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Vertical cylindrical enclosures are used in the crystal growth industry as crystal growth reactors. Gallium nitride crystals are grown in ammonothermal growth reactors that requires temperatures in the range of 600–1000 K (620–1340 °F) and pressures in the range of 1000–6000 bar (14,504–87,022 psi). The process conditions make any experimental measurements difficult, in particular, flow pattern visualization and velocity measurements. A geometrically similar transparent reactor and similar fluid dynamic conditions for crystal growth were used in the process of experimental observation and measurement of the flow. Boundary and fluid temperatures along with velocity vectors were recorded. The flow proved to be oscillating in nature with steady and uniform time-averaged patterns. Fluid temperatures showed a higher standard deviation in the mixing region suggesting that the fluctuations are stronger at this location. Flow patterns and fluid temperatures indicated that the heat transfer between the two sections is driven by conduction and advection.

The voltage-gated sodium channel NaV1.7 has received much attention from the scientific community due to compelling human genetic data linking gain- and loss-of-function mutations to pain phenotypes. Despite this genetic validation of NaV1.7 as a target for pain, high quality pharmacological tools facilitate further understanding of target biology, establishment of target coverage requirements and subsequent progression into the clinic. Within the sulfonamide class of inhibitors, reduced potency on rat NaV1.7 versus human NaV1.7 was observed, rendering in vivo rat pharmacology studies challenging. Herein, we report the discovery and optimization of novel benzoxazine sulfonamide inhibitors of human, rat and mouse NaV1.7 which enabled pharmacological assessment in traditional behavioral rodent models of pain and in turn, established a connection between formalin-induced pain and histamine-induced pruritus in mice. The latter represents a simple and efficient means of measuring target engagement.

Current therapies to treat persistent pain and neuropathic pain are limited by poor efficacy, side effects and risk of addiction. Here, we present a novel class of potent selective, central nervous system (CNS)-penetrant potentiators of glycine receptors (GlyRs), ligand-gated ion channels expressed in the CNS. AM-1488 increased the response to exogenous glycine in mouse spinal cord and significantly reversed mechanical allodynia induced by nerve injury in a mouse model of neuropathic pain. We obtained an X-ray crystal structure of human homopentameric GlyRα3 in complex with AM-3607, a potentiator of the same class with increased potency, and the agonist glycine, at 2.6-Å resolution. AM-3607 binds a novel allosteric site between subunits, which is adjacent to the orthosteric site where glycine binds. Our results provide new insights into the potentiation of cysteine-loop receptors by positive allosteric modulators and hold promise in structure-based design of GlyR modulators for the treatment of neuropathic pain.

Many efforts are underway to develop selective inhibitors of the voltage-gated sodium channel Na V 1.7 as new analgesics. Thus far, however, in vitro selectivity has proved difficult for small molecules, and peptides generally lack appropriate pharmacokinetic properties. We previously identified the Na V 1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity via structure-guided analoging. To further understand GpTx-1 binding to Na V 1.7, we have mapped the binding site to transmembrane segments 1–4 of the second pseudosubunit internal repeat (commonly referred to as Site 4) using Na V 1.5/Na V 1.7 chimeric protein constructs. We also report that select GpTx-1 amino acid residues apparently not contacting Na V 1.7 can be derivatized with a hydrophilic polymer without adversely affecting peptide potency. Homodimerization of GpTx-1 with a bifunctional polyethylene glycol (PEG) linker resulted in a compound with increased potency and a significantly reduced off-rate, demonstrating the ability to modulate the function and properties of GpTx-1 by linking to additional molecules.

Significance: Aging and oxidative stress are complex phenomena, and their understanding is of enormous theoretical and practical significance. Recent Advances: Numerous hypotheses and theories that attempt to explain these phenomena have been developed. These hypotheses and theories compete with each other, with each claiming to be the correct one, while significantly contradicting each other. Critical Issues: It is important to develop a maximally correct theory that may then trigger significant practical breakthroughs. Future Directions: None of these theories is entirely correct or close enough to the truth. However, most of them contain many correct elements (CE). Finding these CE is possible by analysis of these theories. Once the CE are found, they can be merged by synthesis in a better new theory. An analysis of some of the theories of aging followed by synthesis is attempted. Antioxid. Redox Signal. 23, 187–207.

NaV1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34-residue peptide, termed GpTx-1, with potent activity on NaV1.7 (IC50 = 10 nM) and promising selectivity against key NaV subtypes (20× and 1000× over NaV1.4 and NaV1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp(29), Lys(31), and Phe(34) near the C-terminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against NaV1.4. A structure-guided campaign afforded additive improvements in potency and NaV subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a NaV1.7 IC50 value of 1.6 nM and1000× selectivity against NaV1.4 and NaV1.5.

A significant number of low molecular weight metal complexes as well as metal-free compounds that are capable of scavenging superoxide and/or other radicals and reactive species in simple systems have been proposed to be used as potential drugs in the case of various diseases and/or as antiaging agents. Some have been used or suggested to be used as diagnostic tools for the involvement of such species in biological processes. In the present work, analysis of such claims indicates that their use as specific detectors of superoxide or of other reactive oxygen species is unsupported and might be confusing. Many of these compounds exert beneficial effects by counteracting the toxic effects of oxidative stress in a significant number of models of pathological processes. However, it is concluded that these actions are more likely due to other effects including prooxidant actions and that their beneficial effects also may be exerted in pathological processes that do not practically involve reactive oxygen species. Adaptation may be a common mode of action explaining a sizable portion of the beneficial effect of the so-called mimics and other compounds including prooxidants.

The traditional view in the field of free radical biology is that free radicals and reactive oxygen species (ROS) are toxic, mostly owing to direct damage of sensitive and biologically significant targets, and are thus a major cause of oxidative stress; that complex enzymatic and nonenzymatic systems act in concert to counteract this toxicity; and that a major protective role is played by the phenomenon of adaptation. Another part of the traditional view is that the process of aging is at least partly due to accumulated damage done by these harmful species. However, recent workers in this and in related fields are exploring the view that superoxide radical and reactive oxygen species exert beneficial effects. Thus, such ROS are viewed as involved in cellular regulation by acting as (redox) signals, and their harmful effects are seen mostly as a result of compromised signaling, rather than due to direct damage to sensitive targets. According to some followers of this view, ROS such as hydrogen peroxide and superoxide are not just causative agents of aging but may also be agents that increase the life span by acting, for example, as prosurvival signals. The goal of this review is to recall that many of the effects of ROS that are interpreted as beneficial may actually represent adaptations to toxicity and that some of the most extravagant recent claims may be due to misinterpretation, oversimplification, and ignoring the wealth of knowledge supporting the traditional view. Whether it is time to abandon the free radical (oxidative stress) theory of aging is considered.

The aim of this study was to synthesize a series of quinoline-pyrimidine hybrids and to evaluate their in vitro antimalarial activity as well as cytotoxicity. The hybrids were brought about in a two-step nucleophilic substitution process involving quinoline and pyrimidine moieties. They were screened alongside chloroquine (CQ), pyrimethamine (PM) and fixed combinations thereof against the D10 and Dd2 strains of Plasmodium falciparum. The cytotoxicity was determined against the mammalian Chinese Hamster Ovarian cell line. The compounds were all active against both strains. However, hybrid (21) featuring piperazine linker stood as the most active of all. It was found as potent as CQ and PM against the D10 strain, and possessed a moderately superior potency over CQ against the Dd2 strain (IC(50): 0.157 vs 0.417 μM, ∼threefold), and also displayed activity comparable to that of the equimolar fixed combination of CQ and PM against both strains.

There is interest in the identification and optimization of new molecular entities selectively targeting ion channels of therapeutic relevance. Peptide toxins represent a rich source of pharmacology for ion channels, and we recently reported GpTx-1 analogs that inhibit NaV1.7, a voltage-gated sodium ion channel that is a compelling target for improved treatment of pain. Here we utilize multi-attribute positional scan (MAPS) analoging, combining high-throughput synthesis and electrophysiology, to interrogate the interaction of GpTx-1 with NaV1.7 and related NaV subtypes. After one round of MAPS analoging, we found novel substitutions at multiple residue positions not previously identified, specifically glutamic acid at positions 10 or 11 or lysine at position 18, that produce peptides with single digit nanomolar potency on NaV1.7 and 500-fold selectivity against off-target sodium channels. Docking studies with a NaV1.7 homology model and peptide NMR structure generated a model consistent with the key potency and selectivity modifications mapped in this work.

There are both opportunities and challenges in developing improved or novel therapeutics based on targeting voltage-gated calcium channels. Calcium channels are technically difficult with respect to target-based drug discovery but unquestionably are key points for influencing human physiology and pathophysiology. Many marketed drugs, including nifedipine, nimodipine, ethosuximide, pregabalin, and ziconotide, exert therapeutic efficacy via inhibition of calcium channels. Although there is no obvious path to improving these existing drugs, recent clinical results with dihydropyridine drugs point to testable strategies for treatment of Parkinson's disease and possibly other CNS disorders by inhibiting L-type calcium channels. Roles of T-type calcium channels in pain and in abnormal sleep are plausible and may soon be tested with clinical trials of novel therapeutics. Success with any of these trials certainly will be followed by intensive efforts for further refinements around the target class. WIREs Membr Transp Signal 2013, 2:85–104. doi: 10.1002/wmts.71 For further resources related to this article, please visit the WIREs website.

Herein the discovery of a novel class of aminoheterocyclic Na(v)1.7 antagonists is reported. Hit compound 1 was potent but suffered from poor pharmacokinetics and selectivity. The compact structure of 1 offered a modular synthetic strategy towards a broad structure-activity relationship analysis. This analysis led to the identification of aminopyrazine 41, which had vastly improved hERG selectivity and pharmacokinetic properties.

Thermoelectric (TE) semiconductor materials are widely used for miniaturized versatile cooling devices in a wide spectrum of equipments and energy generation in space vehicles. The bismuth telluride pseudobinary alloy family presents the best characteristics for room temperature TE cooling applications. Using appropriately oriented single crystals instead of the well known polycrystalline materials made by powder metallurgy methods, the efficiency of the TE device (Thermoelectric Cooler – TEC or Thermoelectric Generator – TEG) could be almost doubled. For having good quality TE material it is required to produce equally doped single crystals by the controlled crystallization process, namely with the Bridgman-Stockbarber method. Our experiments were made in the Universal Multizone Crystallizator Type UMC, developed by the ADMATIS Ltd., Miskolc, using a quartz tube under high vacuum conditions and automatically controlled thermal field parameters. The crystallographic analysis of the obtained samples was made by Scanning Electron Microscopy – (SEM), X Ray Diffraction – (XRD), and neutron diffraction (TOF spectrometry).

The discovery of a series of small molecule alpha4beta2 nAChR potentiators is reported. The structure-activity relationship leads to potent compounds selective against nAChRs including alpha3beta2 and alpha3beta4 and optimized for CNS penetrance. Compounds increased currents through recombinant alpha4beta2 nAChRs, yet did not compete for binding with the orthosteric ligand cytisine. High potency and efficacy on the rat channel combined with good PK properties will allow testing of the alpha4beta2 potentiator mechanism in animal models of disease.

The synthesis and structure-activity relationship of a series of carbamate potentiators of alpha4beta2 nAChR is reported herein. These compounds were highly selective for alpha4beta2 over other nAChR subtypes. In addition, compounds increased the response of alpha4beta2 nAChRs to acetylcholine, as measured with patch-clamp electrophysiology.

The present study focuses on the application of a therapeutic strategy in patients with chronic severe lower limb ischaemia using a plasmid vector encoding the vascular endothelial growth factor (phVEGF165). It has been shown that VEGF promotes neo-vascularization and blood vessel network formation and thus might have the ability to improve blood-flow at the level of the affected limbs. However, little information is available regarding the necessary level of expression of VEGF and its possible related adverse effects. We have subcloned VEGF 165 isoform into pCMV-Script expression vector (Stratagene) under the control of the CMV promoter. Three patients with chronic ischaemia of the lower limb, considered as not suitable for surgical re-vascularization, received intramuscular injection with 0.5 ml saline solution containing 1011 copies of VEGF 165 plasmid. The clinical evolution has been monitored by angiography and estimated by walking time on the rolling carpet (Gardner protocol). Two months after therapy, all three patients showed complete relief of rest pain, improvement of ischaemic ulcer lesions and increased walking distance on the rolling carpet most probably due to appearance of newly formed collateral vessels.

We report interstellar FeII and SiII column densities toward six translucent sight lines (A_V >~ 1) observed with the Space Telescope Imaging Spectrograph (STIS). The abundances were determined from the absorption of SiII] at 2335 Angstroms, and several weak Fe transitions including the first reported detections of the 2234 Angstrom line. We derive an empirical f-value for the FeII 2234 Angstrom transition of log(f lambda) = -1.54 +/- 0.05. The observed sight lines sample a variety of extinction characteristics as indicated by their R_V values, which range from 2.6 - 5.8. The dust-phase abundances of both Si and Fe are positively correlated with the small-grain population (effective radii smaller than a few hundred micron) toward the targets. The physical conditions along the sight lines suggest that this relationship may be due to differences in the survival of small particles in some interstellar environments. The chemical composition of the small grains could either resemble dust mantles or be silicate rich., accepted for publication in ApJ

We present interstellar elemental abundance measurements derived from Space Telescope Imaging Spectrograph echelle observations of 47 sight lines extending up to 6.5 kpc through the Galactic disk. These paths probe a variety of interstellar environments, covering ranges of nearly four orders of magnitude in molecular hydrogen fraction f(H_2) and more than two in mean hydrogen sight line density . Coupling the current data with Goddard High Resolution Spectrograph data from 17 additional sight lines and the corresponding Far Ultraviolet Spectroscopic Explorer and Copernicus observations of H_2 absorption features, we explore magnesium, phosphorus, manganese, nickel, copper, and germanium gas-phase abundance variations as a function of : density-dependent depletion is noted for each element, consistent with a smooth transition between two abundance plateaus identified with warm and cold neutral interstellar medium depletion levels. The observed scatter with respect to an analytic description of these transitions implies that total elemental abundances are homogeneous on length scales of hundreds of parsecs, to the limits of abundance measurement uncertainty. The probable upper limit we determine for intrinsic variability at any is 0.04 dex, aside from an apparent 0.10 dex deficit in copper (and oxygen) abundances within 800 pc of the Sun. Magnesium dust abundances are shown to scale with the amount of silicon in dust and, in combination with a similar relationship between iron and silicon, these data appear to favor the young F and G star values of Sofia & Meyer (2001; ApJ 554, L221) as an elemental abundance standard for the Galaxy., Comment: 58 pages, including 11 figures and 9 tables, accepted by ApJ

Mn(II) can catalyze the decomposition of H 2 O 2 and, in the presence of H 2 O 2 , can catalyze the oxidation of NADH. Strikingly, these processes depend on the simultaneous presence of both CO 2 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} . This explains the exponential dependence of the rates on [ \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} ], previously noted by other workers. These processes are inhibited by Mn-superoxide dismutase, establishing the generation of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{-}\end{equation*}\end{document} and its role as an essential reactant. A scheme of reactions, consistent with the known properties of this system, is proposed. The large rate enhancements provided by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} + CO 2 , and the abundance of both of these species in vivo , suggest that similar reactions have relevance to the oxidative stress imposed by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{-}\end{equation*}\end{document} and H 2 O 2 .

Copper,zinc superoxide dismutase (Cu,Zn-SOD) catalyzes the HCO(3)(-)-dependent oxidation of diverse substrates. The mechanism of these oxidations involves the generation of a strong oxidant, derived from H(2)O(2), at the active site copper. This bound oxidant then oxidizes HCO(3)(-) to a strong and diffusible oxidant, presumably the carbonate anion radical that leaves the active site and then oxidizes the diverse substrates. Cu,Zn-SOD is also subject to inactivation by H(2)O(2). It is now demonstrated that the rates of HCO(3)(-)-dependent oxidations of NADPH and urate exceed the rate of inactivation of the enzyme by approximately 100-fold. Cu,Zn-SOD is also seen to catalyze a HCO(3)(-)-dependent consumption of the H(2)O(2) and that HCO(3)(-) does not protect Cu,Zn-SOD against inactivation by H(2)O(2). A scheme of reactions is offered in explanation of these observations.

Simple algebraic manipulations and steady state assumptions have been applied to the elucidation of the effects of superoxide dismutase (SOD) on [NO] and [O 2 − ], and on the rates of production of H 2 O 2 and of ONOO − . The deductions help explain seemingly discordant published results and predictions.

The interactions of Cu, Zn superoxide dismutase (SOD) with nitroxyl (NO-) and nitric oxide (NO), both of which are thought to be biologically significant, have been studied but remain undefined. Having previously noted that NO- can reduce Cu (II), Zn SOD aerobically, we now report that it also can do so anaerobically and that Cu, Zn SOD can catalyze the elimination of NO(-) in the absence of O2.NO- acts as a reductant of ferricytochrome c anaerobically, but in the presence of O2 causes the oxidation of ferrocytochrome c and NADPH. Equivalent fluxes of NO-, and NO + O2- were able to comparably oxidize NADPH, but the oxidation by NO + O2- was more than fivefold more sensitive to inhibition by Cu, Zn SOD than was the oxidation by NO-. Thus Cu, Zn SOD inhibited NADPH oxidation by NO- by a route independent of catalyzing the dismutation of O2. Plausible mechanisms for those observations are offered and rate constants are estimated.

It has been reported in the literature that the endogenous estrogen metabolite 2-methoxyestradiol (2-ME) inhibits both manganese and copper,zinc superoxide dismutases (Mn and Cu,Zn SODs) and that this mechanism is responsible for 2-ME's ability to kill cancer cells. In fact, as demonstrated using several SOD assays including pulse radiolysis, 2-ME does not inhibit SOD but rather interferes with the SOD assay originally used. Nevertheless, as confirmed by aconitase inactivation measurements and lactate dehydrogenase release in human leukemia HL-60 cells, 2-ME does increase superoxide production in these cells and is more toxic than its non-O-methylated precursor 2-hydroxyestradiol. Other mechanisms previously suggested in the literature may explain 2-ME's ability to increase intracellular superoxide levels in tumor cells.

The use of lucigenin luminescence as a measure of [O−2] has been questioned because lucigenin has been shown to be capable of mediating the production of O−2. This being the case, lucigenin can signal the presence of O−2 even in systems not producing it in the absence of lucigenin. The reduction potential of lucigenin should be in accord with its ability to mediate O−2 production; but it has not heretofore been measured in aqueous media. The problems facing such measurement are the insolubility of the divalently reduced form, which deposits on the electrode, and the slow conformational transition that follows the second electron transfer and which interferes with reversibility. We have now used rapid scan cyclic voltammetry to determine that the reduction potential for lucigenin is −0.14 ± 0.02 V versus the normal hydrogen electrode. This value applies to both the first and the second electron transfers to lucigenin and it is in accord with the facile mediation of O−2 production by this compound.

To investigate the involvement of K+ efflux in apoptotic cell shrinkage, we monitored efflux of the K+ congener,86 Rb+, and cell volume during CD95-mediated apoptosis in Jurkat cells. An anti-CD95 antibody caused apoptosis associated with intracellular GSH depletion, a significant increase in 86Rb+ efflux, and a decrease in cell volume compared with control cells. Preincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed 86Rb+ efflux and cell shrinkage induced by the anti- CD95 antibody. A wide range of inhibitors against most types of K+ channels could not inhibit CD95-mediated efflux of86 Rb+, however, the uptake of86 Rb+ by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of86 Rb+ in Jurkat cells was sensitive to ouabain (a specific Na+/K+-ATPase inhibitor), demonstrating Na+/K+-ATPase dependent K+ uptake. Ouabain induced significant86 Rb+ efflux in untreated cells, as well as it seemed to compete with86 Rb+ efflux induced by the anti-CD95 antibody, supporting a role for Na+/K+-ATPase in the CD95-mediated86 Rb+ efflux. Ouabain treatment of Jurkat cells did not cause a reduction in cell volume, although together with the anti-CD95 antibody, ouabain potentiated CD95-mediated cell shrinkage. This suggests that the observed inhibition of Na++/K+-ATPase during apoptosis may also facilitate apoptotic cell shrinkage.

The interaction of Cu,ZnSOD with H2O2 generates an oxidant at the active site that can then cause either the inactivation of this enzyme or the oxidation of a variety of exogenous substrates. We show that the rate of inactivation, imposed by 10-mM H2O2 at 25 degrees C and pH 7.2, is not influenced by 10-mM HCO3-; whereas the oxidation of 2,2'-azino-bis-[3-ethylbenzothiazoline sulfonate] (ABTS=) is virtually completely dependent upon HCO3-. The reduction of the active site Cu(II) by H2O2, which precedes inactivation of the enzyme, occurred at the same rate in phosphate buffer with or without bicarbonate added. These results indicate that HCO3- does not play a role in facilitating the interaction of H2O2 with the active site copper, but they can be accommodated by the proposal that HCO3- is oxidized to HCO3*, which then diffuses from that site and causes the oxidation of substrates, such as ABTS=, that are too large to traverse the solvent access channel to the Cu(II).

Superoxide (O2-) poses multiple threats, which are diminished by a family of metalloenzymes, the superoxide dismutases. Among the damaging effects of O2- are direct oxidation of low-molecular-weight reductants; inactivation of a select group of enzymes; and reaction with NO to yield the strong oxidant, peroxynitrite. Of even greater import is the ability of O2- to univalently oxidize the [4 Fe-4 S] clusters of dehydratases, which causes release of iron. The "free" iron, which is kept reduced by cellular reductants, then reduces hydroperoxides to hydroxyl or alkoxyl radicals. Because the "free" iron will preferentially bind to anionic polymers, such as nucleic acids, or to anionic surfaces, such as cell membranes, these radicals will be generated adjacent to these vital targets and will preferentially attack them. O2- and iron can thus be viewed as partners in crime, and reciprocal regulatory effects between iron and O2- may be anticipated. These are discussed.

detectors. The logical conclusion is that ME is not an inhibitor of SOD. Later, two groups (Golab et al. 2003; Florczak et al. 2009) concluded that their findings support the view that ME inhibits SOD. Let us examine whether this is the case. They used essentially the same method as the one used by Huang et al. except that XTT (2,3-bis[2-methoxy4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide); was replaced by similar tetrazolium salts such as INT (2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride). However, they did not use other methods. In addition, in some of the cases SOD activity from cancer cells incubated with ME was studied and in such cases the effect of ME might be indirect as actually suggested (Golab et al. 2003). Therefore, the data in these two papers do not refute any of the conflicting views. On the other hand, our results do not contradict the thesis that ME decreases the activity of SODs in vivo or in cultured (cancer) cells by thus far an unknown mechanism. The most recent claim that ME inhibits SOD (Mn SOD from Clostridium difficile) was made by Li et al. (2015). While in both, Huang’s et al. and Li’s et al. assays superoxide anion radical (O2 ·−) was generated by xanthine oxidase (XO) plus xanthine (X), the first group used XTT, and the second, cytochrome c (cyt c), as detectors for O2 ·−. A more detailed inspection of the Li et al. paper follows. In Fig. 4 panel B, Li et al. followed the reduction of cytochrome c at 550 nm (A550) as a function of the concentration of SOD (Mn SOD) after 30 min of reaction. In the absence of ME, A550 nm was ~0.17 in the absence of SOD, while in the presence of ~0.02 μg/ml SOD A550 nm was slightly over 0.11 and in the presence of ~0.16 μg/ml SOD A550 was also slightly over 0.11. Thus, these authors report that an eightfold range SOD concentration inhibited the reduction of cytochrome

The antihypertensive agent mibefradil completely and reversibly inhibited T-type calcium channels in freshly isolated rat cerebellar Purkinje neurons. The potency of mibefradil was increased at less hyperpolarized holding potentials, and the apparent affinity was correlated with the degree of channel inactivation. At 35 degrees, the apparent dissociation constant Kapp was 1 microM at a holding voltage of -110 mV (corresponding to noninactivated channels) and 83 nM at a holding voltage of -70 mV (corresponding to 65% inactivation). The increased affinity was attributable mainly to a decreased off-rate. Mibefradil also inhibited P-type calcium channels in Purkinje neurons, but inhibition was much less potent. At a holding potential of -70 mV, the Kapp for mibefradil inhibition of P-type channels was approximately 200-fold higher than that for inhibition of T-type channels. Mibefradil should be a useful compound for distinguishing T-type channels from high voltage-activated calcium channels in neurons studied in vitro.

Clinical genetic studies have shown that loss of Nav1.7 function leads to the complete loss of acute pain perception. The global deletion is reported lethal in mice, however, and studies of mice with promoter-specific deletions of Nav1.7 have suggested that the role of Nav1.7 in pain transduction depends on the precise form of pain. We developed genetic and animal husbandry strategies that overcame the neonatal-lethal phenotype and enabled construction of a global Nav1.7 knockout mouse. Knockouts were anatomically normal, reached adulthood, and had phenotype wholly analogous to human congenital indifference to pain (CIP): compared to littermates, knockouts showed no defects in mechanical sensitivity or overall movement yet were completely insensitive to painful tactile, thermal, and chemical stimuli and were anosmic. Knockouts also showed no painful behaviors resulting from peripheral injection of nonselective sodium channel activators, did not develop complete Freund’s adjuvant-induced thermal hyperalgesia, and were insensitive to intra-dermal histamine injection. Tetrodotoxin-sensitive sodium current recorded from cell bodies of isolated sensory neurons and the mechanically-evoked spiking of C-fibers in a skin-nerve preparation each were reduced but not eliminated in tissue from knockouts compared to littermates. Results support a role for Nav1.7 that is conserved between rodents and humans and suggest several possibly translatable biomarkers for the study of Nav1.7-targeted therapeutics. Results further suggest that Nav1.7 may retain its key role in persistent as well as acute forms of pain.

We studied the mechanism by which the peptide omega-grammotoxin-SIA inhibits voltage-dependent calcium channels. Grammotoxin at concentrations of50 nM completely inhibited inward current carried by 2 mM barium through P-type channels in rat cerebellar Purkinje neurons when current was elicited by depolarizations up to +40 mV. However, outward current (carried by internal cesium) elicited by depolarizations to+100 mV was either unaffected or enhanced in the presence of toxin. Tail current activation curves showed that grammotoxin shifted the steady state voltage dependence of channel activation by approximately +40 mV. Activation in the presence of toxin was far slower in addition to having altered voltage dependence. Grammotoxin also inhibited N-type calcium channels in rat and frog sympathetic neurons, with changes in channel voltage dependence and kinetics nearly identical to those of P-type channels. Experiments with monovalent ions as the only charge carriers showed that toxin effects on channel activation and kinetics depended on voltage, not on direction of current flow or on the current-carrying ion. Repeated trains of large depolarizations relieved toxin inhibition, as if toxin affinity for activated channels were low. The effects of grammotoxin on gating of P-type channels are very similar to those of omega-Aga-IVA, but combined application of the two toxins showed that grammotoxin binding is not prevented by saturating binding of omega-Aga-IVA. We conclude that grammotoxin potently inhibits both P-type and N-type channels by impeding channel gating and that grammotoxin binds to distinct or additional sites on P-type channels compared with omega-Aga-IVA.

We studied the mechanism of inhibition of P-type calcium channels in rat cerebellar Purkinje neurons by the peptide toxin omega-Aga-IVA. Saturating concentrations of omega-Aga-IVA (> 50 nM) inhibited inward current carried by 2-5 mM Ba almost completely. However, outward current at depolarizations of > +60 mV, carried by internal Cs, was inhibited much less, as was the tail current after such depolarizations. omega-Aga-IVA shifted the midpoint of the tail current activation curve by about +50 mV and made the curve less steep. The inactivation curve was also shifted in the depolarized direction and was made less steep. With omega-Aga-IVA, channels activated more slowly and deactivated more quickly than in control. Trains of repeated large depolarizations relieved the inhibition of current (as tested with moderate depolarizations), probably reflecting the unbinding of toxin. The relief of inhibition was faster with increasing depolarization, but did not require internal permeant ions. We conclude that omega-Aga-IVA alters voltage-dependent gating by stabilizing closed states of the channel and that omega-Aga-IVA dissociates much more rapidly from open channels than from closed.

Biologically significant conclusions have been based on the use of fluorogenic and luminogenic probes for the detection of reactive species. The basic mechanisms of the processes involved have not been satisfactorily elucidated. In the present work, the mechanism of the enzyme and photosensitized oxidation of NAD(P)H by resorufin is analyzed and appears to involve both aerobic and anaerobic free radical chain reactions. There are two major fallouts of this analysis. Many of the conclusions about the participation of radicals based on the use of probes such as resorufin and Amplex red need reevaluation. It is also concluded that anaerobic free radical reactions may be biologically significant, and the possible existence of enzymatic systems to eliminate certain free radicals is discussed.

The giant hemoglobin, which occurs in free solution in the blood of earthworms, contains copper and zinc and exhibits superoxide dismutase activity as assessed by competition assays using cytochrome c or nitroblue tetrazolium. On a molar basis, the activity of this hemoglobin was approximately 10% that of the mammalian copper, zinc superoxide dismutase. The dodecamer, which contains the globin chains but lacks the linker subunits, had very little activity when compared to the complete native molecule. This suggests that the superoxide dismutase activity resides in one of the linker subunits. This is the first report of a superoxide dismutase bound to a respiratory pigment.

The in vivo production of HO- requires iron ions, H2O2 and O2- or other oxidants but probably does not occur through the Haber-Weiss reaction. Instead oxidants, such as O2-, increase free iron by releasing Fe(II) from the iron-sulfur clusters of dehydratases and by interfering with the iron-sulfur clusters reassembly. Fe(II) then reduces H2O2, and in turn Fe(III) and the oxidized cluster are re-reduced by cellular reductants such as NADPH and glutathione. In this way, SOD cooperates with cellular reductants in keeping the iron-sulfur clusters intact and the rate of HO- production to a minimum.O2- and other oxidants can release iron from Fe(II)-containing enzymes as well as copper from thionein. The released Fe(III) and Cu(II) are then reduced to Fe(II) and Cu(I) and can then participate in the Fenton reaction.In mammalian cells oxidants are able to convert cytosolic aconitase into active IRE-BP, which increases the “free” iron concentration intracellularly both by decreasing the biosynthesis of ferritin a...

Interleukin-1 is a macrophage-derived cytokine, which can also be synthesized in other cell types. It has been shown to exert several activities in the nervous and endocrine system, including a potent

Reversal of the superoxide dismutase (SOD) reaction was measured in terms of the reduction of tetranitromethane (TNM) by O2−. Cu,ZnSOD caused a biphasic reduction of TNM by H2O2. The rapid initial phase was stoichiometric with the enzyme and was followed by a slower catalytic phase that was oxygen dependent and was augmented by HCO3−. The reaction scheme explaining this behavior is presented and a rate constant for the reduction of O2 by the cuprous enzyme is estimated. This rate constant is so low that it precludes significant O2− production by the reduced enzyme under the conditions explored.

Several manganic porphyrins, with substituents on the methine bridge carbons, were prepared and examined for stability, redox behavior, catalysis of the dismutation of superoxide radical (O2-), and the ability to protect a superoxide dismutase (SOD)-null strain of E. coli against dissolved oxygen and a SOD-competent strain against paraquat. All of the compounds tested exhibited reversible redox behavior and were stable to EDTA in both the oxidized and reduced states, and several were able to catalyze the dismutation of O2- with the rate constants of approximately 10(7) M-1 s-1. The marked protective effects of some of these compounds exceeded that which could be anticipated on the basis of such rate constants. The tetrakis (1-methyl-4-pyridyl) compound was reduced enzymatically at the expense of NADPH and nonenzymatically by GSH and was kept in the reduced state within E. coli. Since the rate constant for reoxidation of the reduced form by O2- is 4 x 10(9) M-1 s-1, it appears that this compound acts in vivo as an NADPH/GSH:O2- oxidoreductase rather than as an SOD mimic. Its ability to facilitate aerobic growth of the SOD-null strain can be explained on this basis.

Extracts of E. coli contain at least three easily separable NAD(P)H:paraquat diaphorases. One of these is identified as thioredoxin reductase, which accounts for most of the PQ++ diaphorase in a thioredoxin reductase overproducer but is only 25% of this activity in a wild type. NADP+, but not NAD+, inhibited the diaphorase activity of thioredoxin reductase. All of the soluble PQ++ diaphorases of E. coli are stable during fractionation by HPLC and none depend upon the cooperative action of components separable by this technique. GSSG reductase is inhibited by PQ++ and is not, to any significant degree, a contributor to the diaphorase activity of E. coli.

Herein we describe the discovery, optimization, and structure-activity relationships of novel potent pyrrolopyrimidine Na(v)1.7 antagonists. Hit-to-lead SAR studies of the pyrrolopyrimidine core, head, and tail groups of the molecule led to the identification of pyrrolopyrimidine 48 as exceptionally potent Na(v)1.7 blocker with good selectivity over hERG and improved microsomal stability relative to our hit molecule and pyrazolopyrimidine 8 as a promising starting point for future optimization efforts.

Clinical genetic data have shown that the product of the SCN9A gene, voltage-gated sodium ion channel Nav1.7, is a key control point for pain perception and a possible target for a next generation of analgesics. Sodium channels, however, historically have been difficult drug targets, and many of the existing structure-activity relationships (SAR) have been defined on pharmacologically modified channels with indirect reporter assays. Herein we describe the discovery, optimization, and SAR of potent aminopyrimidinone Nav1.7 antagonists using electrophysiology-based assays that measure the ligand-receptor interaction directly. Within this series, rapid functionalization at the polysubstituted aminopyrimidinone head group enabled exploration of SAR and of pharmacokinetic properties. Lead optimized N-Me-aminopyrimidinone 9 exhibited improved Nav1.7 potency, minimal off-target hERG liability, and improved rat PK properties.

Clinical human genetic studies have recently identified the tetrodotoxin (TTX) sensitive neuronal voltage gated sodium channel Nav1.7 (SCN9A) as a critical mediator of pain sensitization. Herein, we report structure-activity relationships for a novel series of 2,4-diaminotriazines that inhibit hNav1.7. Optimization efforts culminated in compound 52, which demonstrated pharmacokinetic properties appropriate for in vivo testing in rats. The binding site of compound 52 on Nav1.7 was determined to be distinct from that of local anesthetics. Compound 52 inhibited tetrodotoxin-sensitive sodium channels recorded from rat sensory neurons and exhibited modest selectivity against the hERG potassium channel and against cloned and native tetrodotoxin-resistant sodium channels. Upon oral administration to rats, compound 52 produced dose- and exposure-dependent efficacy in the formalin model of pain.

To investigate the distribution of vascular endothelial growth factor (VEGF) isoforms and soluble form of VEGF receptor 1 (sFlt-1) in the follicular fluid (FF) of in vitro fertilization (IVF) patients in relationship to age, body mass index (BMI), diagnosis of polycystic ovary syndrome (PCOS), and their correlation with IVF outcomes.Prospective study.VEGF( 121) and VEGF(165) isoforms were detected using Western blotting and pixel density analysis. The concentration of sFlt-1 was determined by enzyme-linked immunosorbent assay (ELISA). In vitro fertilization outcomes measured included number of oocytes retrieved, fertilization rate, and clinical pregnancy. Statistical analysis used the Kruskal-Wallis and Mann-Whitney U test where appropriate.There was a statistically significant association between higher VEGF(165) levels and the diagnosis of PCOS, BMI ≥ 30, and age ≥40 years. In vitro fertilization cycles resulting in pregnancy were linked to statistically lower VEGF(165) levels in the FF. No statistically significant trend was identified in levels of VEGF(121) or sFlt-1 relative to patient characteristics or IVF outcomes.Our results suggest that elevated VEGF(165) levels are associated with less favorable patient characteristics and clinical IVF outcomes.

Fumarase C was strongly induced by paraquat in a parental strain of Escherichia coli but was not induced in a strain lacking the soxRS response. Moreover, a strain that constitutively expresses the soxRS regulon contained more fumarase C than did the parental strain. The Mn-containing superoxide dismutase and glucose-6-phosphate dehydrogenase, members of the soxRS regulon, were similarly induced by paraquat. Mutational defects in glucose-6-phosphate dehydrogenase increased the induction of fumarase C by paraquat. For Mn-containing superoxide dismutase, responsiveness to paraquat was also enhanced in the glucose-6-phosphate dehydrogenase-defective strains. Overproduction of the Mn-containing superoxide dismutase, elicited by isopropyl beta-D-thiogalactoside in a tac-sodA fusion strain, did not diminish induction of fumarase C or of glucose-6-phosphate dehydrogenase by paraquat, and induction of these enzymes was more sensitive to paraquat when the cells were growing on succinate rather than on LB medium. These results indicate that fumarase C is a member of the soxRS regulon and that this regulon does not respond to changes in O2- concentration but perhaps does respond to some consequence of a decrease in the ratio of NADPH to NADP+.

In addition to its very efficient catalysis of the dismutation of superoxide ( O(2)(-) ) into O(2) plus H(2)O(2), Cu, Zn SOD acts less efficiently as a non-specific peroxidase. This peroxidase activity is CO(2) dependent although very slow peroxidation of some substrates occurs in the absence of CO2. The mechanism of that CO(2) dependence is explained by the generation of a strong oxidant at the copper site by two sequential reactions with H(2)O(2), followed by the oxidation of CO(2) to the carbonate radical that then diffuses into the bulk solution. This diffusible carbonate radical is then responsible for the diverse oxidations that have been reported. A different mechanism that involves the reduction of peroxymonocarbonate by the reduced superoxide dismutase to yield carbonate radical has been proposed. We will demonstrate that this mechanism is not supported by the available data. It seems likely that generation of the carbonate radical has relevance to the oxidative stress faced by aerobic organisms.