess that the great talents of both companies did a good job together, but now it seems that BOTH camps are empty. AMD has no gpu and no cpu talents from old times. Reply
mxnerd - Saturday, November 05, 2011 - linkAMD really sholud have licensed ARM three years ago. But with 10% workforce and lead engineer gone, really wonder AMD's future. Reply
Penti - Saturday, November 05, 2011 - linkI think we can safely say that there will be continued strive for smaller GPU-architecture design thanks to their current plans and products with integrated gpus and APUs where they just can't spend a billion transistors on the graphics. They will have to re-think again to pull it off in our mobile world where mobile gpus for laptops and Fusion APUs will be their biggest graphics silicon market. They sold off the tech ATi had acquired in the field though, but a fresh start might be good. However the mobile and embedded players will get a larger role in this I would guess, their gpus and drivers have to be pretty good and suitable for all types of multimedia. I guess there will be some convergence there at least all platforms will support pretty much the same features though AMD probably won't go for the handset market but I can see them delivering low-power chips for Tablet PCs or some such. Intel hasn't really come up with a GPU for their embedded market either so. But they will have to do more with less (transistors) definitively. CPUs and custom logic gets faster, to be a compute engine they will have to do more with what they got or they will simply be framebuffers plus lots of drivercode and not the high-tech industry it can be.
I think high-end gpus will be pretty much out even when it comes to games in the future though. Games have been pretty stuck as well as the tech under it all it's simply a matter of targeting now, even integrated graphics is pretty competent now. Mid-end should have a great future in the notebook computer space for some time to come though. You still need an architecture that scales though. But just not so badly downwards smaller versions/platforms. Reply
fdfsxcvdcfdh - Monday, November 07, 2011 - link
ONLINE STOR good shopping Reply


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London, June 24 (ANI): Andy Murray has admitted he may never win Wimbledon, as he prepares for another shot at the title.
The British tennis ace will be facing Benjamin Becker on Centre Court.
According to the Sun, the 26-year-old said he has never thought about ending his career not winning Wimbledon, but added he was comfortable with that.
The World no. 2 said that he was going to win Wimbledon, and that was his number one priority.
He added that he would do all he can to give the best possible shot. (ANI)


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I have eaten here several times as I have not gotten gluten. Food is great and staff really seem to care.
Great GF dedicated menu. We always get gf chicken fried rice and my non-gf family members can't tell difference! Sweet and sour gf chicken good too.
Have had one cross contamination issue in 2012 but went back and haven't had an issue since. Update: April 2015. I think their chef changed and the food isn't as good as it used to be. Had cc issue in September. Will not return. hasent tasted the same for years.
Loved it. I had the sesame chicken. Very tasty and no reaction!! It will be my go to Chinese place from now on.
I just went gluten free and loved Chinese food and it tasted no different then non-gluten-free. I loved it! Best food I've had.
Much better than average Chinese food and a good gluten-free selection. Good grasp of gluten-free safety. I have eaten there many times with no gluten reactions. I am not sure whether they have gluten-free soy sauce, because it is not an ingredient in the dishes I order (lemon chicken and sweet and sour chicken). Our celiac support group has had several group parties there. Mid-scale decor. I highly recommend this place. They have another location on 135th Street.
On the menu it says "Not recommended for Celiac Disease" due to involuntary cross contamination. So eat at your own risk. I, personally, have never had any sensitivity issues with anything I have ordered off the GF menu. It's nice to find Chinese food with the GF menu option!!
Try the 135th location if you want the good stuff. This location doesn't taste the same.
"Not recommended for people with full on celiac disease" says this on menu
They have GF items in a section on their dinner menu. I tried the chicken and vegetables. The veggies were nice and crisp. The food had no flavor and was completely bland. I would like to try another entree and see if it was any more flavorful. Bring your own soy sauce, as theirs has wheat. It was about $11 for my entree.
We love this restaurant. They have a great selection of gluten free items. And they taste delicious. We have eaten here 20+ times over the years and have always felt good afterwards. The owner and staff are very nice. Did I mention that we love this place?!
Great! I have eaten here a dozen times and always feel great afterwards! I love their Philadelphia sushi roll and gluten free lemon chicken! Bring your own soy sauce though!
This is one of our favorite gluten free restaurants. They have a designated gluten free menu on the regular menu. We especially love the lemon shrimp, sesame chicken and lettuce wraps. Rice noodle dishes are good too. We love to eat here!
I love it! I am recently gluten-free and loved Chinese and have been overly pleased with the food and service.
I have eaten here several times. Love this place.
I loved it! Kinda wish i could do half orders so I could try more.
11 am - 9:30 pm
11 am - 9:30 pm
11 am - 9:30 pm
11 am - 9:30 pm
11 am - 10 pm
11 am - 10 pm
11 am - 9:30 pm


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H. H. Karmapa - Refuge Prayers, Four Boundless Mind
Refuge Prayers
Chanted by H. H. Karmapa.
In the Buddha, the Dharma and the Sangha,
I go for Refuge until Enlightenment is reached.
By the merit of practicing generosity and the other virtues
May Buddhahood be realized for the benefit of all beings.
Four Boundless Mind
Chanted by H. H. Karmapa.
May all sentient beings be happy and have the cause of happiness,
May they be separated from suffering and the cause of suffering,
Be inseparable from the perfect bliss which is completely free from suffering
May they all without least exception, dwell in the universal equanimity which is unbiased by attachment for dear and near ones and aversion from distant ones.
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Father: Greenberry Bishop
Mother: Ann Eliza (Unknown)
...Napoleon (d 1878) & Martha (d 1890) children...
|Shelby Wright Bishop (1853-1918) married Martha (Young-Bishop) (1846-1890). Note that Shelby married his brother's widow, Martha, on 29 September 1878 after Napoleon died 2 March 1878. Napoleon and Martha's children then lived with Shelby and Martha. Shelby and Martha had daughters Doll b 1885 and Mattie b 1887. After Martha died in 1890 Shelby married Powellton (Fitts) (1872-) on 3 November 1892. Shelby and Powettan had one child that died young.
Obituary Oglesby Bishop, Montgomery County, Missouri, 22 Jul 1925
Obituary Doll Bishop, St Louis, Missouri, 17 Sep 1959
Notes Nancy (Hale) Lee - Shamrock, Callaway County, Missouri
Descendants of Napoleon Bishop, Warren County, Missouri
Notes Shelby R Bishop - Audrain County, Missouri
Notes Sisters Doll M Bishop and Martha Bishop - Kingshighway Blvd, St Louis, Missouri
Census Records | Vital Records | Family Trees & Communities | Immigration Records | Military Records Directories & Member Lists | Family & Local Histories | Newspapers & Periodicals | Court, Land & Probate | Finding Aids


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NEW YORK (May. 25)
The United Jewish War Effort today made public the texts of messages received from Gen. Douglas Macarthur and Admiral William F.Halsey, American commanders in the Pacific area, Paying tribute to the Jews in the American armed forces on the occasion of American Jewish Heroes Day, which was observed throughout the United States last Sunday.
Gen. MacArthur said in his cable: "Every page of American history belies the enemy claim that one race is superior to another. That doctrine is as wrong morally as it is false scientifically. In peace and in war our country's noblest tradition as well as the best security for our future is the doctrine that each citizen shall be weighed on his merits and not by his inheritance. The searing fires of this war have again and again and again put to the test the fighting qualities of our men and women. As colin kelly and his bombardier, Meyer Levin, so well exemplified, we have met the challenge, whether Catholic or Protestant, Jew or Gentile,On May 23, specially set aside in their honor, I am proud to join with you in saluting the memory of fallen American heroes of the Jewish faith."
Admiral Halsey's airmailgram read: "The Jewish heritage has been one of suffering and sorrow. Jews have always fought against oppression, seeking freedom for themselves and for all peoples. Today, the whole civilized fights against history's greatest tyrants and oppressors. We are proud to have the Jews in our ranks. Their deeds furnish unshakable proof of their courage and loyalty to our common cause – the world wide preservation of freedom."


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On the Internet, A Tangled Web Of Classified Ads
This push into online classifieds - a business dominated by newspapers for more than a century... While classified revenue has been shrinking at newspapers across the country, a growing number of Web companies, both established and new, are moving into the business, although not necessarily dominating the market.
A few years ago, a classified ad for Andrew Davis's 2001 Montero SUV might have been limited to two lines of descriptive shorthand: A/C, pwr pkg, 6cyl AT, 2wd, $9k firm.
Today, in ads on the Internet, Davis is posting pictures of his car in an off-road setting and sharing such details as the replacement of the timing belt and water pump 5,000 miles ago. He has even included a link to a feature article about his car's model in Popular Mechanics. Davis, a Clemson University graduate who moved to the Washington area only a few weeks ago, posted free classified ads on Craigslist and the Marketplace area on Facebook, the social-networking site. He also paid for an ad in The Washington Post, a bundled deal that also put his listing on Cars.com and other Web sites.
His approach of putting his vehicle in front of as many potential buyers as possible illustrates how dynamic the process of classified advertising has become. For sellers, the options have moved beyond newspaper ads and fliers on coffee-shop bulletin boards. And for buyers looking for a car, an apartment, a job or a new puppy, it means better chances of seeing relevant ads on a variety of sites.
This push into online classifieds -- a business dominated by newspapers for more than a century -- is still very much in flux, said Greg Sterling, principal analyst with Sterling Market Intelligence in Oakland, Calif.
While classified revenue has been shrinking at newspapers across the country, a growing number of Web companies, both established and new, are moving into the business, although not necessarily dominating the market. Newspapers have lost their grip on classifieds in recent years. Once a steady source of revenue, classified advertising at some of the larger chains has dropped 14 to 20 percent over the past year, notably in once-lucrative segments such as automotive, real estate and employment ads, according to Fitch Ratings. Fitch, a credit ratings agency in New York, said this week that newspaper performance has been weaker than it originally forecast for the year.
EBay recently introduced its U.S. users to two similar sites, Kijiji and Gumtree, both of which started overseas. Geebo, based in McLean, is another online listing site trying to compete against Craigslist by creating a brand name that will draw buyers and sellers to its site. "When I think about classified sites, outside of Geebo, I can only think of one, and that's Craigslist," said Geebo chief executive Greg Collier. "It's not like I can think of 10 or 12 of these right off the top of my head. There may be a lot out there, but nobody knows they're there." In contrast, such companies as Edgeio and Oodle are less concerned with creating a site that buyers will view as a destination.
Instead, they are pushing their way into the marketplace by acting as a go-between, cutting deals with newspapers, as well as Web sites, to increase the number of places where their listings appear. Edgeio collects classified listings through individual sellers, as well as listings on sites like Geebo, or from traditional publications. Using software, it then distributes those ads to Web sites with specialized audiences. For example, a niche Web site for fans of cocker spaniels might partner with Edgeio to display ads from a local breeder selling puppies.
The ads might come from an individual or an ad posted in the community newspaper, but are then distributed to the cocker spaniel site through Edgeio. "Our goal is to get more traffic to the [Web site] publishers," said Edgeio co-founder Keith Teare. "As money flows through the system, we take our share." Like Edgeio, Oodle acts as a middleman, but is positioning itself to be more like a buyer's agent, offering such tools as e-mail alerts. Oodle's service searches classifieds and finds products a prospective buyer is looking for. In addition, it analyzes its listings for trends and other market data for buyers to use in their research. For example, someone shopping for a 2002 Honda Accord will also get information about that car, such as the average price for that year and model, in Oodle's listings.
Oodle chief executive Craig Donato said his model is a new approach to classified advertising. The challenge now is to get those ads in front of a local audience in a creative way so that buyer and seller can find each other and complete the sale.
"What's happening essentially is that companies are trying to do things online that lead to offline transactions," he said.
"The transaction always occurs between two humans offline. . . . You would never rent an apartment without seeing it first or take a job without interviewing. That defines classified ads." For that reason, local papers say they will be key players, even as classifieds find more venues online. Forging partnerships with sites like Oodle have allowed newspapers to test some different approaches without having to build their own technologies, said Mort Goldstrom, vice president of advertising for the Newspaper Association of America in Arlington.
But Fitch Ratings analyst Mike Simonton said newspapers have a long way to go in making up for eroding classified revenue. "We don't think that online classifieds have replaced or are successfully replacing the print classifieds," he said. "There's just a lot more competition in that market."
Source: http://www.washingtonpost.com/wp-dyn/content/article/2007/08/30/AR2007083002046.html?hpid=sec-tech
By Sam Diaz, Washington Post Staff Writer, Friday, August 31, 2007


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Free-riding, carbon treaties, and trade wars: the role of domestic environmental policies
This paper uses a calibrated general-equilibrium model of North-South trade with carbon emissions to explore the strategic, open-economy implications of price and quantity based instruments for CO2 emission reduction. We compute non-cooperative environmental and trade policy equilibria and Nash bargaining outcomes in environmental policies with side payments of cash. Results show that quotas can lead to higher internalization levels in a non- cooperative zero-tariff equilibrium in comparison with emission fees. If tariffs are also chosen non-cooperatively, the form of policy instrument used affects equilibrium tariffs, with quotas leading to lower trade barriers, particularly under a regional carbon treaty.
(This abstract was borrowed from another version of this item.)
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Volume (Year): 58 (1999)
Issue (Month): 2 (April)
Pages: 463-483
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References listed on IDEAS
Please report citation or reference errors to , or , if you are the registered author of the cited work, log in to your RePEc Author Service profile, click on "citations" and make appropriate adjustments.:
- Martin L. Weitzman, 1974.
"Prices vs. Quantities,"
Review of Economic Studies,
Oxford University Press, vol. 41(4), pages 477-491.
- M. L. Weitzman, 1973. "Prices vs. Quantities," Working papers 106, Massachusetts Institute of Technology (MIT), Department of Economics.
- Buchanan, James M & Tullock, Gordon, 1975. "Polluters' Profits and Political Response: Direct Controls Versus Taxes," American Economic Review, American Economic Association, vol. 65(1), pages 139-147, March.
- Fishelson, Gideon, 1976. "Emission control policies under uncertainty," Journal of Environmental Economics and Management, Elsevier, vol. 3(3), pages 189-197, October.
- Nash, John, 1953. "Two-Person Cooperative Games," Econometrica, Econometric Society, vol. 21(1), pages 128-140, April.
- Baumol, William J, 1972. "On Taxation and the Control of Externalities," American Economic Review, American Economic Association, vol. 62(3), pages 307-322, June.
- Rodney D. Ludema & Ian Wooton, 1994. "Cross-Border Externalities and Trade Liberalization: The Strategic Control of Pollution," Canadian Journal of Economics, Canadian Economics Association, vol. 27(4), pages 950-966, November.
- Ludema, R.D. & Wooton, I., 1992. "Cross-Border Externalities and trade Liberalization: The Strategic Control of Pollution," UWO Department of Economics Working Papers 9202, University of Western Ontario, Department of Economics.
- Mestelman, Stuart, 1989. "Taxes, Subsidies, Standards, and Social Choices," Public Finance = Finances publiques, , vol. 44(2), pages 268-284.
- Rodriguez, Carlos Alfredo, 1974. "The non-equivalence of tariffs and quotas under retaliation," Journal of International Economics, Elsevier, vol. 4(3), pages 295-298, August.
- Carlo Perroni & Randall M. Wigle, 1994. "International Trade and Environmental Quality: How Important Are the Linkages?," Canadian Journal of Economics, Canadian Economics Association, vol. 27(3), pages 551-567, August.
- Perroni, Carlo & Rutherford, Thomas F, 1993. " International Trade in Carbon Emission Rights and Basic Materials: General Equilibrium Calculations for 2020," Scandinavian Journal of Economics, Wiley Blackwell, vol. 95(3), pages 257-278.
- Spulber, Daniel F., 1985. "Effluent regulation and long-run optimality," Journal of Environmental Economics and Management, Elsevier, vol. 12(2), pages 103-116, June.
- Adar, Zvi & Griffin, James M., 1976. "Uncertainty and the choice of pollution control instruments," Journal of Environmental Economics and Management, Elsevier, vol. 3(3), pages 178-188, October.
- Wallace Oates & William Baumol, 1975. "The Instruments for Environmental Policy," NBER Chapters,in: Economic Analysis of Environmental Problems, pages 95-132 National Bureau of Economic Research, Inc.
- Desvousges, William H. & Smith, V. Kerry & Fisher, Ann, 1987. "Option price estimates for water quality improvements: A contingent valuation study for the monongahela river," Journal of Environmental Economics and Management, Elsevier, vol. 14(3), pages 248-267, September.
- Copeland, Brian R & Taylor, M Scott, 1995. "Trade and Transboundary Pollution," American Economic Review, American Economic Association, vol. 85(4), pages 716-737, September.
- Markusen, James R., 1975. "International externalities and optimal tax structures," Journal of International Economics, Elsevier, vol. 5(1), pages 15-29, February. Full references (including those not matched with items on IDEAS)
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text:
Staff writer
A high-ranking West Virginia Division of Highways administrator abruptly resigned Friday amid a federal investigation of the agency's equipment division.
Bob Andrew, a 16-year DOH executive who formerly headed the equipment division in Buckhannon, officially stepped down at 4 p.m. Friday, after turning in his resignation earlier in the day.
Since January, Andrew had been reassigned as special executive assistant to Transportation Secretary Paul Mattox.
Asked if Andrew's sudden resignation was linked to the federal investigation, DOH spokesman Brent Walker said, "Those are personnel issues. We have cooperated fully, and will continue to cooperate with this investigation."
Last week, a former DOH supervisor who worked for years under Andrew in Buckhannon pleaded guilty to making a false statement to federal investigators during the investigation. Edward Tuttle, who resigned last April, admitted that he lied about his role in delivering a surplus DOH truck to a buyer at the Port of Baltimore, in Baltimore. The sold truck was to be shipped to Africa.
Tuttle, who faces up to five years in prison, also was the DOH highway administrator for Upshur County.
A second DOH equipment division supervisor, Barry D. Thompson, is scheduled to go on trial next month in Elkins. Thompson is charged with lying to a federal agent about DOH employees "repairing and replacing parts for a dump truck on state time and at state expense," according to a federal indictment. He resigned in April.
Federal authorities have been investigating the DOH's equipment division for more than a year over allegations of bid rigging, misuse of taxpayer funds and political activities by DOH employees on state time.
In August 2013, the U.S. Attorneys Office simultaneously served search warrants at the DOH's equipment office in Buckhannon and at Mo Trim Inc., in Cambridge, Ohio.
The DOH equipment division buys trucks, bulldozers, graders, excavators, mowers, parts and other equipment for state highway garages throughout West Virginia. The office has about 90 employees.
Most recently, Andrew, who made $82,764 a year as Mattox's special assistant, was heading up a study about converting the state vehicle fleet to natural gas.
In 2006, the West Virginia Ethics Commission and the Legislative Auditor investigated truck and equipment purchases at the DOH equipment office. At the time, Andrew called the investigation "a bunch of malarkey." The investigation's findings were never made public.
Andrew could not be reached for comment Friday.
The equipment division case is being investigated by the U.S. Attorney's Office of Public Corruption Unit, the West Virginia State Police, the FBI and the state Legislature's Commission on Special Investigations.
Reach Eric Eyre at email@example.com, 304-348-4869 or follow @EricEyre on Twitter.


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Orwell Wheelers will be sending a strong team to this year's An Post Rás, with four riders making their debut in the premier event on the Irish cycling calendar.
This marks a welcome return to the Rás for Orwell, after a gap of two years. The team representing the club in this years race will be Brian Ahern, David Coughlan, Patrick O'Brien, Daniel Whiting and Aidan Collins.
For the first time ever, the club has had more riders ready to ride the Rás than places on the team, with selection based on performance in Rás Mumhan and the Tour of Ulster.
Odhran Connors, who narrowly missed out on selection for the Orwell team, will also be featuring in this year's Rás, having secured a spot as a guest rider with the LK Bikes Donegal team. This will be Connors' fourth consecutive Rás.
Orwell's challenge will be lead by Brian Ahern who will be participating in his seventh Rás, between the ones he's ridden and his times as a manager. Ahern's experience should prove valuable for the team, with Collins, Coughlan, Whiting and O'Brien all making their Rás debuts, having moved up the ranks to A2 level over the past year.
Supporting the Orwell challenge is a strong backroom team, with Ger McNamara acting as team manager and Tom Blennerhassett as mechanic. Blennerhassett's own bid for a Rás spot was stymied earlier this year by injury and his participation in this year's race will no doubt whet his appetite to complete future editions as a rider. After some sterling work supporting the Orwell team at last year's Rás na mBan, Rachel Glendon will once again act as team masseuse.
The club is hosting a barbecue on Tuesday, May 15 to coincide with the last night of the Women's Criterium League that will act as a fundraiser for the Rás team.
The barbecue will take place a the Blue Light pub in Sandyford from 8pm. Entry is €10 for adults and €5 for children, with an "all you can eat" policy for hungry cyclists.
Entertainment on the night will include a raffle and the Rás team doing a TT on a monkey bike around a 300 metre course in the car park. Team members have promised to do this in full time trial kit, including skin suits and helmets.


text:
Have Jeep Questions? Ask a Jeep Repair Expert for Answers
No not all 3.7 L are the same, there are minor internal timing gear differences and there are also set up differences for emissions and the cylinder heads are different depending on year
In the Chrysler's parts catalog there are four different complete 3.7 L engines listed
2003-2002 3.7L V-6 KJ Body (w/JTEC Controller)
2004 3.7L V-6 KJ Body (w/NGC Controller)
2006-2005 3.7L V6 All
2007 3.7L V6 All
As for cylinder heads
2004-2002 3.7L V-6 (right)
2004-2002 3.7L V-6 (left)
2006-2005 3.7L V-6 (left)
2007-2005 3.7L V-6 (right)
2007 3.7L V-6 (left)
For an 2002 you will need a 2002 or a 2003 engine to work correctly with no modifications
I hope that this helps you
Thank you
The 2004 engine uses a different type of engine controller. This will require that the complete engine harness and controller from the donor vehicle be used in your 02 vehicle.
There are likely some other things that may need to be considered but those are the MAJOR ones off the top of my head.
Depending on the amount of internal damage you may be better off to repair the engine you have and save yourself all the hassles.
Oh, and by the way an excessive plug gap could never cause this. If anything it might be a maintenace issue with sludge and carbon build-up.
This is a PAY SITE so if I have answered your question to your satisfaction a simple ACCEPT would go along way. Feedback and Bonuses are also welcome.
I would go with the newer engine
You will need to swap the right hand camshaft timing gear from the 2002 into the 2004 for the 2002 camshaft sensor to work
You will also need to put a 2002 crankshaft sensor into the 2004 engine
When doing the swap use the 2002 fuel rail and injectors and swap all of the electrical sensors on the top end, MAP, TPS, KNOCK (under the intake), IAT, ETC,AND Idle air control, and any other bolt in sensor
This will give your 2002 KJ a slightly different emissions profile, but ultimately it should be cleaner due to the head redesign in the 2004
You do not need to swap harnesses and controllers, just make the 2004 engine appear to be a 2002 engine to the controllers and you should be fine
It is hard to say, not knowing what you have for tools and equipment or hands on experience, but if you are comfortable with the use of tools and working on cars then:
With the tear down of both engines, swapping parts and removal and re installation, with out a lift I would think that you could get it done with two solid days of work, the exhaust is going to be a bear to get apart lying on your back, be sure to use stands and safety devices when doing the job, please be safe
BONUSES are always welcome!
I suggested fixing what you have........depending on the amount of internal damage.
The other expert is the one who is suggesting making another fit. His way will break federal emmissions laws and I don't wish to be any part of that.
So, to answer your question...............the other expert will have to confirm that this will work for you because I will not.
Have a great night.
I can only advise you the same way I would treat my own vehicle.
Granted I may have a better understanding of the overall condition of the engine just by opening it up and looking at the condition of it. Is it clean? Does it have sludge build-up? etc.
Too many times I respond to questions about people doing engine swaps involving different year vehicles and there are always stumbling blocks and hurdles you have to go through to get it to run.
So........my philosophy is fix what you have or get a direct reman replacement.
As for butting in just look at the thread and you will see who answered first and whose answers contain real information and not just fluff
When you buy a reman motor from a high quality manufacturer, like ATK, they give you instructions in the crate on how to verify the new engine compared to your original and how to make the newer engine work in an older Jeep, and those where what I passed along to you
I have done this myself in the past
A high quality reman while being more costly then a junk yard motor normally will carry a very good warranty, Chrysler has a 3 year 100,000 mile warranty on theirs, and most of the other major manufactures have the same or better
List Price:5,440.00 USD
I know that you can get this from an outside vendor for much less money
Good luck with your Jeep


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ibis Plymouth2
Longbridge Road, Marsh Mills, Forder Valley, PLYMOUTH, PL6 8LR
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About the hotel
ibis Plymouth is a budget hotel, perfectly located for easy access to the A38 and the shopping and entertainment districts of Plymouth. The hotel has 52 modern guest rooms, each with an en-suite bathroom, tea/coffee making facilities and satellite TV. You can enjoy breakfast or a light meal at the Café, or a nightcap in the friendly bar.
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- Main
- Disabled Facilities, 24 Hour Reception, Safety Deposit Box, Pets Allowed
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- Restaurant
- Business
- Wi-Fi Available In Public Areas
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- From 14:00 hours
- By 12:00 hours
- Up to 8 years old
- The price shown is based on the occupancy and dates you have searched for. Prices and availability are subject to change based on the arrival date, number of nights and occupancy searched for. All prices are inclusive of all local taxes.
- Pets are allowed
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