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Purified, soluble recombinant bacterial long chain and short chain PHA synthases are described, with methods and materials for overexpression and purification.
1. A method for the overproduction and purification of soluble polyhydroxyalkanoate (PHA) synthase expressed from an isolated structural gene comprising expressing the isolated structural gene encoding the synthase in a vector in an expression system not expressing an enzyme cleaving the synthase selected from the group consisting of bacteria, yeast, fungi, and plants.
2. The method of claim 1 further comprising placing the PHA synthase gene under the transcriptional control of a regulatable promoter.
3. The method of claim 2 wherein the promoter is selected from the group consisting of an IPTG inducible promoter, lacZ, tac, trc, the trp promoter, the temperature inducible phage promoter lambda PL, and a phage T7 promoter/T7 RNA polymerase system.
4. The method of claim 1 where the PHA synthase is a polyhydroxyalkamoate (SCPHA) synthase.
5. The method of claim 1 where the PHA synthase is a SCPHA synthase from Alcaligenes eutrophus.
6. The method of claim 1 where the PHA synthase is a polyhydroxyalkanoate (LCPHA) synthase.
7. The method of claim 1 where the PHA synthase is a LCPHA synthase encoded by a gene from Pseudomonas.
8. The method of claim 1 where the PHA synthase is overproduced in a host selected from the group including bacteria, yeast, fungi and plants and the vector comprises a ribosome binding site enhancing expression in the host.
9. The method of claim 1 where the PHA synthase is overproduced in a bacterial host.
10. The method of claim 1 where the PHA synthase is overproduced in Escherichia coli.
11. The method of claim 10 where the PHA synthase is overproduced in a protease deficient strain of Escherichia coli.
12. The method of claim 11 where the PHA synthase is expressed in an ompT deficient Escherichia coli strain.
13. The method of claim 10 where the PHA synthase is engineered so as to remove cleavage sites that are normally cleaved by proteases in the naturally occurring synthase.
14. The method of claim 1 further comprising purifying the PHA synthase by chromatography using buffer solutions containing detergents.
15. The method of claim 14 where the PHA synthase is purified by chromatography using buffer solutions containing 6-O-(N-heptylcarbamoyl)-methyl-α-D-glucopyranoside.
16. The method of claim 15 wherein the synthase is purified by chromatography on hydroxyapatite.
17. An expression system for the overproduction and purification of soluble PHA synthase expressed from an isolated structural gene comprising
a host not expressing an enzyme cleaving the synthase selected from the group consisting of bacteria, yeast, fungi, and plants, and
the isolated structural gene encoding PHA synthase in a vector for expression of the synthase in the host.
18. The system of claim 17 wherein the PHA synthase gene is under the transcriptional control of a regulatable promoter.
19. The system of claim 17 where the PHA synthase is selected from the group consisting of SCPHA synthase and LCPHA synthase.
20. The system of claim 17 where the PHA synthase is overproduced in a bacterial host.
21. The system of claim 17 where the PHA synthase is overproduced in Escherichia coli.
22. The system of claim 17 where the PHA synthase is overproduced in a protease deficient strain of Escherichia coli.
23. The method of claim 17 where the PHA synthase is engineered so as to remove cleavage sites that are normally cleaved by proteases in the naturally occurring synthase.
This is a continuation in part of U.S. Ser. No. 08/234,721 filed Apr. 28, 1994, abandoned, which is a continuation of U.S. Ser. No. 08/073,603 filed Jun. 7, 1993, abandoned, which is a continuation of U.S. Ser. No. 07/944,881 filed Sep. 14, 1992, abandoned, which is a divisional of U.S. Ser. No. 07/700,109 filed May 8, 1991, issued as U.S. Pat. No. 5,245,023, which is a continuation of U.S. Ser. No. 07/378,155 filed Jul. 10, 1989, abandoned, which is a continuation in part of U.S. Ser. No. 07/067,695 filed Jun. 29, 1987, by Oliver P. Peoples and Anthony J. Sinskey, abandoned. This is also a continuation in part of U.S. Ser. No. 08/071,574 filed Jun. 3, 1993, by Oliver P. Peoples and Anthony J. Sinskey. The teachings of these applications are specifically incorporated by reference herein.
The United States government has rights in this invention by virtue of National Science Foundation grant 9018546-BMB to J. Stubbe, S. Masamune, O. Peoples and A. J. Sinskey and ONR-N00014-90-J-1529 to Oliver Peoples and A. J. Sinskey.
The use of enzymes as organic synthetic reagents is an area of increasing industrial interest (Jones, (1986) Tetrahedron. 42, 3351-3403; Wong, (1989) Science 244, 1145-1152). Cloning of the genes encoding individual enzymes enables production of reagent-type quantities of enzymes and modification of their properties, for example, temperature stability, pH stability, and substrate utilization, through the use of recombinant DNA technology.
The polyhydroxyalkanoate ("PHA") polymerases are unique biocatalysts capable of polymerizing up to thousands of monomeric units in a head to tail condensation to produce high molecular weight polymers. To date there have been no reports of the preparation of reagent quantities of the PHA polymerases, separated from the PHA granules to which they become attached in vivo, for use as biocatalysts to prepare novel polyester polymers, although the genes encoding these polymerases and their use are described in U.S. Pat. No. 5,245,023 to Peoples and Sinskey. By making these biocatalysts available, a range of polyesters can be prepared by combining organic synthesis of unique precursors with enzymatic polymerization.
The potential for utilizing biological systems as a source of biodegradable materials is becoming increasingly attractive in view of the environmental problems associated with disposal of traditional oil based polymers (Chu, (1985) CRC Crit. Rev. Biocompatibil., 1, 261-322; Agrawal, et al., (1992) Biomaterials, 13, 176-182; Tamada & Langer, (1993) Proc. Natl. Acad. Sci. U.S.A., 90, 552-556).
Poly-D-3-hydroxyalkanoates (PHAs) are polyoxoesters synthesized from optically active thiol esters that are produced in many types of bacteria (Anderson & Dawes, (1990) Microbiological Reviews, 54, 450-472); FEMS Microbiological Reviews, vol. 103 Schlegel, H. G. and Steinbuchel, A., editors (1992). Polyoxoesters have recently been found in mammalian systems as well (Reusch, et al., (1992) Biochim. Biophys. Acta, 1123, 33-40). Thus, these polymers are of interest from both biological and applied perspectives (Seebach, (1992) FEMS Microbiol. Rev., 103, 215); Muller & Seebach, (1993) Angew. Chem. Int. Ed. Engl., 32, 477-502).
Short chain PHAs, homo or co-polymers of four to five carbon units in which the monomeric building blocks are provided by (D)-3-hydroxybutyryl- and (D)-3-hydroxyvaleryl-CoA, are particularly of interest. The biosynthetic pathways for these polyoxoesters have been most extensively investigated in Zoogloea ramigera (Tomita, et al., (1983) In: Biochemistry of Metabolic Processes., Leeon, D., Stratman, F. and Zahlten, R. (eds.), Elsevier Press: Holland, p. 353) and Alcaligenes eutrophus H16 (Slater, et al., (1988) J. Bacteriol., 170, 4431-4436; Schubert, et al., (1988) J. Bacteriol., 170, 5837-5847; Peoples & Sinskey, (1989) Molecular Microbiol., 3, 349-357; J. Biol. Chem., 264, 15293-15297; J. Biol. Chem., 264, 15298-15303, and U.S. Pat. No. 5,245,023). As discussed in more detail below, their biosynthesis involves three proteins: a thiolase, a reductase and a synthase for polymerase. ##STR1##
A large number of bacterial species have the ability to accumulate intracellular reserves of poly D-3-hydroxyalkanoates (PHAs). These polymers may constitute up to 80% of the dry cell weight of the bacterium and are found in storage granules (Anderson and Dawes, 1990). The polymers can be divided into two classes based on the number of carbon atoms in the monomeric units. Short chain PHAs (SCPHAs) are homo/copolymers of three to five carbon units and are typified by the PHAs produced by Alcaligenes eutrophus H16. Long chain PHAs (LCPHAs) contain subunits of six to 12 carbon atoms and are produced by Pseudomonads.
In the past few years, Peoples et al., (1987) J. Biol. Chem., 262, 97-102; (1989) J. Biol. Chem. 264, 15298-15303; J. Biol. Chem. 264, 15293-15297; Molecular Microbiol. 3, 349-357; Peoples and Sinskey, 1990; U.S. Pat. No. 5,245,023) have been successful in identifying both the genes and gene products responsible for the biosynthesis of SCPHAs from Zoogloea ramigera, A. eutrophus H16 and LCPHAs from Pseudomonas oleovorans. ##STR2##
The thiolases and reductases from A. eutrophus and Z. ramigera have been extensively studied from both biochemical and mechanistic perspectives (Masamune, et al., (1989) Pure Appl. Chem., 61, 303-312); Ploux, et al., (1988) Eur. J. Biochem., 174, 177-182; Peoples, et al., (1987) J. Biol. Chem., 262, 97-102). However, both the soluble and granule bound forms of the naturally occurring PHA synthase have resisted purification to a stable form which can be investigated (Haywood, et al., (1989) FEMS Microbiol. Lett. 57:1-6; Tomita, et al., (1983)).
The genes for all three enzymes from A. eutrophus have been cloned and expressed in E. coli. Sequence analysis reveals that the gene encoding the synthase is part of a biosynthetic operon containing the phbC-phbA-phbB genes coding for synthase-thiolase-reductase, respectively. The synthase is a protein of 63.9 kDa and its DNA sequence exhibits no homology with the DNA sequence of any previously reported proteins. The presence of all three genes in E. coli results in accumulation of large quantities of PHA (50% of the dry weight of the cell) (Slater, et al., (1988); Schubert, et al., (1988); Peoples & Sinskey, (1989)). Until recently, efforts to overexpress the synthase in the absence of the thiolase and reductase have proven unsuccessful.
It is therefore an object of the present invention to provide a method and means for overexpression of the soluble form of the synthase.
It is a further object of the present invention to provide a method for isolating, and the isolated, purified synthase in active form.
Two forms of soluble short chain PHA synthase (SCPHA) are disclosed in purified form, as well as a method for purification of recombinant SCPHA synthase overexpressed in another bacteria such as E. coli. As described in the example, the synthase is overexpressed by insertion of the gene encoding the synthase, under the control of a regulatable promoter, such as the IPTG inducible promoter, into an appropriate host, such as an ompT deficient E. coli strain, or E. coli, bacteria, plants, yeast, or fungi, which either do not produce protease cleaving the synthase or where the synthase is engineered to remove protease sensitive cleavage sites.
FIG. 1a and 1b is a schematic of the construction of pKAS4 containing phbC from Alcaligenes eutrophus.
FIG. 2 is a graph of the molecular weight determination of native PHA synthase using Superdex™ G-200 size exclusion chromatography, plotting log (molecular weight) versus volume of elution from the Superdex™ column, in comparison with molecular weight standards (methods). Two different protein containing fractions containing synthase activity and cross-reacting with PHA synthase antibodies are shown by the arrows.
FIG. 3a is a graph of the rate of PHA formation as a function of time (minutes) and enzyme concentration: 0.07 μg (open diamonds); 0.17 μg (open squares); 0.35 μg (circles); 0.23 μg (open triangles); 0.07 μg (closed triangles); 1.4 μg (closed squares).
FIG. 3b is a graph of the determination of the specific activity of PHA synthase as a function of protein concentration.
FIG. 4 is a graph of the sucrose-density centrifugation of PHA synthase incubated with 0, 1000, and 10,000-fold excess of [3 H]-HBCoA relative to protein. For the 1000-fold excess substrate, the scale is on the left (open squares). For the 10,000-fold excess, the scale is on the right (open diamonds). The bottom panel uses dot blots and PHA synthase antibody to determine the location of the synthase.
The functional expression in E. coli of the phbABC gene products encoding thiolase, reductase and synthase, respectively, required for the biosynthesis of PHA from A. eutrophus, has been described by Slater, et al., 1988; Schubert, et al., 1988; and Peoples & Sinskey, 1989. The transformation of E. coli with the phbC gene alone, does not result, however, in the production of polymer in a nitrogen starved medium. Using 35 S-labeling studies in maxicells, Peoples and Sinskey (1989) reported that low levels of expression of a protein of approximately 64 kDa was observable by SDS-PAGE and that expression of this protein appeared to be correlated with production of very low levels of CHCl3 extractable material upon incubation of [3 H]-HBCoA with these crude cell extracts. It would be desirable to overexpress the synthase in soluble form, in the absence of the thiolase and acetoacetyl CoA reductase, since the absence of these latter two gene products is desirable as they can use cellular acetylCoA to initiate the polymerization process leading to granule bound, insoluble synthase. Expression in the soluble form offers the advantage that the priming and initiation processes required for polymerization can be controlled.
The genes encoding both the SCPHA synthase and the LCPHA syntase are known. As described herein, however, are methods to increase production of the soluble synthase in the absence of the reductase and thiolase. These consist of:
1. Expression in an appropriate system
expression in a host expression system not including proteases cleaving the soluble syntase, including bacteria other than E. coli which express the ompT gene such as Bacilllus, Pseudomonads, and Coryneforms (Methods in Enzymology, vol. 185), yeast such as Saccharomyces and Kluvyeromyces for which methods are known to those skilled in the art (Methods in Enzymology vol. 194); and fungi such as Aspergillus, Neurospora and Trichoderma for which methods are known to those skilled in the art, as described in Applied Molecular Genetics of Fungi, Peberdy, Caten, Ogden, and Bennett, eds. (Cambridge University Press, N.Y. 1991), many of which can be obtained from the American Type Culture Collection, Rockville, Md., which are routinely used by those skilled in the art; and plants such as oilseed crops including rapeseed, canola, sunflower, soybean, cottonseed, and safflower, for which methods are known to those skilled in the art (Gasser and Fraley, 1989 Science 244:1293-1299; or
expression in a host system which expresses protease where the synthase gene is modified to not include proteolytic cleavage sites, as described in detail below in the examples below.
2. Expression under the control of a regulatable promoter
many regulatable promoters and chemically inducible promoters such as the IPTG inducible promoters are known, including the lacZ, tac, the trp promoter, the temperature inducible phage promoter lambda PL and the phage T7 promoter/T7 RNA polymerase system, and osmotic sensitive promoters.
3. Other modifications to increase efficiency of expression
alteration of the ribosome binding site to substitute a ribosome binding site specific to the host expression system.
Genetic Engineering of Organisms to overexpress soluble SCPHA Polymerase Biocatalyst
The identification of the phbC gene encoding the A. eutrophus H16 SCPHA polymerase, described in U.S. Pat. No. 5,245,023, enables the overproduction of the functional gene product in Escherichia coli. As described in detail in the following examples, plasmid pKAS4 was constructed as illustrated below. The ribosome-binding (RBS) region of the phbC structural gene was modified using PCR to optimise this region for translation in E. coli and inserted into a modified version of the trc expression vector pTrc99A (Amann et. al., 1988). The original vector was modified by digesting with the restriction enzyme Nco 1 and treatment with S1-nuclease to generate blunt ends prior to religating. The net effect of these manipulations is to destroy the Nco 1 site and remove the ATG start codon contained within it. The phbC structural gene with the added efficient ribosome binding site ("RBS") was inserted into the Eco R1 site of this plasmid to obtain plasmid pKAS4.
E. coli JM105 cells carrying plasmid pKAS4 express significant levels of functional SCPHA polymerase following induction with isopropyl-β-D-thiogalactoside (IPTG). This activity decreases rapidly in lysed cells and immunoblot analysis of the overproduced SCPHA polymerase protein indicates that the full-length, 64 kd polypeptide is being specifically cleaved to yield a 50 kd protein. N-terminal amino acid sequence analysis of the truncated polypeptide identified the site of cleavage between the arginine residues, Arg 100 and Arg 101, of the polypeptide sequence. This sequence of residues is one of the sequences cleaved by the E. coli outer membrane protease VII encoded by the ompT gene (Sugimara and Nishihara, (1988) J. Bacteriol. 170, 5625-5632).
The plasmid pKAS4 was therefore transferred into the ompT deletion mutant strain UT5600 (Fiss et. al., (1979) Biochem. Biophys. Res. Commun. 91, 29-34). In this strain, rapid degradation of the SCPHA polymerase protein is eliminated, resulting in lysed cells with high levels of stable SCPHA polymerase activity suitable for use as a biosynthetic reagent for the synthesis of novel polyesters.
As described in Example 1, PHA synthase was expressed in Escherichia coli by re-engineering the 5'-end of the wild-type (wt) gene and subsequent transformation of this gene into protease deficient E. coli UT5600 (ompT-). Induction with IPTG results in soluble PHA synthase which is approximately 5% of the total protein.
Purification of Soluble PHA synthase
A cell lysate is prepared at 4� C. The supernatant or crude filtrate is isolated, filtered through a 0.45 μm membrane, made 0.05% (w/v) in Hecameg™ and loaded directly onto a hydroxylapatite column equilibrated in glycerol (one to fifteen percent/volume to volume, preferably 5%) KPi (10 to 50 mM, pH 6-8, preferably 7), and Hecameg™ (0.01 to 2% w/v, preferably 0.05%). The column is washed with Hecameg™-glycerol buffer and eluted with a linear gradient from 10 to 300 mM KPi in Hecameg™-glycerol buffer. The synthase elutes between 110 and 150 mM KPi. The fractions containing activity can be pooled and rapidly frozen (liquid nitrogen) without significant loss in activity. This material can be directly loaded onto a Q-Sepharose™ high performance FPLC column equilibrated with Hecameg™-glycerol buffer, pH 8.5 at 4� C. Subsequent to loading, the column is washed and a linear gradient from 0 to 1.5 M NaCl in Hecameg™-glycerol is then applied. Greater than 90% of PHA synthase elutes in fractions 2 and 3. Approximately 10% of the protein elutes in fractions 11 and 12 at 500 mM NaCl. The fractions are pooled and concentrated using a YM 30 membrane and an Amicon filtration apparatus. The protein is then rapidly frozen in liquid nitrogen and stored at -80� C. The protein is stable for at least a month under these conditions.
FPLC, providing rapidity of purification, and a variety of anion exchange materials including hydroxylapatite and Q-Sepharose™, can be used to purify the synthase to greater than 90% homogeneity with a 40% recovery of overall units. Based on Coomassie staining of the protein in the crude cell extracts subsequent to SDS-PAGE, it was anticipated that a purification of approximately 20 fold would be required to obtain homogeneous protein. The protein isolated from the Q Sepharose™ column has a specific activity of 5 μmol min-1 mg-1 and upon freezing and thawing its activity increases to 8 μmol min-1 mg-1, a 16-fold overall purification. The enzyme is stable when rapidly frozen in liquid nitrogen for a period of at least a month.
This procedure yields a soluble synthase purified to greater than 90% homogeneity with a specific activity of 5 μmol min-1 mg-1. The molecular weight of the PHA product is approximately 106 Da based on PlGel chromatography and calibration using polystyrene molecular weight markers. The synthase, in the absence of substrate, appears to exist in both monomeric and dimeric forms. Incubation of the synthase with an excess of substrate converts it into a form that is now extractable into CHCl3 and sediments on sucrose density ultracentrifugation with PHA. Studies in which the ratio of substrate, 3-D-hydroxybutyrylCoA, to synthase is varied suggest that during polymerization, the elongation process occurs at a rate much faster than the initiation process.
In contrast to expectations based on previous attempts to purify the soluble synthase (Haywood, et al., 1989; Tomita, et al., 1983), the enzyme is stable when rapidly frozen at liquid N2 temperatures and stored at -80� C.
Initial efforts to characterize the synthase focused on determination of the size distribution of [3 H]-PHA polymers using a variety of size exclusion chromatographic systems including Showdex™, PlGel™ and/or Sephacryl™ columns. These efforts resulted in minimal recovery of [3 H]-polymer from these columns. Apparently the protein-polymer complex either precipitates on the top of the columns and/or tightly binds to a wide variety of different column backbone matrices. However, treatment of the PHA-synthase complex with pronase followed by analysis on a PlGel™ column resulted in recovery of 40% of the radioactivity in a polymer of molecular weight - 106 Da. This molecular weight has been assigned based on comparison with polystyrene polymer standards. Additional studies on the PHA�synthase complex using sucrose gradient ultracentrifugation revealed formation of very large molecular weight aggregates as well. Thus, the synthase in vitro is capable of producing polymers at least as large as those isolated from A. eutrophus and these polymers appear to behave as a granule bound synthase.
The kinetics of PHB formation obtained using the standard assay gave very different results from those previously reported in the literature by Tomita, et al., 1983; Haywood, et al., 1989. The formation of polymer is preceded by a long and variable lag time. Furthermore, the rate of polymer formation, measured subsequent to the lag, has a non-linear dependence on enzyme concentration up to 0.2 μM PHA synthase. This lag phase can be associated in some way with the priming and initiation process required for polymerization. Similar lag phases have been detected with glycogen synthase and rubber polymerase when the appropriate primers are lacking (Smythe and Cohen, (1991) Eur. J. Biochem., 200, 625-631; Light, et al., (1989) J. Biol. Chem., 264, 18589-18597; Light, et al. (1989) J. Biol. Chem., 264, 18598-18607). The priming process might well require conversion of a monomeric to a dimeric form of the synthase, consistent with two forms of the protein detected by size exclusion chromatography. In synthases isolated from their normal hosts, either PHB granules, in the case of the insoluble synthases, or small amounts of oligomers of PHB, in the case of the soluble synthases, could be present and serve as primers. Thus, the enzymes as isolated from their natural hosts may have been preprimed and thus, no lag phase is detectable (Tomita, et al., 1983). The observation that the lag phase is removed by preincubation of the recombinant synthase with cold 3-D-HBCoA for 10 minutes prior to addition of the [3 H]-HBCoA is consistent with this model.
A set of experiments in which the synthase is incubated with varying ratios of 3-D-HBCoA per synthase (up to 10,000 to 1), followed by analysis of the products by ultracentrifugation, indicates that most of the synthase is active and can produce polymer.
The present invention will be further understood by reference to the following non-limiting examples.
Example 1: Modification and overexpression of PHA synthase in protease-deficient E. coli.
Materials and Methods
Coenzyme A, glucose-6-P dehydrogenase (specific activity 50 to 150 μmol min-1 mg-1), pronase, and NADPH, were obtained from Sigma. NcoI, EcoRI, NotI, SmaI, molecular weight standards, and T4 DNA Ligase were obtained from Gibco BRL. [1-3 HI-Glucose (15.5 curie/mmol) and [35 S] ATPΥS (1000 millicurie/mmol) were obtained from New England Nuclear. IPTG was obtained from Boehringer Mannheim. 6-O-(N-heptylcarbamoyl)-methyl-α-D-glucopyrano-side (Hecameg™) was obtained from Vegatec, Villejuif, France. Q-Sepharose™ was purchased from Pharmacia and Macro-prep™ hydroxylapatite 18/30 (binding capacity of 12 mg of lysozyme/g) was a gift of Dr. Larry Cummings, Bio-Rad Laboratories Inc. PlGel Mixed C (5μ) column was purchased from Polymer Laboratories (Amherst, Mass.). ClS-Sep-Pak cartridges were from Waters Inc. The Klenow fragment of DNA polymerase was obtained from Amersham. The oligonucleotides were synthesized by the MIT Biopolymers Laboratory and used without further purification. HydroxybutyrylCoA (HBCoA) was synthesized using A. eutrophus acetoacetylCoA reductase, (20 units mg-1) as described by Ploux, et al., 1988).
E. coli strains JM105 (Yanisch-Perron, et al., (1985) Gene, 33, 103-119) and UT5600 (Earhart, et al., (1979) FEMS Microbiol. Lett., 6, 277-280) were used. Plasmid pAeT42 containing the A. eutrophus H16 phbC gene coding for the PHA synthase is described in U.S. Pat. No. 5,245,023 to Peoples and Sinskey. pTrc99A was obtained from Pharmacia. Bacteria were grown in 2� TY media (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl) at 30� C. Ampicillin was used in a final concentration of 100 μg/mL. Antibodies to the PHA synthase-glutathione transferase fusion protein were prepared as described by Gerngross, et al., (1993) J. Bacteriol., 175, 5289-5293. The polymerase chain reaction (PCR) was performed using an Ericomp™ thermal cycler and Amplitaq™ reagent kit from Perkin Elmer. Sequencing of protein was carried out by the MIT Biopolymer Laboratory. FPLC chromatography was carried out on a system purchased from Pharmacia.
Preparation of [3-3 H]HBCoA2 : [1-3 H]-D-Glucose (100 μcurie), 0.2 μmol NADP+, 3 μmol acetoacetyl-CoA and 3.5 μmol D-glucose were dissolved in 1 mL of 20 mM Tris�HCl (pH 8.0) at 25� C. Glucose dehydrogenase (5 units) was added followed by 20 units of acetoacetylCoA reductase. The mixture was incubated for 2 h at room temperature. The pH of the solution was then adjusted to 4.5 with 1 M HCl and the reaction mixture was purified in 200 μL aliquots by loading onto 1 mL C18 Sep-Pak™ cartridges and washing with 1 mL of 50 mM KPi 9pH 4.7). The product was eluted with 1 mL of the same buffer containing 25% methanol. The fractions containing [3-3 H]-HBCoA were pooled, lyophilized, and stored as a 12 mM stock solution in 50% ethanol at -20� C. The concentration was determined by A260nm (ε=16.4 mM-1) (Ploux, et al., 1988) and by the coupled assay procedure described by Saito, et al., (1977) Arch. Microbiol., 114, 211-217.
I. Redesigning the PHA Synthase Gene for Overexpression
Routine DNA manipulations were carried out as described by Sambrook, et al., (1987) Molecular Cloning: a Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor, N.Y. pTrc99A (1 μg) was digested with NcoI (10 units) for 1 h as described by the vendor. NcoI is a unique restriction site in the polylinker region of this plasmid. The resulting linearized DNA with recessed ends was made blunt ended using the Klenow fragment (2 units) after incubation with 0.2 mM dNTPs for 15 min. The DNA was then recircularized using T4 DNA ligase (2 units) and the resulting plasmid pTrcN transformed into E. coli JM105. This process removes the ATG start codon immediately downstream of the ribosomal binding site in the vector. pTrcN was characterized by restriction digest analysis subsequent to its isolation and used in the development of the new expression vector described subsequently. The 5'-region of the phbC gene was modified using the PCR. Primer 1: CCGAATTCAGGAGGTTTTTATTATGGCTACCGGCAAAGGCGCGG CAGCTTCCACGC (Sequence ID No. 1) and primer 2: CGTGCAGCGGA CCGGTGGCCTCGGC CTTGCCC (Sequence ID No. 2) were incubated with pAeT42 DNA linearized by digestion with SmaI. Primer 1 was designed to re-engineer the 5' end of the phbC gene and contains an EcoRI restriction site immediately upstream of an efficient E. coli ribosome binding region. Primer 2 is downstream of the NotI site (bps 265-297). The 316 bp amplified fragment was prepared using 35 cycles of the following program: denaturation at 95� C., 30 sec; annealing at 55� C., 60 sec; elongation at 72� C., 60 sec. Following amplification, the DNA was purified by phenol extraction and ethanol precipitation in the presence of 0.3 M NaOAc. The DNA (1 μg) was digested to completion with EcoRI (20 units) and NotI (20 units) for 2 h. Following electrophoresis in a 1.8% agarose gel, the DNA band was visualized by ethidium bromide staining and UV light, and the gel slice was excised.
The 161 bp EcoRI-NotI DNA fragment was recovered from the gel slice and purified using a Qiagen™ kit (Qiagen Inc., Calif.). The 161 bp piece (200 ng) was used in a three piece ligation with the 1560 bp fragment (200 ng) isolated from the restriction digest of pAeT42 with NotI and StuI and the 4.2 kb fragment (100 ng) of DNA resulting from restriction digestion of pTrcN with EcoRI and SmaI followed by alkaline phosphatase treatment. The fragments were ligated with 2 units of T4 DNA ligase for 16 h at 22� C. This reaction mixture was used directly to transform competent E. coli JM105 cells and the resulting transformants selected on 2xTY agar plates containing 100 μg/mL of ampicillin.
The resulting plasmid, pKAS4, was isolated and characterized by both restriction digest maps and DNA sequencing using the dideoxy chain termination method and Sequenase™ II kits (United States Biochemical Corp.).
Optimization of the Expression of PHA Synthase Analyzed by Immunoblotting
E. coli JM105 harboring pKAS4 was grown at 30� C. At various times during the growth 100 μL aliquots were removed and the cells pelleted by centrifugation. The cells were suspended in 25 μL of SDS-PAGE buffer (Laemlli, 1970) and incubated for 5 min in a boiling H2 O bath. The entire sample was then loaded onto a 4-15% gradient polyacrylamide gel (Biorad, Richmond, Calif.) and the proteins separated by SDS-PAGE. The proteins were transferred onto nitrocellulose filters (BA85, 0.45 μm pore size, Schleicher and Schuell) using a Mini Trans-Blot Cell (Biorad). Electroelution proceeded for 1 h at 300 mA in 25 mM Tris, 192 mM glycine, 20% (v/v) methanol. Following transfer, the filters were blocked with TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween™ 20) containing 3% nonfat dry milk for 1 h. The anti-PHA synthase antibody (diluted 1 to 2000) in TBST was then incubated with the filters for 1 h and washed three times with TBS (TBST minus Tween™ 20). The protein cross reacting with the anti-PHA synthase antibodies was visualized using goat anti-rabbit IgG horse radish peroxidase (HRP) conjugate and the standard HRP color development reagent (Biorad).
Alternatively, proteins were electroblotted onto polyvinylidene difluoride (PVDF) membranes in 10 mM CHAPS (pH 11) and 10% CH3 OH and were subjected to sequence analysis by the automated gas phase Edman degradation sequencing method (Matsudaira, (1987) J. Biol. Chem., 262, 10035-10038).
Results of Overproduction of the A. eutrophus PMA Synthase in E. coli
In an effort to increase the level of expression of the phbC gene product in E. coli, the A. eutrophus ribosome binding site was replaced with a strong consensus E. coli ribosome binding site and an eight base pair spacer, rich in Ts and As, upstream of the ATG initiation codon. Other appropriate consensus ribosome binding sites for other expression systems are known and could have been substituted for the E. coli ribosome binding sites. In addition, the third nucleotide of the second codon was changed from GCG (ala) to GCT (ala) (Bagdasarian, et al., (1983) Gene, 26, 273-282). PCR primers were used to generate a 161 bp fragment which was then used to modify the N terminus of the PHA synthase gene. The modified PHA synthase gene was then placed downstream of-the IPTG-inducible trc promoter in the vector pTrcN to produce a new vector pKAS4, as shown in FIG. 1. Other inducible promoters are also known and could have been substituted for IPTG. This plasmid was transformed into E. coli JM105 and upon induction with IPTG, both analysis by Western blotting subsequent to SDS-PAGE and assay for catalytic activity revealed production of significant levels of soluble PHA synthase.
However, Western blotting analysis of the crude cell lysates revealed that subsequent to induction, in addition to the expected 64 kDa protein, there was the unexpected production of a second major protein of Mr=50 kDa and several other smaller proteins. When this experiment was repeated using cells directly boiled in SDS and not subjected to cell lysis, only the 64 kda protein was apparent. These results suggested that subsequent to cell lysis, proteolysis was occurring to degrade the PHA synthase. To test this hypothesis and to define the position of proteolysis, the 50 kDa protein was electroblotted onto a PVDF membrane (Matsudaira, 1987) and the first ten amino acids of its N-terminal sequence determined. As predicted by the antibody analysis, the 50 kDa protein results from a proteolytic clip of the 64 kDa protein between residues 100 and 101 (L95 -H-D-R101 -F-A-G). A search of the sequence specificity of the known E. coli proteases suggested that this cleavage could have been perpetrated by the ompT gene product, a protease found in the E. coli outer membrane (Sugimura & Nishihara, (1988) J. Bacter., 170, 5625-5632; Earhart, et al., 1979). Degradation of PHA synthase by this protease is consistent with the observation that proteolysis appears to occur only after cell lysis.
In order to test this hypothesis and more importantly to prevent proteolysis of the synthase, plasmid pKAS4 was transformed into the E. coli ompT-strain, UT5600. The cells were grown and induced with IPTG and analyzed for proteolysis by Western blotting. The results indicate that the proteolysis has been eliminated. Furthermore, these results have been substantiated by the increase in specific activity of the synthase in crude cell extracts relative to E. coli JM105 as described subsequently.
Example 2: Purification and characterization of PHA Synthase from E. coli UT5600
The electroblotting procedure accompanied by analysis with anti-PHA synthase antibodies or protein sequencing revealed that in E. coli JM105, extensive proteolysis accompanied the overexpression of the synthase. pKAS4 was therefore transformed into E. coli UT5600 and isolated from this strain. The cells were grown at 30� C. to an A600 of 0.5 to 0.6 and subsequently induced by the addition of IPTG to a final concentration of 0.4 mM. The cells were then grown for an additional 4 h at 30� C. The doubling time is typically 50 min and yields of 8.2 g of cells per L of culture were obtained. The cells were harvested by centrifugation at 5000� g for 10 min at 4� C. and stored at -80� C. Cells can be stored under these conditions for months without detectable loss of activity. All purification steps were carried out at 4� C. unless otherwise stated. Cells (8.2 g) were suspended in 55 mL of 50 mM KPi (pH 7.0) containing 5% glycerol and lysed by either sonication or by use of the French Press. In the former case, lysis occurred using a Sonicator (Ultrasonic Processor xL; Farmingdale, N.Y.) turned on for 0.8 sec on a power setting of 9 to 10, then, turned off for 0.2 sec, and this process was repeated over a total period of 90 sec. The sample was continually cooled in an ice-salt bath and the temperature was monitored and not allowed to exceed 4� C.
Alternatively, the cells can be lysed by two passages through a French pressure cell at 16,000 psi and 4� C. The cell debris is then removed by centrifugation at 12,000 xg for 15 min. The supernatant is isolated, filtered through a 0.45 μm membrane, made 0.05% (w/v) in Hecameg™ and loaded directly onto a hydroxylapatite column (1.6�37 cm) which has been previously equilibrated for 30 min with 10 mM KPi (pH 7), 5% glycerol and 0.05% Hecameg™ (Buffer A) (flow rate 3 mL/min). The column is washed with 140 mL of Buffer A subsequent to loading and eluted with a 200�200 mL linear gradient from 10 to 300 mM KPi-Buffer A. Fractions, 10 mL, were collected and the synthase eluted between 110 and 150 mM KPi in 40 to 50 mL. The fractions containing activity can be pooled and rapidly frozen (liquid nitrogen) without significant loss in activity. This material (10 mL) in 130 mM KPi can be directly loaded onto a Q-Sepharose™ high performance FPLC column (t.25 mL) which has been previously equilibrated with 40 mL of 50 mM Tris�HCl (pH 8.5 at pH 4� C.), 5% glycerol and 0.05% Hecameg™ (Buffer B). Subsequent to loading, the column is washed with 25 mL of Buffer B at a flow rate of 1.2 mL/min and 5 mL fractions are collected. A 32.5�32.5 mL linear gradient from 0 to 1.5 M NaCl in Buffer B is then applied. Greater than 90% of PHA synthase elutes in fractions 2 and 3. Approximately 10% of the protein elutes in fractions 11 and 12 at 500 mM NaCl. The fractions are pooled and concentrated to 2.2 mg/mL using a YM 30 membrane and an Amicon filtration apparatus. The protein is then rapidly frozen in liquid nitrogen and stored at -80� C. The protein is stable for at least a month under these conditions. An identical protocol was used to purify the mutant synthases described below.
Results of Purification of PHA Synthase
Numerous investigators over the past decades have attempted to purify PHA synthases, both the soluble and granule bound forms, from a variety of sources. The most successful attempt at purification of the soluble form from A. eutrophus was reported by Haywood, et al. (1989). Subsequent to two column chromatographic procedures, they reported a specific activity of 0.05 μmol min-1 mg-1 with a 2% recovery of total units. Tomita, et al. (1983) have partially purified the soluble form of synthase from Z. ramigera, also with a 2% recovery of total units and a specific activity of 1.1 μmol min-1 mg-1. In both cases the enzyme preparation was reported to be unstable, precluding its characterization or mechanistic studies.
The results of the purification of the A. eutrophus synthase expressed in E. coli are summarized in Table I. SDS-gradient gels (4 to 15%) were also used to characterize the purification of PHA synthase.
In the crude extracts the activity is 10-fold higher than the "purified" preparation of Haywood, et al. (1989) and within a factor of two of the "purified" soluble synthase from Z. ramigera (Tomita, et al., 1983). With the re-engineered gene, in the crude extract, the activity is remarkably stable, losing only 50% of the initial activity after standing at 4� C. for 24 hours. Initial efforts to purify this protein resulted in substantial losses in activity and very broad peaks of activity from almost all chromatographic materials investigated. After extensive experimentation with a variety of detergents, it was discovered that addition of Hecameg™ enhanced recovery of material from the columns and decreased the elution volumes. Therefore, all buffers were made 0.05% in Hecameg™.
FPLC, providing rapidity of purification, and a variety of anion exchange materials including hydroxylapatite and Q-Sepharose™, were used to purify the synthase to greater than 90% homogeneity with a 40% recovery of overall units. Based on Coomassie staining of the protein in the crude cell extracts subsequent to SDS-PAGE, it was anticipated that a purification of approximately 20 fold would be required to obtain homogeneous protein. The protein isolated from the Q Sepharose™ column has a specific activity of 5 μmol min-1 mg-1 and upon freezing and thawing its activity increases to 8 μmol min-1 mg-1, a 16-fold overall purification. The enzyme is stable when rapidly frozen in liquid nitrogen for a period of at least a month.
TABLE 1__________________________________________________________________________Purification of PHA SynthaseaVolume Total Specific Protein Protein RecoveryStep mL Units Activity mg mg/mL % Purification__________________________________________________________________________crude50 166 0.51 325 6.5 --HAb40 70 2.5 28 0.7 42 4.9A-Sepc40 40 5.0 8 0.14 24 9.8frozen &40 64 8.0 8 0.14 38.5 15.7reassayed__________________________________________________________________________ a from 8.2 g of wet bacteria. b hydroxylapatite. c normalized as only 1/4 of the active HA fractions were loaded on the QSepharose column at a time.
Molecular Weight Determinations
Subunit molecular weight was determined using SDS-PAGE by the procedure of Laemmli (1970). The molecular weight standards included phosphorylase b (97.4 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and β-lactalbumin (18.4 kDa). The native molecular weight was examined using FPLC chromatography on a Superdex 200 (Pharmacia) column. Synthase (2.2 mg/ml or 0.1 mg/mL) was loaded onto the column equilibrated in 150 mM NaCl, 25 mM Tris (pH 7.5) and 5% glycerol. The following molecular weight standards were used: ribonuclease A (13.7 kDa), chymotrypsinogen (25 kDa), bovine serum albumin (68 kDa), aldolase (158 kDa), catalase (232 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa). The results are shown in FIG. 2.
The subunit molecular weight of the synthase based on SDS-PAGE is 64 kDa, similar to the Mr of 63.9 kDa defined by the gene sequence. Previous studies by Haywood, et al. (1989) to assess the native molecular weight of the synthase used crude enzyme isolated from A. eutrophus. Values of 150 to 170 kDa using gel filtration analysis and sucrose gradient ultracentrifugation, respectively, were reported. The molecular weight of the synthase isolated from its natural host, however, could be problematic as tightly bound proteins or tightly or covalently bound oligomeric or polymeric-β-hydroxybutyrate (PHB) could co-purify. Isolation of the enzyme from E. coli eliminates this complication.
Studies of the native molecular weight based on Superdex™ 200 size exclusion chromatography reveals molecular weights for the synthase of 60 KDa and 139 KDa. The fractions containing the putative monomer and dimer were concentrated and further analyzed by Western blotting. The proteins in both fractions cross react with antibodies raised to the PHA synthase and indicated a protein of 64 kDa. In addition, each fraction contained polymerase activity. Concentration of each fraction followed by rechromatography revealed the same monomer, dimer mixture. Thus, the protein as isolated from E. coli exists as an equilibrium between monomeric and dimeric forms.
PHA Synthase Assays
(a) Extraction with CHCl3
This method is a modification of the one described by Fukui, et al. (1976) Arch. Microbiol., 110, 149-156. A typical assay mixture contained in a final volume of 40 μL: 143 mM KPi (pH 7.0), 0.62 mM [3-3 H]-3-D-HBCoA (specific activity 1.1�106 cpm/μmol) and variable amounts of PHA synthase (0.07 to 10 μg), depending on the purity. The reactions were started by addition of enzyme after equilibration of the substrate in the buffer for 10 min at 25� C. The reaction mixtures were then incubated at 25� C. for 1 to 30 min. The reactions are stopped by addition of 100 μL of ice-cold 5% trichloroacetic acid with vortexing. The solution is then extracted with 0.5 mL of CHCl3. The layers are allowed to separate, the protein is at the interface, and 0.4 mL of the CHCl3 layer is transferred to a scintillation vial and the CHCl3 evaporated under a stream of argon. The PHA extracted is then quantified by scintillation counting after addition of 8 mL of Scint A-XF scintillation fluid.
(b) Loss of HBCoA monitored spectrophotometrically
HBCoA has been reported by Fukui, et al., (1976) to have an absorption max at 232 nm and an ε=4.5�103 M-1 cm-1. With purified synthase the decrease in absorbance at 232 nm can be monitored spectrophotometrically and continually.
(c) Formation of -SCOA monitored using Ellman's Reagent
Coenzyme A released during the PHA synthase catalyzed reaction can be measured using Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) (Ellman, (1959) Arch. Biochem. Biophys., 82, 70-77). A typical reaction mixture (360 μL) contained 1.0 mM HBCoA and 143 mM KPi (pH 7.0). The assay mixture was preincubated for ten min at 25� C. after which time the reaction was started by the addition of enzyme. Aliquots (40 μL) were removed at timed intervals and stopped by addition of 100 μL of 5% trichloroacetic acid. The precipitated protein was pelleted by centrifugation for 10 min and an aliquot (125 μL) of supernatant was added to 675 μL of 500 mM KPi (pH 7.5)/ DTNB [10 μL of a 10 mM stock solution in 500 mM KPi (pH 7.5)] was added to this mixture and incubated for two min at room temperature. The absorbance at 410 nm (ε=13.7 mM-1) was measured.
Previous workers have used a discontinuous assay to monitor PHA production incubation of [3 H]-HBCoA with synthase for various time periods (Fukui, et al., 1976). The reactions are stopped by addition of trichloroacetic acid and then the product is extracted into CHCl3. The CHCl3 is then removed and the residue quantitated by scintillation counting. Results of a typical assay are shown in FIG. 3a. At each concentration of protein examined, there is a lag phase which precedes the linear phase of product formation. The lower the concentration of the protein, the longer the lag phase.
The rates of the reaction used to calculate the specific activities reported in Table I have been determined by analyzing the linear part of the curve subsequent to the lag phase. The dependence of the specific activity of the protein on the protein concentration is shown in FIG. 3b. These results establish that during the purification of the synthase care must be taken to appropriately adjust the concentrations of synthase in the assay mixture and a full-time course for PHA production is required to insure that one avoids measuring the turnover in the lag phase of the reaction. The assays using purified synthase monitoring CHCl3 extractable polymer agree within 10% with the assays monitoring hydrolysis of 3-D-HBCoA as a change in the absorbance at 232 nm or formation of thiolate of coenzyme A using Ellman's reagent (1959).
Characterization of the PHA Product by Size Exclusion Chromatography
Assay mixtures, 50 μL, containing PHA synthase and [3 H]-HBCoA, subsequent to reaction for 30 min, were incubated with pronase (0.1 unit) in 140 mM KPi (pH 7.0) for 10 min at 25� C. The reaction was stopped by the addition of 0.5 mL CHCl3 and the polymer was extracted by vortexing for 1 min. The CHCl3 phase was dried over MgSO4, filtered through glass wool, and evaporated to less than 100 μL. This solution was injected directly onto a PlGel Mixed C column, and chromatographed using CHCl3 as eluate at a flow rate of 1 mL/min. For calibration, polystyrene standards of low polydispersity (Mr 600 Da to 1,860 kDa) were used (Polysciences, Inc., Warrington, Pa.). Fractions eluted from the column were analyzed by scintillation counting.
Abbreviations used in the foregoing description are: PHA, polyhydroxyalkanoate; PHB, polyhydroxybutyrate; wt, wild-type; IPTG, isopropylthio-β-D-galactosidase; Hecameg™, 6-O-(N-heptylcarbamoyl)-methyl-α-D-glucopyranoside; 3-D-HBCoA, hydroxybutyrylCoA; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecylsulfatepolyacrylamide gel electrophoresis; TBST, 10 mM Tris, 150 mM NaCl, 0.05% Tween™ 20; HRP, horse radish peroxidase; PVDF, polyvinylidene difluoride; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); KPi, potassium phosphate.
Modifications and variations of the present invention will be obvious to those skilled in the art from the foregoing detailed description and are intended to come within the scope of the following claims.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 2(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 56 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (synthetic)(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:CCGAATTCAGGAGGTTTTTATTATGGCTACCGGCAAAGGCGCGGCAGCTTCCACGC56(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (synthetic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:CGTGCAGCGGACCGGTGGCCTCGGCCTTGCCC32
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text:
I wonder if Yablo is going to do another one.
I wonder if Yablo is going to do another one.
'you people' (thats right, I said it) make me smile = )
party at my house!
cherry goes pop
it's time we let dolphins have a go at it
...also, duh.
I don't think that's true. several people (Hannah, Courtney, Corrina, MP, etc) have been likeable from the outset. Meanwhile, supre, you yourself are no longer despised... you've been promoted to indifference.
I never gave a shit one way or the other. Not on my radar screen.
You did?
When I first joined my first post was regarding getting alcohol into the campground. I was one of those people. It was on the old board. Closure.
white power?!

subset: commoncrawl


text:
Updated 03/25/2014
One of Rome's more celebrated outposts of luxe, El Toulà has the warm, welcoming comforts of a 19th-century country house: white walls, antique furniture in dark wood, framed prints, vaulted ceilings, and Venetian-style lamp shades. Along with the usual suspects of high-roller choices (caviar and blini), the chef offers a menu of contemporary interpretations of Italian classics, as well as those special El Toulà dishes with a Venetian slant (the mother restaurant is in Treviso): delicious, expertly prepared risottos, and a nod to the various sea creatures of the Adriatic coast and Venetian lagoon. Note that jacket and tie are required November through February.
Via della Lupa 29/b, Rome, Latium, 00186, Italy
Website: www.toula.it
Restaurant Details:
Updated 03/25/2014
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text:
News 13 - 24 of 28
Final 'Zero Dark Thirty' Trailer Shows More Desperation in Finding bin Laden2012-12-14 09:12:15
The dramatic trailer uncovers more intense action scenes, but at the same time also highlights the frustrating atmosphere surrounding the project to take down the Al Qaeda leader.
Jessica Chastain Looks Fabulous in Blue at 'Zero Dark Thirty' Hollywood Premiere2012-12-11 03:12:51
Donning a blue mini dress with nude-colored details on the front, Jessica becomes the belle of the ball during the premiere taking place at the Dolby Theatres, Hollywood.
Jessica Chastain Denies 'Zero Dark Thirty' Contains Pro-Torture Propaganda2012-12-11 09:12:08
Kathryn Bigelow and Mark Boal also weigh in on criticisms that the film presents some 'enhanced interrogation methods' used by the CIA on al Qaeda detainees.
'Zero Dark Thirty' to Be Premiered in Washington D.C. for Politicians2012-12-07 02:12:24
The Kathryn Bigelow-directed political thriller will hold its screening in the U.S. capital on January 8, 2013, a few days before its nationwide release.
Bret Easton Ellis Brands 'Zero Dark Thirty' Helmer Kathryn Bigelow 'Really Overrated'2012-12-07 08:12:11
The author of 'American Psycho' tweets sexist remark about Bigelow, saying that she'd be 'considered a mildly interesting filmmaker if she was a man.'
'Zero Dark Thirty' Is National Board of Review's Best Film of the Year2012-12-06 08:12:30
The Kathryn Bigelow-directed political thriller nabs the top prize from NBR, marking its second prestigious Best Film award in less than a week.
'Zero Dark Thirty' and 'Lincoln' Are Biggest Winners of 2012 NY Film Critics Awards2012-12-04 10:12:40
The Osama bin Laden hunt film claims the coveted Best Picture title in addition to two other prizes, while the Abraham Lincoln pic wins the Best Actor award for Daniel Day-Lewis.
Kathryn Bigelow and Mark Boal Clarify 'Zero Dark Thirty' Political Controversy2012-11-27 08:11:32
Speaking to 'Nightline' host Martha Raddatz, the 'Hurt Locker' director and screenwriter deny the Obama administration gave them access to top-secret info about Osama bin Laden attack in Pakistan.
Kathryn Bigelow's 'Zero Dark Thirty' Gets Intense New Trailer2012-10-12 09:10:43
It highlights the frustration faced by the military operatives and intelligence during the great hunt for then leader of Al Qaeda, Osama bin Laden.
Newly-Revealed Documents Show CIA's Involvement in 'Zero Dark Thirty'2012-08-29 11:08:40
Obtained by government watchdog Judicial Watch, the documents unveil that the CIA public information office is aware of 'Zero Dark Thirty' and possibly gives access to details of Bin Laden raid.
First Gritty Trailer for Bin Laden Hunt Film 'Zero Dark Thirty' Premiered2012-08-07 01:08:43
The trailer for Kathryn Bigelow's thriller shows random black lines on a map that end in Abbottabad, which was Osama Bin Laden's last hideout before he's killed in a massive raid by the U.S. army.
Kathryn Bigelow's Bin Laden Film Gets New Working Title, Sparks Protests in India2012-03-03 12:03:08
Being given a new moniker 'Zero Dark Thirty', the true story movie draws protests from Indian hardliners over the use of Pakistan flags on the set of the movie at a Chandigarh market.


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I had to post this after awful showing the last 3 days!!!
I had to post this after awful showing the last 3 days!!!
hmm i wanted it to pop up in the page.. hmm now sure what i did wrong
Oh, joy, yet another reactionary thread that could have been posted in the game thread.
"No matter how good you are, you're going to lose one-third of your games. No matter how bad you are you're going to win one-third of your games. It's the other third that makes the difference." ~Tommy Lasorda
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Board Moderators may, at their discretion and judgment, delete and/or edit any messages that violate any of the following guidelines: 1. Explicit references to alleged illegal or unlawful acts. 2. Graphic sexual descriptions. 3. Racial or ethnic slurs. 4. Use of edgy language (including masked profanity). 5. Direct personal attacks, flames, fights, trolling, baiting, name-calling, general nuisance, excessive player criticism or anything along those lines. 6. Posting spam. 7. Each person may have only one user account. It is fine to be critical here - that's what this board is for. But let's not beat a subject or a player to death, please.


text:
A new group calling itself Friends of the Bandshell has organized in an attempt to raise an estimated $150,000 needed to repair and build a foundation for the bandshell in Valley City's City Park.
Becky Heise, a member of the group, said the group met Thursday in the Barnes County Historical Society Museum to talk about what was needed and "to divvy up some tasks" on what will be needed.
Heise said the bandshell is currently on top of a collapsing basement damaged by floods in 2009 and 2011.
Read more in Friday's Times-Record.
Rally in the Valley community days weekend will have no shortage of wheels. A car show and vehicle events are set for Saturday, and a hill climb is planned for Sunday.
The Valley City Cruisers will hold its 15th annual car show Saturday at Rally in the Valley.
According to club president Terry Johnson, the group expects to attract about 120 cars, motorcycles, tractors and more at Central Avenue and Main Street for the event.
The Dakota Riders Motorcycle Club will hold its 35th annual Father's Day hill climb in Valley City Sunday as part of Rally in the Valley.
Ron Tolstad, left, and Tom Nix, Korean War veterans and members of Valley City's Veterans of Foreign Wars club, are the reason Main Street is lined with American flags this summer. The two hung flags on light posts down the street for Memorial Day, and they will keep them up until Labor Day.
Nix said, "I think they should be put up to remind everybody this is America and we're not ashamed of them."
The Barnes County Sheriff's Department has two new vehicles, this AWD Taurus and a four-wheel-drive Tahoe, which will remain unmarked, replaced older vehicles in the fleet. The new vehicles have the advantage of being able to go through snow, the Tahoe allows extra storage space for necessary equipment.
A Region VI housing study commissioned by South Central Dakota Regional Council (SCDRC) reiterates the need for senior friendly housing options throughout the nine-county region.
Becky Thatcher-Kellerm of the South Central Dakota Regional Council said Wednesday the study includes Barnes County, Stutsman County and surrounding counties, "and mentions a specific area in Barnes County.
More in Thursday's issue of the Times-Record.
An Oriska man was arrested late Monday for terrorizing after he discharged a weapon within his home while people were present.
According to Barnes County Sheriff Randy McClaflin, at about 10:30 p.m. he and four deputies responded to a 9-1-1 call from Oriska after a 54-year-old man fired four shots from a handgun into a wall.
Read more in Thursday's Times-Record
Valley City High School speech coach Eydie Wagar has been named North Dakota Class A Speech Coach of the Year for 2013 along with EDC coach of the year.
Wagar has been coaching speech at Valley City High School since the 2007-2008 school year.
More in Thursday's issue of the Times-Record.
With almost 7 billion people living on Earth, there's a lot going on in this world. In fact, there may be so much happening that leaves people asking, "What in the world is going on?" That's a question a local group hopes to answer each time they host an event in the community. The group that started 12 years ago aims to give people a broader understanding of Thomas Jefferson when they bring in an impersonator this fall.
Learn more about how the group started and some of the events they've hosted in Thursday's Times-Record.
All terrain vehicles; North Dakotans use them for work, for play and sometimes, for transportation. But as convenient and useful as an ATV may be, it's important to remember they can also be unsafe.
Riders who know and follow the rules are less likely to be injured or killed in an ATV accident.
The All Terrain Vehicle Safety Institute, a not-for-profit organization that plans and implements nation-wide programs of safety education and awareness, offers its eight golden rules for ATV safety in Wednesday's Times-Record.
Saturday will be a big day for Barnes County youth in 4-H, with the organization's annual expo planned for 1 p.m. to 4 p.m. at the Barnes County Historical Society Museum.
Tammy Jacobson, administrative assistant for the Barnes County Extension Service office, said Tuesday judging will take place from about 1 p.m. to 3 p.m., and the public is invited to check out projects this year's 14 entrants will have on display.
This year the expo is taking place during Valley City's annual Rally in the Valley.
Read more in Wednesday's Times-Record.


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text:
- Review: 'Zoo' Is a James Patterson Novel Brought to TV
- Jack Carter, Comedian Who Brought His Rapid-Fire Delivery to TV, Dies at 93
- Discovery Gains European Rights to 4 Olympics
- ArtsBeat: Merritt Wever on the 'Nurse Jackie' Series Finale and Zoey's Final Line
- ArtsBeat: 'True Detective' Recap: Big Blasts and Diminished Men
Life and Times of Willie Nelson
TV Special
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A daily updated summary of the week in TV.
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Executive Producers: Brian Hughes, Craig Haffner, Donna Lusitana
Genre: Documentary
Genre: Documentary
Program Synopsis
Biographical documentary of country music star, Willie Nelson.
What's On Tonight
- Amy Winehouse Documentary Lets Nobody Off the Hook
- Chris Squire, Founding Bassist With the Rock Band Yes, Dies at 67
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- Magali Noël, a French Singer and Actress and Muse to Fellini, Dies at 83
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- Royal Ballet's Focus on British Choreography Exposes Its Limits
- Marc Maron, at BAM, Finds That Happy Also Gets Laughs
- Review: 'Larry Kramer in Love & Anger,' Portrait of the Rebel as Gay Activist
- Harold Feinstein Dies at 84; Froze New York Moments in Black and White


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38 year old woman | Somerville, MA, USA | Seeking men 31-43 within 25 miles
Gamer girl
I am an energetic gamer girl looking for someone to share adventures with. I love playing games (both video games and board games), traveling (the crazier the place the better - think India and Cuba), reading, watching movies, being active outside (hiking, boating, etc.), exploring new places and trying new things. My ideal weekend has one busy day packed with fun activities and one lazy day spent snuggling on the couch watching movies or playing games. I'm a little bit sarcastic but I have a huge heart. I'm fiercely loyal to my friends and loved ones. I can be very silly. I don't get embarrassed easily. I eat lemons and think blueberries are the greatest. I've never met a hoodie I didn't like. I've traveled all around the world, and can't wait to see even more places. Halloween is BY FAR my favorite holiday. I'm proud to call myself a dork: I love scifi and fantasy novels, movies, and television. I have two tattoos and intend to get a third someday. I'm trying to turn myself into a runner. Or a dancer. Or both :) I make video games for a living and I LOVE it. I am SO lucky to have a job that I love.
Coffee and conversation , Dining out , Hobbies and crafts , Movies/Videos , Exploring new areas , Playing cards , Travel/Sightseeing , Video games
Aerobics, Dancing, Other types of exercise, Running, Swimming, Walking / Hiking, Weights / Machines
I exercise 3-4 times per week.
I have Cats
I love geeky things: books, television shows, movies, board games, and video games. George R.R. Martin is at the top of the list. The Wire is my all-time favorite show. I love lemons. I also love being outside: hiking, boating, travel.
I love playing video games, board games, being outside, going to the movies, travelling, eating ethic food, reading, watching tv, dancing, and doing crafts. Honestly, there aren't enough hours in the day for all the things I want to do.
The Hunger Games Trilogy.
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'2" (157cms)
|5'6" (167cms) to 8'11" (271cms)
|Body type:
|About average
|Slender, About average, Athletic and toned, A few extra pounds
|No preference
|Dark brown
|No preference
|No Way
|No Way
|Social Drinker
|Never, Social Drinker, Regularly
|Other profession
|No preference
Video Game Developer
|I'll tell you later
|No preference
|No preference
|Have kids:
|No preference
|Wants kids:
|Not sure
|No preference
|White / Caucasian
|No preference
|No preference
Humanistic Judaism (so more culturally Jewish than religiously Jewish, plus I'm agnostic)
|Graduate degree
|Bachelors degree, Graduate degree, PhD / Post Doctoral


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SVCC Theatre Department
2014-2015 Season
News & Announcements
- Shakespeare in the Park Broadens its Horizons!
- Sauk Valley Community College Theatre and Woodlawn Arts Academy with Centennial Auditorium team up to present one of musical theatre's greatest works. Join us for "Into the Woods" outside in the woods at Hoover Park in Sterling. Performances are Wednesday, July 8 through Sunday, July 12 at 7PM. Bring snacks, bring chairs, bring blankets, and for this event only, bring some adult beverages and enjoy a live theatrical performance under the stars. Thank you to our title sponsor Savant Capital Management! Please click the link for more information. In case of rain: the show will go on- join us for an "Into the Woods" musical revue inside Woodlawn Arts Academy. A note for parents: This production is suitable for ages 10 and up; however, parents are encouraged to use their own discretion as they know their children's sensitivities and maturity levels best. The first act is lighter in tone than the second. The show contains some mature content.
About the SVCC Theatre Department
Thank you for your interest in the Sauk Valley Community College theatre program! Our program offers a 2-year Associate of Arts and provides opportunities for you to perform as well as work behind the scenes on several productions throughout each academic year including a fall production, a spring production, and a summer Shakespeare production. After completing the 2-year program, you will have the fundamental experience and requirements to facilitate your transfer to a 4-year program. Come join us at SVCC and continue to build your theatre repertoire. If you are interested in learning more about our program email our Instructor of Theatre, Anna K. Kurtz at email@example.com.


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Thread: contest
July 2nd, 2010 #1
hey guys !! who know what link is the best where for finding contest about concept art and character design???
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Hide this ad by registering as a memberJuly 10th, 2010 #2
try this
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Pacific Fund Grant Applications Due May 28
Attending the Bioneers Conference in San Rafael to gain inspiration on green solutions...
Conducting experiments with photoelectron photoion coincidence spectroscopy at the Paul Scherrer Institute in Switzerland...
A combined fashion show and urban art exhibit at the Reynolds Gallery conceived of and carried out by students...
Hosting a hip-hop artist and medieval scholar to rap the Canterbury Tales...
Using a protein concentrator to make synthetic spider silk fibers...
What do all of these activities have in common? Aside from creating amazing learning experiences, they were all funded by Pacific Fund grants applied for through the College of the Pacific.
Field trips, travel related to student and faculty research, on-campus performances and guest speakers, equipment and lab supplies represent just some of the many ways that Pacific Fund grants enhance learning at Pacific. Students and faculty in the College of the Pacific are encouraged to apply for Pacific Fund grants that are awarded by the College in three cycles throughout the year: fall, winter and spring.
The next deadline for submitting your application is coming up soon: Friday, May 28, 2010.
To apply, download and complete the appropriate application form and submit it to Dr. Edie Sparks, Senior Associate Dean (WPC 111).
Click here for a Student Application
Click here for a Faculty Application
Qualifying projects will:
Advance the specific aims of the University's commitment to distinctiveness: investments to expand/improve existing programs; create new academic hallmarks; advance faculty teaching, scholarship and research; develop leadership, experiential learning, and international opportunities for students; promote/support student achievement.
Involve at least one College of the Pacific faculty or student member; projects involving more than one faculty member or student, or involving a faculty member and a student from the College are especially encouraged.
Projects must have measurable results within one academic year. In the application, the grant requester must provide clear, measurable goals and a plan for assessing the results as well as a plan for disseminating the results of the grant investment.
Student requesters must name a faculty sponsor who can comment on the scope and significance of their project. Faculty requesters must seek out a faculty member from another department in the University who is willing to serve as a "sounding board" for the project and provide feedback about the project.
Award Decisions
Pacific Fund grants will be awarded by a selection committee. Grant winners will be broadly representative of all departments and programs in the College. Multiple applications from a single department in a single cycle are unlikely to be funded. However, size of department (number of majors), as well as the type of funding sought in each application will be taken into consideration in determining whether a single department wins more than one grant. Faculty are encouraged to involve students in their projects.
Please note that Pacific Fund grants are reimbursement-based. Funds are not issued up front; rather, expenses must be documented and submitted for reimbursement.
Learn more about Pacific Fund Grants in the College


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Volkswagen Passat Problem Report
Volkswagen Passat Transmission damage due to faild transmission oil cooler
(7 reports)
Extensive transmission damage can occur in some of the automatic transmission models when the transmission oil cooler fails and allows coolant to mix with the automatic transmission fluid.
repair proposal exceeds the value of the car, until 3 days ago this extremely low mileage car ran beautifully, now it costs more to fix than what a trade is worth. - Visitor
1 person likes this
I had several problems With thi vehicle. The water pump Was replaced 3 times due to coolant leak and now my transmission slips. I may need to have it rebuilt or replaced. - MsVW
coolant mixed with transmission fluid in the transmission - Visitor
Related Items:
Hello, Lots of Smoke coming from the bottom right side of the engine compartment. It is not from the wheel, but the smoke comes from the wheel opening or the wheel well. There is also a burning sme...
In February 2010 at 114,000 miles, I had my valve cover gaskets and tensioners replaced because...
Why is my car using too much oil?
I had an oil change about 2 weeks ago. They used 20-50 oil when they replaced it. I had no war...
How much fluid needed to change oil


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26 year old man | Winter Garden, FL, USA | Seeking women 21-27 within 50 miles
Active within 24 hours
This is your life, are you who you want to be?" - Switchfoot
I'm really not a huge fan of dating sites because I feel it promotes more of a superficial form than anything. Think about it, you look at pictures before you read profiles. But, I do like it because it is another tool to meet cool people, or creepy people. Regardless, I'm excited to chat with you! Even though I don't take them often, I love taking adventures to different random places, doing different and random things. I hope to start doing that again more often. Graduated from UCF in psychology and moved to Winter Garden. Currently, I'm working for a local theme park in Event Management. People say they love working with me because I tell it like it is, but have fun at everything I do. My job gets crazy at times, comes with the territory. I really enjoy hanging out with friends, regardless of what we are doing. What am I looking for? Someone I can share life with. Someone I can call up and be excited about something going on, and vise versa. Someone I can plan trips and vacations with, and go out and enjoy what life has in store for us. Basically, I want someone who I can share life with, laugh with, you know. Let's chat?
Alumni connections , Coffee and conversation , Business networking , Cooking , Dining out , Movies/Videos , Music and concerts , Exploring new areas , Nightclubs/Dancing , Performing arts , Playing sports , Religion/Spiritual , Video games , Volunteering , Watching sports
Auto racing / Motorcross, Baseball, Billiards / Pool, Bowling, Football, Golf, Martial arts, Tennis / Racquet sports, Weights / Machines
I exercise 3-4 times per week.
I like Dogs, Fish
Middle of the Road
Beach and night is awesome.I love making small week/weekend trips and going on vacation.
University of Central Florida, Orlando, FL
Spending time with friends, there is nothing better!
If it involves hanging out with my friends, I'll do anything. I love traveling, movies, beach at night, music, concerts, baseball and football games, mall. Last minute random trips are awesome. I love just hanging out with people.
Textbooks!! I'm not much of a reader
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|5'10" (177cms)
|3'0" (91cms) to 8'11" (271cms)
|Body type:
|About average
|Slender, About average, Curvy, Athletic and toned, Full-figured, A few extra pounds
|No preference
|Light brown
|No preference
|No Way
|No Way, Cigar aficionado
|Social Drinker
|No preference
|Travel / Hospitality / Transportation
|No preference
|$50,001 to $75,000
|No preference
|Never Married
|Never Married
|Have kids:
|No preference
|Wants kids:
|Definitely, Someday, Not sure, Probably not
|White / Caucasian
|No preference
|Christian / Other
|No preference
|Bachelors degree
|Some college, Associates degree, Bachelors degree, Graduate degree, PhD / Post Doctoral
I have a Bachelors Degree in Psychology and Behavioral Forensics


text:
Submission Number:
New York
Initiative Name:
Proposed Consent Agreement In the Matter of Eminent, Inc., D/b/a Revolve Clothing, a corporation, File No. 122 3065
Matter Number:
122 3065
REJECT these consent orders in favor of meaningful consequences for mislabeling actual fur products as "faux." Companies must be held accountable for the products they market. Ignorance should not be a defense.


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Parsons Parking
100 W. Walnut St., Pasadena, CA 91124
International Champions Cup: LA Galaxy vs FC Barcelona
Parking Tue, Jul 21 1:00pm + 4hr after event
ParkWhiz worry-free cancellation (cancel anytime up to your start time)
Shuttle passes are free.
Amenities Included:
Upon arrival please place your ParkWhiz parking pass on the dashboard. Enter through the gate off of Fair Oaks Ave. Follow the attendant's directions. Clear signage will be in place directing you to the shuttle location.
446 People Recommend Parking Here
300 Left Comments
This parking facility consists of 2 multi level structures and one flat lot approximately 1 mile from the Rose Bowl (with a shuttle service that might cost $10).
The shuttle runs continuously from 2.5 hours prior to kick off and until one hour after the end of the Game.
The shuttle is free for all UCLA fotball games at the Rose Bowl, while for all other events at the Rose Bowl, the shuttle will be available for a fee.
Shuttle pick up location is on Pasadena Avenue on the West side of the Parsons lot. The shuttle takes you right down to the Rose Bowl Stadium. Pickup will be at the drop-off point so just return to that same location after the game.
Shuttles are equipped with wheelchair lifts.
Managed by LAZ Parking (Pasadena). This location is a commercial parking garage.
This parking lot is located on the southwest corner of W Walnut St and N Fair Oaks Ave, 3 blocks north of Colorado Blvd and one block south of the I-210. Look for the one of the tallest buildings in the area, 12 stories high.


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Public schools across the state perform one-time criminal history checks for teachers and other school employees as required by Pennsylvania law, but crimes committed by school staff during their employment may easily go undetected. Why?
Public schools across the state perform one-time criminal history checks for teachers and other school employees as required by Pennsylvania law, but crimes committed by school staff during their employment may easily go undetected. Why?
Despite a short time line and a potentially big price tag, several lawmakers on the Legislature's Education and Cultural Affairs Committee said they plan to introduce an education reform measure in the upcoming session that will incorporate recommendations from a $450,000 outside analysis of how the state funds local schools.
Lakeshore's Math Plinko and Launch It! Language Games have won the Teachers' Choice Award for the Classroom, making this the second year in a row that the company has won in this category.
With respect to these results, it's important to realize that children in the United States are not falling behind.
McGraw-Hill Education has announced the expansion of McGraw-Hill networks, the company's digital social studies program for middle and high school students, with the introduction of two new interactive geography titles.
A new Iowa media campaign aims to boost awareness of the state's efforts to encourage students to pursue STEM careers. Visiting Greenwood Elementary School in Des Moines, Gov. Terry Branstad unveiled a new logo for the STEM program, which is being led by Lt. Gov. Kim Reynolds.
ASCD recently announced the pre-conference session lineup for its 69th Annual Conference and Exhibit Show, being held in Los Angeles, Calif. The three-, two-, and one-day Pre-Conference Institutes will be held March 12–14, 2014, and offer intensive learning experiences on curriculum, instruction, assessment, and more.
March 19-22, 2014
Washington, D.C.
Through OverDrive, K-12 school libraries and public libraries will be able to offer 24/7 access to McGraw-Hill Professional digital titles in four broad categories: Business/Consumer, Science/Technical, Test Prep/Education, and Medical.
The solution was selected for its innovative new technology by a panel of industry experts working in conjunction with the editors of Live Design.
This week marks the official launch of Hours of Code, a campaign asking every U.S. teacher to dedicate one hour of class time to educating students about computer science and programming. Founded by brothers Ali and Hadi Partovi and their nonprofit Code.org, the event is timed in conjunction with Computer Science Education Week.
These leveled books in print improve students' comprehension of diverse genres, teach them to analyze and respond to text, and expand their ability to write in Spanish.
They added an hour to every school day and gave students laptops to continue their studies at home. They hired more teachers and worked with veterans and rookies alike to improve their classroom strategies. Three years later, student achievement at West Caldwell Tech has soared, with dramatic increases in state test scores, an increased graduation rate and more students applying for college.
STI, the 30-year-old maker of education management solutions for K12 schools, has scooped up Chalkable, an education-focused app store, as more venture capital flows into the education market.
3D-Hub, a provider of interactive 3D teaching software and virtual classrooms, has partnered with It Is 3D, which supplies 3D digital technologies to educational and industrial organizations around the world.


text:
God of War: Ascension Boxshots
NTSC-U (North America)
Login to submit missing boxshots.
God of War: Ascension Forum Posts
- Buy God of War: Ascension and get the God of War saga for free [2]
- God of War: Ascension multiplayer co-op weapons introduced, over 10 co-op combos via 4 weapons [5]
- My FINAL thoughts *spoilers* [4]
- God of War: Top Ten Deaths [Infographic] [1]
- Trial of arcamedes help [6]
- God of War: Ascension Bosses [5]
- God of War Neowiki [1]
- i am looking for a double parter [1]
- Glitchy? [1]
- Official "I own the game" Thread [5]
- All Collectibles Locations Guide [1]
- Looking to trade my King Leonidas (Gamestop) pre-order code for a Blade of Judgement (wal-mart) code [1]
God of War Series
- God of War: Ascension
- God of War Saga
- God of War: Ghost of Sparta HD
- God of War Origins Collection
- God of War III
- God of War Collection


text:
Judge not: GOP blocks dozens
of Obama court picks
The standoff could diminish the stamp that Obama leaves on the judiciary.
The standoff could diminish the stamp that Obama leaves on the judiciary.
One party's talking points are just an election cycle from turning over completely.
Republicans are prepared to bring up a rewrite of the Elementary and Secondary Education Act.
Ayatollah Khamenei is appeasing his hard-liners with tough rhetoric, but he keeps reviving the talks for a reason.
The Eurozone isn't on the verge of collapse, yet.
For GOP presidential hopefuls, there was no holiday from the nation's downward spiral.
DNC chair Debbie Wasserman Schultz has already been raising money for Clinton.
Over July 4, 1940, Roosevelt came up with the idea for one of his most famous speeches.
We'd be less violent, more mobile and in general more normal if not for Dixie.
We all know the warrior president kicked Indians off their land. What's less known is why.
We won't all be celebrating the same thing this July 4th.
One of America's biggest ideologues knows how to make a deal.
If the GOP wants to stay relevant, it has to become less hateful.


text:
59,334 original miles. Recent paint and interior. Beautiful woodgrained dash and gauges. Everything works, ready to drive and enjoy! While these were surely popular cars... More
Very original well maintained car that runs and drives perfectly. Other than an aluminum radiator for improved cooling, this car is all stock and... More
1950 Plymouth 2 door fastback Frame off restoration rebuilt engine all new cloth wiring new interior and carpet runs and drives well 65 mph... More
1950 Plymouth Special DeluxeThis Plymouth from 1950 is a really nice classic oldtimer. The 6 cylinder engine runs excellent and the manual gearbox shifts... More
1950 Plymouth P20, New radiator, water pump, fuel pump and wheel brake cylinders. A California "No Rust" car. Looks great and drives the same.... More


text:
Originally written on The Penalty Flag | Last updated 2/7/13
Just when you thought the Jets 2012 season couldn't look worse, the 2013 season match-ups were released. Here is the breakdown: Home: Patriots, Bills, Dolphins, Saints, Buccaneers, Steelers, Browns, Raiders Road: Patriots, Bills, Dolphins, Falcons, Panthers, Bengals, Ravens, Titans If the Jets can fix their offense, improve special teams, and develop their defense, there is a chance they will not repeat the performance 2012 and a step forward in 2013. To do that though, they would have to pull off a couple of tough wins along the way and win the games they can win... Read more
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The Global FlyFisher - The Largest and Best Place to go for Online Fly Fishing and Fly Tying
GFF Videos
Dark Hendrichson
Originator:Jim Misiura
Submitter:Martin Joergensen
|More great articles on The Global FlyFisher
|A couple of random articles
The ZuddlerQuestion: What to you get when you combine elements of a Muddler Minnow and a Zonker? Answer: A Zuddler - an excellent steelhead fly. Joe Emery and John Rode have combined a zonker strip, a muddler head and a cone head into a killer pattern.
August 14th 2004


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You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
39 year old man | Brentwood (SoCal), CA, USA | Seeking women 28-40 within 50 miles
Total disclosure, truth and honesty
Hello out there ...What do I write here? Who am I and what words describe me best. That's hard to do...I am a chef, a baseball player, good with numbers, a huge Chicago Bears fan. I am loyal and honest to the end. I am spontaneous and impulsive, I wear my heart on my sleeve. I was raised with traditional values, my parents are still together and my Dad is my idol. I think you must earn respect every day, i believe that you must pay your dues and start at the bottom. I love Chicago, its sports, geography, people and food. I believe a true friend is someone who when you talk to them it seems you pick up just where you left off, like no time has passed. I believe in showing affection, its like water, a person needs it to survive. I am romantic....gestures feel good to both parties. I believe in myself first, I have faith I will rise to any challenge. I regret nothing....I am happy the with the man in the mirror each morning and all my mistakes and failures played a part, why take them back. I believe passion comes from the soul and there is no way to fake it. I want from this site a way to meet someone I can marry someday....my free time was chafed by my career and exhaustion usually made the "get up and go meet someone" mindset seem miles away and impossible. Finally I am in a place where I have the time to be selfish. I am a good provider, selfless and generous. I want someone who wants to BE loved, it seems many don't care or have forgotten the importance of having that one person who is always on your team, by your side, who'll fight for you as I would for her. True companionship seems lost in text messenging, iphones, tweeting and so forth. I want someone who wants to put the work INTO a relationship as well, they are never easy. I want someone I can trust, who is honest with me and themselves, openminded to new things and not chained to past routines or notions. I am a chef, I am italian, I have a huge heart and a dry sense of humor. Friends always called me the witty one. I don't look for salaries, savings, possessions or stature. I need someone who is real and comfortable in their own skin. Skeptic as I am about this site but I haven't found any other options at this point. I am a keeper, so judge if you like from the few pictures I posted....I would expect nothing less. But if there is even an inkling of a connection then lets meet for coffee, what can that hurt. Meeting new people is fun and if it leads no where but a new acquaintance then that's still a win. D-
Camping , Coffee and conversation , Cooking , Dining out , Movies/Videos , Museums and art , Music and concerts , Exploring new areas , Playing sports , Travel/Sightseeing , Watching sports
Baseball, Billiards / Pool, Football, Golf, Skiing, Walking / Hiking, Weights / Machines
I exercise 1-2 times per week.
I have Cats
I like Dogs, Fish
My cat's name is Pocho.....and she is definitely a princess
Middle of the Road
I love disneyland, santa monica beach, los feliz, dodger stadium, san vicente, malibu creek state park, and my neighborhood.
Western Washington University, Bellingham, WA
I love mexican food, fresh and vibrant colors and flavors. I cook at home often. I love biggest loser, hells kitchen, person of interest, and the audition portion of talent shows.
In my less than frequent free time I like hiking, especially Los Leones trail up to Parker mesa overlook, and just getting out into the surrounding area. I like watching sports especially NFL and College, was head chef for OSU Beavs!
I recently read Raving fans and .... devil in the white city
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|5'10" (177cms)
|4'11" (149cms) to 5'10" (177cms)
|Body type:
|About average
|Slender, About average, Curvy, Athletic and toned
|Black, Blue, Brown, Grey, Green, Hazel
|Dark brown
|Auburn / Red, Black, Light brown, Dark brown, Blonde, Salt and pepper, Dark blonde, Platinum
|No Way
|No Way
|Social Drinker
|Social Drinker
|Retail / Food services
|Administrative / Secretarial, Artistic / Creative / Performance, Executive / Management, Financial / Accounting / Real Estate, Labor / Construction, Legal, Medical / Dental / Veterinary / Fitness, Political / Govt / Civil Service / Military, Retail / Food services, Sales / Marketing, Self-Employed / Entrepreneur, Student, Education / Teacher / Professor, Technical / Science / Computers / Engineering, Travel / Hospitality / Transportation, Nonprofit / Volunteer / Activist, Law enforcement / Security / Military, Fashion / Model / Beauty, Architecture / Interior design
I am the Chef de Cuisine-East Campus for LMU
|$50,001 to $75,000
|$35,001 to $50,000, $50,001 to $75,000, $75,001 to $100,000, $100,001 to $150,000, $150,001+
|Never Married
|Never Married, Widow / Widower, Divorced
|Have kids:
|Yes, and they sometimes live at home, No, Yes, and they live away from home, Yes, and they live at home
|Wants kids:
|White / Caucasian
|Asian, East Indian, Latino / Hispanic, Middle Eastern, Pacific Islander, White / Caucasian, Other
I am half Italian and half Irish...
|Spiritual but not religious
|No preference
|English, Spanish
|Chinese, English, French, German, Japanese, Russian, Spanish, Swedish, Tagalog
|Bachelors degree
|Some college, Associates degree, Bachelors degree, Graduate degree, PhD / Post Doctoral


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Sunlight is a vital component of good health. Like plants that thrive in the sun, we humans too depend on sunlight, in our case for the production of vitamin D. In the past few decades, however, cultural trends have steered us away from sun exposure. From fear of the potential dangers of UV radiation and the heavy promotion of sunscreen products to artificial work and recreational environments centered on virtual reality, we are all spending much more time indoors and away from the sun. In THE SUNLIGHT SOLUTION: WHY MORE SUN EXPOSURE AND VITAMIN D ARE ESSENTIAL TO YOUR HEALTH (Prometheus Books, $19.98), Laurie Winn Carlson delves into benefits of sunlight and the consequences we would face without it.
In this informative overview of an often-neglected topic, Carlson examines the historical and cultural factors that have created our indoor lifestyles and the medical evidence that suggests we need to get out in the sun.
"The Sunlight Solution is a wealth of knowledge about the history of vitamin D," says Bruce W. Hollis, Ph.D. Professor of Pediatrics, Biochemistry and Molecular Biology and Director of Pediatric Nutritional Sciences at the Medical University of South Carolina, Charleston. He adds, "Even I, who have studied this topic my whole career, learned a significant amount about the history. Also, the practical knowledge in this text will aid in maintaining the health of the general public."
Carlson begins by tracing the behavior patterns that have caused a shift indoors. She notes that it was common decades ago for children to spend hours playing outside. The lure of video games and computers has changed all that. Adults also live and work in the perpetual twilight of electric lighting. Though we feel comfortable, there is evidence that our bodies have not really adjusted to a lifestyle that is less than a century old. In fact, we may be suffering from the unnatural hours we work and sleep. The sun was once our
|Contact: Lynn Pasquale
Prometheus Books


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Featured News
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Atherton Hotel at OSU
Stillwater, OK 74078
Phone: 405-744-6835
Fax: 405-744-7652
The Atherton Hotel at OSU is a Georgian-style gem that opened in 1950 as the Union Club. The Atherton offers a warm and inviting atmosphere in stately elegance and sophistication. All 81 guest rooms feature a cozy down bedding system, high speed internet connections, and are adjacent to the Ranchers Club restaurant. The hotel is centrally located in the heart of the OSU campus.
Facility Amenities: ADA Compliant, Credit Cards Accepted, Exercise/Fitness Facilities, Gift Shop, Interior Corridors, Meeting Space Available, Restaurant on site
General Information: Number of Units: 81
Group Amenities: Baggage Handling, Group Rates Available, Meal Functions, Tour Group Welcome Reception
Room Amenities: Cable/Satellite TV, Coffee Maker, Hair Dryer, Iron/Ironing Board
Room Type: Suites
From I-35, go E on Hwy 51, turn N on Hester St. Go to University Ave and turn W into parking garage.
Primary Contact:
Lyn Putnam
Enjoy luxurious amenities and grand decor at Oklahoma's historic boutique hotels, where impeccable style and service will make you feel right at home.
Email: email@example.com