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The present invention is directed to a constitutively active G protein coupled receptor of human herpesvirus 8, as well as a method of identifying negative antagonists of a constitutively active G protein coupled receptor. The method comprises co-expressing in a host cell a constitutively active G protein coupled receptor and a reporter protein, wherein expression of the reporter protein is controlled by a promoter responsive to a signalling pathway activated by the constitutively active G protein coupled receptor; exposing the host cell to a test substance; and determining a level of reporter protein activity, wherein the level of reporter protein activity indicates effectiveness of the test substance as a negative antagonist of the constitutively active G protein coupled receptor. The invention further provides a method of preventing tumor formation or cell proliferation caused by a constitutively active G protein coupled receptor. This method comprises administering an amount of the negative antagonist so identified to a subject in an amount effective to prevent tumor formation or cell proliferation.
1. A method of identifying a negative antagonist of a constitutively active G protein coupled receptor of human herpesvirus 8, said method comprising:
co-expressing in a host cell a constitutively active G protein coupled receptor of human herpesvirus 8 and a reporter protein, wherein expression of the reporter protein is controlled by a promoter responsive to a signaling pathway activated by the constitutively active G protein coupled receptor of human herpesvirus 8;
determining a first activity level of the reporter protein;
exposing the host cell to a test substance;
determining a second activity level of the reporter protein after said exposing the host cell to the test substance; and
identifying the test substance, where the second activity level is less than the first activity level, as a negative antagonist of the constitutively active G protein coupled receptor of human herpesvirus 8.
2. The method of claim 1 wherein said promoter is a cyclic AMP-responsive promoter.
3. The method of claim 1 wherein said promoter is a protein kinase C-responsive promoter.
4. The method of claim 1 wherein said reporter protein is luciferase.
5. The method of claim 4 wherein said determining the first and second activity levels is carried out using a luminescent assay.
6. The method of claim 1 wherein said host cell is a mammalian cell.
7. The method of claim 6 wherein said mammalian cell is a COS cell.
The subject matter of this application was made with support from the United States Government under National Institutes of Health Grant Nos. DK43036 and DK46652.
The present invention relates to a constitutively active G protein coupled receptor of human herpesvirus 8. The invention further relates to a method of identifying negative antagonists of constitutively active G protein coupled receptors, and to a method of preventing tumor formation or cell proliferation caused by the constitutively active G protein coupled receptor by administering an effective amount of the identified negative antagonist.
Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description of the Invention. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
The family of guanine nucleotide-binding (G) protein-coupled receptors (GPCRs) has been estimated to be comprised of as many as 2,000 members, fully more than 1.5% of all the proteins encoded in the human genome, that are thought to regulate function of virtually every cell in the body. Furthermore, it has been estimated that more than 50% of the drugs in use clinically in humans at the present time are directed at GPCRs.
In general, GPCRs require agonist binding for activation. Constitutive (or agonist-independent) signalling activity in mutant receptors has been well documented but only a few GPCRs have been shown to exhibit agonist-independent activity in the wild type (or native) form. For example, native dopamine D1B and prostaglandin EP1b receptors have been found to possess constitutive activity (Tiberi and Caron 1994; Hasegawa et al. 1996). A number of GPCRs, for example, receptors for thyroid-stimulating hormone (Vassart et al. 1995), have been found to be mutated to exhibit agonist-independent activity and cause disease in humans. Experimentally, several single amino acid mutations have produced agonist independent activity. β2 and α2 adrenergic receptors, for example, mutated at single sites in the third cytoplasmic loop show constitutive activity (Ren et al. 1993; Samama et al. 1994). In some cases, a large deletion mutation in the carboxy tail or in the intracellular loops of GPCRs has led to constitutive activity. For example, in the thyrotropin releasing hormone receptor a truncation deletion of the carboxyl terminus renders the receptor constitutively active (Nussenzveig et al. 1993; Matus-Leibovitch et al. 1995) and a smaller deletion in the second extracellular loop of the thrombin receptor causes constitutive activity (Nanevicz et al. 1995) also.
The finding that certain receptors exhibit constitutive activity has led to a modification of traditional receptor theory (Samama et al. 1993). It is now thought that receptors can exist in at least two conformations, an inactive conformation (R) and an activated conformation (R*), and that an equilibrium exists between these two states that markedly favors R over R* in the majority of receptors. In some native receptors and in the mutants described above, it has been proposed that in the absence of agonist there is a shift in equilibrium that allows a sufficient number of receptors to be in the active R* state to initiate signalling.
As many more GPCRs with constitutive activity are found, both native as well as mutated receptors, a newly recognized class of drugs termed negative antagonists (or inverse agonists) will become important therapeutic agents. Negative antagonism is demonstrated when a drug binds to a receptor that exhibits constitutive activity and reduces this activity. Negative antagonists appear to act by constraining receptors in an inactive state (Samama et al. 1994). Although first described in other receptor systems (Schutz and Freissmuth 1992), negative antagonism has been shown to occur with guanine nucleotide-binding (G) protein-coupled receptors (GPCRs), for example, opoid (Costa and Herz 1989; Costa et al. 1992), β2 -adrenergic (Samama et al. 1994; Pei et al. 1994; Chidiac et al. 1994), serotonin type 2C (Barker et al. 1994), bradykinin (Leeb-Lundberg et al. 1994), and D1B dopamine receptors (Tiberi and Caron 1994). Thus, a technology for identifying negative antagonists of native and mutated GPCRs has important predictable as well as not yet realized pharmaceutical applications. Furthermore, because constitutively active GPCRs are tumorigenic, the identification of negative antagonists for these GPCRs can lead to the development of anti-tumor and/or anti-cell proliferation drugs.
To this end, the subject invention provides a constitutively active G protein coupled receptor of human herpesvirus 8, and a method of identifying negative antagonists of a constitutively active G protein coupled receptor. The method comprises co-expressing in a host cell a constitutively active G protein coupled receptor and a reporter protein, wherein expression of the reporter protein is controlled by a promoter responsive to a signalling pathway activated by the constitutively active G protein coupled receptor; exposing the host cell to a test substance; and determining a level of reporter protein activity, wherein the level of reporter protein activity indicates effectiveness of the test substance as a negative antagonist of the constitutively active G protein coupled receptor.
The invention further provides a method of preventing tumor formation or cell proliferation in a subject caused by a constitutively active G protein coupled receptor. This method comprises identifying a negative antagonist of a constitutively active G protein coupled receptor; and administering to the subject an amount of the negative antagonist effective to prevent tumor formation or cell proliferation caused by the constitutively active G protein coupled receptor.
These and other features and advantages of this invention will be evident from the following detailed description of preferred embodiments when read in conjunction with the accompanying drawings in which:
FIG. 1 illustrates the putative two-dimensional topology and amino acid sequence (SEQ. ID. No. 1) of the HHV 8 GPCR;
FIGS. 2 and 3 illustrate competition binding of human recombinant 125 I-IL-8 by unlabelled chemokines to COS-1 cells transfected with pcKSHV GPCR (10 mg/ml). (2) C-X-C chemokines (Kd or Ki): IL-8 (25 nM), NAP-2 (23 nM) PF-4 (150 nM) and MGSA (270 nM). (3) C-C chemokines (Kd or Ki): I-309 (110 nM) and RANTES (520 nM). Data represent the mean of triplicate determinations in a representative of two experiments;
FIGS. 4 and 5 illustrate the characterization of inositol phosphate (IP) accumulation in COS-1 cells transfected with pcKSHV GPCR. (4) Comparison with COS-1 cells transfected with plasmids for human IL-8 receptors (type A or B; 10 mg/ml) in the presence or absence of plasmid encoding Ga16 (2 mg/ml) after 90 minutes. (5) Time course in COS cells transfected with 10 mg/ml pcKSHV GPCR. Data represent the mean � S.D. of triplicate determinations in a representative of three experiments; IP accumulation of mock-transfected, untreated cells has been subtracted from all points;
FIG. 6 illustrates the dose dependence of inositol phosphate (IP) accumulation in COS-1 cells after transfection with various amounts of pcKSHV GPCR (from 0.003 to 10 mg/ml), measured at 90 min. Data represent the mean � S.D. of triplicate determinations in a representative of three experiments;
FIG. 7 illustrates the transcription of luciferase driven by a PKC-responsive promoter in COS-1 cells transfected with pcKSHV GPCR. Data represent the mean � S.D. of triplicate determinations in a representative of three experiments; and
FIG. 8 illustrates cellular proliferation in NRK-49F cells transfected with control plasmid or pcKSHV GPCR. After 5 days in culture in medium supplemented with calf serum or fetal calf serum, transfected cells were trypsinized and counted. Cell counts; data represent the mean � S.D. of triplicate culture dishes of three transfections.
The subject invention provides a constitutively active G protein coupled receptor of human herpesvirus 8.
The invention further provides a method of identifying negative antagonists of a constitutively active G protein coupled receptor. The method comprises co-expressing in a host cell a constitutively active G protein coupled receptor and a reporter protein, wherein expression of the reporter protein is controlled by a promoter responsive to a signalling pathway activated by the constitutively active G protein coupled receptor; exposing the host cell to a test substance; and determining a level of reporter protein activity, wherein the level of reporter protein activity indicates effectiveness of the test substance as a negative antagonist of the constitutively active G protein coupled receptor.
The constitutively active G protein coupled receptor can be any such receptor which one desires to turn off. For example, GPCRs which are tumorigenic or which cause cells to proliferate could be of interest for application of the method of the subject invention. By identifying negative antagonists of such GPCRs, the negative antagonists could be used to turn off the GPCR and thereby eliminate the GPCR's tumorigenic or cell proliferative effects. An example of such a constitutively active GPCR is the GPCR of human herpesvirus 8 (HHV 8) (also known as Kaposi's sarcoma associated herpesvirus or KSHV) (Cesarman et al. 1996).
Having identified a GPCR of interest, the signalling pathway activated by the GPCR is identified and a promoter responsive to the signalling pathway is selected. Such a "responsive" promoter will only promote expression of a downstream gene if the GPCR is functioning (i.e., signalling through its pathway). GPCRs use various signalling pathways, including cyclic AMP pathways and protein kinase C pathways. Suitable promoters for use with such GPCRs are cyclic AMP-responsive promoters and protein kinase C-responsive promoters, respectively. For a review of the cyclic AMP signalling pathway see Habener 1995, and for a review of the protein kinase C signalling pathway see Gershengorn and Perlman 1995.
A reporter protein is then chosen. The activity levels of the reporter protein will indicate the levels of GPCR signalling, since the reporter protein will only be expressed if the GPCR is signalling through its pathway to which the selected promoter is responsive. A reporter protein should be chosen which is not generally expressed in the host cell to be utilized for co-expression, to eliminate any false indications of GPCR signalling. A suitable reporter protein, as disclosed in the following examples, is the firefly luciferase gene. The activity of this reporter protein can be readily assayed using a luminescent assay. Other reporter proteins could also be readily used in the method of the subject invention, such as β-galactosidase and growth hormone.
Various host cells can be utilzed in the method of the subject invention. The choice of a host cell is based on several factors, including the absence of endogenous expression of the reporter protein in the host cell, presence of the GPCR signalling pathway in the host cell, and capability of co-expressing the GPCR and the reporter protein in the host cell. For the analysis of human GPCRs, mammalian cells are preferred, such as COS cells.
Generally the co-expression of the constitutively active G protein coupled receptor and the reporter protein in the host cell will involve introduction of two constructs into the host cell. The first construct is a nucleic acid molecule encoding the G protein coupled receptor under the control of suitable regulatory molecules for expression of the GPCR in the host cell. The second construct is a nucleic acid molecule encoding the reporter protein under the control of the promoter responsive to the signalling pathway of the GPCR. Each construct can be provided in the form of a plasmid, or a single plasmid comprising both constructs could be used. Techniques for introducing the construct(s) into the host cells may involve the use of expression vectors (such as plasmids and viruses) which comprise the nucleic acid molecule encoding the construct(s). DNA encoding the construct can be injected into the nucleus of a host cell or transformed into the host cell using a suitable vector, or mRNA encoding the construct can be injected directly into the host cell, in order to obtain expression of the GPCR and the reporter protein in the host cell.
Various methods are known in the art for introducing nucleic acid molecules into host cells. One method is microinjection, in which DNA is injected directly into the nucleus of cells through fine glass needles (or RNA is injected directly into the cytoplasm of cells). Alternatively, DNA can be incubated with an inert carbohydrate polymer (e.g. dextran) to which a positively charged chemical group (e.g. diethylaminoethyl ("DEAE")) has been coupled. The DNA sticks to the DEAE-dextran via its negatively charged phosphate groups. These large DNA-containing particles, in turn, stick to the surfaces of cells, which are thought to take them in by a process known as endocytosis. Some of the DNA evades destruction in the cytoplasm of the cell and escapes to the nucleus, where it can be transcribed into RNA like any other gene in the cell. In another method, cells efficiently take in DNA in the form of a precipitate with calcium phosphate. In electroporation, cells are placed in a solution containing DNA and subjected to a brief electrical pulse that causes holes to open transiently in their membranes. DNA enters through the holes directly into the cytoplasm, bypassing the endocytotic vesicles through which they pass in the DEAE-dextran and calcium phosphate procedures (passage through these vesicles may sometimes destroy or damage DNA). DNA can also be incorporated into artificial lipid vesicles, liposomes, which fuse with the cell membrane, delivering their contents directly into the cytoplasm. In an even more direct approach, DNA is absorbed to the surface of tungsten microprojectiles and fired into cells with a device resembling a shotgun.
Further methods for introducing nucleic acid molecules into cells involve the use of viral vectors. Since viral growth depends on the ability to get the viral genome into cells, viruses have devised clever and efficient methods for doing it. Various viral vectors have been used to transform mammalian cells, such as vaccinia virus, adenovirus, and retrovirus.
As indicated, some of these methods of transforming a cell require the use of an intermediate plasmid vector. U.S. Pat. No. 4,237,224 to Cohen and Boyer describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture. The DNA sequences are cloned into the plasmid vector using standard cloning procedures known in the art, as described by Sambrook et al. (1989).
Various test substances can be screened for their effectiveness as negative antagonists of a constitutively active GPCR. Test substances can be, for example, traditional chemical compounds (such as benzodiazepines) or peptides. Any suitable test substance can be screened in the subject method, as long as the host cell can be exposed to the test substance.
Having thus identified negative antagonists of a constitutively active G protein coupled receptor, and knowing that constitutively active G protein coupled receptors cause tumor formation or cell proliferation, the invention further provides a method of preventing tumor formation or cell proliferation in a subject caused by a constitutively active G protein coupled receptor. The method comprises identifying a negative antagonist of a constitutively active G protein coupled receptor (as discussed above); and administering to the subject an amount of the negative antagonist effective to prevent tumor formation or cell proliferation caused by the constitutively active G protein coupled receptor. In one embodiment, the tumor formation or cell proliferation is caused by a constitutively active G protein coupled receptor of a virus such as human herpesvirus 8.
The following examples relate to the identification of a constitutively active GPCR of human herpesvirus 8, and the assaying of the GPCR activity by co-expressing a reporter protein under the control of a promoter responsive to the GPCR signalling pathway. By then exposing the host cell to a test substance, the effectiveness of the test substance as a negative antagonist of the constitutively active GPCR can be determined (by determining the effect of the test substance on reporter protein activity). The identified negative antagonists could serve as probes to determine the role of agonist-independent HHV8 GPCR activity in diseases associated with HHV 8 infections or as lead compounds for drugs (Navia and Murcko 1992) if a role for HHV8 GPCR in a disease process(es) is demonstrated. In particular, because constitutively active GPCRs have been found to be tumorigenic in mice (Young et al. 1986) or to mediate proliferation or transformation of cells infected with HHV 8 (Julius et al. 1989; Allen et al. 1991; Gutkind et al. 1991), these negative antagonists could lead to the development of anti-tumor drugs.
Competition binding. The coding sequence of KSHV GPCR was cleaved from its original subcloning plasmid (Cesarman et al. 1996) by digestion with NcoI and inserted into the EcoRV site of the eukaryotic expression vector pcDNA3.1(+) (Invitrogen), producing the pcKSHV GPCR used for expression in mammalian cells. COS-1 cells (American Type Culture Collection) were grown in 100 mm petri dishes in Dulbecco's modified Eagle's medium (DMEM; GIBCO-BRL) with 5% Nu-Serum (Collaborative Research) in a humidified incubator in an atmosphere of 5% CO2 at 37� C. DEAE-dextran transfections were carried out as previously described (Nussenzveig et al. 1994) COS-1 cells were transfected with 10 mg/ml of pcKSHV GPCR and tested for IL-8 binding 48 hrs post-transfection. Approximately 100,000 cells per well (in 12-well plates) were washed with Hank's balanced salt solution (HBSS) containing 25 mM HEPES, pH 7.4 (GIBCO-BRL). Between 0.3 and 0.5 nM 125 I-IL-8 (DuPont-NEN) in the absence or presence of various concentrations of unlabelled chemokines was added and binding was allowed to proceed at 4� C. for 2 hrs with gentle rocking. The binding buffer contained bovine serum albumin (BSA; 1 mg/ml), bacitracin (1 mg/ml), and phenyl methyl sulphonyl fluoride (PMSF; 1 mM). Assays were terminated by aspirating the binding buffer, washing three times with chilled HBSS and solubilizing the cells with 0.4 N NaOH. An aliquot of this solution was counted in a gamma counter and the data expressed as percent of maximum bound. Binding and dose-response curves were analyzed using Prism software (GraphPad, Inc.).
Inositol phosphate accumulation. COS-1 cells were transfected with pcKSHV GPCR or plasmids encoding human IL-8 receptor types A or B and either Ga16 or Ga15 (Wu et al. 1993). All transfection cocktails were supplemented to contain equal total plasmid DNA by the addition of appropriate amounts of vector (pcDNA3.1). Twenty four hrs following transfection, cells were harvested and reseeded in 12-well plates in DMEM with 10% Nu serum and 1 mCi/ml myo-[3 H]inositol (DuPont-NEN), and maintained in culture as before for an additional 24 hrs. After removing the culture media and rinsing the cells, cells were incubated in HBSS containing 25 mM HEPES, pH 7.4 with 10 mM LiCl in the absence or presence of human recombinant IL-8 (R&D Systems) at 0.25 mg/ml. Cells were harvested after 90 min or other time points, and IP accumulation measured using ion exchange chromatography as described previously (Straub et al. 1990).
Luciferase assay. Cells were transfected with increasing amounts of pcKSHV-GPCR (0.003 to 0.100 mg/ml) and 5 mg/ml pAP1-fos-LUC (Schadlow et al. 1992), supplemented to contain equal total plasmid DNA by the addition of appropriate amounts of vector (pcDNA3.1). Cells were washed with phosphate-buffered saline (PBS) and lysed with 0.5 ml/10 cm dish of lysis buffer (25 mM GlyGly, 15 mM MgSO4, 4 mM EGTA, 1 mM dithiothreitol (DTT), 1% Triton X-100, pH 7.8). Cell extracts were diluted 1:10 or 1:20 in reaction buffer (25 mM GlyGly, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT, 15 mM KH2 PO4, 2 mM ATP, pH 7.8), combined with equal volumes of 0.4 mM luciferin in reaction buffer and luminescence measured for 10 sec.
Cell proliferation assay. "Normal rat kidney" fibroblasts (NRK-49F; ATCC) were seeded at 2�105 /100 mm dish, grown in DMEM with 10% fetal calf serum, and transfected with control vector. (20 mg/plate), pcKSHV GPCR (20 mg/plate) or pRas-Zip (10 mg/plate; positive control) (Dotto et al. 1985) using the calcium phosphate method as previously described (Loyter et al. 1982). Cells were harvested 24 hrs post-transfection and seeded at a density of 5,000 cells/100 mm dish in DMEM supplemented with either 10% calf serum or fetal calf serum. On day 5 post-transfection, cells were photographed for qualitative assessment (Nikon inverted phase microscope) and then trypsinized to determine total cell number using a hemocytometer.
Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV 8) is a recently described virus consistently present in Kaposi's sarcoma (KS) (Chang et al. 1994; Moore and Chang 1995) and primary effusion (body cavity-based) lymphomas (PEL) (Cesarman et al. 1995) which are malignancies occurring frequently, but not exclusively, in AIDS patients. KSHV is a gammaherpesvirus with homology to Herpesvirus Saimiri (HVS) and Epstein-Barr virus (EBV) (Chang et al. 1995; Moore et al. 1996), both of which are viruses capable of transforming lymphocytes (Roizman 1996). Cloning of a viral genome fragment revealed the presence of an open reading frame encoding a putative G protein-coupled receptor (KSHV GPCR) (Cesarman et al. 1996) that is homologous to a GPCR encoded by HVS (Nicholas et al. 1992; Ahuja and Murphy 1993) and to human interleukin 8 (IL-8) receptors (Holmes et al. 1991; Murphy and Tiffany 1991). In the studies that follow, it is shown that KSHV GPCR is a bona fide signaling receptor with constitutive (agonist-independent) activity via the phosphoinositide-1,4,5 inositoltrisphosphate-protein kinase C (PKC) cascade. Furthermore, KSHV GPCR is shown to stimulate cellular. proliferation, making it a candidate viral oncogene. The nucleic acid and amino acid sequences of the HHV 8 GPCR are disclosed in co-pending, co-assigned U.S. Ser. No. 08/728,603, filed Oct. 10, 1996, the contents of which are hereby incorporated by reference.
FIG. 1 illustrates the putative two-dimensional topology of HHV 8 GPCR. The top of the diagram represents the extracellular space (E), the middle portion represents the transmembrane (TM) domain, and the bottom portion represents the intracellular space (C). Each circle represents an amino acid residue designated by the single letter code.
As is evident, HHV 8 GPCR can be represented with the signature seven TM-spanning domains of all GPCRs. This, however, is not sufficient to classify this protein as a receptor. Moreover, it is lacking some of the residues that are highly conserved in GPCRs. The most relevant to this application relate to amino acid residues that have been found to be involved in activation of other GPCRs. For example, an Asp residue is usually present in putative TM-2 that has been shown to be critical for activation of a number of GPCRs (Baldwin 1994). There is an Asp at this position in both IL8-Rs but not in the GPCR in the Herpesvirus saimiri genome and not in HHV 8 GPCR. In HHV 8 GPCR this Asp has been replaced by Asn. A similar replacement of Asp by Asn in TM-2 is present in the receptor for gonadotropin-releasing hormone (GnRH) but in the GnRH receptor there is a complementary change of a highly conserved Asn in TM-7, which is in a Asn-Pro-Xaa-Xaa-Tyr motif (Baldwin 1993), to Asp (Zhou et al. 1994) that is not present in HHV 8 GPCR. It has been postulated that interactions between these two residues, Asp and Asn, may be necessary for receptor activation (van Rhee and Jacobson 1996). There is a Val at this position in TM-7 in HHV 8 GPCR that will not be able to form a hydrogen bond with the Asn in TM-2. The absence of this potential interaction between TM-2 and TM-7 may render HHV 8 GPCR constitutively active, but this cannot be predicted from the sequence.
A number of experiments were performed to show that HHV 8 GPCR is a functioning receptor. To investigate whether the KSHV GPCR gene encodes a functional receptor, the KSHV GPCR was expressed in COS cells and a determination whether the receptor binds IL-8 and other chemokines was made. IL-8, which is a member of the family of C-X-C chemokines (Baggiolini et al. 1994), was chosen as radioligand because of the homology of KSHV GPCR to cellular IL-8 receptors. Furthermore, since KS lesions are sites of active inflammation and have been shown to produce IL-8 and other cytokines (Sciacca et al. 1994), it is possible that such areas could provide stimulatory signals for the KSHV GPCR. The binding characteristics of KSHV GPCR were investigated using 125 I-IL-8 and various concentrations of other chemokines in a competition analysis. As shown in FIG. 2, KSHV GPCR binds IL-8 with an apparent equilibrium dissociation constant (Kd) of 30 nM, which is lower affinity than the values previously reported for binding of IL-8 to the human IL-8 receptor types A or B (Kd of 0.8-1.0 nM) (Holmes et al. 1991; Murphy and Tiffany 1991). Since chemokine receptors may bind more than one ligand, several other C-X-C chemokines were tested, namely MGSA, NAP-2 and PF-4 (Baggiolini et al. 1994), and it was found that they also bind to KSHV GPCR (FIG. 2). Although chemokines of the C-C family do not bind to IL-8 receptors (Baggiolini et al. 1994), it was found that I-309 and RANTES bind to KSHV GPCR whereas MIP-1b and MCP-1 do not (FIG. 3). The rank order of binding was: IL-8=NAP-2>PF-4=I-309=MGSA>RANTES>>MIP-1b and MCP-1. Thus, KSHV GPCR exhibits binding characteristics of a chemokine receptor with affinity for a range of chemokines spanning the C-X-C and C-C families.
To assess whether KSHV GPCR is a signal transducing receptor, the formation of intracellular second messengers in COS cells transiently expressing KSHV GPCR was measured. Inositol phosphate (IP) accumulation was measured because IL-8 receptors signal via the phosphoinositide-IP-calcium/diacylglycerol-PKC cascade (Holmes et al. 1991; Murphy and Tiffany 1991). Stimulated IP formation can be measured in COS cells co-expressing IL-8 receptors and the a subunits of the heterotrimeric proteins G16 (Ga16) or G15 (Ga15) in the presence but not in the absence of IL-8 (Wu et al. 1993). In agreement with that report, little measurable accumulation of IPs in COS cells expressing IL-8 receptors in the absence or presence of IL-8 was found, unless Ga16 or Ga15 was cotransfected (FIG. 4). In cells expressing IL-8 receptors and Ga16 or Ga15, IL-8 stimulated IP formation. In contrast, and under these experimental conditions, cells transiently expressing KSHV GPCR showed IP accumulation which was higher than that obtained with human IL-8 receptors activated by IL-8. In cells transiently expressing KSHV GPCR, IP formation was elevated in the absence of added ligand and without co-expression of Ga16 or Ga15, and was not increased by IL-8 or any of the chemokines that showed binding to KSHV GPCR. There was a constant rate of IP accumulation for at least 4 hours (FIG. 5); IL-8 had no effect at any time point. Transfection of COS-1 cells with increasing amounts of plasmid encoding KSHV GPCR showed that IP accumulation was directly proportional to the amount of plasmid used for transfection (between 0.1 and 3 mg/ml plasmid) (FIG. 6). Again, there was no effect of IL-8 on IP accumulation at any level of KSHV GPCR expression. Thus, KSHV GPCR exhibits agonist-independent (or constitutive) activity in COS-1 cells. In contrast to IL-8 receptors, KSHV GPCR couples effectively to the G protein(s) endogenous in COS-1 cells. Since IP accumulation from the KSHV GPCR is resistant to pertussis toxin, this suggests that the receptor may signal through the Gq family of G-proteins.
To determine whether the constitutive signaling activity of KSHV GPCR leads to stimulation of downstream processes, the effect of basal second messenger formation on gene transcription was measured. A reporter gene, firefly luciferase, under the control of a PKC-responsive promoter containing an AP-1 binding motif (Schadlow et al. 1992) was used to measure activity of the diacylglycerol/PKC limb of the phosphoinositide signaling pathway. In these experiments, COS-1 cells were co-transfected with plasmid encoding KSHV GPCR and a plasmid containing a minimal promoter derived from the human c-fos gene to which a PKC-responsive element was inserted to drive luciferase transcription (Schadlow et al. 1992). As shown in FIG. 7, there was a direct relationship between the amount of plasmid encoding KSHV GPCR used for transfection and the level of luciferase activity. Thus, second messenger formation caused by the basal activity of KSHV GPCR is sufficient to induce gene transcription for a PKC-responsive promoter. This finding is important given the wide range of genes, including those for inflammatory and angiogenic agents, whose transcription is regulated by PKC-responsive promoters (Karin 1995). Of note is the observation that luciferase activity (FIG. 7) is stimulated at lower plasmid levels than IP accumulation (FIG. 6). This likely occurred because induction of gene transcription is a more amplified response occurring downstream of second messenger formation.
To test whether the constitutive KSHV GPCR signaling activity affects cellular proliferation, NRK-49F cells were transiently transfected with plasmid encoding KSHV GPCR and monitored for proliferation as compared to cells transfected with control vector (negative control) or with a plasmid encoding the Ras oncogene (positive control) (Dotto et al. 1985). Culture dishes transfected with Ras-encoding plasmids contained more cells than control dishes within 24 to 48 hrs. As early as 72 hrs post-transfection, the KSHV GPCR transfectants exhibited increased cell number as compared to the control transfectants. This response was confirmed by phase contrast microscopy and quantified after 5 days in culture (FIG. 8). All clusters transfected with pcKSHV GPCR contained many more, closely apposed cells than clusters of cells transfected with control plasmid.
Since gammaherpesviruses cause tranformation (Roisman 1996) and GPCRs have been found to be oncogenic under some circumstances (Julius et al. 1989), it is possible that KSHV may be a transforming agent that acts, at least in part, through KSHV GPCR. This possibility is further strengthened by the specific association of KSHV with KS and PEL, and the fact that KSHV GPCR is expressed at the RNA level in both of these types of malignancies (Cesarman et al. 1996; Arvanitakis et al. 1996). These results have now demonstrated that KSHV GPCR is a bona fide receptor that signals constitutively via the phosphoinositide transduction pathway. Although it has an amino acid sequence that is homologous to IL-8 receptors, binds IL-8 with relatively high affinity and activates the same signal transduction cascade, KSHV GPCR has two characteristics that make it a more promiscuous receptor and one more likely to be involved in pathogenesis than IL-8 receptors. KSHV GPCR signals constitutively, that is, it does not require activation by an agonist(s) and is capable of coupling to a wider variety of G proteins than IL-8 receptors. Coupling of KSHV GPCR to G proteins other than Ga16, as shown by signaling in COS cells that do not express this G protein, would allow KSHV GPCR to signal in a broader range of cell types. Constitutive signaling activity, which is exhibited by some native GPCRs (Milano et al. 1994), has been observed more commonly with mutated GPCRs (Lefkowitz et al. 1993; Alblas et al. 1996) in an increasing number of human diseases, including tumors (Coughlin 1994; Sande et al. 1995). Finally, the enhanced proliferation exhibited by KSHV GPCR transfectants supports a role for this receptor in tumorigenesis.
The presence of this functional, constitutively active GPCR in the genome of a virus that is so tightly associated with specific malignancies provides circumstantial evidence toward its pathogenetic role. Two alternative, or perhaps complementary, hypotheses can be proposed to explain the role of the viral GPCR in the development of KSHV-associated diseases: a direct mechanism, in which the constitutive signaling activity of KSHV GPCR leads to altered growth and neoplastic transformation, and/or an indirect mechanism, where signaling by KSHV GPCR induces the expression of other genes which are in turn involved in pathogenetic events. For example, the latter model could apply to the case of basic fibroblast growth factor (bFGF) which has previously been shown to play a pivotal role in the development of KS lesions (Ensoli et al. 1989), and whose expression is driven by an AP-1 binding, and probably PKC-responsive promoter (Erdos et al. 1995).
The determination of the effect on cells expressing HHV 8 GPCRs of negative antagonists of HHV 8 GPCRs will delineate the role(s) of HHV 8 GPCRs in infected cells. In this regard, the two most important downstream effects are the stimulation of cell proliferation or if HHV 8 GPCR activity caused cell transformation. One can determine whether HHV 8 GPCR can induce cell transformation and whether antagonists can inhibit this effect. Transformation in NIH 3T3 fibroblasts can be used in a protocol used to show that the agonist-activated serotonin 1c receptor, another GPCR, is capable of triggering transformation (Julius et al. 1989). In this assay, HHV 8 GPCR expression will be produced by transfection with plasmids encoding HHV 8 GPCR, not by infection with HHV-8. This assay will show whether HHV 8 GPCR expression leads to a transformed phenotype and provide a quantitative assessment of the effect of antagonist on transforming activity of HHV 8 GPCR because the number of foci of transformed cells in the absence versus the presence of antagonist can be counted. One can also determine whether HHV-8 infection will transform CD19+ B-cells in experiments similar to those performed to show lymphoblastoid transformation by Herpesvirus saimiri and Epstein-Barr viruses (Rangan et al. 1977; Miller 1974). If this series of experiments shows that HHV 8 GPCR expression causes transformation in these in vitro assays, one can determine whether cells from these foci will generate tumors in nude mice and whether this can be inhibited by antagonists.
B-cell lines will be used for studies of cell proliferation because of ease of their experimental manipulation. These B-cell lines may not be good models for study of proliferation because they may have acquired other changes (mutations) that allow them to proliferate without the signalling activity of HHV 8 GPCRs. Acutely infected cells, such as B-cells, will be used and one can study other cell lines including human lymphoblastoid cell lines UH-1 and CB-33, and endothelial and fibroblast cell lines derived from KS lesions. An effect of antagonists of HHV 8 GPCR to inhibit the rates of cell proliferation will be monitored. This will be tested in a standard assay by measuring the rate of proliferation of cells incubated without (control) and with antagonists. By counting cells, which are seeded at low density, over time, one can determine the effect of these antagonists on the generation times of these cells. Different incubation mediums can be tested and, in particular, attempt to adjust the type and concentration of the serum supplement so that there is a moderate generation time for cells in the absence of antagonists. This will allow for the highest likelihood of observing an inhibitory effect on proliferation. These experiments will be complemented with those that measure DNA synthesis more directly, for example, by measuring [3 H]thymidine incorporation into DNA and by measuring DNA content, for example with 5-bromodeoxyuridine. The most definitive measurement of cell proliferation is, however, the rate of increase in cell number over time.
These experiments will establish whether HHV 8 GPCR is expressed at the protein level in infected cells, whether HHV 8 GPCR stimulates cellular transformation and/or proliferation and whether the negative antagonists discovered according to the method of the subject invention will inhibit these effects.
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.
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__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 1 - - - - (2) INFORMATION FOR SEQ ID NO:1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 342 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - - Met Ala Ala Glu Asp Phe Leu Thr Ile Phe Le - #u Asp Asp Asp GluSer 1 5 - # 10 - # 15 - - Trp Asn Glu Thr Leu Asn Met Ser Gly Tyr As - #p Tyr Ser Gly Asn Phe 20 - # 25 - # 30 - - Ser Leu Glu Val Ser Val Cys Glu Met Thr Th - #r Val Val Pro Tyr Thr 35 - # 40 - # 45 - - Trp Asn Val Gly Ile Leu Ser Leu Ile Phe Le - #u Ile Asn Val Leu Gly50 - # 55 - # 60 - - Asn Gly Leu Val Thr Tyr Ile Phe Cys Lys Hi - #s Arg Ser Arg Ala Gly 65 - #70 - #75 - #80 - - Ala Ile Asp Ile Leu Leu Leu Gly Ile Cys Le - #u Asn Ser Leu Cys Leu 85 - # 90 - # 95 - - Ser Ile Ser Leu Leu Ala Glu Val Leu Met Ph - #e Leu Phe Pro Asn Ile 100 - # 105 - # 110 - - Ile Ser Thr Gly Leu Cys Arg Leu Glu Ile Ph - #e Phe Tyr Tyr Leu Tyr 115 - # 120 - # 125 - - Val Tyr Leu Asp Ile Phe Ser Val Val Cys Va - #l Ser Leu Val Arg Tyr130 - # 135 - # 140 - - Leu Leu Val Ala Tyr Ser Thr Arg Ser Trp Pr - #o Lys Lys Gln Ser Leu 145 1 - #50 1 - #55 1 -#60 - - Gly Trp Val Leu Thr Ser Ala Ala Leu Leu Il - #e Ala Leu Val LeuSer 165 - # 170 - # 175 - - Gly Asp Ala Cys Arg His Arg Ser Arg Val Va - #l Asp Pro Val Ser Lys 180 - # 185 - # 190 - - Gln Ala Met Cys Tyr Glu Asn Ala Gly Asn Me - #t Thr Ala Asp Trp Arg 195 - # 200 - # 205 - - Leu His Val Arg Thr Val Ser Val Thr Ala Gl - #y Phe Leu Leu Pro Leu210 - # 215 - # 220 - - Ala Leu Leu Ile Leu Phe Tyr Ala Leu Thr Tr - #p Cys Val Val Arg Arg 225 2 - #30 2 - #35 2 -#40 - - Thr Lys Leu Gln Ala Arg Arg Lys Val Arg Gl - #y Val Ile Val AlaVal 245 - # 250 - # 255 - - Val Leu Leu Phe Phe Val Phe Cys Phe Pro Ty - #r His Val Leu Asn Leu 260 - # 265 - # 270 - - Leu Asp Thr Leu Leu Arg Arg Arg Trp Ile Ar - #g Asp Ser Cys Tyr Thr 275 - # 280 - # 285 - - Arg Gly Leu Ile Asn Val Gly Leu Ala Val Th - #r Ser Leu Leu Gln Ala290 - # 295 - # 300 - - Leu Tyr Ser Ala Val Val Pro Leu Ile Tyr Se - #r Cys Leu Gly Ser Leu 305 3 - #10 3 - #15 3 -#20 - - Phe Arg Gln Arg Met Tyr Gly Leu Phe Gln Se - #r Leu Arg Gln SerPhe 325 - # 330 - # 335 - - Met Ser Gly Ala Thr Thr 340__________________________________________________________________________
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By far the most significant of tonight's three NBA games is a must-win for Indiana, at home against the Wizards. Boston has clinched a postseason spot but the Pacers are still in a battle with Brooklyn to determine the eighth team in the East. [Read more…]
Fantasy Spin: Busy NBA Night & Huge Masters Contest
A 13-game slate means plenty of choices in daily NBA contests at DraftKings. Of the four players with 5-digit salaries, James Harden $11000 is probably the safest play, as he leads the Rockets into San Antonio. [Read more…]
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No fooling, there are a dozen NBA games and DraftKings offers four different starting times for daily fantasy fun and profit. The four-game Turbo contests at 8:00 ET are fascinating. [Read more…]
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Teams in the NBA playoff races aren't the only ones watching scoreboards this time of year. Take the Philadelphia 76ers, for example. The Sixers lost their first 17 games, were mathematically eliminated from the playoff race nearly a month ago and almost certainly will lose 60-plus games for the second straight season. But GM Sam Hinkie and the rest of the Sixers' front office are tracking the results of other games almost as closely as their own. That's because Philadelphia could have
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With 13 NBA games tonight, there are several excellent players to choose from in every salary range at DraftKings but strategy begins at the highest level. [Read more…]
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Another six-game NBA slate gets under way at 7:30 ET, with the Westbrook Decision looming large in DFS contests. Russell Westbrook $13200 has been cranking out triple-doubles and averaging 65.3 FPTS over his last ten. It's also been an incredibly consistent streak — only the Clippers kept him from dominating. [Read more…]
Fantasy Spin: Will The Thunder Keep Rolling?
It's a quiet Sunday in the NBA — only three games — on the eve of a busy holiday Monday. There are two start times at DraftKings; contests at 3:30 EST include the Pelicans game in Toronto, where Anthony Davis $11200 (toe) remains questionable. You can also play at 6:00, focusing on just two games. [Read more…]
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Toronto Raptors basketball was non-existent for a long time. There was more booing than cheering, the word "retooling" became too familiar, and games rarely gathered genuine excitement. That was their identity, or lack thereof. I went to my first Raptors game at the age of 13. Toronto had a good team that year, they won 47 games and finished first in the Atlantic Division. The Raps were hosting the Denver Nuggets at Air Canada Centre, a hot team with Carmelo Anthony and

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Peter Poole
Peter Poole (c. 1932 – 18 August 1960 in Nairobi, Kenya) was a British-born Kenyan engineer and shop owner. He was the only white in Kenya to be executed for killing an indigenous African person.[1][2]
On 12 October 1959 he was charged with killing Kamawe Musunge, Poole's houseboy,[1] in Gordon Road, Nairobi. Musunge had been riding a bicycle when Poole's two dogs stopped him. Musunge threw stones at one dog, for which Poole shot Musunge dead with a Luger pistol.[3] Poole was executed on 18 August 1960. At the time Kenya was still under British rule, and the verdict was received dismally by white settlers in Kenya, who could not accept that a white man could be sentenced for killing an indigenous African.[3]
Poole had emigrated to Kenya from Essex and was an engineer by profession.[4] He owned an electrical shop on Nairobi's Government Road (now Moi Avenue).[2] Poole served in the British Army during the Mau Mau Uprising.[1] Poole was married with two children. His father was Norman Poole.[3]
See also[edit]
- Thomas P. G. Cholmondeley, white Kenyan found guilty of manslaughter
- ^ a b c "KENYA: White Man Hangs - TIME". content.time.com. August 29, 1960. Retrieved 4 March 2014.
- ^ a b Mwaura, Peter (7 May 2009). "Year when all men became equal at the end of a rope - News - nation.co.ke". nation.co.ke. Retrieved 4 March 2014.
- ^ a b c Christine Stephanie Nicholls: Red strangers: the white tribe of Kenya Timewell Press, 2005. ISBN 1-85725-206-3
- ^ Truecrimelibrary.com: Peter Poole – Kenya


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HOME College Kansas State Wildcats Gear Kansas State Wildcats Accessories (419 items) Product ID: 1191380
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Make sure you're showing maximum Kansas State spirit with this Wordmark scarf. It features two woven team logos and "K-State" lettering for optimal team pride. This scarf will keep you warm while you cheer on your Wildcats!
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High View Homes for Rent
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With a month or so of downtime between the Gold Coast and Bells Beach events, France's favourite backhand tube hound Jeremy Flores made good use of his time with a free surfing trip to the Mentawais.
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Pulaski Furniture Accents Bar Cabinet with Wine Storage
Pulaski Furniture Accents Bar Cabinet with Wine Storage
Weights & Dimensions
|Product Type: Wine bar; Bar cabinet
|Style: Contemporary
|Bar Shape: Straight
|Finish: Brown and black
|Distressed: Yes
|Countertop Material: Manufactured wood; Wood
|Base Material: Wood; Manufactured wood
|Number of Items Included: 1
|Solid Wood Construction: No
|Cabinets Included: Yes
|Shelving Included: Yes
|Wheels Included: No
|Drawers Included: No
|Stools Included: No
|Wine Rack Included: Yes
|Wine Glass Storage: Yes
|Dry Sink Included: No
|Expandable: No
|Foldable: No
|Footrail Included: No
|Hand painted: Yes
|Eco-Friendly: Yes
|Product Care: Wipe clean with a dry cloth
|Country of Manufacture: China
|Assembly Required: Yes
|Installation Required: No
|Additional Parts Required: No
|Product Warranty: 1 year against material manufacturing defects
About the ManufacturerAs one of the country's best known furniture brands, Pulaski Furniture is synonymous with outstanding craftsmanship and refined styling. Offering a wide range of bedroom, dining, accents and display cabinets, Pulaski Furniture covers the complete design spectrum: traditional, contemporary and transitional. With 60 years of furnishing homes across America, Pulaski Furniture continues its heritage of outstanding value, quality and design.
Pulaski Furniture Accents Bar Cabinet with Wine Storage
Customer Reviews
|5 star
|4 star
|3 star
|2 star
|1 star
Wine cabinet
Renee from Connersville, IN – Verified Buyer
We absolutely love our new wine cabinet. It is large enough to hold everything, including our wine glasses. It came assemble except for the feet, which took no trouble to attach. The delivery was not a good experience. We were given a time frame for delivery. They showed up almost 2 hours early. There was no one at our house to meet them because we had scheduled everything around their delivery time frame. We were left with a "sorry we missed you" note in our door. Upon calling the delivery company several times (with conversations and unreturned phone calls), we were told that we would have to wait for another week because they weren't scheduled to be in our area again for a week. My husband finally called and told them that that was not acceptable, that he expected it the next day or we would cancel the order. I contacted Wayfair as well and they said that they would make sure it got to us sooner. After more phone calls that went in to early afternoon the next day, we got delivery scheduled for that afternoon/evening. The driver showed up and my husband told him to back in to our yard and turn around. It was rainy and, instead of doing as my husband indicated, he pulled in to the side yard, got stuck and tore up our yard as well as our neighbor's, before he just drove across the neighbors yard. (Needless to say we had to devote some time and resources to repairing our neighbor's yard as well as ours.) There was only one person in the vehicle so my husband had to help unload the cabinet. The driver's dolly had a flat tire so we now have marks all the way up our front walk and to our porch where it just scraped the metal wheel on the ground. At this point we were just glad to get it in the house and the driver had us sign and took off. Again, we LOVE this cabinet but we almost didn't get it because of the delivery. Wayfair did do a good job of helping us schedule the delivery after all of our phone call. For that we are thankful.
Wonderful accent piece
Axel from Cedar Park, TX – Verified Buyer
This cabinet is well built, sturdy, and it defines a special accent in our home. We even modified the interior slightly by removing the horizontal bottle storage portion and replacing it with a glass bottom to store larger bottles in an upright fashion. Wayfair had by far the best price on this, and shipping and communication was excellent. We love to shop at Wayfair. So far ... no problems :-)
great product
paula from New Hope, PA – Verified Buyer
Great product. Was a big hit for Thanksgiving Dinner! Fits tons of stuff from bottles to glasses….wished it had an interior drawer but besides that we are very happy. Was a bit worried about shipping and delivery but it worked out great!
Excellent piece of furniture
Elizabeth from Deland, FL – Verified Buyer
This is a solid wood furniture piece. Quality is very good. Item looks exactly as pictured, very roomy inside and accommodates even larger sized wine bottles and a large amount of wine glasses in the holders. Everything from ordering to delivery was very smooth, item arrived timely.
Funky and practical
Rosa from Lacey, WA – Verified Buyer
First one. arrived damaged, Broker hinge and damaged paint on top . Immediately called Wayfair and they sent us a new one couple of weeks later Pros: No assembly necessary, just legs which we decided not to install. Second one looks just like picture and fits nicely in family room. Cons: Having to wait for replacement.
An excellent Wine Bar
Minnie from Bend, OR – Verified Buyer
The unique wine bar makes the perfect accent, yet functional conversation piece.. It is very well constructed and functional with inside shelves for storage and holds 14 bottles of wine...with plenty of room for glasses and bottles of liquor.
Great product.
Diane from Wyoming, MI – Verified Buyer
Great product that matches the description. Bought the wine cabinet to use in the family room, the style fits beautifully. Absolutely love it!
Very pleased with product...
Raymond Doane from Tucson, AZ – Verified Buyer
Very pleased with quality of product. Well made fits perfectly. Excellent storage and quirky with language on the front. Use for wine storage and glasses fit perfectly
Wine Cabinet
Larry from Seymour, TN – Verified Buyer
Pleasantly surprised at the quality. Only had to screw on the legs. Other than that, no assembly. Can tell it was designed to last and it was cheaper than I could find the same cabinet anywhere else.
Love it! Very unique
Kathleen from Perry Hall, MD – Verified Buyer
Only reason I didn't get it 5 stars is because the wine glass holders do not fit a lot of bigger stemmed glasses and also doesn't fit taller glasses - only smaller/shorter ones.
OUR SKU # PU4022Accents Bar Cabinet with Wine Storage is also sometimes listed under the following manufacturer numbers: 549159


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Create a logo for a Recipes Wiki. It´s not acctually a corporate Kit, but a Logo Design, and a Card (like business card) with the Logo, URL and email only.
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If you are looking to update your living-room interiors but don't want to spend lots on a new set of furniture, the Better Homes a...nd Gardens Stretch Pearson Sofa Slipcover may just be what you need. This item is a practical solution to changing your decor's look and feel without breaking the bank. This stretch couch slipcover has a unique two-way stretch material and goes on easily and stays in place. The fabric is made with 94 percent polyester and four percent spandex and is designed to weather repeated use. This furniture slipcover can be machine washed, so maintenance is also not a problem. This item gives your living room a fresh new look, while ensuring that your original furniture stays protected from possible damage. read more


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Snow White And The Seven DwarfsSnow White And The Seven Dwarfs (1973) - Festival of Family Classics Cartoon Episode Guide
This page has been viewed 14 times this month, and 840 times total.
- Festival of Family Classics Cartoon Episode Guide
- Rankin Bass Productions, Mushi Studios
- Distributed by: Videocraft International
- Cartoon Characters: Snow White, Seven Dwarfs, Queen, Prince, Magic Mirror, Owl.
- Originally Released in 1973.
- Color
- Running Time: 22:47 minutes.
Cartoon Summary:
The familiar plot points of the timeless tale of Snow White -- pursuit by the jealous queen, undercover with the Dwarfs, the poison apple snack and the Prince's wake-up kiss -- are all present and accounted for in this charming retelling.
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text:
8 Reasons We Need Women Leaders
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text:
Hey, Im a source mapper interested in writing, gaming, filming, anything with the word 'steampunk' in it, and modding for Half Life 2. Thanks for dropping by!
Welcome To Our Profile Page, we hope you enjoyed the mods we have created so far
Desura is a community driven digital distribution service for gamers, putting the best games, mods and downloadable content from developers at gamers...
A group dedicated to the civilized debate and discussion of a wide range of topics, from the political to gaming and so on.
This is a group for those of us that enjoy leaving comments and reviews for HL2 mods. We encourage you to leave comments, images, or even videos on how...
seriously, theres barely any good Horror-Mods, and there should be. this, in short, is a group for all the gamers who feel left out of the mod-playing...
A group for those interested in science from the vast expanse of the universe or technology.
For people and teams developing mods and games with Valve's Source engine.
A place where ModDB members can debate civilly, and learn from each other's views.
The Fellowship is an unofficial Tolkien lore group that brings every fan together from his great works in the Middle-Earth universe!


text:
Broccoli plugin to merge multiple trees into one
Copy multiple trees on top of each other, resulting in a single merged tree.
npm install --save-dev broccoli-merge-trees
var mergeTrees = require'broccoli-merge-trees'; var outputTree = mergeTreesinputTrees options;
inputTrees: An array of trees
options: A hash of options
overwrite: By default, broccoli-merge-trees throws an error when a file
exists in multiple trees. If you pass
{ overwrite: true }, the resulting
tree will contain the version of the file as it exists in the last input
tree that contains it.
Clone this repo and run the tests like so:
npm installnpm test
Issues and pull requests are welcome. If you change code, be sure to re-run
npm test. Oftentimes it's useful to add or update tests as well.


text:
How to integrate resources in an ActionLink? I want the title to to display a translation when I navigate to a route where I inject the culture-language. But I do not know how to get the translation into the ActionLink.
Take the 2-minute tour ×
Stack Overflow is a question and answer site for professional and enthusiast programmers. It's 100% free, no registration required.
Maybe I'm missing something. But you should add a simple route as follows.
Afterward, you have to create an ActionFilter to set the culture on request.
At last in the view, use ActionLink as follows.
The above code snippet demonstrated a simple one. You can find more information here and a simple way to use resources in ASP.NET MVC view, here.


text:
What happened to the men who signed the Declaration of Independence
Click here for our listing
Click here for a list of local libraries in our area.
"Signing Their Lives Away: The Fame and Misfortune of the Men Who Signed the Declaration of Independence" by Denise Kiernan and Joseph D'Agnese; Quirk Books, Philadelphia (256 pages, $19.95)
"Signing their Lives Away" is a good road trip read based on a pivot point of American history.
In the hot summer of 1776, fifty-six men in Philadelphia voted to break with Great Britain and declare the American Colonies free. We know these men who left their mark on a moment of time. But what happened next?
That's what "Signing Their Lives Away" tells — what happened to the men after the ink dried on the Declaration of Independence.
Beyond the famous names of John Hancock, Benjamin Franklin, Thomas Jefferson and John Adams, who were these guys?
William Paca was a lawyer remembered mainly for supporting the troops and becoming a chief justice in Maryland. He also had a "child out of wedlock with a free black woman" and acknowledged the daughter, Hester, sending her to the finest schools.
Sam Adams, of Boston was a lousy businessman. Even before his fame at rabble rousing, he inherited a brewery that "went bust shortly after he took over and Sammy was back in debt, where he remained for the rest of his life."
Another Massachusetts delegate, Elbridge Gerry, gave us the political term "gerrymandering." After being elected governor in 1812, Gerry began to "creatively redraw the state senate voting districts to favor his party. A political cartoonist thought one of the districts resembled a salamander, and the process was quickly dubbed 'gerrymandering."' (This still happens in today's politics, with districts being redrawn to give undue advantage to one political party or the other.)
Richard Stockton of New Jersey recanted signing the Declaration but, since at the time he was imprisoned by the British, George Washington forgave him.
George Wythe, a mentor of Thomas Jefferson, lawyer and all-around legal eagle, helped build the Virginia constitution. In his 80s, he was poisoned by his sister's grandson, who hoped to inherit the estate. He didn't; Wythe lived long enough to disinherit him.
"Signing" is told in a brisk entertaining fashion, with various accompanying essays including a timeline, "The Madness of King George" and "Immigrant Signers." Its greatest liability is the layout. Instead of listing each man alphabetically or even by state, the entries are laid out in the order they signed in 1776; geographically from the North to South along the Eastern Seaboard. Thus the index of names becomes vital.
Some of the signers died in their 80s and 90s, long enough to see the consequences of their actions. They were men of courage who, to quote the Declaration, put their "Lives, our Fortunes, and our sacred Honor," on the line. And this is a good way to meet them.
(c) 2009, McClatchy-Tribune Information Services.
Visit the McClatchy Washington Bureau on the World Wide Web at www.mcclatchydc.com.
PHOTO (from MCT Photo Service, 202-383-6099):
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text:
Contractors say homeowners with this trait are the most satisfied with home improvement projects.
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Don't get burned by failing to read the fine print of a home remodeling contract. Check out these things every remodeling contract should contain.
Houston roofing complaint | Contractor claims leaks are coming from plumbing, not roof.
Angie's Answers
An itemized list / cost breakdown, more often than not is used against the contractor when it is shared with other builders who will then beat it.
Good contractors use good people, and good people cost more. Just the cost of having the appropriate insurance / bond can be the difference between winning a job or losing it ot a 'lower bid'.
It is the rule of three; there is Good, Cheap and Fast. You can have any two: Good and Cheap, won't be Fast; Good and Fast, won't be Cheap; Cheap and Fast, won't be Good!
When comparing bids, it isn't the cheapest or the 'nicest' person you should select. You should understand why there is a large price difference (it shows there are gaps in your design program or what you have asked for specifically, which means there may be arguments later). If most of the bids are in line, and one is way high or way low, you want to know why before dismissing or selecting them.
A price-only decision almost always costs more in the long run.
Good luck!
No. Heck no. Here's a good example. We very recently needed to find someone to install about 500 square feet of exotic wood flooring (we already have the materials). We contacted about 12-15 top-rated Angieslist contractors. Out of the few who did get back to us, we got 5 quotes, 2 of them were literally just over the phone. They "didn't feel it would be necessary to even see the space".
Here were the bids:
$4000 (sight unseen), $2800 (sight unseen), $2500, $1500, $1450
We didn't "share our budget for this". Why would we? We asked them to bid the job. That's it. All of them should be well-qualified and they are all highly rated. We were interested in how THEY value their time/resources - for an apples/apples job.
Do you still think that you should tell them about your budget? Your choice. From my standpoint it isn't their business. I'm asking them to bid on a project. Invariably I'll get some very high bids, medium bids and a few more reasonable ones - ALL from "highly rated contractors".
Next photograph the work you feel is sub-par. If work has been corrected, photograph it now to have a record of its condition. If you have any "original" or "before work began" pictures get those together, too.
If you do have written contract, see what it says about warranties, complaints, failure to finish / comply, etc. Holding the money may end up with the contractor taking YOU to court for the funds - you cannot just hold the money. You need to document in writing what is wrong, what you expect to happen (be specific) and when it should happen by. A good contract will explain if and how money can be held, how the arbitration or complaint is filed, etc.
You should also invite another contractor to come in and bid the work to finish the job. They can confirm the quality of the work and give you a price to fix / finish the job. This gives you justification for holding the funds and an option to finish the job.
If the contractor is not willing to fix the work or listen to direction, do not allow them back in the house. A judge will ask you why you let them continue to do work if you found it unacceptable. Take back the key or access to the building - you can also attempt holding any materials or tools as collateral if the cost of repair is higher than the amount owed. Again, document what you are holding, its estimated value (google or ebay search), etc.
Finally, in writing you should fire the contractor and state the exact reason(s). Document everything; if it is done in person after they leave make notes of what was said, agreed upon and disputed. If you are satisfied that what you have paid is fair compensation for the work done, make sure this is noted in the letter firing the contractor. If you feel money is owed, you will need to file a small claim in your local court. Include the documentation you made, notes, letters, etc. when you file your claim so the judge will have a copy of everything. Don't forget to contact the BBB.
Do not wait for the court date; go ahead and hire the other contractor and have the work completed. Bring this invoice to court with you (file it before the court date if you can). Bring photos of the finished work (again, file it with the court before the date if possible). You must show what good quality work looks like vs the poor quality work.
Otherwise it will be a your word against the contractor and you will most likely lose, (the contract is a promise to pay for work) or even if you "win" you will most likely split the difference between the argued amount of money. Also be prepared for the contractor to file a small court claim against you. Same process as above, except now you will respond to the summons with a copy of your stuff to defend your reason for holding funds instead of asking for money back.
Good luck!
Herlonginc's answer stated that it is not the contractor's job to pay for materials and labor to do the job. I say baloney - a reputable, established contractor has the funds (or a business operations line of credit) to "carry" the job between interim or partial payments, each of which should be keyed to completion of distinct easily measured mileposts in the job, and for a homeowner I would say should be in not more than 20% increments for jobs exceeding a week or so. For shorter jobs, then an initial payment, 50% completion, and completion would be normal. His cost of carry funds is part of his cost of doing business, and is figured as part of his overhead.Bear in mind when he is buying materials and paying labor, his materials he typically pays for on a 10-30 day invoice, and his labor typically a week or two after they work, so he is not really "fronting" that much money if you are giving him weekly or biweekly interim payments, on a typical residential job.
If he does not have the funds to buy materials (excepting possibly deposit on special-order or luxury items, which still typically are 10-30 day invoiceable to him) and hire personnel then he is a fly-by-night operation, and he should not be bidding that size job. You should never (other than MAYBE an earnest deposit of not more than the LESSER of 10% or $5000) let the payments get ahead of the approved/inspected work progress - typically payment should be 10-20% BEHIND the progress, with at least 10% retained at the effective end of work until final inspections and completion of the final "punchlist".
That promotes rapid continuation of the work, discourages the all-too common nightmare of contractors taking on more work than they can handle so they leave your job for weeks or months to go work on someone else's job (frequently to start that someone else's new job so he can get the job), and does not leave you out a tremendous amount of cash if he does not finish and you have to hire another contractor to finish the job. Remember, if you have to hire a new contractor to finish the job, he will charge you a lot more than the original bid to finish someone else's unfinished mess.
This may seem cynical, but having started in the construction business about 50 years ago and seeing the shenanigans that a lot of contractors pull you cannot be too safe. You have to remember contractors are like any other people - I would say maybe 10% are outright crooks, another 25% or so will pull a fast one or overcharge if the opportunity presents itself, maybe 30% will do the work but not any better than they are forced to, about 25% are good conscientious reputable workmen, and the last 10% or so are really spectacular - conscientious, fair, and efficient craftsmen. This top 35% are the only ones you should have bidding in the first place. Therefore, only get bids from long-term reputable firms (so you shake out the marginal short-timers with less experience and also generally less ability to finish the job on budget and schedule), only those that have good RECENT references, and preferably with excellent word-of-mouth recommendation from people you know and trust. That way, you are starting right off with the cream of the crop, so hopefully whichever one bids low should be a good choice.
NEVER start with bids, then check the references of the low bidder - why even consider a vendor or contractor who you do not have faith in from the start ? Get references and short-list you possibles BEFORE you ask for bids.
Low bids - that is another matter - commonly the low bidder is NOT who you want, especially if he is significantly lower than several others, which might mean he is desperate for work, made a math error, or did not correctly figure the entire scope of work. You want a reasonable bid with someone you connect with and trust - that is worth a lot more in the success of the job than the absolute lowest bid.
The real question is why do you need this break down? If you are thinking you will do the demo yourself to save money, you can certainly tell your contractor this - but I would be willing to bet once you buy or rent the tools, haul the trash to the correct disposal dump (many trash dumps will not take home building materials anymore) and clean up / prep for the new work - you will have spent more and delayed the project more than just letting the professionals do it. Plus, do not be surprised when they still have to do additional demo work that you didn't know would be needed to complete the job, etc.
Also keep in mind that cutting portions of the work out of the job to do later is not a money saving move. You will find that the cost for the individual items go up when done seperately - the contractor has to come back multiple times, has to set the equipment back up, possibly pull seperate permits, schedule the work crew / subs, etc.
If you are asking for the break down to compare bids, then again, tell the contractors what numbers you want to see. If you are doing it because you feel the total price is too high, have a discussion with your contractor; they may be able to suggest ways to save costs, etc. Ultimately if you know the materail costs, and have the total figure, you can do a pretty good estimate of the percentage for labor and profit in the job.
It is your project and your contract, so you can ask for anything you want on the quote - just be clear on why you want the information so the contractor can work with you.
Good luck!
Mobile Home Remodeling reviews in Folsom
When the house was almost finished,
Upon asking about other items that needed attention he decided that I needed to figure those items out myself. One was a faulty thermostat. He left after receiving full payment by check. Within roughly forty-eight hours while showering a hose blew off the water heater. I contacted his office(Wife) and stated that there was still a problem and we no longer had no hot water. She stated that it would cost me another service call for her husband to come out no earlier than a week later. When I became more insistent she proceeded to tell me that my attitude was poor and hung up on me.
It is my contention that this service company and parts department are under-qualified and charge excessively due to the circumstances of each client. The owner's wife even informed me that other dealers would not deal with me because I was too demanding.
Please stay clear of using this
I have gone back and forth with
I would definitely use them again and would highly recommend them.
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Handyman Contractor
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Performance Restoration Inc.
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Precision Builders
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Prime Cut Construction
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RCS Builders
Redline Electric
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Robert M. Jones Construction #387479
Scott's Home Improvement and Repair Services
See It N 3D Design & Technical Illustrations
ServiceMaster by Benevento
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Skillz Construction
Smart Choice Remodeling Inc
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