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|Jan 11, 1994
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|May 29, 1992
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|Nov 14, 1990
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|Joseph S. Beckman, Haralambos Ischiropoulos, Craig D. Smith
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|The Uab Research Foundation
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This application is a continuation of application Ser. No. 07/613,213, filed Nov. 14, 1990, now abandoned.
The present invention relates generally to the field of administration of superoxide dismutase (SOD) to reduce ischemic injury or injury following sepsis or inflammation. In particular, this invention relates to a method and compositions for reducing toxic side effects caused by the reaction of SOD and peroxynitrite. Specifically, an effective amount of SOD, modified by substituting amino acid residues close to the active site of the SOD with amino acid residues, such as tyrosine, methionine, or cysteine residues, that can trap the toxic side products of the reaction of SOD with peroxynitrite, such as nitronium ion, is administered to an animal. This invention has particular relevance in the treatment of stroke and head trauma, myocardial ischemia, sepsis, inflammation, adult respiratory distress syndrome, and bronchiopulmonary dysplasia.
Peroxynitrite anion (ONOO31) is a potent oxidant. Peroxynitrite is formed by the reaction of superoxide (O2 31) and nitric oxide in tissues subjected to ischemic, inflammatory or septic conditions. Nitric oxide is present in such tissues. For example, in ischemic injury, ischemia allows calcium entry into endothelial cytoplasm due to failure of ionic pumps and opening of ion channels. Endothelium and neurons produce nitric oxide by an oxygen (O2) dependent calmodulin activated nitric oxide synthetase which oxidizes arginine in the presence of NADPH (Palmer et al., Nature (London) 333:664-666 (1988); Knowles et al., Proc. Natl. Acad. Sci. (USA) 86:5159-62 (1989); Marletta et al., Biochem., 27:8706-8711 (1988)). Reperfusion allows rapid nitric oxide synthesis by providing O2 to the enzyme and other substrates already present as a result of ischemia.
Superoxide is also present in injured tissue. For example, ischemia induces intracellular O2 production by xanthine oxidase, mitochondria and other sources. The O2 can escape into the extracellular millieu through anion channels (Lynch et al., J. Biol. Chem., 253: 4697-4699 (1978)). Extracellular O2 - and nitric oxide are also produced in the vascular lumen by activated neutrophils and macrophages, and by circulating xanthine oxidase released from liver (Yokoyama et al., Amer. J. Physiol., 258:G564-G570 (1990); Moncada et al., Biochem. Pharmacol., 38:1709-1715 (1989)). The superoxide radical is also an important mediator of both the inflammatory response of neutrophils and of the damage that occurs during reperfusion of anoxic tissue after organ transplantation or when a blood clot is removed. (Petrone et al., Proc. Natl. Acad. Sci (USA), 77:1159-163 (1980)).
NO reacts rapidly with O2 - both intracellularly and in the vascular lumen to form peroxynitrite (Blough et al., Inorg. Chem., 24:3504-3505 (1985); Beckman et al., Proc. Natl. Acad. Sci. (USA), 87:1620-1624 (1990)). The rate of peroxynitrite formation depends upon O2 - and nitric oxide concentrations. Peroxynitrite can be toxic by at least three mechanisms: hydrogen ion-catalyzed homolytic cleavage to form hydroxyl radical (•OH) and nitrogen dioxide (NO2), direct reaction with sulfhydryl groups, and reactions with SOD and transition metals to form hydroxyl ion (- OH) and nitronium ion (NO2 +) a potent nitrating agent (Beckman et al., Nature (London) 345:27-28 (1990). Thus, peroxynitrite is a reactive species which can produce other highly reactive species such as •OH, NO2, and NO2 +.
Superoxide dismutases consist of several distinct families of metal-containing enzymes that catalyze the dismutation of the oxygen radical superoxide by the following two step reaction:
O2 - +Me-SOD====→O2 +Me-1 -SOD
2H+ +O2 - +Me-1 -SOD====→H2 O2 +Me-SOD
where Me refers to the metal bound in the active site. This metal undergoes repeated cycles of oxidation and reduction in the reactions given above. Many compounds can either reduce or oxidize superoxide, but the distinguishing feature of a superoxide dismutase is the catalysis of both reactions given above. A series of positively charged amino acids positioned near the active site of the SODs generates an electrostatic gradient which attracts the negatively charged O2 - into the active site.
Three families of SODs are distinguished by the metal in the active site: the copper+zinc (Cu,Zn) SOD family, the manganese (Mn) SOD family and the iron (Fe) SOD family. The vast majority of therapeutic studies have been performed using Cu,Zn SODs, which naturally occur in the cytoplasm of eukaryotic cells as a dimer of two identical 16 Kd peptides. There is also a distinct Cu,Zn SOD found in plasma, which is a tetramer. Another form of SOD contains manganese (Mn) and is found in mitochondria. This protein has an amino acid sequence distinct from the Cu,Zn SOD but is similar to the sequence for Mn and Fe SODs found in bacteria.
SOD's have been commonly utilized to prevent or reduce oxidation injury in the treatment of stroke and head trauma, myocardial ischemia, abdominal vascular occlusion, cystitis, and a variety of inflammatory conditions (Greenwald, Free Radical Biol. and Med., 8:201-209 (1990); McCord, New Eng. J. Med., 312:159-183 (1985); U.S. Pat. No. 4,695,456; U.S. Pat. No. 4,656,034).
However, there have been several disappointing results in humans treated with native human Cu,Zn SOD. One explanation for these results may be that the native enzyme has a circulatory half life of only minutes because of rapid clearance by the kidneys (Petkau et al., Res. Commun. Chem. Pathol. Pharmacol., 15:641-657 (1976)).
To address this problem, several longer-lived derivatives of SODs have been developed. Human Mn SOD has been cloned and human trials have been reported by Nimrod et al., in Medical Biochem. and Chem. Aspects of Free Radicals, N.Y.: Elsevier Science Pub., 743-746 (1989). Genetic engineering has produced a long-lived modification of human cytoplasmic Cu,Zn SOD (Hallewell et al., J. Biol. Chem., 264:5260-5268 (1989)). Other modifications, such as polyethylene glycol conjugates of both human and bovine Cu,Zn SOD have longer half lives and are less immunogenic than unmodified forms (Pyatak et al., Res. Commun. Chem. Pathol. Pharmacol., 29:113-127 (1980); Saifer et al., Proc. Fifth Internatl. Conf. on Suoeroxide and Suoeroxide Dismutase (Jerusalem) (1989)).
Another problem with in vivo SOD therapy has recently been reported in which treatment with higher SOD dosage levels to reduce ischemic injury resulted in an increased infarct size (Omar et al., Circulation 80:SII-294 (1989); Werns et al., Tr. Pharmacol. Sci., 1:161-166 (1988)). Further, the use of SOD in tissues containing ONOO- can lead to the production of other destructive species, such as NO2 +. Thus, there exists a need for an improved therapy to treat ischemic, inflammatory or septic conditions.
The above deficiencies in the prior art are solved by the present invention, which provides for an improved superoxide dismutase therapy. This invention comprises a method and composition for reducing ischemic injury, or injury associated with sepsis or inflammation, by reducing the toxic side effects caused by the reaction of SOD and peroxynitrite. A SOD is modified either chemically or by site-directed mutagenesis, by substituting at least one amino acid residue close to the active site of SOD, particularly close to the rim around the active site, most particularly within 3-12 Å of the active site, with an amino acid residue, such as a tyrosine, a methionine residue or cysteine residue, that can trap the toxic side products, such as NO2 +, of the reaction of SOD with peroxynitrite. An effective amount of modified SOD is administered, such as parenterally, particularly intravenously, intraarticularly, intramuscularly, or subcutaneously, or by intratracheal infusion, inhalation, or intranasally to an animal to treat ischemic, inflammatory or septic conditions, such as stroke, head trauma, or arthritis. Such effective amount preferably ranges from 100 to 10,000 units of activity/kilogram of body weight, and more preferably ranges from 1000 to 10,000 units of activity/kilogram of body weight.
Therefore, it is an object of this invention to provide a means for protecting tissue from oxidative damage associated with ischemia, sepsis and inflammation. It is a further object of this invention to prevent injury to tissue caused by toxic side reactions of superoxide dismutase.
Furthermore, an objective of this invention is to provide a method and compositions that reduce the injury or side effects associated with treatment therapy utilizing SOD. It is a particular object of this invention to provide a SOD that is modified so as to trap the toxic products formed in vivo upon treatment with SOD. In addition, it is an object of this invention to provide for a SOD that is modified by replacing at least one amino acid residue close to the active site of SOD with an amino acid residue that can trap the toxic side products formed during SOD therapy. It is a further object of this invention to provide a method for treating ischemic, inflammatory or septic conditions comprising administering the modified SOD to an animal.
These and other objects and advantages of this invention are described in the following detailed description, which is not meant to be limiting.
Although SODs comprise a family of several distinct enzymes that vary in amino acid sequence, their catalytic mechanisms are very similar. Thus, the tertiary structure surrounding the active sites of the various SODs are sufficiently homologous to permit effective modification by the same or very similar methods.
For example, all Cu,Zn superoxide dismutase molecules are dimers of identical subunits and each subunit contains about 153 amino acids (depending on the species) and one copper ion and one zinc ion. The crystallographic structure of bovine Cu,Zn superoxide dismutase, refined to a resolution of 2.0 Å, shows that the structural core of the subunit is a flattened Greek key β-barrel motif consisting of eight antiparallel beta-strands joined by seven turns or loops. The catalytic copper ion is ligated to the surface of the β-barrel by four histidine residues and occurs at the base of a channel formed by two loops extending from the β-barrel. In addition to forming the superoxide channel, the two loops encode specific functional subdomains involved in pre-collision electrostatic guidance of substrate (loop VII), zinc binding (loop IV), and dimer contact (loop V) (Getzoff et al., Nature, 306:287-290 (1983)).
Superoxide is drawn into the active site by electrostatic fields generated by the spatial arrangement of positively charged amino acids forming a ring near the active site and negatively charged amino acids more distant from the active site. This tends to draw the negatively charged superoxide into the active site and accelerates the reaction rate to near the theoretical diffusion limit.
Generally, the catalytic mechanism of SODs involve two sequential reactions. First, one molecule of superoxide transfers an electron to the metal in the active site of SOD and is released as oxygen. A second superoxide entering the active site of SOD picks up the electron from the metal, thereby becoming reduced to hydrogen peroxide while regenerating native SOD. Coordination of the metal in the SOD protein alters the redox potential favoring the repeated reduction and oxidation by superoxide. For example, the midpoint potential of Cu in Cu,Zn SOD is about 0.4 volts, and undergoes the following reactions:
O2 - +Cu+2 -SOD====→O2 +Cu+1 -SOD
O2 - +Cu+1 -SOD====→H2 O2 +Cu+2 -SOD
Peroxynitrite also reacts with the active site of SOD to produce NO2 +, which is a highly reactive species that can cause considerable physical damage in vivo and may account for the apparent toxicity of SOD in high doses to ischemic heart. Peroxynitrite also is attracted to the active site by the same electrostatic field that draws O2 - into the active site (Getzoff et al., Nature. 306:287-290 (1983); Tainer et al., Mol. Biol., 160:181-217 (1982)). The O-O part of peroxynitrite will fit into the hydrophobic pocket of the active site, while NO2 - extends into the solvent space.
Cu,Zn SODs catalyze the decomposition of ONOO- to produce NO2 + and OH- from peroxynitrite with copper playing an essential role in this reaction. Once in the active site, - OONO decomposes to give OH- and the nitronium ion (NO2 +), apparently by forming a transient cuprous adduct as shown: ##STR1## The nitronium ion, which has a high heat of formation, might be transiently stabilized as a salt, such as by complexing with phosphate or chloride anion (X-). The positively charged nitronium ion will be repelled by the same electrical field that drew ONOO- into the active site, arcing towards the negatively charged distal regions where the sole tyrosine is located. This positioning allows NO2 + to nitrate the tyrosine residue to form 3-nitrotyrosine similar to the well-known nitronium ion displacement of hydrogen ion from the ring of tyrosine to form 3-nitrotyrosine. The X- may serve as a carrier anion for the nitronium ion in aqueous solutions, allowing it to exist long enough in aqueous solution to nitrate tyrosine.
Evidence that SOD produces NO2 + when it reacts with peroxynitrite is verified by the isolation of a stable yellow adduct, which is formed when peroxynitrite is added to a solution of bovine Cu,Zn SOD. As discussed below, the yellow adduct corresponds to the formation of 3-nitrotyrosine on the tyrosine residue of SOD. The yellow adduct is typical of nitrated phenols such as p-nitrophenol (Halfpenny et al., J. Chem. Soc., 1952:939-946 (1952)). Furthermore, SOD catalyzes the nitration of other phenols as well as tyrosines on other proteins at a rate which could be biologically significant.
The yellow adduct produced from bovine Cu,Zn SOD reacting with ONOO- was crystallized and used to derive the x-ray structure. Electron density maps calculated from x-ray diffraction from these crystals show an increase in electron density near the ortho position of the sole tyrosine residue 108 on the bovine Cu,Zn SOD, indicating that 3-nitrotyrosine is formed. This position is located between 18-21 Å from the active site on the same subunit and considerably further from the opposing subunit of the dimer, indicating that the positively charged nitronium ion was formed in the SOD active site and then directed to the distal tyrosine by the electrostatic fields generated from charged amino acids that form a rim around the active site of SOD.
The pH-dependent spectra of the SOD-peroxynitrite adduct are also consistent with formation of 3-nitrotyrosine, exhibiting a pKa of 7.6 between an absorbance maximum in the alkaline range at 438 nm (E=4,600 M-1 cm-1) and an absorbance maximum at pH 6.0 of 356 nm (E356 nm=3,500 M-1 cm-1). Similar spectra are obtained by treating native SOD with tetranitromethane, which is a standard reagent used by protein chemists to produce 3-nitrotyrosine in proteins. The pKa of 3-nitrotyrosine is near 7.0 with E350 nm=3,400 M-1 cm-1 in acid and E438 nm=4,200 M-1 cm-1 in alkali. The SOD-- OONO adduct maxima at 350 and 438 nm are lost with reduction by dithionite (sodium hydrosulfide), but not ascorbate, glutathione or borohydride. This is consistent with the established reaction of dithionite reducing 3-nitrotyrosine to a colorless amine. Laser Raman spectra of the peroxynitrite-treated SOD are nearly identical to authentic 3-nitrotyrosine. The only subtle differences between the spectra are a broadening of a major peak at 1340 cm-1 and the splitting of a minor peak at 830 cm-1, both of which are consistent with the restricted rotation of 3-nitrotyrosine constrained within the SOD protein.
The reaction of peroxynitrite with Cu,Zn SOD also produces a series of increasingly negatively charged SOD variants on native polyacrylamide gel electrophoresis, indicative of 2-5 additional negative charges being present on the protein. Thus, multiple sites on SOD are modified during the reaction with peroxynitrite, suggesting that the formation of nitrotyrosine is simply the most visible of several changes to the SOD protein. The number of peroxynitrite-mediated modifications to SOD could be reduced in a concentration-dependent manner by either phenol or the tyrosine-containing protein lysozyme. Evidence is presented below that both phenol and lysozyme are also nitrated. Thus, the self-nitration of SOD involves a process that is competitively inhibited by adding exogenous phenolic compounds.
SOD also catalyzes the nitration of a wide range of phenolic compounds, including tyrosines, in other proteins like egg white lysozyme. HPLC analysis of the products from the SOD-catalyzed reaction of ONOO- with 1 mM phenol yields both 2- and 4-nitrophenol but no biphenol. The increase in phenol nitration measured by HPLC was linearly related to SOD concentration.
The rate of phenol nitration by peroxynitrite in the presence of Cu,Zn SOD can be measured by stopped flow spectroscopy at 412 nm. At moderately alkaline pH (between pH 8-10), the kinetics of SOD catalyzed nitration are relatively simple to interpret because peroxynitrite decomposes slowly, the phenol nitration products have larger extinction coefficients, and the spontaneous rate of phenol nitration by proton-catalyzed decomposition of ONOO- is greatly reduced. The rate of absorbance increase at 412 nm was linear for the first 3-5 seconds at pH 9 and directly proportional to SOD concentration. At pH 9, a minimum estimate for the apparent second order rate constant for phenol nitration by SOD and ONOO. is approximately 105 M-1 s-1. This rate is about 104 fold slower than the reaction with O2 -, but demonstrates that the peroxynitrite reaction proceeds at a significant rate.
The Mn SOD catalyzes the nitration of phenol much like Cu,Zn SODs. Several tyrosines are present on the Mn SOD; at least 3-4 of the 7 tyrosines are also nitrated when the enzyme is treated with peroxynitrite in the absence of phenol. Unlike the Cu,Zn SOD, the Mn SOD is inactivated by large concentrations of peroxynitrite. If the Mn SOD is first treated with 15 mM peroxynitrite at pH 7.8, catalytic activities are lost for both the superoxide and peroxynitrite reactions. An essential tyrosine near the active site (tyrosine 34 in the bacterial sequences; tyrosine 58 in the human sequence) of Mn SOD is slowly being modified by a small percentage of enzyme turnover rate (Stallings et al., J. Biol. Chem. 260:16424-16432 (1989). This suggests that substituting an amino acid to place tyrosine near the active site at some other position could be effective. Thus, improvements by the addition of sulfhydryl groups or tyrosine near the active site could be made on both the Mn and Cu,Zn SOD.
The Cu,Zn SOD-- OONO adduct had normal catalytic activity when measured by a standard SOD assay employing inhibition of cytochrome c reduction by xanthine oxidase (McCord et al., J. Biol. Chem., 244:6049-6055 (1969), suggesting that the - OONO does not remain bound in the active site. However, 3-nitrotyrosine has a pKa near 7.5 and the resulting negative charge in the active site could reduce the activity of SOD for both superoxide and peroxynitrite. Copper in the SOD active site is necessary for the formation of the adduct, as evidenced by the lack of nitration of 1 mM phenol by 2.0 mg/ml Cu-free SOD with 1 mM - OONO at pH 7.4. The Cu-free SOD can be obtained by reversible removal of copper with KCN; this type of metal replacement experiment with Cu,Zn SOD has been performed frequently over the past twenty years. Following use of a slightly modified procedure developed by Rotili et al., in which the copper is removed by reduction with borohydride followed by dialysis against 50 mM KCN, the Zn-containing apoprotein does not form the characteristic 3-nitrotyrosine when peroxynitrite is added. This is evidenced by the absence of a large peak of absorbance at 412 nm. Replacement of copper at the active site restored normal enzymatic activity for both O2 - and ONOO-. Cyanide is also a weak inhibitor of Cu,Zn SOD and also inhibits SOD-catalyzed nitration by peroxynitrite. These results show that SOD-catalyzes nitration by peroxynitrite.
A potential nitration mechanism might involve the formation of nitrogen dioxide (NO2) catalyzed by SOD or SOD may catalyze the formation of a strong oxidant, like hydroxyl radical, as well as nitrogen dioxide. We could find no evidence to support either of these two mechanisms to explain the SOD-catalyzed nitration of a phenolic ring.
The nitration reaction with SOD was specific for ONOO-, as evidenced by little or no complex formation from treating either reduced or oxidized SOD with up to 4 mM nitric oxide, NO2 NO2 - or NO3 -. Furthermore, only 0.29�0.09 μM NO2 was formed by SOD reacting with 1000 μM ONOO-. Also, addition of 100 mM dimethylsulfoxide to trap any hydroxyl radical formed by decomposition of ONOOH increased NO2 by 250 fold without affecting the amount of phenol nitration catalyzed by SOD. However, the background level of nitration was greater in the presence of DMSO without SOD. Finally, nitration by NO2 proceeds through phenyl radical intermediates and produces significant amounts of biphenols. We could detect no biphenol formation by HPLC from phenol nitration by SOD plus peroxynitrite at alkaline pH (pH>7.5). At neutral to acidic pH, some biphenyl formation takes place by the proton-catalyzed decomposition of peroxynitrite, which forms •HO and NO2 as intermediates.
As discussed above, SOD can react with ONOO- producing toxic side products. This invention is designed to render these products, particularly NO2 +, less harmful by modifying the SOD to react with the toxic side product. For instance, SOD can be modified to position a tyrosine residue near the active site so that the NO2 + that is produced will nitrate the tyrosine, thereby eliminating the toxic NO2 + species.
It is desirable to position a tyrosine residue near the active site where the ortho position of the tyrosine ring can interact with the ONOO- moiety. When peroxynitrite reacts with the copper in the active site to form nitronium ion, the nitronium ion will attack the phenolic ring of tyrosine to form 3-nitrotyrosine rather than being released into solution.
Also, by positioning the nucleophilic sulfur of cysteine or methionine residues near the active site, one may effect a trap for nitronium ion or peroxynitrite. The intermediate nitrothiol will be unstable and may be able to regenerate the sulfhydryl group with a spontaneous reaction with low molecular weight thiols.
Peroxynitrite anion reacts rapidly with sulfhydryl groups, most likely according to the following series of reactions: ##STR2## The resulting sulfonic acid may be regenerated to a sulfhydryl group by reaction with glutathione or other thiol agents as follows:
where GSH is glutathione or some other low molecular weight sulfhydryl agent.
Several threonines exist in or near the active site of Cu,Zn SOD, which could be substituted with a cysteine residue with minor effects upon structure. A limitation to cysteine substitutions is that the SH group is susceptible to autoxidation or may be oxidized during the catalytic cycle of superoxide.
Substitution of a methionine residue near the active site may be more useful. The sulfur of methionine should be far less susceptible to autoxidation, but is still highly nucleophilic and susceptible to attack by nitronium or peroxynitrite. Methionine is slightly larger and more hydrophobic than cysteine, imposing greater constraints upon where methionine may be substituted on the protein.
The preparation of site directed mutants is a standard procedure. For example, basic procedures for the preparation of site-direct mutants near the active site of human Cu,Zn SOD is similar to that used by Beyer et al (Beyer et al., J. Biol. Chem., 282:11182-11187 (1987)). DNA manipulations are performed using standard conditions as described by Maniatis et al. (Cold Springs Harbor Laboratory, Cold Springs Harbor, N.Y., (1982)). For example, in preparing modified human cytoplasmic Cu,Zn SOD, a 1.8 Kbp BamHI DNA fragment containing the wild type HSOD (human Cu,Zn SOD sequence) is subcloned in M13 (mp10) and a single stranded template DNA is prepared containing the coding strand of HSOD. Mutagenic oligonucleotide primers containing the appropriate anticodon for the desired amino acid substitution are synthesized with an automated DNA synthesizer (such as the Chiron Gene-O-Matic) using standard phosphoramidite synthetic techniques. The mutagenic oligonucleotide primer is constructed to contain 8-15 bp of the matching sequences for HSOD on both sides of the mutated sequence. The mutagenic oligonucleotide is then hybridized to the single strand M13 DNA containing the HSOD fragment and a covalently closed double stranded DNA is synthesized by primer extension. Mutant plaques from E. coli JM101 transformed with the M13 virus are identified using the radiolabeled mutagenic oligonucleotide as a hybridization probe and then confirmed by DNA sequencing. Double stranded DNA is prepared from the sequenced isolates and digested with NcoI and SalI to isolate a 520 bp fragment containing the HSOD cDNA, which is purified by PAGE electrophoresis. This purified fragment is cloned into the yeast expression plasmid (pCl/lPGAPSOD) containing the glyceraldehyde phosphate dehydrogenase gene 49 promotor as described by Maniatis et al.
Yeast strain AB110 is transformed with Pcl/lPGAPSOD and the corresponding mutated plasmid. Transformed colonies are selected on agar plates lacking leucine. These colonies are then used to inoculate larger fermentation flasks in nonselective YEPD media supplemented with 3 mM CuSO4, and the final cultures are lysed with glass beads. The Cu,Zn SOD is isolated by ammonium sulfate precipitation and DEAE chromatography as described by Beyer et al above.
The mutated HSOD may also be expressed in E. coli for the initial screening of mutations to select the best scavenger of peroxynitrite. For human usage, the final product is prepared from yeast to avoid endotoxin contamination from the bacterial coat.
The DNA sequences encoding both human Mn SOD and extracellular Cu,Zn SODs have been cloned. With slight changes in the restriction nucleases used to excise the initial DNA fragment to be mutated, the procedures given above are readily adaptable to generate desired site-directed mutants for all SODs. To express the Mn SOD, Mn will be added to the culture medium.
The artificial construction of a tyrosine-like residue may also be accomplished by direct chemical modification. This is a standard modification performed by protein chemists and involves adding a phenolic group to the ε-amino group of lysine with the Hunter-Bolton reagent (Thompson et al., Biochem., 26:743-750 (1987)). This procedure is frequently utilized to radiolabel small peptides by reacting the phenolic ring with 125 I. Cu,Zn SOD contains approximately 19 free amino groups, many of which are clustered near the active site to create an electrostatic field drawing the negatively charged superoxide and peroxynitrite anions into the active site. Thus, it may be possible to use a simple chemical reaction to place several phenolic rings near the active site. A major limitation of direct chemical modification is that multiple sites on the protein may undergo modification, which can reduce enzymatic activity or create new antigenic sites.
For cytoplasmic and extracellular human SODs, all amino acid residues within 12 Å of the copper center are suitable candidates for substitution with tyrosine, cysteine or methionine residues. The distances from the copper to these residues varies from 5 to 10.7 Å. The preferred amino acid candidates for substitution in the human cytoplasmic Cu,Zn SOD are residues 132-142, which form a loop or rim around the active site. The equivalent positions for the human extracellular Cu,Zn SOD are residues 172-185. Residue 48, which is a phenylalanine, may be modified to a tyrosine. Threonine 58, alanine 60 to proline 62 and comprise part of the active site. Threonine 58, alanine 60, and glutamine 165 could be replaced by cysteine or methionine; arginine 143 is also a good candidate for substitution.
In bovine Cu,Zn SOD, the amino acids number 56, 58 to 60, 63, 131 and 134 to 140 form a rim around the active site that could be modified without necessarily disrupting the binding site for superoxide. Residue 135 is a threonine that is only 5.3 Å from the copper and might be substituted with a cysteine. Residue 48 is a phenylalanine which could be changed to a tyrosine with minimal affects upon structure, but it is somewhat removed from the active site. The amino terminal lysine 151 of the other monomer is close enough to the active site that it may also be modified.
The human extracellular SOD is closely related to the cytoplasmic enzymes (Hjalmarsson et al., Proc. Natl. Acad. Sci., 84:6340-6344 (1987)), except for the presence of an extensive amino terminal tail that is probably needed for assembly of the tetramer and a carboxy-terminal sequence responsible for binding the extracellular SOD to heparin-sulfate groups present on endothelial cell surfaces. The positions listed for the extracellular SOD are inferred from analogy to the bovine cytoplasmic SOD sequence, which seems reasonable because of the high sequence homology in the active site region.
The following amino acid substitutions for Mn SOD were determined by homology to the Bacillus stearothermophilus and consideration of the x-ray structure (Parker et al., J. Mol. Biol., 199:649-661 (1988)) The numbering refers to the human liver Mn-SOD with the N-terminal signal sequence. The active site region consists of part of the first alpha-helix (αl) from leucine 49 to glutamine 59. This region lines one side of the active site and is the most likely path for superoxide entering the active site. The sequence from alanine 96 to phenylalanine 101 of the third alpha helix (α3) lines the opposite side of the active site but is slightly deeper and forms the pocket near the superoxide binding site. A fold in the sequence from serine 139 to serine 146 joins the fifth alpha helix (α5) to the tail of beta sheet (β1) and forms another part of the active site. The sequence from cysteine 164 to leucine 170 forms a bend between the head of beta sheet (β2) leading to the tail of β3. Glutamine 166 in this sequence is important for hydrogen bonding to tyrosine 58. The final sequence contributing to the active site is from valine 184 to glutamine 192, which is highly conserved in all species.
Once the site-directed mutants of SOD are purified, a sample is treated with peroxynitrite to test whether the mutant SOD is inactivated. The catalytic activity for the superoxide dismutase is determined by the standard assay of inhibition-of-cytochrome c-reduction by xanthine oxidase before and after treatment with peroxynitrite. The rate of phenol nitration by peroxynitrite is also determined by stopped flow spectroscopy, monitoring the absorbance increase at 412 nm. For the wild-type SOD, phenol nitration is linear for the first 2-6 seconds, depending upon the assay pH. A nonlinear decrease in the rate of phenol nitration is indicative of progressive inactivation of the SOD. From these data, the reaction rates are calculated for superoxide dismutation, peroxynitrite-mediated nitration and inactivation of SOD by peroxynitrite for each mutant SOD. The mutants that retain high superoxide dismutation activity and low rates of phenol nitration are selected for further animal experimentation to determine their efficacy at reducing ischemic injury. In particular, we use a highly reproducible surgical procedure to induce a stroke in rats and measure the reduction of infarct volume (dead brain tissue) as a measure of efficacy. This procedure comprises occlusion of the middle cerebral artery and a ninety-minute occlusion of the common carotid artery followed by reperfusion for 24 hours (Liu et al., Am. J. Physiol., 256:H589-H593 (1989). An effective dose of modified SOD is administered to the animal for treatment of ischemic, inflammatory or septic conditions. Such administration may be accomplished by any effective route, preferably parenterally, such as intravenously, intraarticularly, intramuscularly or subcutaneously, or by intratracheal infusion, inhalation or intranasally. An effective dose preferably ranges from 100 to 10,000 units of activity/kilogram of body weight, and more preferably ranges from 1000 to 10,000 units of activity/kilogram of body weight. The relevant dosage rate depends on the particular symptom or organ being treated.
An important modification of the Cu,Zn SOD has been to bind 5-17 molecules of polyethylene glycol (PEG) (--O--(CH2 --CH2 --O)n --CH3) to lysine on the SOD surface. PEG is an ideal polymer because it has little or no toxicity or reactivity. The PEG modification of proteins reduces immunogenicity, increases circulatory half-life from a few minutes to an upper limit of 7 days in humans, and can increase cell association to cultured endothelial cells. An appropriate PEG modification of SOD will most likely continue to be appropriate for a therapeutically useful SOD.
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|Blough and Zafiriou, Reaction of Superoxide with Nitric Oxide to Form Peroxonitrite in Alkaline Aqueous Solution, Inorg. Chem. 24:3502 3504, 1985.
|Getzoff, et al., "Evolution of CuZn Superoxide Dismutase and the Greek Key β-Barrel Structural Motif," Proteins: Structure, Function and Genetics 5:322-336, 1989.
|Getzoff, et al., Evolution of CuZn Superoxide Dismutase and the Greek Key Barrel Structural Motif, Proteins: Structure, Function and Genetics 5:322 336, 1989.
|Greewald, "Superoxide dismutase and catalase as therapeutic agents for human diseases: a critical review," Free Radical Biology & Medicine 8:201-209, 1990.
|Greewald, Superoxide dismutase and catalase as therapeutic agents for human diseases: a critical review, Free Radical Biology & Medicine 8:201 209, 1990.
|Halfpenny and Robinson, "The Nitration and Hydroxylation of Aromatic Compounds by Pernitrous Acid," J. Chem. Soc. 939-946, 1952.
|Halfpenny and Robinson, The Nitration and Hydroxylation of Aromatic Compounds by Pernitrous Acid, J. Chem. Soc. 939 946, 1952.
|Hallewell, et al., "Genetically Engineered Polymers of Human CuZn Superoxide Dismutase," The Journal of Biological Chemistry 264:5260-5268, Mar. 25, 1989.
|Hallewell, et al., Genetically Engineered Polymers of Human CuZn Superoxide Dismutase, The Journal of Biological Chemistry 264:5260 5268, Mar. 25, 1989.
|Hjalmarsson, et al., "Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase," Proc. Natl. Acad. Sci. USA 84:6340-6344, Sep. 1987.
|Hjalmarsson, et al., Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase, Proc. Natl. Acad. Sci. USA 84:6340 6344, Sep. 1987.
|Inoue, et al., "Synthesis of a Superoxide Dismutase Derivative that Circulates Bound to Albumin and Accumulates in Tissues Whose pH is Decreased," Biochemistry 28:6619-6624, 1989.
|Inoue, et al., Synthesis of a Superoxide Dismutase Derivative that Circulates Bound to Albumin and Accumulates in Tissues Whose pH is Decreased, Biochemistry 28:6619 6624, 1989.
|J. of Biological Chemistry, Hallewell et al., vol. 264, No. 9, Mar. 25, 1989 pp. 5260 5268.
|J. of Biological Chemistry, Hallewell et al., vol. 264, No. 9, Mar. 25, 1989 pp. 5260-5268.
|Knowles, et al., "Formation of nitric oxide from L-arginine in the central nervous system: A transduction mechanism for stimulation of the soluble guanylate cyclase," Proc. Natl. Acad. Sci. USA 86:5159-5162, Jul. 1989.
|Knowles, et al., Formation of nitric oxide from L arginine in the central nervous system: A transduction mechanism for stimulation of the soluble guanylate cyclase, Proc. Natl. Acad. Sci. USA 86:5159 5162, Jul. 1989.
|Liu, et al., "Polyethylene glycol-conjugated superoxide dismutase and catalase reduce ischemic brain injury," American Journal of Physiology 256:H589-593, 1989.
|Liu, et al., Polyethylene glycol conjugated superoxide dismutase and catalase reduce ischemic brain injury, American Journal of Physiology 256:H589 593, 1989.
|Lynch and Fridovich, "Permeation of the Erythrocyte Stroma by Superoxide Radical," The Journal of Biological Chemistry 253:4697-4699, Jul., 1978.
|Lynch and Fridovich, Permeation of the Erythrocyte Stroma by Superoxide Radical, The Journal of Biological Chemistry 253:4697 4699, Jul., 1978.
|Marletta, et al., "Macrophage Oxidation of L-Arginine to Nitrite and Nitrate: Nitric Oxide is an Intermediate," Biochemistry 27:8706-8711, 1988.
|Marletta, et al., Macrophage Oxidation of L Arginine to Nitrite and Nitrate: Nitric Oxide is an Intermediate, Biochemistry 27:8706 8711, 1988.
|McCord, "Oxygen-Derived Free Radicals in Postischemic Tissue Injury," Free Radicals and Ischemia 312:159-163, Jan., 1985.
|McCord, Oxygen Derived Free Radicals in Postischemic Tissue Injury, Free Radicals and Ischemia 312:159 163, Jan., 1985.
|Moncada, et al., "Endothelium-derived relaxing factor is identified as nitric oxide," TIPS 8:365-368, Oct., 1987.
|Moncada, et al., Endothelium derived relaxing factor is identified as nitric oxide, TIPS 8:365 368, Oct., 1987.
|Omar et al., "Protection Afforded by Superoxide Dismutase is Dose Dependent in the in situ Reperfused Rabbit Heart," Circulation 80:II-294, Oct., 1989.
|Omar et al., Protection Afforded by Superoxide Dismutase is Dose Dependent in the in situ Reperfused Rabbit Heart, Circulation 80:II 294, Oct., 1989.
|Omar, et al., "Cardioprotection by Cu,Zn-superoxide dismutase is lost at high doses in the reoxygenated heart," Free Radical Biology & Medicine 9:465-471, 1990.
|Omar, et al., Cardioprotection by Cu,Zn superoxide dismutase is lost at high doses in the reoxygenated heart, Free Radical Biology & Medicine 9:465 471, 1990.
|Palmer, et al., "Vascular endothelial cells synthesize nitric oxide from L-arginine," Nature 333:664-666, Jun. 1988.
|Palmer, et al., Vascular endothelial cells synthesize nitric oxide from L arginine, Nature 333:664 666, Jun. 1988.
|Parker and Blake, "Crystal Structure of Manganese Superoxide Dismutase from Bacillus stearothermophilus at 2.4 A Resolution," J. Mol. Biol. 199:649-661, 1988.
|Parker and Blake, Crystal Structure of Manganese Superoxide Dismutase from Bacillus stearothermophilus at 2.4 A Resolution, J. Mol. Biol. 199:649 661, 1988.
|Petrone, et al., "Free radicals and inflammation: Superoxide-dependent activation of a neutrophil chemotactic factor in plasma," Proc. Natl. Acad. Sci. USA 77:1159-1163, Feb. 1980.
|Petrone, et al., Free radicals and inflammation: Superoxide dependent activation of a neutrophil chemotactic factor in plasma, Proc. Natl. Acad. Sci. USA 77:1159 1163, Feb. 1980.
|Pyatak, et al., "Preparation of a Polyethylene Glycol: Superoxide Dismutase Adduct, and an Examination of its Blood Circulation Life and Anti-Inflammatory Activity," Research Communications in Chemical Pathology and Pharmacology 29:113-127, Jul. 1980.
|Pyatak, et al., Preparation of a Polyethylene Glycol: Superoxide Dismutase Adduct, and an Examination of its Blood Circulation Life and Anti Inflammatory Activity, Research Communications in Chemical Pathology and Pharmacology 29:113 127, Jul. 1980.
|Stallings, et al., "The Structure of Manganese Superoxide Dismutase from Thermus thermophilus HB8 at 2.4-Å Resolution," The Journal of Biological Chemistry 260:16424-16432, 1985.
|Stallings, et al., The Structure of Manganese Superoxide Dismutase from Thermus thermophilus HB 8 at 2.4 Resolution, The Journal of Biological Chemistry 260:16424 16432, 1985.
|Tainer, et al., "Structure and mechanism of copper, zinc superoxide dismutase," Nature 306:284-290, Nov., 1983.
|Tainer, et al., Structure and mechanism of copper, zinc superoxide dismutase, Nature 306:284 290, Nov., 1983.
|Werns and Lucchesi, "Free radicals and ischemic tissue injury,"TIPS 11:161-166, Apr., 1990.
|Werns and Lucchesi, Free radicals and ischemic tissue injury, TIPS 11:161 166, Apr., 1990.
|Yokoyama, et al., "Circulating xanthine oxidase: potential mediator of ischemic injury," American Journal of Physiology 258:G564-570, 1990.
|Yokoyama, et al., Circulating xanthine oxidase: potential mediator of ischemic injury, American Journal of Physiology 258:G564 570, 1990.
|Citing Patent
|Filing date
|Publication date
|Jul 31, 1995
|Jun 9, 1998
|Sam Research Corporation
|Anti-motoneuron-disease agent
|Sep 9, 1996
|Jun 12, 2001
|Michael K. Stern
|Methods of use for peroxynitrite decomposition catalysts, pharmaceutical compositions therefor
|Jun 14, 2001
|Jul 12, 2005
|Duke University
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|Dec 30, 2008
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|Feb 15, 2011
|Nov 26, 2013
|Duane C. Keller
|Devices and methods for creating a positive pressure environment for treatment of oral biofilms associated with periodontal disease
|Nov 9, 2006
|Jul 1, 2014
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|Dec 9, 2014
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|Duane C Keller
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|Dec 18, 2014
|Mar 22, 2016
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|Feb 17, 2015
|May 3, 2016
|Duane C. Keller
|Article and method for controlling oral-originated systemic disease
|Jun 13, 2002
|Feb 6, 2003
|Repine John E.
|Diagnostic and prognostic method for evaluating ocular inflammation and oxidative stress and the treatment of the same
|Jun 3, 2002
|Feb 13, 2003
|Piganelli Jon D.
|Oxidant scavengers for treatment of diabetes or use in transplantation or induction of immune tolerance
|Jan 22, 2002
|Mar 13, 2003
|Crapo James D.
|Cancer therapy
|Apr 28, 2003
|Feb 26, 2004
|Duke University And National Jewish Medical And Research Center
|Jun 9, 2003
|Mar 25, 2004
|Duke University
|Substituted porphyrins
|Nov 9, 2006
|Jun 28, 2007
|Aeolus Sciences, Inc.
|Cancer therapy
|Feb 4, 2008
|Aug 28, 2008
|Duke University
|Substituted Porphyrins
|Apr 2, 2009
|May 6, 2010
|Keller Duane C
|Methods and systems for progressively treating and controlling oral periopathogens causing systemic inflammations
|Sep 17, 2010
|Mar 17, 2011
|Aeolus Pharmaceuticals, Inc.
|Substituted Porphyrins
|May 5, 2011
|Aeolus Sciences, Inc.
|Oxidant Scavengers for Treatment of Diabetes or Use in Transplantation or Induction of Immune Tolerance
|U.S. Classification
|424/94.4, 530/350, 530/400, 424/94.1, 435/189
|International Classification
|Cooperative Classification
|European Classification
|Jul 11, 1997
|Fee payment
Year of fee payment: 4
|Aug 10, 2001
|Maintenance fee reminder mailed
|Jan 11, 2002
|Lapse for failure to pay maintenance fees
|Mar 19, 2002
|Expired due to failure to pay maintenance fee
Effective date: 20020111


text:
- Vadim Ghirda / Associated Press
Pictures in the News | January 28, 2013
Posted On: 8:42 a.m. | January 28, 2013
Pictures in the News from around the world. View Post»
- Tags: Learn & Discover :: Gallery :: Pictures in the News :: Daily Life
Posted On: 8:42 a.m. | January 28, 2013
Pictures in the News from around the world. View Post»
Posted On: 12:08 a.m. | January 28, 2013
Snow flurries and a violent 10-minute hailstorm lay a white canopy at Los Angeles City Hall. View Post»
Posted On: 7:35 p.m. | January 27, 2013
Ben Affleck's "Argo" seems to be unstoppable. The film about a CIA plot to rescue American hostages in Iran in 1980 won the 19th Screen Actors Guild Award on Sunday night for ensemble in a motion picture. The win came a day after the film, which also stars Affleck, took the Producers Guild Award on Saturday night — an honor that is one of the leading indicators of Oscar gold. Two... View Post»
Posted On: 4:02 p.m. | January 26, 2013
Colorado has become a nucleus of the rapidly evolving marijuana industry, offering a glimpse at what life might be like if weed is legalized nationwide, with companies, entrepreneurs and investors maneuvering for a piece of the expected boom. View Post»
Posted On: 2:50 p.m. | January 25, 2013
The Week in Pictures from around the world. View Post»
Posted On: 9:27 a.m. | January 25, 2013
Pictures in the News from around the world. View Post»
Posted On: 3:00 a.m. | January 25, 2013
"I've always seen the world differently so I built a camera to share images the way I see them in my mind…growing up I had severe dyslexia and it was always hard to communicate – when I found photography I found my voice." –Ian Ruhter View Post»
Posted On: 12:20 a.m. | January 25, 2013
I miss the good old days, when a camera just took still photographs and a video camera shot video. The choices where simple. But today with video and still photos blurring the line across most cameras, you've got the best of both worlds seamlessly blending together. View Post»
Framework, the photography and video blog of the Los Angeles Times, celebrates the power and explores the craft of visual storytelling. The blog highlights the work of Times photojournalists who frame by frame, document the drama, the emotion and sometimes the humor of life. Framework also aims to serve as a resource hub for photography, multimedia and video enthusiasts who share our passion. We will trade insights and discuss the tools and techniques of telling stories through images. Have a look and let us know what you think.
Need help or have a question?

subset: commoncrawl


text:
There are three things a person needs to secure his or her financial future: an emergency savings account, solid investments and life insurance. Many modern consumers see life insurance as a form of investment, just like betting money on the stock market or buying seafront property. However, some consumers may see life insurance as a good investment, while others may consider it possibly one of the worst types of investments ever. Continue reading →
Business Travel for Less
Some readers may wonder, what's the deal with trying to minimize business travel expenses? Well, not all of us travel on a giant corporate account. If you are an independent insurance agent or other business person, you likely hesitate to take any business trip. The potential for gains almost always offsets all of the costs you will incur, at least in your mind. Continue reading →
How to Identify the Best Identity Theft Protection Services
Today, just about anything is paid for by credit card or debit card. While such payments methods have numerous benefits, they also have disadvantages. When your credit rating is stable, you are secure in the knowledge that you don't have to struggle on a daily basis. But this is one of the reasons why you might become a victim of identity theft and also the reasons why you need the best identity protection service. Continue reading →
ESTA visa waiver service for travel
Planning a trip to the United States should be a fun and easy task. There are so many wonderful travel destinations to visit while you are there, all you have to do is decide on the regions you want to visit and activities you would like to participate in. Continue reading →
Early Retirement: Turn Your Dreams Into Reality
Retiring early in life means that you will have plenty of time to enjoy all the wonderful things you always wanted. However, getting there might be a bit hard, because for some it will mean that they will have to start saving money as early as possible. Nevertheless, dedication, smart saving and good investment can get you there fast. Continue reading →
XFR Financial Ltd – What Does Technical Analysis In Forex Involve?
Technical analysis refers to the activities involved in speculating development of future price by viewing history of market information. Continue reading →
Are your precious pieces of jewelry under insured?
When looking around for contents insurance, many home owners choose a policy which fits their pocket or stay with a company through customer loyalty; both trusting that if the time comes then their provider will compensate them for loss or damage. Continue reading →
Advantages of a Roth IRA over Traditional IRA
The Roth IRA is a newer version of the classic Individual Retirement Account that is becoming far more popular than the latter in the modern day. People are always striving for better deals when it comes to their retirement. The more security and comfort that they are promised when they retire, the better it is for them. This is the main reason that the Roth IRA has come forth as the latest and greatest competitor in the field of retirement accounts. Some of the main benefits of a Roth IRA over a traditional one include: Continue reading →


text:
2nds, barely perceptible blemishes. Cold feet? Not if you're wearing SmartWool's PhD Light Ski socks! Naturally itch-free merino wool fibers regulate temperature, control odor and wick moisture for hours on the slopes.
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text:
I read with consternation an article on page 13 of the 31 January issue of the Wall Street Journal by Stephen J. Hadley, the National Security Advisor in the Administration of George W. Bush. The headline was: "Americans Can Be Proud of What Was Achieved in Iraq." The Iraq war -- what Secretary of Defense Chuck Hagel, in a previous incarnation, characterized as "[a] misstep [that] will play out to be the most dangerous and costly foreign policy debacle in our nation's history" -- has now been spun out by this former White House official as instead a triumph.
Essentially what has changed in Iraq since the American invasion of 2003 is that minority Sunni rule has been replaced by majority Shiite rule, with the latter exhibiting all the winner-take-all behavior that is characteristic in that part of the world. In addition, sectarian killing has returned in force with the departure of American troops at the end of 2011. But most palpably, the legacy of the Iraq War has been the 4,486 American soldiers killed, the many more wounded, and the many thousands of Iraqi soldiers and civilians killed. All this for a war that was conceived on an erroneous assessment that Saddam Hussein was in possession of nuclear weapons.
It is true, as Hadley points out, that a brutal dictator (Saddam) has gone, and that Al Qaeda's resurgence doesn't change that fact. But was it worth all the American blood and treasure, and the many more numerous Iraqi casualties? One can only conclude that some of the officials involved remain in a state of denial; and whatever revisionist arguments are put forward, the judgment of the future on the Iraq War will only be harsh.
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text:
Hotel with Garda lake view
With a superb location on Lake Garda, just a few kilometers from Brenzone and Malcesinethe Hotel Drago is the ideal starting point to visit the historic cities of northern Italy.
At the foot of Monte Baldo, a panoramic hinterland opens up onto hiking in the hills and mountains. The trails of the Nordic Walking Park, sailing and kitesurfing school are just a few minutes away.
Pool, Fitness center, Bar, Restaurant, and Lido Bar right on the beach.
Garage and Parking.
An elegant and relaxing environment, with an ejoyable family atmosphere.
Enjoy the charme of a Hotel with Garda lake view
Book now
The "Hotel Drago" derives its name from mysterious legends that tell of apparitions
of real or imagined monstrous dragons and witches.
A mythology that has sought to explain the lake's natural beauty since ancient times.
Stunning views over the lake 20 meters to lakes edge, views from balconies most rooms, good food buffet… clean stylish hotel with a outdoor nice sized pool well kept gardens… rooms providing all requirements.. Beds twin or double quite basic. Friendly hotel staff. Would go again…
Tripadvisor, August 2015
This hotel is fabulous. All the staff work very hard to keep the rooms and gardens spotless… Regular bus very near the hotel and makes visiting other places around the lake very easy. Have no hesitation to highly recommend – faultless throughout our week stay.
Tripadvisor, July 2015
Elegant and welcoming ambiance, with a relaxing family atmosphere, and prompt courteous service.
The Hotel Drago offers all the services you need to enjoy your holiday on Lake Garda to the fullest!
Special offers
Hotel Drago Gift Box – 3 nights


text:
Published on
How do you achieve outstanding results with small actions? In this Freelunch webinar, Mark Turrell will show you that with minimal efforts, tremendous results can be achieved.
Mark will share practical tools that will help you achieve this. The tools he will discuss, are based on the scientific working of networks and mass movements.
Strategist and entrepreneur, Mark Turrell (@mark_turrel) is a Young Global Leader and a Technology Pioneer, nominated by the World Economic Forum, founder of Orcasci. Together with Menno van Dijk (co-founder & managing director of THNK), he is the author of the book 'Scaling: Small Smart Moves for outsized Results'.


text:
Orange finds SMBs backing flexible working hours
Flexible working is good for business and all staff should be given rights to demand flexible working hours, say a third of UK small companies. This is according to a new survey from Orange and Ipsos Mori.
Flexible working is good for business and all staff should be given rights to demand flexible working hours, say a third of UK small companies. This is according to a new survey from Orange and Ipsos Mori.
Your exclusive guide to CIO trends
A collection of our most popular articles for IT leaders from the first few months of 2016, including: - Corporate giants recruit digitally-minded outsiders to drive transformation - Analytics platforms to drive strategy in 2016 - Next generation: The changing role of IT leaders.
By submitting your email address, you agree to receive emails regarding relevant topic offers from TechTarget and its partners. You can withdraw your consent at any time. Contact TechTarget at 275 Grove Street, Newton, MA.
You also agree that your personal information may be transferred and processed in the United States, and that you have read and agree to the Terms of Use and the Privacy Policy.
A third of businesses (32%) surveyed said that the extension of the right to demand flexible working would benefit the bottom line, while only 22% said that the policy would be bad for business.
The Orange Business Jury, conducted by Ipsos MORI, polled 276 decision-makers in Britain's small businesses and demonstrated that over half of respondents (54%) are currently not aware of existing flexible working policy.
Flexible working is one of the themes of National Work Wise Week which starts on 16 May and calls for widespread adoption of smarter working practices.
"It is encouraging to see initiatives like Work Wise Week promoting better ways of working," said Neil Laidler, head of Orange Business Services in the UK. "Through the Orange Business Jury, we have a unique window on what the UK's smaller companies are thinking on issues like flexible working. The results of this poll show that small businesses share the same view as Orange: flexible working is good for business."
What VoIP can do for smaller firms >>
Comment on this article: firstname.lastname@example.com
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text:
Bless the depressed people to get into Heaven! Harold the Holy Cow must perform good deeds to meet God's quota. The Unholy Cow will try to convert true blessings back to evil, raw blessings. The Holy Scroll can open the pearly gates!
Holy Cow is one of our selected Animal Games. Also very popular on this website right now are My Dolphin Show 8, Monkey Go Happy Ninjas 3, StrikeForce Kitty: Last Stand and Baby Hazel Parrot care.
Report game as broken
Cancel report of broken game


text:
Nate Griffin
Nate Griffin
What I do for 610: Talk show host and sports blogger
Birthday: 11/13
Hometown: Born in Los Angeles, Ca., and claim Hattiesburg, Ms.
College: Southern Miss
High school: Hattiesburg High
How long at SportsRadio 610: Third tour of duty – since 1996 (At one time hosted Fast Break w/Nate that aired on SportsRadio 610)
How long in radio business: 15+ years
Coolest person at SR610: No one certain person…
What I love about Houston: Diversity, culture, and entertainment values. Also has great opportunities for personal and professional growth.
Favorite people to interview: Michael Jordan, Tiger Woods, Warren Moon, Ozzie Smith, Shaq, Otis Birdsong, Hakeem Olajuwon, Bobby Jones, Julius Erving, Magic Johnson, Jeff Bagwell, Craig Biggio, Rudy Tomjanovich, Don Chaney, Kenny Smith, Bubba McDowell, Otis Thorpe, Jack Pardee, Vivian Stringer, George Gervin, Artis Gilmore, Bruce Matthews, Mike Munchak, Andre Johnson, Tommy Lasorda, Art Howe, Bobby Cox, David Justice, etc.
Experience: Have worked as radio and television reporter and anchor. Have covered the NFL, NBA, MLB, College sports, professional tennis. I have worked as radio and TV color analyst for regional and national football and basketball. Covered both Rockets NBA Championships, numerous golf tournaments, have broadcasted "live" from the Shell Houston Open. Currently serving as radio color analyst for Rice Football broadcasts as well as color analyst for Comcast TV Sports. Also worked as NFL Houston Texans TV Reporter.
Teams followed: L.A. Dodgers, San Francisco 49er's, Houston Texans
Favorite sports experiences: So many!
Sports played: Football, track, baseball as a prep in Hattiesburg, Ms. Played Collegiate football and ran track at Los Medanos College and the University of Southern Mississippi.
Family info: Happily married with one child.
Nate and Creight With John McClain 2-28-2016
Nate and Creight talk with John McClain of the Houston Chronicle about the best and worst performances he saw this weekend at the NFL Scouting Combine, if the Texans should trade for Colin Kaepnernick, the biggest team needs for the Texans, and more!
Nate and Creight with Calvin Watkins of ESPN 2-21-2016
Nate and Creight talk to Calvin Watkins of ESPN about the Rockets moves at the NBA trade deadline, about playoff positioning, and much more!
Nate and Creight with Charlie Campbell of Walter Football 2-21-2016
Nate and Creight talk to Charlie Campbell of WalterFootball.com about the Texans, his mock draft selection for Houston, Tom Savage, and some of the players the team might target at quarterback and running back in the upcoming NFL Draft.
Phil Savage Joins Nate and Creight
College football analyst and draft expert Phil Savage joined Nate and Creight to talk about the NFL Draft and how the players the Texans selected will fit on their roster.
Hancock on why TCU is in, Alabama is not
The Top 4 teams in this week's College Football Playoff rankings have been the subject for many debates. However, Bill Hancock's honesty about how and why leaves little doubt.
Bailiff & Owls aim for six straight & Redding on officiating today's football
Head Football Coach David Bailiff & the Owls are quietly preparing to win their 6th straight & 10th consecutive homecoming game. Meanwhile, officiating these games has become big business says Redding.
Heavy Coaches Edition of College Football Today
The University of Houston looks to win three in a row. But, they'll have to win in Tampa against a very hungry South Florida team. Cougars Head Coach Tony Levine shares his thoughts.
Luck; "check biases at the door" ; CBS' O'Connell talks best college QB's
West Virginia AD Oliver Luck and the new College Football Playoff Committee come together in Dallas to formulate their Top 25 while CBS Analyst Kevin O'Connell talks best college QB's in the land.
Notre Dame has a 'Fighting' chance, says Pinkett
The College Football world focuses on Tallahassee, Florida as the Fighting Irish of Notre Dame prepare to face Jameis Winston and the No.2 team in the land, a frustrated Florida State Seminole team.
Kansas OC John Reagan joins CFT; Also, is A&M feeling the pressure?
Ole Miss and Mississippi State are the powers in College Football, at minimum, in the SEC. However, KU Offensive Coordinator, John Reagan joins CFT and feels the Big 12 is also in the mix.


text: Imagine If This Crazy Confederate Helicopter Had Actually Been Able to FlyEric Limer4/20/13 6:00pmFiled to: Past PerfectHelicoptersFlightVehiclesCivil WarconfederatesConfederacy371EditPromoteDismissUndismissHideShare to KinjaToggle Conversation toolsGo to permalink Back in the 1860s, repeating rifles were a pretty new development. There was a still a lot of horse-riding and open field-fighting. And the Ashokan Farewell played under everything. But the south had a high tech trick under its sleeve. A good old-fashioned whirlybird. Advertisement Developed by engineer William C. Powers, who lived in Mobile Alabama, this 1860s beast was intended to fly over the Union Navy and give them a pummeling Confederate ships just couldn't pull off. Like the concept of Da Vinci fame, the airship would have relied on Archimedean screws (four, in fact) to provide lift and thrust, with a rudder for steeringThe idea was plagued with problems though, from the lack of a thorough understanding of aerodynamics to the absence of an engine powerful enough to take it off the ground. As such it never made it past model form. The National Air and Space Museum has more on the 'copter, including some of the early plans. You are now excused to go write your historical fiction. [The National Air and Space Museum via Slate]Gear from Kinja DealsDoes Your Gaming Mouse Need a Bungee?Bestsellers: Dymo LabelManager 160Here's the Best Price We've Ever Seen For a 3TB External Hard DriveReply37 repliesLeave a reply


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text:
a bffl that you met like three days prior to the establishment of gnomie-hood.
yo nigguh we gnomies already we met at the party last week
by nigguhs and gnomies for real July 04, 2009
Trending Now
Can you define these popular missing words?
Top Definition
A person that is like a good friend but is short. Sometimes wears a sweatshirt that has a pointy hood.
"What's up my gnomie?!
by Spartan14 March 27, 2009
Typically a gnomie can be found chilling with his other gnomies. Can also be found stealing rocks from yards, passed out on a bathroom floor, tipping taxi drivers $1 on a $20 ride, etc. A gnomie will NOT be found in Portland.
Q: What are you doing tonight?
A: Going to chill with my gnomie (s).
A: Going to chill with my gnomie (s).
by Chuzzler April 15, 2009
1. A lawn gnome wearing excessive bling.
2. An African-American lawn gnome.
1. A lawn gnome wearing excessive bling.
2. An African-American lawn gnome.
American rap artist T-Pain is said to have multiple gnomies in his lawn.
by Ladderwalker86 February 03, 2009
a small, strange looking animal that appears to be a cross between a shark, a seal, a shrimp, a small dog, a bug, a donkey, and a rabbit. it is extremely attracted to cheese. (aka, my dog)
look at that gnomie! it's kinda cute!
here, gnomie!
here, gnomie!
by Satinka January 22, 2008
Any girl who is very short
And likes calling people poopoo's as a swear
And likes calling people poopoo's as a swear
Gnomie, "Hey your a poopoo"
by MwahahahaahahahahaEM August 18, 2011
Gnomie or a gnomie is a pseudonym for a perfect faithful man. A gnomie makes great husband material.
I'll bet she's glad she left that other wanker now that she found herself a gnomie.
by puddles29729 July 21, 2016


text:
The typical lament of the modern investor who painfully remembers the credit crisis goes something like this: "I like the high return of stocks, but I love the safety of my bonds."
The behavioral-finance guys would describe this as anchoring on a past event, an act that influences future behavior. Sometimes, anchoring is good, and sometimes it isn't. Right now, it's possible for an anchored investor to do rather well, thanks to a conservative options strategy that, in effect, lets you buy good stocks without significantly increasing risk.
It's critical to participate at least selectively in the stock market, because equities keep chugging higher, shrugging off the fiscal cliff, sequestration, and Europe's lingering economic woes—events that many anchored investors think should be pacing the market.
Bonds, meanwhile, are increasingly less appetizing. Yields remain under pressure, and often are declining. And it's now common for banks and asset managers to advise clients that bond returns will be wiped out when the Federal Reserve raises interest rates.
IN SHORT, MANY INVESTORS are paralyzed. But a solution is at hand: By using low-yielding Treasuries as collateral for put trades on quality stocks, you can increase the returns on a quiescent area of your portfolio. Most banks let investors use 90% of the value of their bonds as collateral.
Some people will say that selling puts is risky because the seller could have to buy the associated stock, even if it falls far below the put strike price. The key is never to sell puts on volatile, high-risk stocks, but instead on blue-chips that pay reliable dividends and have solid business models. Many such stocks come to mind, including Coca-Cola KO 0.6899833452295979% Coca-Cola Co. U.S.: NYSE USD42.32 0.29 0.6899833452295979% /Date(1475269211300-0500)/ Volume (Delayed 15m) : 13251015 AFTER HOURS USD42.32 % Volume (Delayed 15m) : 1124687 P/E Ratio 24.32183908045977 Market Cap 182654337862.54 Dividend Yield 3.3081285444234405% Rev. per Employee 347411 More quote details and news » (ticker: KO), General Electric GE 0.3047748052827633% General Electric Co. U.S.: NYSE USD29.62 0.09 0.3047748052827633% /Date(1475269274951-0500)/ Volume (Delayed 15m) : 33314847 AFTER HOURS USD29.65 0.03 0.1012829169480081% Volume (Delayed 15m) : 2805193 P/E Ratio 25.250746890462356 Market Cap 265431740782.285 Dividend Yield 3.1060094530722484% Rev. per Employee 367748 More quote details and news » (GE), Southern Co. SO -1.440922190201729% Southern Co. U.S.: NYSE USD51.3 -0.75 -1.440922190201729% /Date(1475269482910-0500)/ Volume (Delayed 15m) : 6584276 AFTER HOURS USD51.3 % Volume (Delayed 15m) : 464403 P/E Ratio 20.307180745784184 Market Cap 50207616727.6718 Dividend Yield 4.366471734892787% Rev. per Employee 652773 More quote details and news » (SO), Wells Fargo WFC -0.2028397565922921% Wells Fargo & Co. U.S.: NYSE USD44.28 -0.09 -0.2028397565922921% /Date(1475269222408-0500)/ Volume (Delayed 15m) : 52852373 AFTER HOURS USD44.44 0.16 0.36133694670280037% Volume (Delayed 15m) : 1265292 P/E Ratio 10.852941176470589 Market Cap 223416809465.885 Dividend Yield 3.4327009936766033% Rev. per Employee 352599 More quote details and news » (WFC), Corning GLW 0.5527210884353742% Corning Inc. U.S.: NYSE USD23.65 0.13 0.5527210884353742% /Date(1475269221992-0500)/ Volume (Delayed 15m) : 9511734 AFTER HOURS USD23.65 % Volume (Delayed 15m) : 316722 P/E Ratio 12.755514805026698 Market Cap 24522139545.8691 Dividend Yield 2.2832980972515857% Rev. per Employee 249272 More quote details and news » (GLW), and Exxon Mobil XOM 0.9484154522322461% Exxon Mobil Corp. U.S.: NYSE USD87.28 0.82 0.9484154522322461% /Date(1475269309492-0500)/ Volume (Delayed 15m) : 14819239 AFTER HOURS USD87.21 -0.07 -0.08020164986251145% Volume (Delayed 15m) : 1142267 P/E Ratio 34.63492063492063 Market Cap 361919598414.734 Dividend Yield 3.4372135655362053% Rev. per Employee 2716960 More quote details and news » (XOM).
The strategy is simple. Sell puts 5% to 10% below the stock's market price. If the shares fall below the put strike price, you buy the stock. If the stock advances, you keep the premium. Naturally, you must have the cash to purchase the shares; otherwise your Treasuries would be sold to pay the bill.
Now, if you like hefty put premiums, consider a variation of the strategy: Sell big-dollar puts far below a big-dollar stock's price. We've recommended this trade a few weeks ago to good effect with Google (GOOG), and we still like it. (See the Feb. 27 Striking Price Daily.)
Investors who don't own Treasuries, or prefer to leave those bonds unencumbered, must deposit the full value of Google puts, less the money received for selling the put in their cash accounts. Selling a $690 strike put means that an investor would need to produce $69,000, less the put premium.
The January $690 put that expires in 2014 trades for about $26. Many other puts far away from the stock price can be sold to collect similarly hefty premiums. If the stock never crosses the low strike prices, the put premiums are kept.
CAVEAT EMPTOR: Most new financial products fail and are never worth serious mention. But mini-options are attracting so much attention before their March 18 launch that an exception is in order. The press is heralding mini-options, which cover 10 shares as opposed to the standard 100 shares, as a powerful new tool for investors. Maybe the marketing mumbo-jumbo will prove true, but investors are well-advised to be cautious. If you can't afford regular options, much less the associated stock, you probably have no business trading mini-options that will likely become the domain of momentum traders. If you want to trade minis, learn how they behave before plunking down your hard-earned money, lest you painfully learn the old maxim: Buy cheaply, pay dearly.
Steven Sears is the author of The Indomitable Investor: Why a Few Succeed in the Stock Market When Everyone Else Fails.
Comments: firstname.lastname@example.com http://twitter.com/sm_sears


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Zack's Kernel News
220.127.116.11 Released; Users Encouraged To Upgrade
Willy Tarreau has released 188.8.131.52, which includes some important security fixes. Alexander Viro had identified and fixed an SMP problem as well as a dnotify race condition.
UBIFS Official Tree Inclusion
Artem Bityutskiy made a formal request to have UBIFS, a relatively new Flash filesystem, merged into the mainline tree during the next merge window.
Artem asked Stephen Rothwell to include his git tree in the Linux-Next tree, aiming for submission to Linus Torvalds at the next opportunity.
Stephen had no objection to this. In general, Stephen's requirements for inclusion in Linux-Next are that code be reviewed and tested as well as can be managed and that the submitter intends to have the code accepted in the next merge window. Because Artem had done all that, Stephen was happy to include Artem's tree.
Andrew Morton also approved, although he hadn't looked at any of the code himself.
Andrew knew that Christoph Hellwig had made a number of technical suggestions and wanted to know the status of Artem's work in response to that. Artem said that he and the other UBIFS developers were actively addressing Christoph's concerns.
Regarding Christoph's suggestions, Artem did say that NFS support would probably not happen in the near future, but that some ideas were already percolating as to how to fix that in the long term. None of this seemed to pose a significant obstacle, and it looks as though UBIFS will be merged (or at least will go to Linus for consideration) for the 2.6.27 release.
Purging OSS
The final PCI OSS driver has been removed from the Linux tree. In the old days, Muli Ben-Yehuda had resisted taking out the Trident 4DWave/SIS 7018 PCI Audio Core, but he's moved on to other projects and no longer has the hardware to maintain the driver. With his permission, and given that an ALSA driver that supports the same hardware exists, Adrian Bunk submitted the patch to remove it.
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Nextcloud Announces Raspberry Pi-Powered Home Cloud Box
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The Apache Software Foundation considers retiring OpenOffice
Adobe Gives New Life to NPAPI Plugin for Linux
Adobe won't kill the plugin in 2017
LinuxCon North America Convenes in Toronto, Canada
Linux Foundation's big event celebrates the 25th anniversary of Linux
Linux Turns 25
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Mirantis Joins Hands with SUSE To Offer RHEL Support; Red Hat Not Happy
Competitors get in the game with RHEL without Red Hat
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Security researchers have already notified Microsoft; some fixes are available
Mirantis to Refactor OpenStack Fuel for Kubernetes
The company is collaborating with Google and Intel to use Kubernetes as an engine for Fuel
Canonical Joins Document Foundation Advisory Board
The upcoming release of LibreOffice will be available as a snap package


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"When a legislature decides to steal some of our rights and plans to use police force to accomplish it, what's the real difference between them and the thief? Darn little! They hide behind the excuse that they're legislating democratically. The fact they do it by a majority vote has no moral significance whatsoever. Numerical might does not constitute right, no more than a lynch mob can justify its act because a majority participated." ~ H.L. Richardson
Fracking and Radioactivity
Submitted by Persona non grata on Mon, 2012-11-12 01:00
But also released, notes the report, is radioactive material in the shale—including Radium-226 with a half-life of 1,600 years. A half-life is how long it takes for a radioactive substance to lose half its radiation. It is multiplied by between 10 and 20 to determine the "hazardous lifetime" of a radioactive material, how long it takes for it to lose its radioactivity. Thus Radium-226 remains radioactive for between 16,000 and 32,000 years.
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China Approves Wal-Mart's E-Commerce Deal
BEIJING— Wal-Mart Stores Inc. WMT -1.44 % is expanding in China's booming e-commerce sector as rivals increasingly wage price wars to win a bigger share of the growing online retail market.
Chinese regulators approved the U.S. retailer's purchase of a majority stake in Chinese e-commerce company Yihaodian, enabling Wal-Mart to increase its stake to 51% from 18%.
The transaction remains subject to final closing conditions; Wal-Mart didn't disclose financial details of the deal.
Wal-Mart's spokesman said the company will detail later this year its plans for Yihaodian's expansion.
Clearly, though, the Bentonville, Ark., company hopes to expand its e-commerce presence in China, where online sales are expected to explode.
Business-to-consumer transactions are poised to expand fivefold to 665 billion yuan ($100 billion) over the next two years, according to forecasts from Beijing-based research firm Analysys International. And by 2015, China's online shoppers are estimated to reach 329 million, more than doubling from 145 million in 2010, with each one spending on average nearly $1,000 online annually, doubling in the same time period, according to consulting firm Boston Consulting Group.
By 2015, e-commerce sales in China will account for 8% of all of the country's retail sales, up from 3% in 2010, Boston Consulting projects
While Wal-Mart is counting on the boom to help its expansion, others—both with existing bricks-and-mortar stores and without—are flocking to the Web to sell products ranging from cellphones and appliances to clothing and home furnishings.
The buildup of online companies has kicked off intense rivalry in which some companies are waging price wars to win a broader share of consumers.
A vice president of Chinese electronics retailer Suning Appliance Co. 002024 0.18 % said on Sina Weibo, China's Twitter-like microblogging service, that the company would lower its prices online and offline to undercut online rival Beijing Jingdong Century Trading Co.'s 360buy.com. Beijing Jingdong's chief executive announced in July on Sina Weibo that 360buy.com, which has been planning to go public in the U.S., would forgo profits on its electronics for the next three years to offer consumers the lowest prices on products such as television sets and air conditioners.
Many of the sites have been attempting to establish their brands in consumers' minds in order to chip away at a dominance that has been long held by China's Alibaba Group Holding Ltd. The Chinese Internet company's shopping site Taobao Mall holds a roughly 42% share in business-to-consumer transactions online, leading over Beijing Jingdong's 15.5% and well ahead of Yihaodian's 0.3%, according to Analysys.
Unlike Yihaodian, which fills its own orders, Alibaba's Tmall.com is a platform connecting consumers to merchants and doesn't distribute itself.
—Li Yuan contributed to this article.
Write to Laurie Burkitt at email@example.com


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Newest Posts
- Take a closer look at student innovation during the 2016 Engaged Learning Symposium and Big iDeas Pitch Contest, Friday, Sept. 23
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Tag Archives: honorary degees
SMU hosts 2015 Honorary Degree Symposia Friday, May 15
Three international leaders who will receive honorary degrees at SMU's 100th May Commencement will participate in symposia on the main campus Friday, May 15. All symposia are free and open to the public. The symposia will feature 2015 honorees Meave Leakey, a renowned anthropologist whose … Continue reading
Posted in Calendar Highlights, News, Save the Date Tagged Annette Caldwell Simmons School of Education and Human Development, Commencement, Dedman College, earth sciences, free events, Helen LaKelly Hunt, honorary degees, honorary degree recipients, honorary degree symposia, Huffington Department of Earth Sciences, Irene Hirano Inouye, John Goodwin Tower Center for Political Studies, Kimberly Cobb, Maguire Center for Ethics and Public Responsibility, Meave Leakey, political science, psychology, Sun and Star Program on Japan and East Asia, symposia, Tower Center for Political Studies, visitors Comments Off on SMU hosts 2015 Honorary Degree Symposia Friday, May 15
SMU names three honorary degree recipients for 2014
Three outstanding individuals, including a current and a former SMU professor, will receive honorary degrees at the University's 99th Commencement ceremony Saturday, May 17, in Moody Coliseum. Pulitzer Prize-winning journalist Isabel Wilkerson and Methodist historian Richard P. Heitzenrater will receive honorary Doctor of Humane Letters degrees. SMU law … Continue reading
Posted in Calendar Highlights, News Tagged academic ceremonies, awards and honors, Commencement, Dedman School of Law, honorary degees, honorary degree recipients, Isabel Wilkerson, Joe McKnight, Joseph W. McKnight, R. Gerald Turner, Richard Heitzenrater, symposia Comments Off on SMU names three honorary degree recipients for 2014
Nominate 2011 honorary degree candidates by Sept. 27
Posted in News, Save the Date Tagged academic ceremonies, Faculty Senate, honorary degees Comments Off on Nominate 2011 honorary degree candidates by Sept. 27
Three to receive honorary degrees at Commencement 2009
Posted in Calendar Highlights, News Tagged academic calendar, academic ceremonies, Alexander McCall Smith, Arthur Mitchell, awards and honors, Commencement, Harold W. Attridge, honorary degees, Laura Bush, visitors Comments Off on Three to receive honorary degrees at Commencement 2009


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Yesss it's food we're talking about....
- Posted Jan. 11, 2012 by Piyali in Random. Viewed 1749 times
- This is a migrated legacy post. Image resolution is low. Info
After all the discussion in the forum about food photography I couldn't resist posting this picture. I attended a class on food photography back in Florida and this is what I wrote when I posted this picture on flickr long time back…
"Food shoot @ Teatro, Y'bor city.
At least now I know that this is not my cup of tea..I love food too much to see it only through my lenses. It was hilarious how everyone jumped at the sight of the dessert plate during the photoshoot and the enthusiasm was not at all related to photography." :) :)
3 There are 3 comments, add yours!
Sandra Vermeulen
That shot looks indeed yummy! ;-D
Yes, good-looking food does attract a lot of attention! Fantastic image!!!
Giancarlo Fosci
beautiful and Good!!!!


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Look Way Up... By Elizabeth H
The Calgary Tower is probably one of the most photographed buildings in Calgary's downtown... This is my version.
Mar 30, 2011
Arts & Culture, Must-See Spots
Calgary, Alberta
Like this Photo
Suggested Itineraries
Icons & Landmarksview details
Take a tour of Calgary at some of our vintage landmarks, or go straight for the big vistas at attractions like the Calgary Tower and WinSport.
Unexpectedview details
What do ballet, tapas and fly fishing have in common? They're but three of the hidden surprises you'll find within Calgary's city limits.
Report this Photo
Is this photo inappropriate? If so, please let us know.Report ImageCancel


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Oct 2Sunday October 2, 2016 / 3:30pm
Oct 7Friday October 7, 2016 / 7:30pm
Oct 8Saturday October 8, 2016 / 7:30pm
Oct 9Sunday October 9, 2016 / 3:30pm
There is no late seating. Special-access ADA seating is ONLY available directly through Art Centre Theatre's website and cannot be purchased nor requested through Goldstar.
13 Art Centre Theatre Tips
- Casual 2
- Dine before you go 1
The weather was hot. Night time. . I wore jeans and a t-shirt. Very casual venue. .star this tip starred
I wore Summer casual the theater temperature was very comfortable .star this tip starred
If going to the Plano theatre, be sure to show up 20 minutes early or they may give your spot away. I noticed 3 women showing 5 minutes before the presentation and they had to sit separately.star this tip starred
My ticket was a scrap of paper with my seat numbers written in pencil though we were on the last row there were only 7 rows of seating. It was very casual most of audience appeared to be family and friends of actors.star this tip starred
The weather was sunny and hot.star this tip starred
Don't arrive early. They were not set up to have you get your tickets 30 minutes early and no place to standstar this tip starred
They have chips/candy/soda/water for purchase - $2 each. Worth it, since I'm guessing the proceeds go to the theater.star this tip starred
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