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The crystal structures of catalytic domain of HPTPbeta, both ligand-bound and ligan-free are described. These structures are useful in computer aided drug design for identifying compounds that bind or activate HPTPbeta and thereby modulate angiogenesis mediated disorders or diseases.
1. A method of identifying a drug candidate compound for the treatment of an angiogenic mediate disorder, comprising:
a) employing the three-dimensional structural coordinates of the human protein tyrosine phosphatase beta (HPTPbeta) catalytic domain [SEQ ID NO:7] as set forth in
b) selecting one or more compounds which have the best fit with the HPTPbeta catalytic domain [SEQ ID NO:7] as a set forth in
c) assaying analyzing the ability of said drug candidate compound to bind or modulate HPTPbeta activity.
2. A method of identifying a drug candidate compound for the treatment of an angiogenic mediate disorder, comprising:
a) employing the three-dimensional structural coordinates of the HPTPbeta catalytic domain [SEQ ID NO:7] as set forth in
b) selecting one or more compounds which have the best fit with the HPTPbeta catalytic domain [SEQ ID NO:7] as set forth in
c) assaying the ability of said drug candidate compound to bind or modulate HPTPbeta activity.
3. The method according to
4. The method according to
5. The method according to
6. The method according to
7. The method according to
8. The method according to
9. A method of identifying an inhibitor of an HPTPbeta molecule, comprising:
a) providing a crystal of HPTPbeta comprising the amino acid residues of SEQ ID NO:7 which either has space group P212121 and unit cell parameters a=39 Å, b=72 Å, c=120 Å and α=β=γ=90¯, or space group P21 with unit cell parameters of a=62 Å, b=70 Å, c=70 Å and α=90¯, β=93¯, γ=90¯;
b) subjecting the crystal in (a) to X-ray diffraction and determining the three-dimensional structure coordinates of the HPTPbeta catalytic domain;
c) employing the three-dimensional structure coordinates from (b) to graphically display the three-dimensional structure and identifying the active binding site residues of Asn74 Asn75 Ile76, Leu77, Cys152, Pro209, Asp210, His211, Gly212, Va1213, Pro214, Cys244, Ser245, Ala246, Gly247, Va1248, Gly249, Arg250, Thr251, Gly252, Thr253, Gin288, Thr289, Glu290, and Tyr293;
d) using the atomic coordinates of amino acids Asn74 Asn75 Ile76, Leu77, Cys 152, Pro209, Asp210, His211, Gly212, Va1213, Pro214, Cys244, Ser245, Ala246, Gly247, Va1248, Gly249, Arg250, Thr251, Gly252, Thr253, Gin288, Thr289, Glu290, and Tyr293 as determined in c) to generate a three-dimensional structure of a molecule comprising an HPTPbeta active site binding pocket so that the root mean square deviation of conserved residue backbone atoms is less than 1.5 Å as compared to the three-dimensional coordinates in a-c);
e) employing the three-dimensional structure from d) to design or select a potential drug candidate; and
f) contacting the potential drug candidate with the molecule to determine the abilut of the potential inhibitor to associated with the molecule.
10. A method of identifying an inhibitor of an HPTPbeta molecule, comprising:
a) providing a crystal of HPTPbeta comprising the amino acid residues of SEQ ID NO:7 which either has space group P212121 and unit cell parameters a=39 Å, b=72 Å, c=120 Å and α=β=γ=90¯, or space group P21 with unit cell parameters of a=62 Å, b=70 Å, c=70 Å and α=90¯, β=93¯, γ=90¯;
b) subjecting the crystal in (a) to X-ray diffraction and determining the three-dimensional structure coordinates of the HPTPbeta catalytic domain;
c) employing the three-dimensional structure coordinates from (b) to graphically display the three-dimensional structure and identifying the active binding site residues of Glu48, Glu49, Leu50, Lys51, Asp52, Va153, Gly54, Arg55, Asn56, Gln57, Ser58, Cys59, Asp60, Ile61, Ala62, Leu63, Leu64, Pro65, Glu66, Ile76, Leu77, Pro78, Tyr79, Asp80, Gly212, Va1213, Pro214, Va1284, His285, Met286, Va1287, Gln288, Thr289, Glu290, Cys291, and Gln292;
d) using the atomic coordinates of amino acids Glu48, Glu49, Leu50, Lys51, Asp52, Va153, Gly54, Arg55, Asn56, Gln57, Ser58, Cys59, Asp60, Ile61, Ala62, Leu63, Leu64, Pro65, Glu66, Ile76, Leu77, Pro78, Tyr79, Asp80, Gly212, Va1213, Pro214, Va1284, His285, Met286, Va1287, Gln288, Thr289, Glu290, Cys291, and Gln292 as determined in c) to generate a three-dimensional structure of a molecule comprising an HPTPbeta active site binding pocket so that the root mean square deviation of conserved residue backbone atoms is less than 1.5 Å as compared to the three-dimensional coordinates in a-c);
e) employing the three-dimensional structure from d) to design or select a potential drug candidate; and
f) containing the potential drug candidate with the molecule to determine the ability of the potential inhibitor to associate with the molecule.
The disclosure of U.S. Provisional Application Ser. No. 60/413,547 filed 25 Sep. 2002, is herein incorporated by reference in its entirety.
The present invention relates to three-dimensional structures of the catalytic domain of HPTPbeta [SEQ ID NO: 7], and structures derived therefrom.
HPTPbeta (Kruegar et al., EMBO J., 9, (1990)) has been suggested inter alia for modulating the activity of angiopoietin receptor-type tyrosine kinase Tie-2. See PCT Patent Application WO 00/65088. Inventors have shown in the present and concurrently filed patent applications that modulation of HPTPbeta modulates activities of both Tie-2 and VEGFR2. Therefore, HPTPbeta could be a target for the treatment of angiogenesis mediated disorders. However, the crystal structure of HPTPbeta has not been described. High-resolution 3D experimental models are needed to obtain insight into the mechanisms of HPTPbeta activation, the source of interactions between specific ligands and HPTPbeta, and to design better agonists and antagonists of HPTPbeta. Thus, there is a need for crystal structure of HPTPbeta.
The present invention attempts to address this need by providing a 3D structure of the catalytic domain of human HPTPbeta [SEQ ID NO: 7], and suitable means to design and identify potent and selective agonists or antagonist of the HPTPbeta for the treatment of angiogenesis mediated disorders.
In one aspect the invention provides for crystalline forms of the HPTPbeta catalytic domain [SEQ ID NO: 7] having unit cell dimensions of a=62Ý1 Å, b=72Ý1 Å, and c=70Ý1 Å, α=90¯, β=93Ý3¯, γ=90¯ in the space group P21 (monoclinic form) and unit cell dimensions of a=39Ý1 Å, b=71Ý1 Å, and c=120Ý2 Å, α=90¯, β=90¯, γ=90¯ in the space group P212121 (orthorhombic form).
In another aspect the invention provides for a method of identifying a compound useful for the treatment of an angiogenesis mediated disorder, comprising the steps of using a three-dimensional (3D) structure of HPTPbeta as defined by the atomic coordinates of
A method of identifying a compound useful for the treatment of an angiogenesis mediated disorder, comprising the steps of: providing a crystal of the complex between HPTPbeta and compound, or alternately exposing a crystal of HPTPbeta with a compound in aqueous media; exposing the complex to X-rays to generate a diffraction pattern; capturing the pattern to a recording device to generate diffraction data; processing the data to solve the complex structure; determining the locator of compound within complex structure; wherein the compound binding to the binding site of HPTPbeta, wherein the binding site is selected from the group consisting of P0, P+1, P−1, or mixtures thereof, indicates the compound is useful for the treatment of the HPTPbeta mediated disorder.
A method of identifying a compound useful for the treatment of an angiogenesis mediated disorder, comprising the steps of: selecting the compounds based on computer-aided drug design (CADD) using the coordinates from
These and other objects, features, and advantages will become apparent to those of ordinary skill in the art from a reading of the following detailed description and the appended claims.
The data shown in
Each of the nucleotide or amino acid sequences in the sequence listing is shown in Table A.
The present invention is directed to identifying or obtaining compounds useful for modulating HPTPbeta activity. Crystal structure information presented herein is useful in designing compounds and modeling them or their potential interaction with binding site(s) of HPTPbeta. Actual compounds may be identified from following design and model work performed in silico. A compound identified using the present invention may be effective for the treatment of an angiogenesis mediated disorder. These and other aspects and embodiments of the present invention are discussed below.
One aspect of the invention provides for the crystalline form of HPTPbeta. Four crystal structures of HPTPbeta are presented.
The coordinates of
Also, modifications in the HPTPbeta crystal structure due to e.g. mutations, additions, substitutions, and/or deletions of amino acid residues could account for variations in the HPTPbeta atomic coordinates. However, atomic coordinate data of HPTPbeta modified so that a ligand that bound to one or more binding sites of the HPTPbeta binding pocket would also be expected to bind to the corresponding binding sites of the modified HPTPbeta, and therefore are, for the purposes described herein as being aspects of the present invention, also within the scope of the invention. References herein to the coordinates of
Another aspect of the invention provides for the HPTPbeta binding pocket, wherein the binding pocket comprises at least the P(0), P(1) and P(−1) binding sites. The nomenclature of the binding sites is based on binding of phosphorylated peptides to PTPases, for example: P(0) is the active site of the enzyme, which accommodates the phosphotyrosine residue of the phosphopeptide; P(+1) is the site which accommodates the amino acid of the phosphopeptide that is adjacent to the phosphotyrosine in the direction of the carboxy terminus of the phosphopeptide; P(−1) accommodates the amino acid of the phosphopeptide that is adjacent to the phosphotyrosine in the direction of the amino terminus of the phosphopeptide. In HPTPbeta, P(0) is characterized by at least amino acid residues 152, 74-77, 209-214, 244-253, 288-290, and 293 of SEQ ID NO: 7; P(+1) is characterized by at least amino acid residues 76-80, 48-66, 284-292, and 212-214 of SEQ ID NO: 7; P−1 is characterized by at least amino acid residues 69-76, 119-123, and 149-154 of SEQ ID NO: 7.
For the first time, the present invention permits the use of virtual design techniques (i.e., computer modeling or in silico) to design, select, and synthesize compounds capable of inhibiting/stimulating or binding HPTPbeta. In turn, these drug candidates may be effective in the treatment of an angiogenesis mediated disorder.
The term angiogenesis mediated disorder is defined as a disorder that involves a modulation in angiogenic activity resulting in the biological manifestation of a disease, disorder, and/or condition; in the biological cascade leading to the disorder; or as a symptom of the disorder. The Applicants have shown that the process of angiogenesis is modulated by HPTPbeta. This involvement of HPTPbeta in an angiogenesis mediated disorder includes, but is not limited to, the following: (1) The modulation of HPTPbeta activity as a cause of the angiogenesis mediated disorder or biological manifestation, whether the HPTPbeta is modulated genetically, by infection, by autoimmunity, trauma, biomechanical causes, lifestyle, or by some other causes; (2) The modulated HPTPbeta activity is part of the observable manifestation of the disease or disorder. That is, the disease or disorder is measurable in terms of the modulated HPTPbeta activity. From a clinical standpoint, modulated HPTPbeta activity indicates the disease, however, HPTPbeta activity need not be the hallmark of the disease or disorder; (3) The modulated HPTPbeta activity is part of the biochemical or cellular cascade that results in the disease or disorder. In this respect, inhibiting or stimulating of HPTPbeta (per the respective therapeutic goal) interrupts the cascade, and thus controls the disease; (4) The angiogenesis mediated disease or disorder is not the result of modulation in HPTPbeta activity, but modulation of the HPTPbeta activity would result in amelioration of the disease. Modulation in HPTPbeta activity, as used herein, encompasses both unwanted or elevated HPTPbeta activity and desired or reduced HPTPbeta activity. As used herein, angiogenesis mediated disorders include: (1) those disorders, diseases and/or unwanted conditions which are characterized by unwanted or elevated angiogenesis, or (2) those disorders, diseases and/or unwanted conditions which are characterized by wanted or reduced angiogenesis.
The agents screened by the present invention may be used in a method for the treatment of a disorder mediated by elevated angiogenesis. The agents identified by the present invention may be used to treat diseases like diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease, vein or artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, Eales' disease, Behcet's disease, infections causing retinitis or choroiditis, presumed ocular histoplasmosis, Best's disease, myopia, optic pits, Stargardt's disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma, post-laser complications, diseases associated with rubeosis (neovasculariation of the angle) and diseases caused by the abnormal proliferation of fibrovasucular or fibrous tissue including all forms of proliferative viteroretinopathy. Agents screened by of the present invention can also treat diseases associated with chronic inflammation such as Crohn's disease and ulcerative colitis, psoriasis, sarcoidosis and rheumatoid arthritis. Other diseases that can be treated according to the present invention are hemangiomas, Osler-Weber-Rendu disease, or hereditary hemorrhagic telangiectasia, solid or blood borne tumors and acquired immune deficiency syndrome.
In one aspect, an agent may be used in a method for the treatment of a disorder mediated by reduced angiogenesis. As expected, this involves stimulated angiogenesis to treat a disease, disorder, or condition. It is likely that an agent that inhibits HPTPbeta would be used for treatment of an angiogenesis mediated disorder. The disorder is one characterized by tissue that is suffering from or is at risk of suffering from ischemic damage, infection, and/or poor healing, which results when the tissue is deprived of an adequate supply of oxygenated blood due to inadequate circulation (ischemic tissue).
In the present invention, it is possible to carry out virtual screening for drugs using the above-described atomic coordinates or coordinates derived therefrom.
Briefly, the atomic coordinates of the three-dimensional structure elucidated by the invention are input into a computer so that images of the structure and various parameters are shown on the display. Then, other resultant data are input into a virtual compound library. Since a virtual compound library is contained in a virtual screening software such as DOCK-4 (Kuntz, UCSF), the above-described data may be input into such a software. Candidate drugs may be searched for, using a three-dimensional structure database of virtual or no-virtual drug candidate compounds, such as MDDR (Prous Science, Spain).
The potential stimulating/inhibitory or binding effect (i.e., interaction or association) of a compound may be analyzed prior to its actual synthesis and testing by the use of computer modeling techniques. If the theoretical structure of the given compound suggests insufficient interaction and association between it and HPTPbeta, synthesis and testing of the compound may be obviated. However, if computer modeling indicates a strong interaction, the molecule may then be synthesized and tested for its ability to bind to or stimulate/inhibit HPTPbeta using various methods known in the art or as described in the co-pending application. In this manner, synthesis of inoperative compounds may be avoided.
Agonist/antagonist or binding drug candidates of HPTPbeta may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to bind with individual binding sites or combinations thereof (e.g., P0, P+1, and/or P−1) or other areas of HPTPbeta.
One skilled in the art may use any of several methods to screen chemical entities or fragments for their ability to associate with HPTPbeta and more particularly with the specific binding sites. This process may begin by visual inspection of, for example, the active site on the computer screen based on the HPTPbeta coordinates in any of the
Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include: (1) GRID (Goodford, P. J., A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules J. Med. Chem., 28, pp. 849-857 (1985)), available from Oxford University, Oxford, UK; (2) MCSS (Miranker, A. and M. Karplus, Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method Proteins: Structure, Function and Genetics, 11, pp. 29-34 (1991)), available from Molecular Simulations, Burlington, Mass.; (3) AUTODOCK (Goodsell, D. S. and A. J. Olsen, Automated Docking of Substrates to Proteins by Simulated Annealing Proteins: Structure, Function, and Genetics, 8, pp. 195-202 (1990)), available from Scripps Research Institute, La Jolla, Calif.; and (4) DOCK (Kuntz, I. D. et al., A Geometric Approach to Macromolecule-Ligand Interactions J. Mol. Biol., 161, pp. 269-288 (1982)), available from University of California, San Francisco, Calif.; (5) GLIDE available from Schrodinger Inc.; (6) FlexX available from Tripos Inc; (7) GOLD (Jones et al., J. Mol. Biol., 245, 43-53, 1995), available from the Cambridge Crystallographic Data Centre.
Once suitable chemical entities or fragments have been selected, they can be assembled in silico or synthesized into a single drug candidate. In silico assembly may proceed by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of HPTPbeta. This would be followed by manual model building using software such as QUANTA or SYBYL. Chemical syntheses are by those methods well-known in the art.
Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include the following: (1) CAVEAT (Bartlett, P. A. et al, CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules. In Molecular Recognition in Chemical and Biological Problems, Special Pub., Royal Chem. Soc., 78, pp. 182-196 (1989)), available from the University of California, Berkeley, Calif.; (2) 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.). This area is reviewed in Martin, Y. C., 3D Database Searching in Drug Design, J. Med. Chem., 35, pp. 2145-2154 (1992)); and (3) HOOK (available from Molecular Simulations, Burlington, Mass.).
Instead of proceeding to build an HPTPbeta agonist or antagonist in a step-wise fashion one fragment or chemical entity at a time as described above, drug candidates useful for the treatment of angiogenesis mediated disorders may be designed as a whole or de novo using either an empty active site or optionally including some portion(s) of a known agonist or antagonist. These methods include the following: (1) LUDI (Bohm, H. -J., The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors, J. ComR. Aid. Molec. Design, 6, pp. 61-78 (1992)), available from Biosym Technologies, San Diego, Calif.; (2) LEGEND (Nishibata, Y. and A. Itai, Tetrahedron, 47, p. 8985 (1991)), available from Molecular Simulations, Burlington, Mass.; (3) LeapFrog (available from Tripos Associates, St. Louis, Mo.). Examples of known agonist or antagonists are described in WO 02/26774 A2.
Once a compound has been designed or selected by the above methods, the efficiency with which that compound may bind to HPTPbeta may be tested and optimized by computational evaluation. For example, an effective HPTPbeta agonist must preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding). Thus, the most efficient HPTPbeta agonist should preferably be designed with deformation energy of binding of not greater that about 10 kcal/mole, preferably, not greater than 7 kcal/mole. HPTPbeta agonists may interact with the enzyme in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free compound and the average energy of the conformations observed when the agonist binds to the enzyme.
A compound designed or selected, as binding to HPTPbeta may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme. Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole, and charge-dipole interactions. Specifically, the sum of all electrostatic interactions between the compound and the enzyme when the compound is bound to HPTPbeta, preferably make a neutral or favorable contribution to the enthalpy of binding.
Specific computer softwares are available in the art to evaluate compound deformation energy and electrostatic interaction. Examples of programs designed for such uses include: Gaussian 92, revision C [M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa. ˋ1992]; AMBER, version 4.0 [P. A. Kollman, University of California at San Francisco, ˋ1994]; QUANTA/CHARMM [Molecular Simulations, Inc., Burlington, Mass. ˋ1994]; and Insight II/Discover (Biosym Technologies Inc., San Diego, Calif. ˋ1994). Other software packages will be known to those skilled in the art.
Once an HPTPbeta-binding compound has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided. Such substituted chemical compounds may then be analyzed for efficiency of fit to HPTPbeta by the same computer methods described in detail, above.
For the first time, this invention allows one skilled in the art to study the binding of chemical entities to HPTPbeta by exposing either individual compounds or mixtures of compounds (such as may be obtained from combinatorial libraries) into HPTPbeta crystals, or, alternatively, by co-crystallization of the substances of interest with HPTPbeta, using methods known to those of ordinary skill in the art, and the crystallization conditions based on those described in the following examples. Acquisition and analysis of X-ray diffraction data from these crystals can be then performed using standard methods. If substance or substances bind to HPTPbeta then positive difference electron density will be observed in the Fourier maps calculated using the X-ray diffraction intensities and phases obtained from the HPTPbeta models presented in
The intracellular domain (ICD) of HPTPbeta (SEQ ID NO: 3) is cloned from human fetal heart cDNA containing the full gene of HPTPbeta (SEQ ID NO: 1) by PCR using Advantage Polymerase (Clontech) and the primers:
The sequence [SEQ ID NO: 5] encoding base pairs 5014 to 5949 of SEQ ID NO: 1 is cloned into the vector pMALc2x (New England Biolabs) using the following oligonucleotides:
The HPTPbeta truncated gene [SEQ ID NO: 5] (Wang, Y. & Pallen, C. J. The journal of Biological Chemistry, 267(23), pp16696-16702, 1992) is amplified with these primers by PCR using the pPCRScript plasmid described above as the template, digested with SnaB1 and HindIII and ligated to pMAL-c2x that is pre-digested with Asp700 and HindIII to create plasmid pMAL-c2x-PTPbeta(5014-5949). The protein construct thus encoded is maltose-binding protein from Escherichia coli (MBP) followed by a Factor Xa cleavage site followed by base pairs 5014 to 5949 of HPTPbeta SEQ ID NO: 1. A six-histidine tag is added to the carboxy terminus using the QuikChange Site Directed Mutagenesis kit (Stratagene) and the following primers:
The coding sequence of the clone (pMAL-c2x-PTPbeta(5014-5949)His6) is verified by DNA sequence analysis. Finally, the Factor Xa site is changed to a TEV protease cleavage site using the QuikChange Site Directed Mutagenesis kit and the following primers:
The protein construct thus encoded is maltose-binding protein from Escherichia coli (MBP) followed by a TEV protease cleavage site, followed by base pairs 5014 to 5949 of HPTPbeta SEQ ID NO: 1, followed by a six-histidine tag. After TEV protease cleavage, the resulting protein contains a non-native Glycine residue on the amino-terminus and a six-histidine tag on the carboxy-terminus (SEQ ID 7).
The coding sequence of the final clone (pMAL-TEV-PTPbeta(5014-5949)His6) is verified by DNA sequence analysis and used for recombinant protein production, Escherichia coli BL21-RIL cells (Strategene) are used as the host strain. Bacteria are grown in a 10-liter fermenter, using Super Broth medium (30 g tryptone, 20 g yeast extract, 7.5 g NaCl per liter) supplemented with 0.2% glucose and 100 mg/L, ampicillin at 22C to mid-log phase, at which point the bacteria are induced with 0.5 mM β-isopropyl thiogalactopyranoside and harvested by centrifugation 16 hours after induction.
26 grams of cell pellet containing the overexpressed protein are suspended in 287.5 ml of 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol, pH 8.0 (Buffer A) containing 4 Complete-EDTA free protease inhibitor tablets (Roche). Cells are lysed during 2 passes through a French press at 12000 psi, at 4¯ C. The lysate is centrifuged for 40 minutes at 17000 rpm, using a JA-17 (Sorvall) rotor at 4¯ C. Resulting supernatant (295 ml) is loaded at 10 ml/min, 4¯ C. on a 73 ml Ni-NTA column which is pre-equilibrated with Buffer A. The column is washed with 200 ml of 75 mM Imidazole in Buffer A and the protein is eluted with 175 mM of imidazole in Buffer A (6 ml fractions, 10 ml/min). Fractions containing the fusion protein are pooled based on Coomassie Blue-stained SDS-PAGE analysis. The pool is diluted to 515 ml with pure water to final conductivity of 10.42 mS/m (approximately 125-135 mM NaCl).
Diluted fusion protein is loaded onto a 150 ml Resource Q15 (Pharmacia) column pre-equilibrated with 10 mM Tris-HCl, 2 mM DTT, pH 7.2. The protein is eluted with a 22.5-29% linear gradient of 10 mM Tris-HCl, 2 mM DTT, 1 M NaCl, pH 7.2 (10 ml fractions are collected at 10 ml/min flow rate). Fractions are pooled based on SDS-PAGE analysis.
N-Octyl β-glycopyranoside (NOG) and dithiothreitol (DTT) are added to the pool resulting in final concentrations of 0.25% and 2 mM, respectively. Approximately 1.5 mg of recombinant Tobacco Etch Virus protease (TEV protease, Invitrogen) are added to the pool (using 1/200 weight ratio of protease to substrate) and the reaction mixture is incubated with stirring for 17 hrs at 4¯ C. SDS-PAGE analysis reveals 65% cleavage efficiency. Further addition of TEV protease does not result in additional cleavage.
Reaction mixture is adjusted to the final conductivity of 10.4 mS/cm by addition of 10 mM Tris-HCl, 2 mM DTT, 0.25% NOG, pH 7.2 (Buffer B) and loaded on a 150 ml Resource Q15 column pre-equilibrated with Buffer B. Proteins are eluted with a 19-24% linear gradient of 1M NaCl in Buffer B. Unfortunately, the fusion protein and the cleaved PTPβ catalytic domain [SEQ ID NO: 7] do not separate well using this method. Fractions containing the cleaved catalytic domain are pooled based on SDS-PAGE analysis.
The pool containing the PTPbeta catalytic domain [SEQ ID NO: 7] as well as some of the uncleaved fusion protein is concentrated down to 4.5 ml using a YM-10 membrane in an Amicon stirred cell and slowly passed through a 3 ml amylose resin (Quiagen) column pre-equilibrated with Buffer-B. Flow-through is collected and the column is washed with 3 ml of Buffer B. Combined flow-through and wash are loaded on a 48æ5.0 cm Superdex 75 prep grade column, pre-equilibrated with Buffer B. The column is eluted with Buffer B at 2 ml/min while 10 ml fractions are collected. Two peaks are observed indicating good separation between the full-length fusion protein and the HPTPbeta catalytic domain [SEQ ID NO: 7]. Fractions are analyzed by SDS-PAGE and those found to contain pure protein are pooled. HPTPbeta catalytic domain [SEQ ID NO: 7] is concentrated to 9.8 mg/ml using an Amicon stirred cell equipped with YM-10 membrane. Total yield of HPTPbeta catalytic domain [SEQ ID NO: 7] is 58.6 mg based on the OD280 measured in 6M guanidine-hydrochloride, pH 8.0.
HPTPbeta catalytic domain [SEQ ID NO: 7] is crystallized in hanging drops via the sparse-matrix approach using crystallization screens manufactured by Hampton Research and Emerald Biostructures (currently DeCode Genetics). Several polyethylene glycol (PEG)/magnesium chloride conditions are identified as crystallization leads, which are eventually refined to the following condition: 18% PEG8000, 100 mM TRIS-HCl pH 7.5, 1% β-mercaptoethanol (BME), 0.2 M MgCl2 in 6 μl drops containing equal amounts of protein and reservoir solutions. Under these conditions, crystals usually appear after 2-5 days, reach their maximum size of 0.2æ0.2æ0.3 mm in 6-8 days and decay within 3-4 weeks after set-up. The best crystals are grown by streak-seeding, which results in more reliable nucleation. For structural studies, individual protein crystals are cryoprotected by immersion into Paratone-N oil followed by flash cooling in a stream of nitrogen gas at 100K.
Two clearly different crystal morphologies are observedone is subsequently identified as orthorhombic and the other as monoclinic crystal form. Laboratory X-ray source equipped with a CCD X-ray detector is used to collect 2.3 Å data from the orthorhombic crystals, which are found to belong to the space group P212121 with unit cell dimensions of a=39.25 Å, b=71.13 Å, and c=119.91 Å, α=90¯, β=90¯, γ=90¯, and one molecule in the asymmetric unit (a.u.). Programs of the HKL2000 suite are employed to index, integrate, and scale the diffraction data.
The crystal structure is solved via molecular replacement (CCP4 program AMoRe) (Collaborative Computational Project, Number 4. 199 Acta Cryst. D50, 760-763; and Navaza, J. Act Cryst. A50, 157-163 (1994) using the published structure of PTPμ from the Protein Data Bank (PDB), PDB code 1RPM as the search model. After multiple rounds of manual rebuilding (program O) and refinement (programs Refimac and SHELXL), the crystal structure of the enzyme contains residues 24 to 305, as well as 115 water molecules, and had the Rwork of 21.0% and Rfree of 26.2%. Concomitantly we are able to collect high-resolution data for both the orthorhombic and the monoclinic crystal forms of the enzyme using a synchrotron radiation source (beam lines 17-ID and 17-BM of the Advanced Photon Source at the Argon Naturation LaboratoryAPS/ANL). The monoclinic form of the enzyme is found to belong to the space group P21 with unit cell dimensions of a=61.89 Å, b=71.53 Å, and c=70.35 Å, α=90¯, β=93.25¯, γ=90¯. Using synchrotron data we are able to refine the structure of the enzyme in the orthorhombic crystal form to 1.75 Å resolution (Rwork=19.0%, Rfree=22.0%) and the monoclinic crystal form (which has two protein molecules in the a.u.) to 1.9 Å resolution (Rwork=20.0%, Rfree=24.0%). Validation of structures using programs PROCHECK and SFCHECK does not reveal any geometric abnormalities. Finished structures contain residues 19 to 310 as numerous water molecules.
Proprietary X-ray diffraction data were collected at beamlines 17-ID and 17-BM in the facilitates of the Industrial Macromolecular Crystallography Association Collaborative Access Team (IMCA-CAT) at the Advanced Photon Source. These facilities are supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Illinois Institute of Technology (IIT), executed through IIT's Center for Synchrotron Radiation Research and Instrumentation. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under Contract No. W-31-109-Eng-38.
To study the interaction of ligands with PTPβ we expose (soak) various molecules of interest into both the orthorhombic and the monoclinic crystals at a concentration of 1-10 mM in the crystallization buffer, using exposure times from 2 to 24 hours. Monoclinic crystals of the enzyme can be readily soaked (2-4 hours) with compounds of interestand the resulting soaked crystals usually (with a notable exception that is outlined later) do not deteriorate. True ligands and/or inhibitors can be unambiguously identified, by analyzing electron density maps calculated using X-ray diffraction data collected from the soaked crystals. Interestingly, when molecules that belong to the family of Compound 2 are soaked into the monoclinic crystals, the latter deteriorate to the point where collection of X-ray diffraction data is impossible. Fortunately, the orthorhombic crystals of PTPbeta catalytic domain [SEQ ID NO: 7] can be successfully soaked with compounds of this class, which is particularly interesting in view of the fact that the orthorhombic crystals are nigh impossible to soak with many other classes of compoundseven 24-hr soaks do not result in small molecule penetration into the crystal lattice.
X-ray diffraction data from soaked crystals are collected and processed at APS/ANL in the same manner as described above, X-ray crystal structures of the complexes are solved via molecular replacement (AMoRe) using corresponding native structures for each crystal form as search models. Several rounds of manual rebuilding (O) and refinement (Refmac, SHELXL) are employed, after which the inhibitor molecules are built into the electron density (Quanta, SPARTAN) and refined. The geometry of the structures is analyzed using programs PROCHECK and SPFCHECK.
The two final models of the protein contain residues 19-310, which are clearly defined in the electron density maps of the orthorhombic and monoclinic forms, respectively. Loop 89-96 is entirely disordered in the orthorhombic form (and therefore is omitted from the final model) and is mostly disordered in the monoclinic form. The orthorhombic crystal form of the PTPbeta catalytic domain [SEQ ID NO: 7] contains one promoter in a.u. whereas the monoclinic form contains two promoters in the a.u. Root mean square deviation (rmsd) of the protein backbone atoms of the two molecules found in the monoclinic a.u. is 0.45 Å, whereas rmsd of the orthorhombic PTPbeta catalytic domain [SEQ ID NO: 7] and the monoclinic promoters is 0.55-0.66 A. Upon close inspection, it is evident that there are no major structural differences between the three moleculesthe changes are confined to minor movements of the loops and rearrangements of conformationally unrestrained side chains of surface-exposed amino-acids. Therefore it is sufficient to supply the overall description of the unliganded PTPbeta catalytic domain [SEQ ID NO: 7] performed using the best-defined (highest resolution) promoterthe one found in the orthorhombic crystal form.
PTPbeta belongs to a broad family of PTPases that are extremely dissimilar in terms of their biological function, intracellular localization, and domain structure. These very diverse enzymes have one feature in common, namely their catalytic domain, the fold of which is preserved throughout this whole class of PTPases,. Historically, the first human PTPase to be discovered and studied is PTP-1B, which is a soluble single-domain phosphatase that was first identified in human placenta in 1989. Because of its early discovery and biological significance, PTP-1B is the most studied PTPase to-date, with over 40 X-ray crystal structures, both unliganded and liganded, available in the PDB. PTP-1B structure is therefore considered to be an archetype of the catalytic domain of this class of PTPases. Here, we use comparisons between PTPbeta catalytic domain [SEQ ID NO: 7] and PTP-1B (PDB code 2HNP) structures to illustrate the relationship between our crystal structure of the PTPbeta catalytic domain [SEQ ID NO: 7] and the rest of the PTPases of this class.
Similar to PTP-1B, the crystal structure of PTPbeta catalytic domain [SEQ ID NO: 7] reveals a common PTPase fold, consisting of two closely-packed compartments: the alpha-helical domain and the beta-sheet one (
The active site of the enzyme (residues 152-153, 244-253, 288-290, and 293) is located at the junction of the two domains and is occupied by four water molecules. A characteristic loop, containing a Trp-Pro-Asp triad (the WPD loop, residues 208-214) is located near the binding site. In PTP-1B and other phosphatases this loop is known to adopt a different conformation upon binding of ligands in the active site. As will be shown in the next example this conformation change also takes place when PTPbeta catalytic domain [SEQ ID NO: 7] binds to ligands (
Additional small, but important distinctions between PTPbeta catalytic domain [SEQ ID NO: 7] and PTP-1B are further discussed in the example 7.
Boc-D-Phe(4-NO2)-NMe: Boc-D-Phe(4-NO2)-OH (4.0 g, 12.9 mmol) is dissolved in anhydrous tetrahydrofuran (20 mL) with 4-methylmorpholine (1.56 mL, 14.2 mmol). Isobutylchloroformate (1.84 mL, 14.2 mmol) is dropwise added at 0¯ C. and the mixture is stirred for 1 hr. at 0¯ C. Methylamine (12.9 mL, 2.0 M in tetrahydrofuran) is added dropwise at 0¯ C. and the mixture is stirred for 18 hr. at room temperature. The mixture is then recrystallized from 1:1 DCM:methanol to give a white solid.
H-D-Phe(4-NO2)-NMe: Boc-D-Phe(4-NO2)-NMe (1.5 g, 4.64 mmol) is dissolved in HCl (10 mL, 4.0 M in 1,4-dioxane), and the resulting mixture is stirred at room temperature for 1 hr. Ether (60 mL) is added to the mixture and the resulting precipitate is collected by filtration to yield pure white product.
CBZ-D-Phe(4-NO2)-NMe: H-D-Phe(4-NO2)-NMe (410 mg, 1.84 mmol) is dissolved in anhydrous DCM (10 mL) and diisopropylethylamine (0.352 mL, 2.02 mmol). Benzyl chloroformate (0.263 mL, 1.84 mmol) is added dropwise at 0¯ C., the mixture is allowed to warm to room temperature and is stirred for 72 hr. The solution is partitioned between DCM and 1N HCl. The organic layer is washed with brine, dried over MgSO4, filtered and evaporated to give crude white solid.
CBZ-D Phe(4-NH2)-NMe: CBZ-D-Phe(4-NO2)-NMe (80 mg, 0.224 mmol) is dissolved in EtOAc:ethanol (1:1, 2 mL) and tin(II) chloride dihydrate (252 mg, 1.12 mmol) is added. The mixture is stirred at room temperature for 18 h. The reaction is partitioned between EtOAc (25 mL) and 1N NaOH (25 mL). The organic layer is washed twice more with 1N NaOH (25 mL). The combined organics were dried over MgSO4, filtered and evaporated to give pure yellow oil.
(S)-[1-Methylcarbamoyl-2-(4-sulfoamino-phenyl)-ethyl]-carbamic acid benzyl ester: In a dry flask 0.107 g of the aniline compound is dissolved in 2 mL pyridine. To this solution is added 0.156 g of sulfurtrioxide-pyridine complex. The mixture is stirred 5 minutes then diluted with 25 mL of 7% ammonium hydroxide. The mixture is evaporated down to an off-white solid and purified to provide 0.056 g of product as its ammonium salt. 1H(D2O): δ7.26-7.20 (m, 3H), 7.11-6.96 (m, 6H), 4.90-4.78 (m, 2H), 4.08 (t, 1H, J=8.3 Hz), 2.84-2.66 (m, 2H) 2.50 (s, 3H)
Boc-Phe(4-NO2)-NMe: Boc-Phe(4-NO2)-OH (10.0 g, 32.3 mmol) is dissolved in anhydrous tetrahydrofuran (32.2 mL) with 4-methylmorpholine (3.90 mL, 35.4 mmol). Isobutylchloroformate (4.18 mL, 32.3 mmol) is dropwise added at 0¯ C. and the mixture is stirred for 1 hr. at 0¯ C. Methylamine (332.3 mL, 2.0 M in tetrahydrofuran) is added dropwise at 0¯ C. and the mixture is stirred for 18 hr. at room temperature. The mixture is then recrystallized from 1:1 DCM:methanol to give 6.69 g pure white solid.
H-Phe(4-NO2)-NMe: Boc-D-Phe(4-NO2)-NMe (1.5 g, 4.64 mmol) is dissolved in HCl (10 mL, 4.0 M in 1,4-dioxane) and the resulting mixture is stirred at room temperature for 1 hr. Ether (60 mL) is added to the mixture and the resulting precipitate is collected by filtration to yield pure white product.
{2-(4-Hydroxy-phenyl)-1-[1-methylcarbamoyl-2-(4-nitro-phenyl)-ethylcarbamoyl]-ethyl} carbamic acid tert-butyl ester: H-L-Phe(4-NO2)-NMe (200 mg, 0.770 mmol) is dissolved in 1 mL DMF. Diisopropylethylamine (209 mg, 1.62 mmol), EDC (162 mg, 0.847 mmol), HOBt.H2O (130 mg, 0.847 mmol), and Boc-Tyr (238 mg, 0.847 mmol) are added and the mixture is stirred for 18 hr. at 20 ¯ C. The mixture is partitioned between water and EtOAc (2æ60 mL). The organics are combined and washed with brine, dried over MgSO4, filtered and evaporated to give crude product. Purification by flash chromatography, which is eluted with 97:3 DCM:methanol to give pure white solid.
{2-(4-Hydroxy-pyenyl)-1-[1-methylcarbamoyl-2-(4-amino-phenyl)-ethylcarbamoyl]-ethyl}-carbamic acid tert-butyl ester: {2-(4-Hyrdroxy-phenyl)-1-[1-methylcarbamoyl-2-(4-nitro-phenyl)-ethylcarbamoyl]-ethyl} carbamic acid tert-butyl ester (300, 0.617 mmol) is dissolved in methanol (10 mL). To this was added palladium on carbon (10% by weight, 100 mg). The reaction is placed under a hydrogen atmosphere until reaction is complete (tlc). The catalyst is removed by filtration and the filtrate is concentrated to provide the amine, which is used without purification.
{2-(4-Hydroxy-phenyl)-1-[1-methylcarbamoyl-2-(4-sulfoamino-phenyl)-ethylcarbamoyl]-ethyl}-carbamic acid tert-butyl ester: In a dry flask the aniline compound is dissolved in 2 mL pyridine. To this solution is added 0.295 g of sulfurtrioxide-pyridine complex. The mixture is stirred 5 minutes then diluted with 50 mL of 7% ammonium hydroxide. The mixture is evaporated down to an off-white solid and purified to provide 0.062 g of product as its ammonium salt. 1H(D2O): δ7.00-6.87 (m, 6H), 6.69 and 6.66 (d, 2H, J=9.3 Hz), 4.28 (t, 1H, J=8.0), 4.05 (t, 1H, J=8.7 Hz), 2.81-2.65 (m, 4H), 2.47 (s, 3H), 1.21 (s, 9H)
Crystal structures are a very useful tool for design of PTPbeta inhibitors. As an illustration, we describe here two different modes of binding of PTPbeta inhibitors, determined through solving high-resolution crystal structures of PTPbeta catalytic domain [SEQ ID NO: 7] complexes with the representative members of these classesCompound 1 and Compound 2.
Additional similarities and differences between the structure of the ligand-binding site of PTP-1B and PTPbeta catalytic domain [SEQ ID NO: 7] become apparent upon comparison of the ligand-bound structures of PTPbeta catalytic domain [SEQ ID NO: 7] with the ligand-bound structure of PTP-1B (PDB code 2HNP).
On the one hand, as can be seen from
On the other hand, there are significant differences in the structure of the binding sites between PTPbeta catalytic domain [SEQ ID NO: 7] and PTP-1B. In particular these include:
In both PTPbeta catalytic domain [SEQ ID NO: 7] complex structures presented here, the sulfamic acid moiety and the phenyl ring bind to the enzyme in a very similar manner, however the binding mode of the remainder of these two inhibitors to the protein is quite different, as follows:
P(0) pocket: Whereas Compound 1 interacts with His212 through the C-terminal amide carbonyl and through the phenyl ring, in the structure of the Compound 2 complex, His212 side chain is abstracted away from the binding site as a result of an interaction with the tyrosine side chain of the inhibitor (
P(−1) pocket: The N-terminal carbobenzoxy group of Compound 1 participates in extensive Van der Waal's contacts with Arg73 and Tyr74. In contrast, the C-terminal amide of Compound 2 interacts with Tyr74 and to a lesser extent with Arg150.
P(+1) pocket: The only interaction of Compound 1 with this pocket of the enzyme is a superficial VdW contact with Gin289Compound 2 interacts with the P(+1) pocket through hydrogen bonding of the t-BOC carbonyl with Gln289 side chain, and via extensive VdW interactions of the tert-butyl group with Ile77, Val249, Gln289, Val285, His286, and even Arg56. Notably, His286 is misplaced from its position in the both the unliganded enzyme and in the complex of PTPbeta catalytic domain [SEQ ID NO: 7] with Compound 1. In its new position, His286 pi-stacks with the guanidine of Arg281 and forms potential hydrogen bonds with main-chain carbonyls of Val288 and Lys52 or, depending on its protonation state, an ion-pair with Arg56.
In view of the above, it is not surprising that Compound 2, which forms an impressive array of interactions with the enzyme is a more potent inhibitor than Compound 1 which has a much more modest number of interactions with PTPbeta catalytic domain [SEQ ID NO: 7].
Except as otherwise noted, all amounts including quantities, percentages, portions, and proportions, are understood to be modified by the word about, and amounts are not intended to indicate significant digits.
Except as otherwise noted, the articles a, an, and the mean one or more.
All documents cited are, in relevant part, incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.
While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope.
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A beautiful, natural and young girl shows herself naked, open and uninhibited while she smokes one cigarette after another.
The beautiful female smoker Candela has a fantastic body and beautiful long hair. She doesn't shave her pubic hair nor her legs. I guess, I'm not the only one who likes her fine downy blond hair :-) Due to her thick furry pubic hair, I publish this series here without marking it as "explicit", although some shots are very direct and open.
It is a quite revealing series of artistic nudes. Who doesn't like to see any intimate parts, or is set fanatically against smoking will not be happy with this series. But all others for sure!
The same series of artistic nudes are is also available in black and white for art lovers!
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guapamania photos/series: "young girl smoking (color)" (nudes). Artistic nude photography and lingerie photos with a touch of feminine and sensual eroticism.. The mania of pretty pictures of pretty girls. Collections with sexy stories about women from the world!. Tags for this page: Candela Guapamania art artistic nudes beautiful long hair bodyhair cigarette smoking furry bush girls hairy legs hairy vulva hisute girls hirsute long haired naked natural woman nude female smokers nude photography nude smoking girls nudes photos pubes pubic hair sensual charisma series tiny hair unshaven girls wild long mane artistic nude photography sensual artistic nudes erotic art nude models art nudes
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subset: commoncrawl


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Brandon Sun - ONLINE EDITION
Flooding expected close to 2009 levels, major in some areas
Threatened communities begin preparations
Provincial flood forecasters are expected to upgrade the threat of spring flooding in southern Manitoba today as communities throughout the flood zone prepare for what could be headed their way.
In its first flood outlook of the season last month, the province said it expected only minor to moderate flooding along the Red, Assiniboine, Pembina and Souris rivers and in the Interlake.
But that changed earlier this month when a late winter storm dumped about 20 centimetres more snow on southern Manitoba and areas of Saskatchewan and North Dakota. That thick snowpack across the region, plus a major-flood warning from the U.S. last week, now has flood forecasters here reworking their predictions.
"If you look at the forecasting, it's changed pretty well around the table for most," St. Clements Mayor Steve Strang said Monday. "Every one of them is getting a little bit worse."
How severe flooding will be on the Red and Assiniboine rivers depends on one thing -- whether Mother Nature pitches us a curveball over the next six weeks or throws us a walk.
Colder-than-normal temperatures have delayed the spring thaw, but at the same time have allowed for the snowpack that covers the region to slowly evaporate. The fear is if temperatures rise too quickly, snow will melt that much faster and, if combined with rain, it's a whole new ball game.
Provincial officials met with municipal leaders last week in Selkirk to outline various flooding scenarios under different weather conditions. The consensus, according to those who were there, isn't whether there will flooding, but how much.
"The rumour mill has it it's going to be worse than first anticipated," RM of St. Andrews emergency measures co-ordinator Darcy Hardman said Monday. "They're going to say we're going to have to start mitigating for situations that we might have in the future."
That's already started up and down the Red River Valley, but out west along the Assiniboine River it's a bit of a different story -- all eyes are focused on the Portage Diversion and whether it will be called into service as it was in 2011, the year Lake Manitoba flooded with catastrophic results.
"We're trying to keep an even head with this," Twin Lakes Beach resident Jack King said. "But I'll tell you, a lot of people are so discouraged. All the work we've put in trying to get back, lifting places and doing landscaping and all the rest of it, and now the prospects of another flood, it's just so disheartening."
King lost his retirement home in the 2011 flood and has since rebuilt.
The province over the winter months has reinforced and dredged part of the diversion, but King and RM of St. Laurent Reeve Earl Zotter said it has done precious little to increase outflow from the lake at the Fairford River Water Control Structure into Lake St. Martin or moved on the idea of building a new outflow channel at Watchorn Bay.
"As far as what's going to happen on Lake Manitoba this year, it's anybody's guess," Zotter said. "From what I understand, the whole drainage basin of the Assiniboine River has pretty heavy moisture in it. I don't feel the province will have any qualms about using the diversion and running the amount of water at us like they did in 2011. That will probably be their only strategy to saving Portage and Winnipeg and everyone in between."
Flood preparations for this spring started several weeks ago with Amphibex ice-smashing machines breaking up 30 kilometres of thick ice on the Red River to reduce the risk of ice jams and the reinforcement of a dike in St. Clements near St. Peters Road. Municipalities such as St. Clements and St. Andrews are also busy clearing ditches and culverts of snow and ice to reduce the threat of overland flooding. Both communities are gearing up to stockpile thousands of sandbags with new sandbag-making machines. South of Winnipeg, the border town of Emerson has trucked away piles of snow in preparation for what could be headed its way on the Red River.
We will live stream the news conference at 11 a.m.
Updated on Tuesday, March 26, 2013 at 11:11 AM CDT:
update live
Updated on Tuesday, March 26, 2013 at 12:27 PM CDT:
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text:
Help is coming in for people trying to save an old stone church on the shores of Sydney Harbour.
As confusing as it seems, two groups are competing for the future of St. Alphonsus Church in Victoria Mines, including one that would like to see it reopen as a place of worship.
The other group, the Stone Church Restoration Society, wants it restored to its former glory for all to see.
"We've been very busy trying to save the stone church, not to have as a church but as a historical landmark on the Cape Breton shores," said spokeswoman Melanie Sampson.
Her group is hoping St. Alphonsus can become a tourism stop on a route that includes nearby Fort Petrie and the Whitney Pier Historical Museum.
"People just like to drive by and have a look at it," said Sampson. "We're really hoping to save this piece of history, but we need funding."
At least $300,000 needs to be raised to obtain, secure and repair the church, said Sampson. So far, a little less than $5,000 has been raised.
On Tuesday, the society was allowed to view the inside of the former church.
Sampson said water damage was found near the bell tower, where a window was left open for almost two years. Her group is planning to repair the window with help from a company that has offered its services for free.
"People are really coming together at this 11th hour," said Sampson.
The society is in negotiations with the Diocese of Antigonish to buy the church, which was slated for demolition.
Meanwhile, members of the Save St. Alphonsus Church Coalition say the diocese has no right to sell the church, as it was never given an official decree of closure.
St. Alphonsus Parish was founded with the building of a mission church in 1853. The original church was destroyed by fire in 1916 and replaced by the stone building. In 2007, the diocese closed St. Alphonsus due, in part, to dwindling attendance.


text:
Property Details
1000 Illinois Lake Forest, IL 60045
Property ID # 08272322
Bedrooms: 4 Bathrooms: 6.1
Year Built: 1903 Lot Size: 3.81 Stories: 2 Fireplaces: 3 Garage Spaces: 2 Includes basement
VALLOMBROSA, designed by noted architects Edwin H. Clark and William A. Otis, was their second Villa built in Lake Forest, the first being the J. Ogden Armour Estate, now Lake Forest Academy. Listed in the National Register of Historic Places and carefully restored to preserve the dignity of its original architecture, VALLOMBROSA remains an exquisite landmark. A stone's throw away from the glorious shores of Lake Michigan, this home has been the gathering place of parties supporting various high profile celebrities and politicians.


text:
Doctoral Seminar Series
Doctoral Seminar Series
The focus of the seminar is to discuss topics related to the four graduate tracks: exercise physiology, motor control, biomechanics, and physical activity epidemiology. Each track invites a minimum of one external speaker each year.
If you are interested in presenting, please contact Dr. Sara Myers.
On September 5th, Dr. Sherry Pagoto will present a seminar on using Social Media in Health Research. Learn more...
Dr. Pagoto is an Associate Professor of Medicine at the University of Massachusetts Medical School and co-founder of the UMass Center for mHealth. Her expertise is in obesity interventions, eating behavior, physical activity, depression, and cancer prevention. She has been continuously funded by the NIH and/or CDC for 10 years and has published 96 papers in scientific journals. Her work has been featured in Time.com, NPR.org, NBC News, and CNN.com just to name a few.
|Sept. 5
|Dr. Sherry Pagoto
|Social Media in Health Research
|Sept. 12
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|Can We Rely on Traditional Methods to Diagnose Ankle Injuries?
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|Dr. James Stimpson
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|Dec. 12
|Dr. Weiwen Chai


text: NWHikers.net - View topic - Mt. Pilchuck (East Route) 7/6/02 Shop FAQ Search Chat / Members / Pics Login Register Active Topics Min 1 Week Min 1 Month Trail Talk Partners Trip Reports Stewardship/History Gear/Food/Photo Support/Links Saloon/Trade Forum Index -> Trip Reports -> Mt. Pilchuck (East Route) 7/6/02 Page 1 of 1 Previous :: Next Topic Author Message TomAdminJoined: 15 Dec 2001Posts: 14170 | TRs | Pics Sun Jul 07, 2002 12:07 am Mt. Pilchuck (East Route) 7/6/02 I hadn't been up to Mt. Pilchuck in several years so I decided to climb it on Saturday via the east route described in the 75 Scrambles book. The first mile up to Pinnacle Lake was a bit muddy with plenty of snow patches. Upon attaining the ridge at 3400' there was solid snow all the way to Pinnacle lake at 3800' which is just starting to thaw. The hike up the steep gully to the Bathtub plateau was rewarded by a refreshingly cool breeze swept down from above. About halfway up I was overtaken by a young couple. I was most impressed by the young lady who kicked steps all the way to the top without as much as dropping a beat - I was tempted to ask if she might have an older sister . The rest of the way up was pretty straight forward, though near the end instead of hanging a left and ascending the steep but non-technical gully the summit, I made the mistake of following tracks right which required backtracking up the steep northern slopes just below the lookout and ascending exposed rock to the summit (definitely not recommended). For those looking to avoid the crowds, the eastern route offers plenty of solitude, at the cost of being extremely steep in sections and twice the workout. Either way, the hordes await you up top. Tom On Mt Pilchuck crw_6333.jpg (38 KB) File downloaded or viewed 687 time(s) Back to top Display: All Posts1 Day7 Days2 Weeks1 Month3 Months6 Months1 Year Oldest FirstNewest First All times are GMT - 8 Hours Page 1 of 1 Forum Index -> Trip Reports -> Mt. Pilchuck (East Route) 7/6/02 Happy Birthday Gimpilator, dazdarren! Jump to: Trail TalkPartners / Trip OrganizationTrip ReportsStewardshipPacific NW HistoryGear TalkFood & GrubPhotography TalkSupport & FeedbackLinksFull Moon SaloonThe Trading Post You cannot post new topics in this forumYou cannot reply to topics in this forumYou cannot edit your posts in this forumYou cannot delete your posts in this forumYou cannot vote in polls in this forumYou cannot attach files in this forumYou can download files in this forum Search this topic: Use Disclaimer Powered by phpBB © phpBB Group Privacy Policy


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Pic: Tim Boetsch broken foot, which "The Barbarian" thinks he cracked kicking the knee of Hector Lombard in the UFC 149 co main event, some time in the second round. To watch the video highlights of the fight, which resulted in a split decision win for Boetsch, click here.
Phil Davis vs Tim Boetsch likely for UFC 123 on Nov. 20
Thiago Silva injured, out of UFC 117 fight against Tim Boetsch -- Todd Brown tapped as replacement
UFC 117: Thiago Silva vs Tim Boetsch possible for August 7 in Oakland
UFC light heavyweight Tim Boetsch is set to make his third straight appearance inside the Octagon when he takes on the well-rounded Jason Brilz at UFC 96 "Jackson vs. Jardine" this Saturday, March...
Click the banner above or right here for up-to-the-minute results and blow-by-blow coverage of UFC 88: "Breakthrough." Quick results of the prelim fights will begin to flow around 8:30 p.m. ET and...
The official weigh-in event for UFC 88: "Breakthrough" at the Philips Arena in Atlanta, Ga., is set to air online via streaming video at 4 p.m. ET TODAY. Those who want to check it out in...
It appears that the injury bug has bitten once again. Yesterday it was Jason Day and today it's James Lee. Tim Patt (11-2) will fill-in for an injured Lee and take on Tim Boetsch (7-2) in a light...
UFC 88: "Breakthrough" is set go off tonight (September 6) LIVE from the Philips Arena in Atlanta, Ga., on pay-per-view (PPV) at 10 p.m. ET. MMAmania.com will provide LIVE blow-by-blow,...
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The Cardinal used to be one of the best in the country in the red zone, but that's no longer the case
We asked fellow SBNation blog Rule of Tree about Stanford's hype, matching up with UW's skill players, stopping Bishop Sankey, Kevin Hogan's evolution, and attacking UW's defense
This week's topics include Kevin Hogan, slowing down Bishop Sankey, big plays from wideouts, the coaching matchup, and whether or not the Huskies can put their man pants on.
WSU will take on Stanford in a matter of hours, but we have some links to pass the time until then.
Ranking and breaking down the Pac-12 football games of week five.
After losing to Stanford last year 20-17, San Jose heads to Palo Alto for revenge in week 2.
Virginia Tech wants Stanford offensive coordinator Pep Hamilton to take the same position under Frank Beamer, hoping he can make a Hokies offense that sputtered this past season despite having...
If you like old-school power rushing, this game is for you.
Kevin Hogan helped transform what could have been a middling year for Stanford to one in which they won a Pac-12 Championship and have a good shot at victory in the Rose Bowl. If they can pull it...
The Rose Bowl gets its traditional Pac-12 champion vs. Big Ten champion matchup when Wisconsin and Stanford square off on New Year's Day.
The Stanford Cardinal are playing in Tuesday's Rose Bowl thanks in large part to quarterback Kevin Hogan. The red-shirt freshman started the Cardinal's last four games, including a Pac-12...
Stanford has been installed as a six-point favorite in the Rose Bowl against Wisconsin.
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Place your bets: Alves the Tapir makes World Cup predictions
A family of Brazilian tapirs are placing their bets about the World Cup.
Alves, the youngest of the three Tapirs at Lake District Wildlife Park, is said to enjoy football. He makes his guesses about the big game by choosing which bucket of bananas to eat out of.
Ryan Dollard went to meet them.


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After months of design and preparation, a dedicated group of CIM students culminated phase one of a special project: the pouring of a concrete rook. The concrete rook is the symbol of the Newark College of Engineering.
Two producers formed a mutually beneficial arrangement that provided expertise on both sides of the river. Massive amounts of concrete were produced for the high-profile, fast-paced project in two states.
StarSeal PS Ultra Stain will penetrate and color any interior or exterior new or old concrete surface prior to being ground and polished, densified or sealed. The product is ideal for high UV exposed environments. When properly installed StarSeal PS Ultra Stain deeply penetrates the surface...
In this month's issue we are drawing attention to a few more folks who are working on industry initiatives that could be about to shape your future. These influencers are not only shaping the North American concrete industry. Thanks to the world wide web, and now with our expansion plans for World...
Las Vegas never sleeps. So for those World of Concrete attendees who arrive early at the Las Vegas Convention Center, we've launched "Breakfast with the Experts".
DUROCK EcoCap self-leveling underlayment is an innovative cementitious floor topping specifically engineered for application over concrete substrates. DUROCK EcoCap, with its breakthrough environmentally sustainable geopolymer binder technology, provides a fast-drying, smooth and monolithic surface...
This special event features contractors who will showcase how they've benefited from using self-consolidating concrete (SCC). It takes place 1-4 p.m., Jan. 20, at the show in Las Vegas.
Rick Yelton has shared the World of Concrete experience far and wide, traveling to Brazil, India, and the Philippines this year. He found producers and contractors share common problems and successes in every part of the world.
When the ASTM Standard Specification for Lightweight Aggregate for Internal Curing of Concrete was published in 2012, the standard change revealed a new understanding about the importance of high-level hydration of concrete. One year later, we look at how ASTM Standards C09 and C09.21 are...
The San Jose, Calif., producer leads the nation in developing Environmental Product Declarations (EPDs). EPDs disclose environmental data such as carbon emissions, water use, and embodied energy.
Jason Weiss' internal curing research reveals small changes that result in big improvements. For more than a decade, Weiss and his Purdue University students have worked to improve concrete with internal curing.
A look at what concrete producers can expect at World of Concrete 2014.
Special resin blend produces a light, strong, and durable film.
One operator can change this twin-shaft mixer's lining plates in one day.
Hydromedia is designed to rapidly direct stormwater from streets and other surfaces.
The Producer Center at World of Concrete is home to exhibitors catering to the interests of producers like you.
The Mobile 5 batch plant is ideal for remote jobsites requiring high-quality concrete.
Want to learn more about SCC? Rich Scezy will be present a World of Concrete Seminar on self-consolidating concrete on Monday 20. His presentation is part of the WOC Education Program Production track. The track includes seminars on mix designs, self-consolidating concrete and chemical admixtures.
The E-ZBar is a revolutionary device that positions your wall steel to your job specific requirements. The purpose of the E-ZBar is to hold correct spacing between your wall matts and maintain minimum concrete coverage on each face of your wall.
The PAM_Dek is a tool holder to perform deck concrete chipping with conventional hammers while maintaining a vertical posture without musculoskeletal strains. It includes a vibration dampening system, ergonomic handles for a comfortable work posture and an engineered mechanism that neutralize the...
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2014 Hanley Wood Media, Inc. All rights reserved.


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Because Hotel Alcantara has so many rooms, you have plenty of options to choose from. Book a stay in one of the 3-star rooms offered here.
You can find a restaurant, a bar/lounge and a smoking area present in Hotel Alcantara. Not only is a dry cleaning/laundry service part of the services here, a concierge service is also available during your stay. A conference space is one of the facilities especially convenient for guests on business trips. 66 rooms are waiting for you to choose from.
Air conditioning makes a stay in Hotel Alcantara more comfortable. A housekeeping service is a convenient inclusion in each of the rooms here. Stay connected: wireless high-speed Internet access (complimentary) is available here.
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Healthy Weight
Find informative articles on Healthy Weight, related to health. At Fitho, you can read reliable health & fitness information on Healthy Weight. Stay updated with the latest and most comprehensive information related to Healthy Weight. Below, you can read all the articles related to Healthy Weight & stay fit.


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WASHINGTON — Fewer Americans applied for unemployment benefits last week, and a private survey showed businesses stepped up hiring in August. The data sketched a brighter outlook for the job market one day before the government reports on August employment.
Weekly applications for unemployment benefits fell by 12,000 to a seasonally adjusted 365,000, the Labor Department said Thursday. The four-week average, a less volatile measure, ticked up to 371,250.
Unemployment benefit applications are a measure of the pace of layoffs. When they consistently fall below 375,000, it suggests that hiring is strong enough to lower the unemployment rate.
Separately, payroll provider ADP said businesses added 201,000 jobs last month. That's the most reported by the survey since March. And ADP said July job growth was stronger than first thought: Employers created 173,000 jobs — 10,000 more jobs than the group reported last month.
"The labor market is getting better and while the pace of improvement is nothing great, the direction is clear," Joel Naroff, president of Naroff Economic Advisors, said in an email to clients.
Despite the positive signals, most economists tempered their expectations for August payroll growth.
The ADP survey only covers hiring in the private sector and excludes government job growth. The Labor Department's report on Friday will offer a more complete picture. The two surveys reported roughly the same private-sector job creation in July. But they have diverged sharply in previous months.
And the drop in applications likely won't affect the August jobs report, set to be released Friday. The data for that report were compiled three weeks ago. But it could signal better hiring in September.
Economists forecast that employers added 135,000 jobs last month, while the unemployment rate is expected to stay unchanged at 8.3 percent.
Weekly unemployment applications "are still stubbornly high, which is consistent with our expectations for a modest employment report in August," said Bricklin Dwyer, an economist at BNP Paribas
In July, employers added 163,000 jobs. It was the best month of hiring since February and an improvement from the average of only 73,000 per month created in the April-June quarter.
Still, the economy grew at a tepid 1.7 percent annual rate in the April-June quarter, down from the 2 percent rate in the January-March quarter and 4.1 percent in the final three months of last year.
Growth at or below 2 percent is typically too weak to lower the unemployment rate. Most economists expect the unemployment rate will remain above 8 percent through the November election.
President Barack Obama will address the Democrat National Convention in Charlotte on Thursday. Polls show most Americans regard Republican nominee Mitt Romney as better able to handle the U.S. economy.
Romney and his Republican allies have pointed to high unemployment as evidence that Obama's policies are not helping the economy. The unemployment rate was 7.8 percent when Obama took office and has been above 8 percent for the past 42 months. Most economists say "normal" unemployment is 6 percent or less.
But Obama and his allies have focused on more than 4 million jobs that the economy has added in the past 2½ years.
A weak employment report could push the Federal Reserve to announce some new action after its meeting next week to try to boost growth.
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Studio equipment
Massive range of equipment and accessories for studio use.
DJ equipment
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The world's largest dance music store featuring the most comprehensive selection of new and back catalogue dance music Vinyl and CDs online.
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Features the latest dance music news, interviews, music and tech reviews, podcasts & more...
To celebrate four years of releases, Hemlock, the label run by Untold and Jack Dunning presents Chapter One, a collection of label highlights and unreleased exclusives. Moving through the glittery funk-leaning post-dubstep of Fantastic Mr Fox, Mount Kimbie and James Blake, through techno contributions from Sei A, Randomer and Guy Andrews, and bass hybrids from Pangaea and Joe, the selections on offer demonstrate why Hemlock has been ahead of the game since its inception. As well as the individual tracks, a continuous mix is also on offer, joining the dots between their diverse releases with style.
Dj Friendly full length tracks; DRM Free; Plays on any mp3 player
Secure Shopping - PayPal, Visa, Maestro, Mastercard, Switch, Solo


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- Mobile site
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Organisers issue rallying call for Keighley people to support event for the town (From Bradford Telegraph and Argus)
Get involved: send your pictures, video, news and views by texting TANEWS to 80360, or email
Organisers issue rallying call for Keighley people to support event for the town
6:00am Wednesday 4th June 2014 in News
A final rallying call has been issued to townspeople ahead of Keighley Gala.
The 138th annual charity extravaganza takes place on Sunday, with a host of attractions lined up.
Now organisers are appealing for the public to turn out in numbers to give its support.
Chairman Shona Brunskill said: "We hope everyone will join us in celebrating the gala – which is run by people of the town, for the town, to raise money for local charities.
"With something for everybody – a procession, live music, stalls, food, rides, games, a baby show, fun dog show and much more – we are really hoping for another successful event to build on for future years.
"Our message is please come along, watch the procession and visit the events in the park, to show your love for our town and the charities the gala supports, including Manorlands.
"Without people's backing, the gala may not survive and it would be a shame to let it go under after all these years"
The event will start with the crowning of the gala queen, Charlotte Lily Carter, in Town Hall Square, from noon. A jazz band will entertain.
The procession will set off at 1pm from Spring Gardens Lane and head to Victoria Park via North Street, Cavendish Street, Lawkholme Lane, Bradford Street, Bingley Street and Bradford Road.
Attractions in the park will include fairground rides and stalls, face painting, charity stalls, sumo-suit wrestling, a ducking stool and food.
Live music will be provided from 2.30pm, with The Elderberries, Jonny B, Emma Feather, Haley Harrison and Kitty Le Mare among the performers.
The fun dog show, sponsored by Cobbydog and Just4Paws, will take entries from 12.30pm. It has an agility course and 16 classes, from waggiest tail to the most beautiful eyes.
And entry to the baby show, which features categories for up to 12 months and 13-24 months, will open at 3pm. There will be prizes for best boy and girl and certificates for each participant.
Procession and stall entries will be accepted up to Saturday, space permitting.
E-mail email@example.com, visit keighleygala.co.uk or call 07891 589539 for details.


text:
- Age / Gender:
- 26, Male
- Location:
- Belgium
- Joined:
- 12/26/12
- All Stats >
User Statshot
- Community Stats
Level 11 Musician
Normal Whistle
Ranked as Scout
Contact Info / Websites
Latest News
Hey guys,
just wanted to let you know I released a new album (it's not the one I am currently working on though!)
It's an album for the game called Sky Jives I was composing for a couple of months back. Unfortunatly I don't think the game will ever be finished due to some problems between our team members, so I decided to release the uncompleted OST on Bandcamp.
It's a shame I had allot of fun working with the creator on this and I probably wont ever see the game completed.
Atleast I can share the music with you, hope you enjoy.
(note that most of these songs are loops so they might end a bit abruptly.)
Latest Audio Submissions
|Tomb Of The Ancients
|Cinematic Song
|The Weapon
|Cinematic Song
|Forest Of Desolation
|World Song
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Latest Movies
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Total Medals Earned: 761 (From 135 different games.)
Favorite Audio
|Cinematic Loop
|ARMA 3: Resist - Tension
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|Cinematic Loop
|The Hero (Western Theme)
|Cinematic Song


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text:
What they think I think:
So today there were some protesters on campus. Did you see them? Man, talk about presenting an extremely convincing argument. I admire their bravery to take a side on such a controversial topic. Ya know, they're much braver than me. Up until I saw them today, I was really undecided on the issue. And, when I saw them, I thought I'd just hurry past like I usually do. But, this time, something caught my eye. One of them had this really frickin' sweet poster. It was so articulate and cool that it just completely blew my mind. Because of that poster, I have completely changed my world view. I can already tell that my life is 110% better. Thank you, Mister Protester. Thank you.
What I actually think:


text:
Tate Donovan / Getty Images
Tate Buckley Donovan is an American actor and director. He is known for his role in the FX drama Damages, as Tom Shayes, and for his role as Jimmy Cooper in the American teen drama television series The O.C.. He voiced the title character Hercules in Disney's thirty-fifth animated feature film, in ...the animated television series and in the video games Kingdom Hearts II and Kingdom Hearts Re:Coded. He currently stars as Brian Sanders on the CBS drama Hostages.more
Full Name: Tate Donovan
Occupation: Actor
Birthdate: 25 Sep 1963 (age 50)
Birthplace: New York City, New York
Relationships: Jennifer Aniston, Sandra Bullock


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Latest headlines
How to internationalise the curriculum
A practical guide to adding an international perspective to taught courses More...
Published: Aug 14, 2014 4:06:39 PM
Highlights of the 2014 NSS results, plus inspiration for improvements in 2015
The 2014 NSS results made for encouraging reading for UCL. Not only did the overall institutional satisfaction score jump to 86 per cent, but departments from across the university made significant advances. More...
Published: Aug 12, 2014 2:10:39 PM
What makes a great CPD course?
UCL Faculty of Laws delivered the first CPD course to win a Provost's Teaching Award. Visiting Lecturer Mark Anderson shares his advice on creating engaging and effective CPD sessions More...
Published: Aug 6, 2014 4:28:43 PM
More than 50 staff gain UCL Arena Fellowships
Fifty-two members of UCL staff received UCL Arena Fellowships in July in recognition of their teaching experience and expertise. More...
Published: Aug 5, 2014 9:58:42 AM
Effective teaching videos
A selection of tips to help create professional-looking, powerful videos that complement face-to-face teaching.
Read more »
Gain teaching qualification
Formal recognition for HE teaching is available through the UCL Arena scheme.
Read more »
Teaching and Learning Desktop: summer upgrade
26 June 2013
ISD will be upgrading the current Myriad and Cluster WTS 2000 services to Desktop@UCL over the summer.
The new desktop service will provide a number of enhancements to the teaching and learning experience, including:
• Modern desktop services providing flexible 'anywhere, anytime' access via the web
• Wide range of applications including Office 2013
Rollout will start in early July and complete before the start of the 2013/2014 academic year.
The project team are seeking help for testing teaching applications on Desktop@UCL. If you would like trial the desktop and assist with the testing, please email email@example.com.
Learning and teaching spaces will start to be converted to the new desktop in mid-July. The project team will be publishing the full schedule shortly.
It's important to note that there will a period of coexistence of current desktop services (Myriad and Cluster WTS 2000) and the new Desktop@UCL over the summer as the rollout progresses. All teaching applications will be made available by the start of the next academic session.
Further details on how the rollout will affect the learning and teaching community and information about available training will follow later in the summer.
If you have any queries related to Desktop@UCL, please get in touch with the project team by emailing email@example.com.
Page last modified on 26 jun 13 16:25
Tell us about the inspiring teaching and learning taking place in your department: email email@example.com


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Report:Quick Take: Apple And IBM Team Up To Create A New Enterprise Mobile Platform
New Partnership Melds Complementary Strengths In Consumer And Enterprise Markets
The leading mobile OS devices from Apple and Google have blossomed in enterprise settings because individuals want the same mobile devices and apps at work that they use in their personal lives. But...
- Downloads: 272
For CIO Professionals
Report:Quick Take: Apple's WWDC 2014 Tour De Force
New Developer Tools And Business Features Will Strengthen Apple's Leading Position In The Age Of The Customer
Introduced by Apple CEO Tim Cook, Apple software leader Craig Federighi motored through a dense list of new operating systems, iOS 8 and OS X Yosemite, compelling new customer features such as the...
- Downloads: 119
For CIO Professionals
Report:Quick Take: Apple Doubles Down On Its Premium Brand With The iPhone 5s And 5c
New Products Reflect The Company's Conviction That It Can Command Growth At The High End Of The Smartphone Market
As the world watched Apple's latest iPhone announcements, the company departed from its previous product strategy. With a saturating high-end smartphone market and growth rapidly shifting to...
- Downloads: 113
For CIO Professionals
Report:Tablets Will Rule The Future Personal Computing Landscape
We'll Use Tablets As The Conductor For PCs, Devices, And Personal Cloud Services At Work And At Home
For a new technology that is just two years old, tablets are huge hit. For CIOs the question is — will they last and what are the long-term implications? How do tablets interact with other...
- Downloads: 2139
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Report:Apple Infiltrates The Enterprise And Reshapes The Markets For Personal Devices At Work
More Than 21% Of Information Workers Use Apple Products For Work
Apple's products are much more visible in business environments than they used to be. To quantify Apple's business presence, Forrester surveyed almost 10,000 information workers — workers that...
- Downloads: 801
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Report:The Age Of Computing Diversity
The ICT Industry Is Broadening Out From The Wintel Monoculture
The computing industry is exiting an era built mostly on general-purpose, good enough central processing units (CPUs), servers, systems designs, software, Web apps, and client devices. These...
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text:
People from across Northern Nevada were in Carson City Saturday morning as the Annual Nevada Day Parade rolled through town.
It lasted more than three hours. Thousands of people grabbed a chair, or got comfortable where they could, and watched while it made its way down Carson Street.
"It is really great," says Denise West. "I cannot believe all the bands and stuff that are in it."
Roughly 40,000 people filled up the heart of our state's capital. Some arrived as early as 4:30 a.m.
"Every year," says Brenda Swart. "Nevada Day is my favorite holiday as well as all my friends' back here."
Things kicked off with a flyover right at 10 a.m. From there, everything really got rolling.
"It's beautiful," says Melody Hoover. "We got to see the flyover, the memorial wall over at Mills Park. All of our friends are here. Everybody has a great time."
Some of our friends were in attendance as well. Several members of the Channel 2 News team waved to the crowds, while John Potter grabbed a microphone and emceed the event.
Folks at the parade for the first time say this community, and especially this event, welcomes everybody.
"I have never left Michigan until last year, and everybody that I've met here has been very, very nice," says West. "They're awesome people."
With more than 200 entries, this year featured a little bit of everything. It's election year, and both the Democrats and Republicans were represented multiple times.
But for people who don't meet the age requirement to vote this year, all that political stuff takes a back seat to what's really important.
"All the cars and blowups and stuff," says 9-year-old Christopher West. "And all the people that are doing it."
Another Nevada Day Parade is in the books. But one thing's for sure: this tradition will continue for years to come.
Hard to believe, but organizers with the parade say planning for next year's event begins as early as Sunday.
Written by Adam Rasmussen
KTVN-TVPhone: (775) 858-2222Fax: (775) 861-4298
Mailing Address:4925 Energy WayReno, NV 89502


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Aug 05 2014
Expensive Judicial Elections are an Assault on Our Courts
Tennessee courts are specifically tasked with deciding "on the constitutionality of legislation passed by the General Assembly" and to "consider the legality of administrative policies and regulations." Why then, are so many politically motivated groups spending so much campaign cash prior to the upcoming Tennessee Supreme Court retention elections?
The answer seems to be related to the Supreme Court justices appointing an attorney general who did not join other attorney generals in a lawsuit opposing the Affordable Care Act. For the record, once the Tennessee Supreme Court appoints an Attorney General, the Attorney General is completely independent and does not answer to the Court.
Regardless of the underlying motivations, the amount of money spent in an attempt to influence the retention elections is emblematic of recent trends of increasingly political and expensive judicial elections. According to Justice at Stake, the Tennessee Supreme Court retention election has set a record by exceeding $500,000 in television advertisement spending. Tennessee Forum, a group that opposes retaining the three justices up for election, has contributed $245,000. Likewise, Keep Tennessee Courts Fair, a group that supports retaining the three justices, has given a considerable contribution amounting to $210,000.
Industrialists Charles and David Koch have even thrown their hats in the ring by having their organization, Americans for Prosperity, contribute significant amounts to the campaign to unseat the three justices.
Judges should be chosen (and retained) based on their qualifications including legal experience, judicial temperament, and quality of their decisions. Choosing judges (or choosing whether to retain judges) through a political process for a nonpartisan job is nonsensical.
Pennsylvania needs to start learning from the examples of states like Tennessee. We need to protect our courts from special interests and politicians that see them as just another tool in the political arsenal. It's time for Harrisburg to stand up to entrenched special interests and have a serious discussion about commonsense reforms like merit selection.


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The Deal
- $10 for one ticket to see A Slice of Comedy with Johnny Osburn and Terry Michaels ($20 value)
- When: Saturday, November 30, at 9 p.m.
- Where: Mother Mary's
- Section: general admission
- Door time: 8 p.m.
- Ticket values include all fees.
Featured Comedians
Warning: contains extreme language, raunchy material, offensive humor
Terry Michaels
Veteran seen at the Ice House, Comedy Store, and Funny Bone, who's shared the stage with Sam Kinison
Warning: contains vivid examples of unhealthy living
A Slice of Comedy
Pizza and comedy: a match made in entertainment heaven. Inside Mother Mary's, chefs sling hearty calzones and crisp pizzas while professional standups sate appetites for humor. And even though the event eschews the typical comedy-club drink minimum, the eatery's lauded ceramic-oven-baked pizza crust may tempt attendees to set their own rules about the minimum number of pies they'll ingest.