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The signaling pathways mediating lysophosphatidic acid (LPA)-stimulated PKD2 activation and the potential contribution of PKD2 in regulating LPA-induced interleukin 8 (IL-8) secretion in nontransformed, human colonic epithelial NCM460 cells were examined. Treatment of serum-deprived NCM460 cells with LPA led to a rapid and striking activation of PKD2, as measured by in vitro kinase assay and phosphorylation at the activation loop (Ser706/710) and autophosphorylation site (Ser876). PKD2 activation induced by LPA was abrogated by preincubation with selective PKC inhibitors GF-I and Ro-31-8220 in a dose-dependent manner. These inhibitors did not have any direct inhibitory effect on PKD2 activity. LPA induced a striking increase in IL-8 production and stimulated NF-κB activation, as measured by NF-κB-DNA binding, NF-κB-driven luciferase reporter activity, and IκBα phosphorylation. PKD2 gene silencing utilizing small interfering RNAs targeting distinct PKD2 sequences dramatically reduced LPA-stimulated NF-κB promoter activity and IL-8 production. PKD2 activation is a novel early event in the biological action of LPA and mediates LPA-stimulated IL-8 secretion in NCM460 cells through a NF-κB-dependent pathway. Our results demonstrate, for the first time, the involvement of a member of the PKD family in the production of IL-8, a potent proinflammatory chemokine, by epithelial cells.
- NCM460 cells
- protein kinase C
- phorbol esters
colonic epithelial cells are uniquely positioned to serve as a direct line of communication between the external environment and the immune system via subepithelial immune cells. These epithelial cells are constantly exposed to luminal contents, including ingested pathogens and commensal microflora that populate the gut. Intestinal epithelial cells have evolved mechanisms for sensing luminal antigens or ligands and can respond to external stimuli by inducing intracellular signaling events, leading to the production and basolateral secretion of inflammatory cytokines (13, 31). In addition, these cells also respond to signals generated in the internal environment. The colonic epithelial cells must then integrate and transduce internal and external signals into biological responses.
PKC, a major target for the tumor-promoting phorbol esters, has been implicated in the signal-transduction pathways that mediate important functions in intestinal epithelial cells, including proliferation (1, 14) and carcinogenesis (35). It is known that intestinal epithelial cells express multiple isoforms of the PKC family, including α, β, δ, ε, and ζ (11). Transgenic overexpression of PKC-βII in murine colonic epithelium (32) or overexpression of certain PKCs in intestinal epithelial cells in culture has been shown to promote growth (26). In addition to its effects on colonic epithelial cell proliferation, multiple PKCs have also been shown to stimulate NF-κB activation (34, 43, 55, 56) in various cell types and thus mediate cell survival as well as proinflammatory signaling (22, 61). Despite the critical role of PKCs in intestinal epithelial cell function and specifically, in initiating the pathway leading to NF-κB activation, downstream signaling targets of PKCs within the NF-κB pathway in intestinal epithelial cells remain poorly understood.
Protein kinase D (PKD; also known initially as PKCμ) has emerged as a major target in the signal-transduction pathways initiated by diacylglycerol and PKC in a variety of cell types (40). PKD is the founding member of a new family of serine/threonine protein kinases, including PKD, PKD2, and PKD3 (reviewed in Ref. 40). These kinases share similarities in overall structure, primary sequence, and enzymological properties (16, 39, 52). PKD has been implicated in the regulation of multiple important cell functions, including signal transduction via MAPK kinase pathways (5, 18, 19), chromatin remodeling via histone deacetylase phosphorylation (10, 20, 27), polarized Golgi function (6, 15, 25), NF-κB activation in response to oxidative stress (29, 47–50), and cell proliferation (46, 64). Although much less is known about the regulation and function of the other PKD isoforms, recent studies revealed interesting differences in the subcellular distribution (38, 39), embryonic expression (33), and regulation (59) of these isoforms. However, studies on the physiological functions of specific PKD isoforms have been hampered by the endogenous expression of more than one isoform in the same cell type and frequently involved the overexpression of wild-type and mutant PKDs in transient transfection systems. The identification of cell types that endogenously express a single member of the PKD family would be of great value to clarify the regulation and physiological function of that isoform.
In this study, we identified PKD2 as the predominant PKD isoform in nontransformed human colonic epithelial NCM460 cells. This provided a unique opportunity to examine the regulation and function of endogenous PKD2 without the potential contribution of the other PKD isoforms. Initially, we showed that stimulation of intact NCM460 cells with the G-protein-coupled receptor (GPCR) agonist lysophosphatidic acid (LPA), a major bioactive lipid of serum (3), induced a rapid and striking PKC-dependent PKD2 activation and promoted the production of the potent proinflammatory chemokine interleukin 8 (IL-8; designated CXCL8 in current chemokine nomenclature). Subsequently, we used small interfering RNA (siRNA)-mediated knockdown of PKD2 to determine the contribution of this PKD isoform in mediating NF-κB activation and IL-8/CXCL8 production in response to LPA in NCM460 cells. Our results demonstrate that endogenous PKD2 mediates a substantial component of LPA-induced IL-8/CXCL8 production in NCM460 cells.
Cell culture.
The nontransformed human colonic epithelial cell line NCM460 was obtained from INCELL (San Antonio, TX). Cultures of these cells were maintained as described in a humidified atmosphere containing 5% CO2 and 95% air at 37°C (30). NCM460 cells were used between passages 35 and 45 in this study. For experimental purposes, cells were plated in medium containing 10% FBS and were allowed to grow to confluency (5–7 days) and then changed to serum-free medium for 18–24 h before the experiment. IEC-18 cells (American Type Culture Collection) were maintained as previously described (9).
Western blot analysis for PKD isoforms and PKD2 phosphorylation.
Serum-starved cultures of NCM460 cells grown on plastic dishes were washed twice with DMEM and then treated as described in the individual experiments. The cells were lysed in 2× SDS-PAGE sample buffer. After SDS-PAGE, proteins were transferred to Immobilon-P membranes (Millipore) and blocked by 3- to 6-h incubation with 5% nonfat milk in PBS (pH 7.2). Membranes were then incubated overnight with the respective primary antibody. Bound primary antibodies to immunoreactive bands were visualized by enhanced chemiluminescence detection with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies. Autoradiograms were scanned with a GS-710 scanner (Bio-Rad), and the labeled bands were quantified with the Quantity One software program (Bio-Rad).
In vitro kinase assay of PKD2.
Cultures of NCM460 cells, treated as described in the individual experiments, were washed and lysed in 50 mM Tris·HCl (pH 7.6), 2 mM EGTA, 2 mM EDTA, 1 mM DTT, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, and 1% Triton X-100 (lysis buffer A). Cell lysates were clarified by centrifugation at 15,000 g for 10 min at 4°C. PKD2 was immunoprecipitated at 4°C for 2–4 h with PKD2 antibody (1:200). The immune complexes were recovered by protein A coupled to agarose.
PKD2 autophosphorylation was determined in an in vitro kinase assay by mixing 20 μl of PKD2 immunocomplexes with 10 μl of a phosphorylation mixture containing (final concentration) 100 μM [γ-32P]ATP (specific activity of 400–600 cpm/pmol), 30 mM Tris·HCl (pH 7.4), 10 mM MgCl2, and 1 mM DTT. After 10 min of incubation at 30°C, the reaction was stopped by washing with 200 μl of kinase buffer and then adding an equal volume of 2× SDS-PAGE sample buffer (200 mM Tris·HCl, pH 6.8, 2 mM EDTA, 0.1 M Na3VO4, 6% SDS, 10% glycerol, and 4% 2-mercaptoethanol), followed by SDS-PAGE analysis. The gels were dried, and the 105-kDa radioactive band corresponding to autophosphorylated PKD2 was visualized by autoradiography. Autoradiographs were scanned in a GS-710 calibrated imaging densitometer (Bio-Rad), and the labeled band was quantified by the Quantity One software program.
Exogenous substrate phosphorylation by purified PKD2 was carried out by mixing 20 μl of the washed immunocomplexes with 20 μl of a phosphorylation mixture containing 2.5 mg/ml syntide-2 (PLARTLSVAGLPGKK), a peptide based on phosphorylation site two of glycogen synthase. After 10 min of incubation at 30°C, the reaction was stopped by adding 100 μl of 75 mM H3PO4 and spotting 75 μl of the supernatant on P-81 phosphocellulose paper. Free [γ-32P]ATP was separated from the labeled substrate by washing the P-81 paper four times for 5 min in 75 mM H3PO4. The papers were dried, and the radioactivity incorporated into syntide-2 was determined by Cerenkov counting.
EMSA procedures.
Nuclear extracts were prepared, and EMSA was performed according to the instructions of the manufacturer (Panomics, Redwood City, CA). The EMSA "Gel-Shift" kit (Panomics) was used. The oligonucleotide probes (biotin labeled and cold) encoding the consensus sequence of NF-κB (5′-AGTTGAGGGGACTTTCCCAGGC-3′) transcription factor was purchased from Panomics. EMSAs were performed by incubating biotin-labeled NF-κB probe with nuclear extracts (5 μg) from cells with or without LPA treatment in binding buffer containing nonspecific blocker poly(dI-dC) at 20°C for 30 min. The protein-DNA complexes were analyzed by electrophoresis through a 6% nondenaturing polyacrylamide gel. The protein-DNA complexes were then transferred onto a nylon membrane using a semi-dry method at 300 mA for 30 min. After transfer, the nylon membrane was subjected to a UV cross-linker oven for 3 min. The membrane was blocked, followed by addition of streptavidin-horseradish peroxidase conjugate (1:1,000). The shifted bands corresponding to the protein-DNA complex were visualized relative to the unbound double-strand DNA after exposure to film.
PKD2 siRNA.
To inhibit PKD2 protein expression, subconfluent cultures of NCM460 cells were transfected with siRNAs. PKD2 siRNAs from Dharmacon (Chicago, IL) and from Ambion (Austin, TX) were used. The sequence for the siRNA oligonucleotides (sense strands) are as follows: UGAGACACCUUCACUUCAUU, CAAGAACAUUGUCCACUGUUU, and GGAAGAUGGGAGAGCGAUAUU. siCONTROL nontargeting siRNA pool (pool of 4 duplexes) was purchased from Dharmacon and used as a control. Transfection of duplex siRNAs was performed with Lipofectamine 2000 according to the manufacturer's protocol. Forty-eight hours after transfection, cells were used for experiments and subsequent analyses.
Luciferase assay.
Cells (4 × 105/well) were plated onto six-well dishes (Nunc). The next day, a transfection mixture was prepared by adding 1 μg of the NF-κB luciferase construct (pNF-κB-Luc plasmid; Stratagene, La Jolla, CA), 0.25 μg of control luciferase construct phRG-TK (Promega, Madison, WI), and either PKD2 siRNA (as indicated above) or siCONTROL nontargeting siRNA pool to serum-free medium. After a 20-min incubation and addition of Lipofectamine 2000 (Invitrogen, Carlsbad, CA), the transfection mixture was combined with 0.2 ml of complete DMEM and placed on the cells in each well.
Two days after transfection, the cells were treated with agonists. After the indicated times, the cells were washed with PBS and then lysed with passive lysis buffer (150 μl; Promega). The relative light units from firefly and Renilla luciferases (20 μl) were measured with the use of the dual-luciferase activity assay kit (100 μl; Promega) on a Turner TD20/20 luminometer. The ratio of firefly to Renilla luciferase activity was calculated. The results are reported as relative changes in the ratio, with a relative-fold increase of 1.0 signifying no change. The results are reported as means ± SE of three or more sets of transfections done in triplicate.
Serum-starved cultures of NCM460 cells were stimulated with LPA. To determine the amount of IL-8/CXCL8 protein secreted by the cells, conditioned medium was collected and centrifuged at 5,000 g for 5 min to remove cell debris. The collected samples were used for IL-8 ELISA (BD Pharmingen).
[γ-32P]ATP (370 MBq/ml) was from Amersham Pharmacia Biotech (Piscataway, NJ). Bisindolylmaleimide I (GF-109203X), bisindolylmaleimide V, AG-1478, LY-294002, PD-98059, U-0126, rapamycin, and Ro-31-8220 were purchased from Calbiochem. LPA, phorbol 12,13-dibutyrate (PDBu), and trypsin-EDTA solution (1×) were obtained from Sigma (St. Louis, MO). Phosphoserine 744/748 PKD antibody, anti-phospho-ERK1/2 (Thr202/Tyr204) MAb, and anti-phospho-p38MAPK (Thr180/Tyr182) MAb were obtained from Cell Signaling Technology (Beverly, MA). Purified PKD2 protein was obtained from Stressgen (Santa Diego, CA). TRIzol reagent, Lipofectamine 2000, and the SuperScript III one-step RT-PCR system with Platinum Taq DNA polymerase were obtained from Invitrogen. Anti-phospho-PKD2 (Ser876) was obtained from Upstate (Lake Placid, NY). PKD2 siRNA oligonucleotides and siCONTROL nontargeting siRNA pool were obtained from Dharmancon and Ambion. The PKD2 polyclonal antibody was used for immunoprecipitation, and Western blotting was generated against the synthetic peptide GLPTDRDLGGACPPQD, corresponding to amino acids 850–865 of PKD2 (by the Antibody Core of CURE: Digestive Diseases Research Center) or obtained from Upstate. Similar results were obtained with the PKD2 antibodies from both sources. The polyclonal anti-PKD3 N17 antibody was raised by immunizing rabbits with a peptide corresponding to the human PKD3 17 amino-terminal residues SANNSPPSAQKSVLPTA using standard procedures (39). Antibodies (PKD C-20, PKC-ζ C-20, PKC-ε C-15, PKC-δ, and PKC-α C-15; anti-ERK2 polyclonal antibody) used in Western blot analysis were obtained from Santa Cruz Biotechnologies (Santa Cruz, California). Caco-2, HT-29, and T84 cells were obtained from American Type Culture Collection. Other items were from standard suppliers or as indicated in the text.
Statistical analysis.
Paired t-test was applied. All data are expressed as means ± SE. Significance at P < 0.05 and P < 0.01 was as indicated.
PKD2 is the only detectable member of the PKD family in NCM460 cells.
To determine which PKD isoform(s) is expressed in the nontransformed human colonic epithelial cell line NCM460, cultures of these cells were lysed and analyzed by SDS-PAGE, followed by Western blot analysis with a polyclonal antibody that recognizes the COOH-terminal region of both PKD and PKD2 (C-20; Santa Cruz Biotechnology) (52). Lysates from rat IEC-18 cells, known to express PKD and PKD2 (9, 38), were analyzed in parallel. Interestingly, NCM460 cells yielded only the faster migrating band compared with IEC-18 cells (Fig. 1A). Similarly, Western blot analysis of NCM460 cell lysates immunoprecipitated with the antibody that recognizes the COOH-terminal region of both PKD and PKD2 yielded only the 105-kDa band (results not shown). In addition to rat IEC-18 and NCM460 cells, we also tested three human colon adenocarcinoma cell lines (Caco-2, HT-29, and T84) and mucosal scrapings of mouse ileum and colon to determine whether PKD2 is also the predominant PKD family isoform in these colon epithelial cells. As shown in Fig. 1A, left, three of four human colonic epithelial cell lines tested, including NCM460 cells, and mouse intestinal lining express predominantly the faster migrating band, corresponding to the apparent molecular mass of PKD2. To further substantiate these results, Western blot analysis of IEC-18 and all four human colon cell line lysates was performed with the use of a different PKD2 antibody that was generated against amino acids 850–865 (GLPTDRDLGGACPPQD) of human PKD2 (either produced in our laboratory or available from Upstate; see materials and methods). This antibody is highly specific for human PKD2 and has weak reactivity against murine PKD2. As shown in Fig. 1A, right, the PKD2-specific antibody detected the faster migrating band of the doublet recognized by C-20 antibody in the cell lines. Together, these results indicate that human colonic epithelial NCM460 cells express predominantly the PKD2 isoform of the PKD family.
To substantiate further this conclusion, we immunoprecipitated cell lysates with the PKD2-specific polyclonal antibody and the immunocomplexes were then analyzed by SDS-PAGE, followed by Western blot analysis with the same PKD2 antibody. As shown in Fig. 1B, left lane, we found a well-defined band migrating with an apparent molecular mass of 105 kDa, corresponding to PKD2. The detection of this band was completely blocked by inclusion of the immunizing peptide during immunoprecipitation, confirming the specificity of the PKD2 antibody (Fig. 1B, right lane).
We next determined whether NCM460 cells endogenously express the PKD3 isoform of the PKD family. As shown in Fig. 1C, we did not detect any immunoreactive signal in lysates of these cells by Western blot analysis using a specific antibody directed against PKD3 (39). As positive controls, the same antibody detected ectopic human PKD3 protein in COS-7 cells transfected with green fluorescent protein-PKD3 and endogenous PKD3 protein in rat IEC-18 cells (Fig. 1C). These results indicate that PKD2 protein is the predominate isoform of the PKD family endogenously expressed by NCM460 cells. Thus these human epithelial cells provide a model system to study the regulation and function of PKD2 without significant contribution of the other PKD isoforms.
LPA stimulates PKD2 activation through a PKC-dependent pathway.
LPA is a physiological ligand that mediates its biological effects predominantly via members of the endothelial differentiation gene subfamily of GPCRs, namely LPA1, LPA2, and LPA3 (3). All three LPA receptor subtypes led to the generation of the second messengers inositol 1,4,5-trisphosphate, which stimulates Ca2+ mobilization from intracellular stores, and diacylglycerol, which activates classic and novel PKCs (3). To determine whether PKD2 is regulated through GPCR-mediated pathways, serum-starved NCM460 cells were treated with LPA (5 μM) for various times, lysed, and immunoprecipitated with PKD2 antibody. The immunocomplexes recovered with protein A agarose were then incubated with [γ-32P]ATP, and the incorporation of 32P into PKD2 was analyzed by SDS-PAGE and autoradiography to examine the level of autophosphorylation.
As shown in Fig. 2A, PKD2 isolated from unstimulated NCM460 cells had low catalytic activity. Treatment of NCM460 cells with LPA induced a rapid and striking increase in PKD2 kinase activity that was maintained during cell lysis and immunoprecipitation. PKD2 activation was detectable in 30 s and reached a maximum (∼10-fold) within 1 min of LPA stimulation (Fig. 2A, top).
PKD2 has two serine residues at 706/710 located within the activation loop of its catalytic domain. The transphosphorylation of these residues by PKCs is thought to stabilize the activation loop in an active conformation (40). Stimulation of NCM460 cells with LPA strikingly stimulated PKD2 phosphorylation at Ser706/710 and also at Ser876, an autophosphorylation site (51), in a time-dependent manner (Fig. 2A). These results indicate that stimulation of NCM460 cells with LPA induces rapid and striking catalytic activation and multisite phosphorylation of endogenous PKD2.
In many cases, PKD is activated through PKC-dependent activation loop phosphorylation, but PKC-independent pathways, including tyrosine phosphorylation of residues in the pleckstrin homology (PH) domain and other less defined pathways, have also been described (40). Much less is known about the mechanisms leading to the activation of PKD2 in normal epithelial cells. To determine whether PKCs mediate PKD2 activation induced by LPA in NCM460 cells, cultures of these cells were treated with various concentrations of the selective PKC inhibitors Ro-31-8220 (58) or GF-I (54) before LPA stimulation. Control cells received either an equivalent amount of solvent or GF-V, a biologically inactive analog of GF-I, before addition of LPA. As shown in Fig. 2B, treatment with either Ro-31-8220 or GF-I potently blocked PKD2 activation induced by subsequent addition of LPA. Because a previous report suggested that GF-I and Ro-31-8220 also inhibit PKD2 activity (51), we tested the effect of these inhibitors on the activity of purified PKD2 and PKC-ε using the exogenous substrate syntide-2. As shown in Fig. 2C, syntide-2 phosphorylation catalyzed by PKD2 was not inhibited by the addition of either Ro-31-8220 or GF-I to the incubation mixture, whereas syntide-2 phosphorylation by PKC-ε was completely blocked. These results indicate that Ro-31-8220 and GF-I interfere with LPA-induced PKD2 activation in intact NCM460 cells by inhibiting PKCs rather than PKD2.
A role of PKCs in PKD2 activation is further substantiated by experiments in which NCM460 cells were exposed to biologically active phorbol esters that directly activate classic and novel PKCs and subsequently, with long-term exposure, promote their downregulation. As shown in Fig. 2D, treatment with 100 nM PDBu induced a rapid and striking PKD2 phosphorylation that remains robust at 30 min. Conversely, downregulation of PKCs by exposure to PDBu for 24 h inhibited PKD2 phosphorylation at Ser876, elicited by subsequent stimulation with LPA (Fig. 2E). Collectively, these results indicate that LPA stimulates PKD2 activation through a PKC-dependent pathway.
LPA stimulates IL-8 secretion and NF-κB activity.
We next investigated whether LPA, which is increasingly implicated in immunoregulation (23, 41), could regulate a known and important biological function of colonic epithelial cells, namely, production of cytokines/chemokines. Initially, using a human cytokine microarray assay, we screened the expression profile of cytokines from serum-starved NCM460 cells. The membranes were hybridized with supernatant from LPA-treated (10 μM) for 24 h or vehicle-treated (control) NCM460 cells. The corresponding proteins were detected by a mixture of antibodies and visualized by an enhanced chemiluminescence system. Interestingly, of the 40 anti-cytokine antibodies on the array, only IL-8/CXCL8 was markedly increased after incubation with LPA for 24 h (Fig. 3A).
To further characterize the kinetics and magnitude of GPCR agonist-induced IL-8/CXCL8 production, we stimulated NCM460 cells with LPA (10 μM) for various times, and the levels of IL-8 in the medium were measured by ELISA. As shown in Fig. 3B, stimulation of NCM460 cells with 10 μM LPA induced a striking increase in IL-8/CXCL8 production in a time-dependent manner. The level of IL-8/CXCL8 increased by over 20-fold (1,444 ± 113 pg/ml) after 6 h of exposure to LPA.
The transcription factor NF-κB plays an important role in the regulation of cytokine/chemokine expression in a variety of cell types (17). Here, we tested whether treatment of NCM460 cells with LPA stimulates NF-κB activation by measuring NF-κB-DNA binding, NF-κB-driven luciferase reporter activity, and IκBα phosphorylation. As shown in Fig. 4A, stimulation of NCM460 cells with 10 μM LPA for 30 min induced NF-κB binding to oligonucleotide probes encoding the consensus sequence of NF-κB. Similarly, LPA increased NF-κB-driven luciferase reporter activity up to approximately fivefold at 4 h (Fig. 4B). Correspondingly, LPA induced IκBα phosphorylation, the prelude to NF-κB nuclear translocation and subsequent activation (Fig. 4C).
PKD2 knockdown decreases LPA-stimulated NF-κB activation and IL-8 secretion.
Given that LPA elicited a striking increase in IL-8/CXCL8 production, NF-κB promoter activity, and PKC-dependent PKD2 activation, we hypothesized that these events lie in a signal-transduction pathway, namely, PKC/PKD2/NF-κB/IL-8 production. To determine directly whether PKD2 mediates NF-κB activation in response to LPA, we decreased the endogenous levels of PKD2 using gene silencing by RNA interference. Subconfluent cultures of NCM460 cells were transiently transfected with PKD2 siRNA or nontargeted negative control duplex. To minimize the possibility that the siRNA oligonucleotide may be affecting the expression of a gene other than PKD2, NCM460 cells were transfected with siRNA targeting distinct regions of PKD2. As shown in Figs. 5A and 6B, the PKD2 protein level in NCM460 cells transfected with PKD2 siRNA was dramatically reduced (∼80%) compared with cells transfected with nontargeted negative control duplex. In contrast, PKC-α protein levels, determined as a control, were not affected, thus further demonstrating the selectivity of PKD2 gene silencing (Figs. 5A and 6B). PKD2 knockdown dramatically reduced LPA-induced IκBα phosphorylation (Fig. 5A), but it did not interfere with the phosphorylation of either ERK1/2 or p38 MAPK stimulated by exposure to LPA for 30 or 240 min (Fig. 5B). Furthermore, PKD2 siRNA significantly reduced NF-κB-driven luciferase reporter activity (Fig. 5C). These results indicate that PKD2 plays a major role in mediating LPA-induced NF-κB activation.
Next, we determined whether PKC/PKD2 mediates LPA-stimulated IL-8/CXCL8 production. As a first line of evidence supporting this hypothesis, we demonstrated that treatment of NCM460 cells with the selective PKC inhibitor GF-I, a potent inhibitor of PKD2 activation (see Fig. 2), dramatically (∼80%) inhibited the production of IL-8/CXCL8 induced by LPA (Fig. 6A).
Very recently, Zhao et al. (62, 63) demonstrated that in normal human bronchial epithelial cells IL-8 production in response to LPA is dependent on EGF receptor transactivation stimulated by PKC-δ, Lyn kinase, matrix metalloproteinases, and human bronchial EGF. Therefore, if LPA stimulates IL-8 production in NCM460 cells through a similar pathway, inhibition of EGF receptor tyrosine kinase should prevent LPA-stimulated IL-8 production in these cells. As shown in Fig. 6A, treatment of NCM460 cells with the selective EGF receptor tyrosine kinase inhibitor AG-1478 attenuated LPA-stimulated IL-8 production only slightly. These results are consistent with the hypothesis that LPA induces IL-8 production through a PKC-dependent but EGF receptor-independent pathway in human intestinal NCM460 cells and imply that IL-8/CXCL8 production is regulated through different pathways in different epithelial cell types.
We next determined the contribution of PKD2 to LPA-induced IL-8/CXCL8 production in NCM460 cells. As shown in Fig. 6B, transfection of NCM460 cells with siRNAs targeting PKD2 resulted in selective knockdown of PKD2, in agreement with results in Fig. 5A. The salient feature of the results shown in Fig. 6 is that PKD2 knockdown markedly reduced LPA-stimulated IL-8/CXCL8 production by 60% (Fig. 6C). Collectively, the results presented here indicate that a substantial component of LPA-stimulated IL-8/CXCL8 production is mediated by endogenous PKD2 through a NF-κB-dependent pathway in normal human intestinal NCM460 cells.
Mucosal surfaces, such as the lung and the intestine, are lined by a single layer of epithelial cells. The human epithelial lining is positioned at the interface between the luminal/external environment and the organism. Not only does the intestinal epithelial lining serve as a physical barrier, but it also serves as a sensor of nutrients, toxins, and microorganisms. Thus colonic epithelial cells can respond to luminal stimuli by inducing intracellular signaling events, leading to the production and release of chemokines through the basolateral surface (13, 31).
LPA has been shown to play a role in immunoregulation, affecting various cell types. In human umbilical endothelial cells, LPA stimulates expression of intercellular adhesion molecule 1, which interacts with activated lymphocytes (23). More recently, LPA has been shown to enhance IL-13 expression in T cells (41). Furthermore, LPA has been shown to stimulate IL-8/CXCL8 production in human bronchial epithelial cells (42) and in human colon cancer cell lines (45, 60). Zhao et al. (62, 63) recently demonstrated that LPA-induced EGF receptor transactivation is a major mediator of IL-8 production, using human bronchial epithelial cells as their model system. In contrast, our study demonstrates that exposure of human intestinal NCM460 cells to the specific EGF receptor tyrosine kinase inhibitor AG-1478 inhibited the production of IL-8/CXCL8 in response to LPA only modestly (∼20%). Thus, although in bronchial epithelial cells the majority of the IL-8/CXCL8 production induced by LPA is mediated by EGF receptor transactivation, this pathway has only a modest contribution to the response in intestinal NCM460 cells, the experimental system used in this study.
There is increasing evidence that the PKD family of protein kinases plays fundamental cellular functions in multiple cell types. However, the preponderance of this data has been generated from studies of PKD, the founding member of this protein kinase family. Although the isoforms of the PKD family share extensive homology in their catalytic domains, recent studies demonstrated that these enzymes localize to different organelles in the cell. Furthermore, many aspects of PKD regulation and physiological functions are based on overexpression of wild-type and mutant PKDs in transient transfection systems, which have inherent limitations. Previous studies implicated PKD in a pathway that promotes NF-κB activation in response to oxidative stress (47–50). However, most of these studies utilized transfection of PKD mutants or tagged PKD in cells, which express both PKD and PKD2 (29, 47–50). None of the previous studies demonstrated a role of endogenous PKDs in mediating GPCR-mediated pathways leading to NF-κB activation (as opposed to oxidative stress), and none identified a role of any of the PKDs in chemokine production.
In this study, we demonstrated that PKD2 is the only PKD family protein detected in nontransformed, human colonic epithelial NCM460 cells. LPA stimulated PKD2 activation in these cells, as judged by measurements of catalytic activity in immunoprecipitates and by phosphorylation of activation loop residues, Ser706/710 and Ser876, the autophosphorylation site in the COOH terminus of PKD2. These results indicate that LPA induces striking endogenous PKD2 activation and multisite phosphorylation in human epithelial cells.
Several lines of evidence indicate that LPA-induced PKD2 activation proceeds via a PKC-dependent pathway. Either pharmacological PKC inhibitors or downregulation of classic and novel isoforms of PKC by long-term exposure to phorbol esters dramatically reduced LPA-stimulated PKD2 phosphorylation at Ser706/710 and Ser876. A prior report suggested that the PKC inhibitors GF-1 and Ro-31-8220 have direct inhibitory effects on immunoprecipitated PKD2 in vitro (51). To clarify the mechanism by which the PKC inhibitors interfere with LPA-induced PKD2 activation in intact cells, we tested the activity of purified PKD2 in the absence or presence of Ro-31-8220 or GF-I. Our results indicate that neither Ro-31-8220 nor GF-I had a direct effect on PKD2 catalytic activity. We verified that Ro-31-8220 or GF-I used in our experiments completely inhibited PKC-ε-catalyzed substrate phosphorylation at concentrations that did not exert any inhibitory effect on PKD2 activity. These results are consistent with the notion that LPA stimulates PKD2 activation through a PKC-dependent pathway.
Human intestinal epithelial cells in vivo are thought to represent a major local source of proinflammatory cytokines and, in particular, for the IL-8/CXCL8 chemokine. Indeed, epithelial-derived IL-8/CXCL8 has been shown to support neutrophil infiltration in human mucosal inflammatory diseases of the colon, including ulcerative colitis (21, 28). In the present study, we demonstrate that LPA potently stimulates IL-8/CXCL8 production in nontransformed human colonic epithelial cells NCM460. The levels of IL-8/CXCL8 production in response to LPA were comparable to those induced by pathogenic bacteria (37, 44). Either inhibition of PKC or PKD2 gene silencing utilizing siRNAs targeting distinct PKD2 sequences markedly reduced LPA-stimulated IL-8/CXCL8 production and NF-κB promoter activity. Our results indicate that PKD2 is a novel signal-transduction element in the pathway that mediates IL-8/CXCL8 production in response to LPA via NF-κB.
In addition to NF-κB, the IL-8 promoter contains binding sites for the transcription factors activator protein-1 (AP-1), C-EBP/NF-IL-6, and Tcf/Lef (17, 24). NF-κB is considered to be essential, whereas the others are dispensable for transcriptional activation in some cells but contribute to activation in others (24). Therefore, in some cell types, optimal IL-8/CXCL8 transcription and subsequent expression require contribution of other transcription factors, such as AP-1, C-EBP/NF-IL-6, and/or Tcf/Lef. Although our results with NCM460 cells demonstrate a prominent role of PKD2 in LPA-stimulated IL-8/CXCL8 production, the AP-1/ERK pathway could be required for promoting a maximal IL-8/CXCL8 response to LPA stimulation.
In different intestinal epithelial cell lines, LPA stimulates intestinal epithelial cell migration (53), proliferation (57), and IL-8/CXCL8 production (45, 60). The serum concentrations of this bioactive lipid range from 2 to 20 μM (3). Major sources of LPA in the vicinity of injured epithelial surfaces and inflammatory processes are activated platelets and stimulated fibroblasts (3). We envisage that, after intestinal epithelial injury, bleeding and blood clots ensue with subsequent activation of platelets, resulting in high concentrations of LPA in the local microenvironment. LPA receptor activation (at concentrations such as those used in this study) could then lead to PKD2-mediated NF-κB activation that results in IL-8/CXCL8 production. This in turn may lead to neutrophil recruitment to counter potential bacterial invasion and stimulation of CXCR1/CXCR2-expressing cells, such as myofibroblasts (36), in the lamina propria. In addition to its neutrophil chemoattractant ability, IL-8/CXCL8 has recently been shown to mediate spreading and wound closure by fibroblasts (12). These are important events in the repair of intestinal injury that compromise the integrity of the basement membrane.
It is conceivable that the PKC/PKD2/NF-κB/IL-8 pathway delineated in this study could also operate in intestinal epithelial cells challenged by other stimuli. Toll-like receptors (TLRs), which recognize microbial products known as pathogen-associated molecular patterns, are expressed in colonic epithelial cells (2). Recently, PKCs have been implicated as a downstream targets of TLR signaling (4, 7, 8). Thus PKD2 may serve as a point of convergence that integrates signals from both TLRs and GPCRs.
In summary, our results indicate that a substantial component of the production of the potent proinflammatory chemokine IL-8/CXCL8 is mediated by PKD2 through a NF-κB-dependent pathway in NCM460 cells. This is the first time that endogenous PKD2, and indeed any member of the PKD family, is shown to play an important role in mediating the production of a chemokine in epithelial cells or in any cell type.
This work was supported by a Howard Hughes Medical Institute Research Resources Faculty Development Award and by National Institute of Diabetes and Digestive and Kidney Diseases Grants K08 DK-063983 to T. T. Chiu and R0–1 DK-55003 and R0–1 DK-56930 to E. Rozengurt.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society


text:
May 5, 2016
Roberto Martinez: Phil Jagielka to return but this Everton star won't play again this term
"We're very confident that Phil Jagielka will be available"
Roberto Martinez
Midfielder Gareth Barry and right-back Seamus Coleman, meanwhile, could return from injury at champions Leicester on Saturday, but winger Gerard Deulofeu's season is over.
Deulofeu has knee ligament damage, while Barry is fit after a groin injury and Coleman is over a hamstring problem.
Martinez said: "The only three players we're keeping a close eye on are Seamus Coleman, Gareth Barry and Phil Jagielka.
"Obviously Gerard Deulofeu, I think it will be very difficult to see in any of the final three games.
"We're very confident that Phil Jagielka will be available for the last game of the season.

subset: commoncrawl


text:
fun fall refreshers
It is always great to find some fun fall refreshers to add to your wardrobe. There are a ton of cute pieces out there right now. The holidays are approaching and it's a great time to plan some festi ... read more »
Casual Autumn Afternoon
Autumn weather is finally here and I am loving pieces for a casual afternoon. In the next few weeks, I plan on picking pumpkins, going to an apple orchard, and enjoying the fall leaves. A look like th ... read more »
late summer must haves
Summer is coming to an end and there are still some darling pieces out these. I love ruffle details and sweet embroidery. There is still time to savor the sunshine and bare your shoulders in the sultr ... read more »
tween summer style
Summer is a fun time to pick out great clothes for my kids. There are also some major sales right now. This makes everything on trend and fun, but not precious. I would hate to get mad at my kids for ... read more »
summer travel daydreams
It's summer and i have a serious case of wanderlust. I have been wanting to travel anywhere and everywhere these days. There are so many cool pieces on the market right now. You can certainly travel ... read more »
fun finds under $40
It is always fun to find a cute piece that can rejuvenate your wardrobe and add a fresh touch! It's even better when you snag an adorable piece that's a bargain. read more »
blue skies ahead
Spring is here and it's time to embrace the season. There are so many cute pieces out there right now and it's super tempting to buy them all! There are so many pretty details to play with. I am l ... read more »
Fetching spring fashions
Spring has sprung early and i am excited about spring fashions! There are so many adorable pieces out there right now! I love the bell sleeves, ruffles and striped details that are everywhere right no ... read more »
date night fashion inspiration
Every monday could be a little a bit more inspired, so i am going to share some fashions that i have been daydreaming of. read more »
winter fashion inspiration
february is here and i'm inspired by some really cool ideas i have seen lately. This edie parker personalized clutch is so adorable. You can choose a ton of different colors, clasps and fonts to cre ... read more »
fashion inspiration
Sometimes i am really taken with fashion. This is one of those times. Are you ever struck by just how lovely some things are and the artistry that goes into creating them? Over the past few months i h ... read more »
Sleek winter finds
With the new year comes winter weather and i have picked some sleek ways to keep warm this season. read more »
flirty holiday fashion
I am loving the cute holiday pieces that are out there these days. I love a bit of lace and some sparkle too. I bought these lace j crew pants and am really excited to wear them. I am pairing them wit ... read more »
daydreaming of
I got a ton of great holiday catalogs in the mail that are so dreamy. This fall i am truly smitten with all things gucci. They have so many butterflies and bees on everything and it is so insanely boh ... read more »
These boots are made for walking
Leaves are starting to fall and it's time for boot shopping again! We all need some great boots that can be dressed up or down. I like pairs that are cute while still comfortable for everyday! I thi ... read more »
Fall jacket roundup
The first few crisp days of fall have arrived and i have so enjoyed them. It's almost time to add a jacket to our fall ensembles and there are a ton of cute ones out there right now. Most of these a ... read more »
Indian summer ideas
Indian summer is here and it really is one of my favorite times of year. The end of summer wildflowers are at their best and there are butterflies and hummingbirds everywhere. I love that the mornings ... read more »
Fall finds under $100
Here are some cute things I found for fall! I love a little leopard in my life. These heels are unbelievably chic for the price! Cute wedge booties are my go to for fall and I am smitten with these on ... read more »
Fall Favorites
Summer is coming to a close and I am always a bit excited for the crisp days ahead. Dan and I are working on some fun projects and these pieces would be perfect for the activities on our fall list. I ... read more »
Just add water
Summer is here and it is time for fun by the pool. We all need a good pool read and some cute ensembles too. read more »
bohemian spring and summer style
Summer is almost here and while it's a little chilly here, i am dreaming of warm summer sunshine. There are so many cute pieces out there to wear to enjoy a nice evening on a patio. I am loving pom ... read more »
fun spring finds
spring has officially sprung and is here to stay! The weather is sensational and looking at spring and summer fashion is so fun. When we were on spring break we found the cutest pieces that worked for ... read more »
spring break daydreams
groundhog day has come and gone and it seems that spring will come early...well, hopefully! the sunny skies and balmy weather have left me daydreaming of spring break and beach vacations. i cannot wai ... read more »
friday night out
looking through the pages of the j crew catalog is a fabulous way to spend time sitting in carpool. i am always smitten with a ton of great pieces. these items are versatile and polished. i love this ... read more »
date night decisions
i am in love with these ideas for date night ensembles! the jumpsuits are perfect for an evening out, wherever you are headed! i love these strappy sandals with one, it adds a sexy vibe. toes in jewel ... read more »
fall fashion 2015
this weekend's dip into chilly temps got me in a fall mood. the leaves are beginning to turn and there is a chill in the air in the morning. at the beginning of any new season i get excited. here ar ... read more »
splish splash
april showers bring may flowers! well, it may be raining today, but at least it's not snow! i got this yellow rain coat and it makes me happy even on rainy days! i am loving these fun hunters and i ... read more »
here comes the sun
today we sprung forward, and to me that is huge! after getting a foot of snow and being housebound with my crew, i am ready for spring! the sun was shining today and i can only imagine the fun and che ... read more »
wish list
the neiman marcus christmas book came out and that really set my mind racing. i have been day dreaming of all sorts of lovely things that i would like to have. the mon monogram pieces at louis vuitton ... read more »
the hamptons shopping list
on our most recent vacation we took a trip to the hamptons. the shopping is heavenly and you can certainly return with chic finds. i found great scents with hints of tuberose and relaxed elegant fashi ... read more »
sunshine and happiness
summer has arrived and i could not be happier! having sundrenched days by the pool with friends makes life fun! fun summer clothes and accessories in sunny yellow are perfect for right now! summery fr ... read more »
nothing but blue skies
here's to the blue skies that await us this spring and summer. i have decided to focus on the good ahead and relish in the balmy spring air. i am loving all things indigo these days and cannot wait ... read more »
spring fever
the sun is out and it has crept past 50 degrees here! this weather seems like a tropical paradise in contrast to the snowy winter we had! this gets me dreaming of some spring outfits and warmth and su ... read more »
chic valentine's day gifts under $150
when thinking about valentine's gifts, you ultimately want to find a gift that is chic and romantic and makes your loved one feel cherished. i found pieces that are super luxe and won't break the ... read more »
festive party dresses
do you have a big night out this weekend? you might need to swing into a shop and pick up the perfect party dress. i like a dress that is flirty, fun and festive! here are my picks for this holiday se ... read more »
chic gifts under $100
these gifts for her are fun and accessible! any lady on your list would be happy with these finds! these pieces add a bit of luxe to everyday! i love the lucite clasp on this clutch and the lizard fin ... read more »
winter warmers
the first few really chilly days have arrived here and i am ready to break out my winter gear. there are a ton of great coats out there to keep you toasty. i love the down coats from uniqlo. they are ... read more »
monogrammed gift guide
i think monogrammed gifts are wonderful! they make the recipient feel special and they look unique and fun! these gift range from stocking stuffers to luxe splurges! this earphone case would be a grea ... read more »
holiday beauty
holiday season is on the horizon and now is a great time to start thinking about gifts for your loved ones. one of my favorite things to buy is beauty collections. i have loved this since i used bonne ... read more »
fall weekend wear
there is definitely a chill to the air these days! this puts me in the mood for new fall weekend ensembles! i have to admit that i am a huge j crew addict. i can walk in and whip together an outfit in ... read more »
everyday updates
i was at a girl's night a bit back and a friend was relating to me that she loved my accessible fashion content. that got me thinking about how i actually spend my wardrobe budget. i want to share s ... read more »
fall leather looks
this fall leather is everywhere and i am loving the fun looks there are! i think quilting and leather leggings are fun and flirty. i love layering a silky blouse over a rock and roll pant for a fun ni ... read more »
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i always find myself needing a polished look for evening events. when going to a book signing, a meeting or a lecture, i want to feel polished and appropriate. date night looks may be too casual or fl ... read more »
fall weekend looks 2013
when going on weekend outings in the fall, looking casual and chic can prove a bit challenging. pairing great boots you'll own for ages with classic chinos or skirts is a great way to go. this can t ... read more »
shoes and handbags for fall 2013
when thinking about your wardrobes in the fall, shoes and handbags are an important staple that can change and elevate your look! there are so many fun jewel tones and rich textures in the fall lines. ... read more »
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when the seasons change and the temperatures drop, i find myself looking for cute new ensembles for evenings out. i love to pair jeans or leggings with cute shoes and a great top. evening makeup and a ... read more »
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the weather was cool and crisp this weekend and was the perfect hint of the fall days ahead. this gets me into the mood for sweaters, boots and vests more than anything. i am seriously obsessed with e ... read more »
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for everyday beauty, i am all about products that are natural looking, quick and effective. this is what my makeup bag has in it these days. i have to admit that i am a creature of habit. like most pe ... read more »
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Summer has been sultry and fun, and the start of the school year is just days away. I adore carefree days at the pool and having the freedom to do whatever I like on a whim. Then, the fashion magazine ... read more »
the short story
this summer i am daydreaming of vacationing in exotic locales. chic shorts paired with a pretty top could take you on a day of exploring or out to dinner. dress it up with a great handbag, cute sandal ... read more »


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Welcome to the website of the San Francisco Chapter of the California Grand Jurors' Association. Our membership is comprised of current and former jurors from San Francisco civil grand juries.
This chapter exists to promote government accountability by improving resources available to San Francisco's civil grand jury and to educate the public about the substantial local government oversight and reporting powers of the grand jury.
The purposes of the San Francisco Chapter are:
- Provide residents and public officials of San Francisco with informational and educational materials and activities on the California grand jury system that will help increase public and official awareness of the valuable role the grand jury plays in our democratic system of government, and
- Review the implementation of past civil grand jury recommendations, issue status reports, and publicize the results, and
- Advocate that social studies and history classes and texts in San Francisco secondary schools contain comprehensive information regarding the role of the grand jury in California, and
- Work with sitting civil grand juries on preparing for their presentations at pertinent city commissions, GAO, and Board of Supervisors hearings and communicate with chapter membership about opportunities to attend and appear before local commissions, boards, task forces and advisory committees to monitor and/or advocate regarding issues of concern to the grand jury, and
- Actively publicize through a broad variety of medium all San Francisco grand jury reports and their official responses in order to inform the public regarding their investigative findings, recommendations, the official reactions to them and the long-term results stemming from the implementation of the recommendations, and
- Engage in annual outreach and recruitment efforts as appropriate for prospective grand jurors, and
- Sponsor general membership meetings of former and current civil grand jurors and others interested in matters pertaining to the preservation and enhancement of the California civil grand jury system and continuing educational opportunities about local government, and
- Promote the preservation of all grand jury reports, and
- Undertake other efforts consistent with the foregoing that will increase the citizens' and public officials' knowledge of the grand jury system and the contributions it has made, and continues to make, to enhance the effectiveness and efficiency of San Francisco's government, and
- Support the work of the state California Grand Jurors' Association toward the preservation and enhancement of the grand jury system.


text:
My Nonprofit Reviews
Review for DO Foundation, Inc., Detroit, MI, USA
As the name says, this organization is not about talking about things, but DOING something to better the issue of homelessness. As the global and Stateside economy suffer from a decline too complex to go into details about, the DO Foundation is right where help is needed: In the streets, shelters, WITH the people in need. If there was proof for "Walk the talk", the DO Foundation is it. The hosts of the American Smooth Jazz Awards must have felt the same way, when giving their founder Brian O'Neal the Humanitarian Award of the Year.
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seeing what they do.
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text:
Lust For Love iii by retrodiva88Watch
©2011-2017 retrodiva88
Harper's Bazaar Indonesia February 2011
Model: YangLuxi & Hale
Stylist: Michael Pondaag
Hair & Make Up: Qiqi Franky & Team
the video
Fabulous! Bravo!
she is stunning!!!
gorgeous series
this reminds me of that movie, i think it's called 'living in color' or something about color...
great photo series. I love this one the most.
This whole series is completely stunning. This is my absolute favorite image though Superb work!
A wonderful series, so Wong Kar Waiesque (hence the music in your video !)
I'm quite happy that you're investigating video as well.
Keep it up!
I'm quite happy that you're investigating video as well.
Keep it up!
I love that series!!! So cineastic!


text:
verb (used without object), solidarized, solidarizing.
to unite or come together; become solidified.
Tagged: s
Read Also:
- Solidarnosc
noun 1. a Polish organization of independent trade unions founded in 1980: outlawed by the government of Poland in 1982. noun (pl) -ties 1. unity of interests, sympathies, etc, as among members of the same class noun 1. the organization of free trade unions in Poland: recognized in 1980; outlawed in 1982; legalized and led […]
- Solidary
adjective 1. characterized by or involving community of responsibilities and interests. adjective 1. marked by unity of interests, responsibilities, etc
- Solider
adjective, solider, solidest. 1. having three dimensions (length, breadth, and thickness), as a geometrical body or figure. 2. of or relating to bodies or figures of three dimensions. 3. having the interior completely filled up, free from cavities, or not hollow: a solid piece of chocolate. 4. without openings or breaks: a solid wall. 5. […]
- Solidi
noun, plural solidi [sol-i-dahy] /ˈsɒl ɪˌdaɪ/ (Show IPA) 1. a gold coin of ancient Rome, introduced by Constantine and continued in the Byzantine Empire; bezant. 2. (in medieval Europe) a money of account equal to 12 denarii. Compare sol2 . 3. virgule. noun, Physical Chemistry. 1. (on a graph of temperature versus composition) the curve […]


text:
With over two years of playing for his national side, Zakir Hossain is back with us after having spent Season One on the sidelines. The most experienced of our three Bangladeshi imports, Arduzzaman aka 'Zaman' has played kabaddi in various Asian cities. Originally from Bagerhat, he took up kabaddi early and got a break when he was picked for the Police team in 2008. Since then, he has represented 'Services' and the Bangladesh team in competitions such as the SAFF Games, Beach Asian Games and Indoor Asian Games - having travelled to Vietnam, China, Thailand, China and Korea.
Zaman stays in Bagerhat with his wife and three-year-old daughter. In PKL Season 4, he wishes to make the most of all the opportunities he gets.


text:
Winds Of Change & The Final Solution – YouTube
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Filed 12/9/98
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54. Exempt Income
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text: the Beat Broker - Classy Dancer IV by the Beat Broker published on 2011-06-03T06:25:05Z Just move to the music and enjoy yourself. Annoying others and misbehaving yourself means just asking for trouble. You might simply get bounced. Be ready to dance when you get on the dance floor. Don't stop or stand on the dance floor at any time. If you need to stop dancing, make your way off the dance floor. If you bump into any dancer, do not stop dancing but apologize (no matter who's fault). Always be polite with other dancers. ( Tracklist and download: http://beatbroker.net/mixes?mix=28 ) Genre DJ mix Comment by the Beat Broker @mr-danomyte: Daco - Drip Dry (YamWho remix) 2015/09/19 18:11:18 +0000 Comment by Mr. Danomyte What is this track? Love it!!!! 2015/09/19 11:59:53 +0000 Comment by Disco Terrorist Wooooo 2015/09/18 23:49:20 +0000 Comment by Disco Terrorist Gah, this is so dope 2015/09/18 23:46:32 +0000 Comment by ROTCIV If you bump into any dancer, do not stop dancing but apologize (no matter who's fault) 2013/04/21 05:58:59 +0000 Comment by the Beat Broker @phgroove: Daco - Drip Dry (YamWho remix). Yeah so good! stoked you like the mix 2012/04/22 01:16:18 +0000 Comment by PH Groove This track is sick! love this mix, been listening to it over and over. 2012/04/21 18:39:25 +0000 Comment by the Beat Broker @josiasleal: thanks! this is "Reverso 68 – Well Heeled" 2012/03/13 00:38:08 +0000 Comment by intergalactic guest track ID pleaase! great mix by the way... 2012/03/12 18:34:04 +0000 Comment by #Ambiance @the beat broker: thanks a lot! 2012/02/27 11:47:14 +0000 Comment by freddtedd Crumbs chief it's good 2011/11/07 17:17:46 +0000 Comment by S.Dejavu @the-beat-broker: very good track 2011/11/05 20:40:31 +0000 Comment by Zatoon @Thebeatbroker Love the mix! I have to share it with all of our followers at our diverse music blog www.zatoonblog.com . I see your from San Fran, me to! Awesome! SAN FRAN love! Cheers to good music. Zatoon 2011/10/04 18:01:23 +0000 Comment by the Beat Broker @djulzzz: Mushrooms Project Present Soupaczar - So Mr Leary (Richard Sen Remix) 2011/09/23 16:47:39 +0000 Comment by #Ambiance track ID please? 2011/09/23 16:32:58 +0000 Comment by CE85 Great mix, waking me up and getting me through Tuesday morning. 2011/09/13 14:07:37 +0000 Comment by Sekkatackk nice Track! 2011/09/13 00:50:05 +0000 Comment by CHill OUt :) nice :) 2011/08/02 22:33:56 +0000 Comment by Karl Spackler Great mix R, still really want to get you up to Van. Can't be that much for a flight from SF? 2011/07/22 07:46:17 +0000 Comment by Karl Spackler Love this tune! Definitely a bit of an odd one to mix. Glad I stumbled across your mix. Always love the Classy Dancers. 2011/07/22 07:37:30 +0000 Comment by Flagrados! very nice! http://flagradoss.blogspot.com/ 2011/07/20 23:52:23 +0000 Comment by Ricksounds TOT DANCE 2011/07/15 05:17:18 +0000 Comment by AISI CRAVID nice 2011/07/06 16:25:34 +0000 Comment by Hypnotic Tamborine great tune! love 2011/07/05 22:08:18 +0000 Comment by COSMIC CRATES Yeah, I like my dancers... classy! 2011/06/15 03:15:41 +0000 Comment by the Beat Broker thanks to all for feedback and listening! ive posted the 320 mp3 download on my site. http://beatbroker.net/mixes.php 2011/06/12 20:49:07 +0000 Comment by sunCHAser Fine Mix, classic!! 2011/06/08 13:45:00 +0000 Comment by Alphabet City Thanks Ryan 2011/06/08 12:27:35 +0000 Comment by Casual Encounters Awesome mix! 2011/06/08 03:02:56 +0000 Comment by Sportloto Thanx, Ryan ) Would be pleased to be your remixer on your next 12'' =) 2011/06/08 01:14:17 +0000


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Espen Egil Hansen
Last quote by Espen Egil Hansen
I am upset, disappointed – well, in fact even afraid – of what you are about to do to a mainstay of our democratic society.feedback
Sep 09 2016 Facebook
Espen Egil Hansen has been quoted 5 times. The one recent article where Espen Egil Hansen has been quoted is Zuckerberg accused of censorsing iconic Viet War photo of naked girl fleeing. Most recently, Espen Egil Hansen was quoted as having said, "I am worried that the world's most important medium is limiting freedom instead of trying to extend it, and that this occasionally happens in an authoritarian way.".
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Sep 09 2016
I am worried that the world's most important medium is limiting freedom instead of trying to extend it, and that this occasionally happens in an authoritarian way.feedback
Sep 09 2016
The free and independent media have an important task in bringing information, even including pictures, which sometimes may be unpleasant, and which the ruling elite and maybe even ordinary citizens cannot bear to see or hear, but which might be important precisely for that reason.feedback
Sep 09 2016
Even though I am editor-in-chief of Norway's largest newspaper, I have to realize that you are restricting my room for exercising my editorial responsibility.feedback
Sep 09 2016
I say loud and clear that what's happening now is dangerous. I want to start a discussion, which is the role of the media, to speak up even when things are uncomfortable and to create discussion. So we'll have to wait and see what the result would be.feedback
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Brenda Starr Trailer (1989)
"Off the comics onto the big screen..." 15 May 1989 Adventure, Fantasy 93 mins
Mike is a struggling artist who draws the 'Brenda Starr' strip for the papers. When Brenda comes to life in the strip and sees how unappreciated she is by Mike, she leaves the strip. To get her back, and keep his job, Mike draws himself into the strip. In her world, Brenda Starr is the Ace Reporter for the New York Flash. She is talented, fearless, smart and a very snappy dresser. The only competition she has is from the rival paper's top reporter Libby Lipscomb. Brenda heads to the Amazon jungle to find a scientist with a secret formula which will create cheap and powerful gas from ordinary water. (This movie was filmed in 1986 but release was delayed for several years.)
Watch the official Brenda Starr 1989 trailer in HD below or find more Brenda Starr videos on Vidimovie.


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Hi, is there a way to stop portfolio items going any further than 1 click. So I click on thumbnail image comes larger at the top with < > x – that's all fine but I don't want the image clickable again, currently when I go over it again I get a 'chain link' icon
I think what you want is accomplished by setting the "Apply Link to Image" for each image in the slideshow to No Link (in the Default Options for the slideshow images). That's what I did.
The topic 'Portfolio items' is closed to new replies.


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Here we go again. You can guess who PRAISED this attack, can't you?
Four Israeli soldiers are dead, and 15 wounded after a truck jumped a curb and smashed through where they were standing.
The Palestinian Hamas movement is praised the attack, but did not take responsibility.
Hamas spokesman Abdul-Latif Qanou called it a 'heroic' act and encouraged other Palestinians to do the same and 'escalate the resistance.'
Qanou said Sunday's attack proves the wave of Palestinian violence has not ended, despite a recent lull. He says 'it may be quiet, it may linger, but it will never end.'
Hamas, which rules the Gaza Strip, is pledged to Israel's destruction. -DailyMail
Those would be the very same Palestinians the UN voted 14-0 (and USA did NOT exercise its veto) to vote in support of. Such a peaceful bunch. That doesn't line up with the 'victim' rhetoric we hear about them in the West, is it? "Coexist" or something like that, right guys?
The killer then stood on his brakes and reversed, trapping some ten of his victims under the truck.
Some of the surviving soldiers managed to jump clear and opened fire, riddling the cab of the truck killing the killer.
It is understood the soldiers fired at least 18 rounds at the terrorist, who is believed to be from an Arab area of east Jerusalem.
The soldiers were on a cultural sightseeing tour of the city when they were attacked. –DailyMail
?‼? #Israel: Terror attack in #Jerusalem. Truck drives into crowd. Kill and injured civilians and soldiers. pic.twitter.com/RMOzRxBKu7
— Onlinemagazin (@OnlineMagazin) January 8, 2017
Is there a connection between the 14-0 Resolution the UN just passed and this attack?
A direct connection? Probably not.
But when the world confirms and approves of a people who have clearly stated that the annihilation of another State is on their bucket list?
That's another story.


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Reisterstown is located in Maryland.
Reisterstown, Maryland 21136 has a population of 27,493.
The median household income in Reisterstown, Maryland 21136 is
The median household income for the surrounding county is $66,940
compared to the national median of $53,482.
The median age of people living in Reisterstown 21136 is
34.6 years.
Sat, 11/25/2017 12:00 PM-5:00 PM


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Monarch Book Readers!
Congratulations to some amazing second grade readers! These dolphins read all 20 Monarch books and had an indoor recess in the LMC to celebrate. After playing games, they even helped up check in the books.
First graders got a chance to explore Dryden's magazine collection this week. They used Vocaroo to hear articles from Ranger Rick Jr. read aloud to create webs organizing animal facts. They also used stories in Click Magazine to read "readers' theater" style with a small group. Students used the table of contents in these magazine to locate the articles they were looking for.
Lookin' For Light in First Grade
After reading the literary non fiction ebook Lookin' For Light, first graders loved doing a Nearpod interactive lesson about the book. The determined the difference between opaque, translucent, and transparent and explored reflection and refraction. Working with a partner and getting instant feedback on the big screen helped to make this NEarpod lesson a hit.
Family Reading Night 2017
Our second annual Family Reading Night at Dryden was a big success. It was great to see students and parents come to school in their jammies to have a cozy night of reading. Thank you to the many teachers and staff who read stories and did crafts in their classrooms. Thank you to the PTA to providing the bedtime snack during our last story of the night in the cafeteria.
Book Fair in the LMC!
Having the book fair in the LMC was such a treat! Thank you to our Dryden PTA for putting on such a wonderful book fair for our students!
The fifth graders had a wonderful leadership opportunity this month. They helped the muti-age first and second graders use their notes from China research to create Adobe Spark videos. These videos about Beijing, Hong Kong, The Terracotta Army, The Forbidden City, The Yangtze River, The Dyjiangyan Panda Base, and The Great Wall of China were shared with the class to teach each other about the places we researched.
Mrs. O'Kelly and
October 2017


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Hey you guys, If you haven't watched this yet you're in for a totally fun romp in the usual Lost Girl world of succubus shenanigans. I'm so excited to share the joy with you that I just can't wait to talk about this episode, "End of a Line." If you've already seen or heard about it than you're probably as drunk as I am just to keep going and we can group hug about it in just a few minutes.
After a week off Tamsin has returned to the apartment of now predominantly dark fae and is planning some quality horror movie time with Kenzi. Before they can get into zombie night Kenzi gets her rant on about Bo's abandonment of their sacred alliance. The succubus in question enters mid-outburst and promises that they will always be Bo and Kenzi. We know she means it because she quotes Jerry Maguire as only a best friend can.
Bo heads downstairs on a snack run and encounters a zombie with the same plan. It seems Bo should have spent a little more time on Kenzi's undead movie collection than yelling "show me the money" because in a rookie error she stabs him in the chest. The zombie is nonplussed and continues towards her brains when an unseen figure decapitates him. As zombie's head slides to the ground Acacia, Tamsin's old valkyrie mentor Acacia introduces herself to Bo.
Tamsin mirrors my emotions about Acacia's return – was she not dead last we heard? It seems sometimes a hand in a box is just a hand in a box as Acacia was alive and kicking, just not juggling until Massimo re-attached her hand. This season's hand re-attachment tally will start to rival it's memory-recovery numbers at this rate. Just kidding, there aren't enough hands in Canada for that.
It turns out that Rainer's crows performed the amputation when Acacia failed to make Tamsin deliver Bo promptly enough last season. She seems only mildly surprised to learn that Bo and Rainer are now going steady and hastily makes to excuse herself, explaining that she was just there for the revenant which is a fancy word for zombie fae. Also the French title of the best show I read last year.
Tamsin inspects the revenant's clothing and finds The Wanderer card. She thinks Rainer is behind the dead raising but Bo insists he's just gone to the battlefields to pay respects to the dead. The valkyries remain unconvinced and remind Bo that really she knows nothing about Rainer. Her assurances that he is just a good guy bring comfort to nobody and the team set out to find out who is really raising the dead.
Trick is spending some quality time with his whiskey flask and a picture of a dragon-pegasus beast when Vex drops in. He recounts some peace-bringing history of Trick's and the blood king explains that Rainer was a constant thorn in his side. He laments the drastic blood journaling measures he took against this defiant challenger while real evil is still a thing. Trick mentions the garuda and this creature in his drawing, called a pyrippus, as examples of actual darkness. He and Vex then take a nice trip down memory lane, as Trick shares his respect for Papa Mesmer who Vex never got along with. If anyone had 'daddy issues' in Vex emotional trauma bingo you can cross that one off right now.
Over at Bo and Kenzi's house of zombie killing, Kenzi has just finished cleaning up the twice dead fae mess and is waiting for her high-pitched beau to arrive. She is more than a little surprised to find not Hale, but her cousin Dimitri and mother at the door. Hale arrives just as the family are having an awkward reunion and proudly declares that he was the one who invited them.
When it comes to hunting zombie summoners it seems the place to go is a rundown part of town that resembles a refugee camp or District 9 but with more fae and less aliens. Bo, Tamsin and Acacia are almost instantly welcomed by a revenant and as Bo knifes up she is intercepted by Dyson who hates to miss out on an accusing-the-ex's-new-man-of-evil-deeds party. They split into pairs, with Bo and Acacia teaming up to bicker at each other and catch up with the valkyries' old revenant-raising friend Harvey. Acacia presents the original revenant's head and Bo works her sleazy magic to persuade him into cooperation.
I must admit I didn't immediately assume that Harvey's method of gaining intel would be gross and I feel incredibly naïve for that. Because how else would he learn about the undead than by pouring the juice from the head into a shot glass and sipping? Sometimes it's like I don't know anything about anything. He helpfully informs that this is the only way to gain dominion over a revenant or to find out who does. After critiquing the tannins in the blood he reveals that this revenant is "hers" – gesturing at Acacia and inciting a cat fight between her and Bo. While he starts in with some amateur film making, the valkyrie claims she killed the man but had no part in his return.
The other half of the revenant investigation team, Tamsin and Dyson, are finally spending some quality time together when they come across a crazy man raving about a woman who is both dead and alive and can control the dead. Tamsin makes her way to Harvey's to find Acacia and Bo in the midst of the lamest fight ever. Are these two not bother warriors? When did they decide hair-pulling was their favorite form of martial arts?
Kenzi's mother comes to talk to her daughter and the most important thing about this scene is that her full name is Mackenzie. Also the incredibly sad story of how she was forced onto the streets by her mother's boyfriend. Mama Kenzi finally kicked him to the curb around six months ago and was super excited by Hale informing her of Kenzi's location.
It's valkyrie bonding time and Tamsin reluctantly admits that it was her that gave Rainer's soul to Trick, rendering him The Wanderer. Acacia realizes that the revenants must be after Tamsin and her close friend-ish relationship with his new squeeze is unlikely to save her. The group find a voodoo priestess named Laveau who with very minimal denial reveals that she is behind the attacks. She says Bo has what she needs – the seed containing the powers of the Una Mens. Laveau states that she needs the power to defeat the pyrippus, which is apparently on its way.


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Recent History
Stoneware Rooster Salad Plate
$7.99 $4.00
Set a sophisticated country table with our Stoneware Rooster Salad Plate, part of our signature Rooster Collection of dishes and table accessories. With a pretty scalloped edge, raised design and handsome rooster image, this plate is full of simple style and special details. Create your own set by adding coordinating plates, bowls, mugs and more, and set the farmhouse table of your dreams!
- Rating
- 5
- Dimensions
- 8"


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(This is the latest installation in a long-running series of introductions to books I recommend. If you would like me to look at a book on business, brands, or the people who work on them, please let me know in the comments section below.) – the book: Words That Work: It's Not What You Say, It's What […]
brand-building expert | speaker | author
Words that Work
Brand-as-Business Briefs™ are periodic emailed briefings for learning how companies operationalize their brands to grow their businesses.
To be added to this exclusive list of CEOs, CMOs, and business leaders, and to receive a free chapter from each of Denise's books — What Great Brands Do and Extraordinary Experiences — please sign-up using the form below.


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Application Process
Application Process Details
Scholarships are offered every two years. The next auditions will be held in May, 2018. Applications can be submitted after January of 2018 when detailed instructions and a new application form will be available.
The Tuesday Musical Club Scholarship is available to anyone of exceptional musical talent who is at least a senior in High School (or its equivalent) and who has not reached the age of 25 by the time of auditions. The Scholarship provides only for music and those subjects related to music. Scholarships may be applied to undergraduate or graduate study. This Scholarship does not provide for living or travel expenses. Applicant's family must have maintained legal residence within a 100 mile radius of Pittsburgh for not less than one year immediately prior to date of auditions.
Applicants will be required to submit a CD or MP3 for preliminary screening. A portion of those initial applicants will then be invited to perform at a live audition. Applicants should provide their own accompanist.
After the Scholarship is awarded:
- The recipient of the scholarship receives no money. All money is paid directly to the school or teacher.
- Since this is a supervised scholarship awarded for two years, term grades from your school and an evaluation from the teacher in your major field will be required yearly.
- Scholarship recipients are eligible to apply a second time for a total of four years on the scholarship program.


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Indigenous architecture's adaption to its climate and its use of local materials has attracted interest in the search for a sustainable built environment. In Chile surviving examples include the iconic Ruka Lafkenche and the little known Fogón Pehuenche. United by the world outlook of the Mapuche people, these two examples are located in different climates and as a result different construction systems have developed.
In this work is analyzed the improvement of comfort conditions using confluent jets ventilation located near the floor level in front to the occupants in an experimental chamber. In this study are evaluated the thermal comfort, the local thermal discomfort and the air quality levels. The thermal comfort level is evaluated using the multi-nodal human thermal comfort numerical model, while the local thermal discomfort and the air quality levels are evaluated by the computational fluid dynamics numerical model.
With the continuous improvement of the energy performance of buildings, ventilation plays a crucial role in the control of pollutants from indoor sources and related comfort and health effects. However, the ventilation system itself could possibly also be a source of indoor air pollutants such as microbial contaminants. Profound scientific and technical knowledge on the impact of the design, installation and maintenance on the real performances of ventilation systems is currently lacking.
In this work the evaluation of indoor air quality in a classroom equipped with cross-flow ventilation is presented. A numerical methodology, based on comparison with experimental data, used in the evaluation of the air exchange rate, airflow rate and the age of the air, was applied in the first phase of this work. The evolution of carbon dioxide inside spaces, with different airflow typologies, was then predicted in the second part. The study was based on a school located in the South of Portugal. In the experimental methodology the tracer gas decay method was applied.
The air quality in laboratories has a profound affect on occupant health and safety. Reducing the timeof exposure and the amount of contaminant can improve the occupant environment and have directimpact on health and safety. In this study, a novel ventilation system which introduces benchexhausts is proposed. The system offers the potential for application in new and existing researchlaboratories to effectively remove airborne contaminants at the bench using a technology that is costeffective and efficient.
In this article the concept of a new energy-efficient office building and results of a 3-year monitoringare described. The monitoring was performed within the German funding programme ENOB.In this building most of the offices are naturally ventilated and passively cooled. Another focus of theenergy concept is on regenerative heating.Monitoring results show that the integrated planning enabled a very low consumption of energy forheating, ventilation, cooling and lighting.
The effect on Indoor Air Quality of painting at home while resident stays at home has been discussed. This paper presents a case study of the effects. A normal Scandinavian apartement has been refurnished with new paint on the walls and ceilings and the VOC emissions have been followed during eight weeks. Two types of low-emitting paint was selected for the study. A paint shop rolled two layers of new paint on the walls and ceiling in two rooms in the flat.
In a test cell equipped with an operating table, a medical lamp and a manikin representing the surgeon, air velocity and tracer-gas concentration were automatically measured at more than 700 points. Numerical simulations were performed too for analyzing air quality in operating rooms. The results showed that the distribution of the contaminants depends strongly on the presence of obstacles like medical equipment and staff.
Until recently, the air quality did not play a major role in the planning of buildings. Air qualitywas simply understood to be synonymous with pollution-free and safe air. This approach,however, omitted consideration of how to achieve the well-being of the occupants. Asresearch has shown, elimination of pollutants is far from being sufficient and does notsignificantly reduce the dissatisfaction rate of building occupants. More recently, research hasaddressed the occupants perception of indoor air with the goal of increasing their satisfactionrate.


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Windows 7: 2.1 or 5.1 headset better?
10 May 2013
Windows 7 Professional 64 bit SP 1
245 posts
2.1 or 5.1 headset better?
Hello guys, I'm want to buy a gaming or normal headset that is 2.1 or 5.1. Maybe the price is around $50-$60 (In my country maybe about MYR 150-160) or higher, I not sure, but can anyone recommend any headset that has good deep bass for gaming and music?
Maybe from Turtle Beach, Logitech (Not so sure, never made any research on logitech products), Razer (Razer Kraken, Electra, Orca)? If you have any other suggestion or the products that you suggest have similar quality or cheaper price that would be good. But if the price is higher a bit but the quality is good, that also can be consider. But my budget is only about $50-$60 (In my country maybe about MYR 150-200?).
Turtle Beach, Inc. - EAR FORCE X12 can be consider too I guess?
Looking forward for anyone's reply, thank you.
My System Specs
System Manufacturer/Model Number
Custom Build
Windows 7 Professional 64 bit SP 1
Intel(R) Core(TM) i5-2500 CPU 3.30GHz
Asus P8H61-MLE
Graphics Card
NVIDIA GeForce 9500 GT
Sound Card
High Definition Audio Codec:
Monitor(s) Displays
SyncMaster (1280x1024@60Hz)
Screen Resolution
Razer Tarantula
Razer Mamba
Cooler Master eXtreme Power Plus 650W
Cooler Master Elite 431 Plus
Hard Drives
1465GB Western Digital WDC WD15EARS-00Z5B1 ATA Device (SATA) 977GB Western Digital WDC WD10EADS-00L5B1 ATA Device (SATA) 1863GB Western Digital WDC WD20EARX-00PASB0 ATA Device (SATA) 156GB Seagate ST3160813AS ATA Device (SATA)
Other Info
Creative SBS A200 speaker
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Pokemon Lightning Yellow is a ROM Hack of Pokemon Fire Red with some changes. Except the graphics, Pokemon Yellow and Pokemon Fire Red are almost the same but in Pokemon Yellow, Pikachu is always follow you. Now the author of Pokemon Lightning Yellow brings this into Pokemon Fire Red. Now when the game starts, you will have Pikachu as your starter. Your Rival Starter is Eevee.
Sponsored Links
The story is almost kept. It is the same as Pokemon Fire Red
- Pikachu, Eevee are Pokemon Starters
- Better Graphics


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Valley of Sabbia rentals, holiday lettings direct from the owners
A good holiday plan to share with family or friends? Destination Valley of Sabbia for holiday letting with IHA, the private letting expert. Enjoy the cheapest accommodation, promotions and last minute in Valley of Sabbia. Book your house, villa, flat and studio letting in Valley of Sabbia direct by owners without fees nor commissions. Take the time of a holiday and enjoy an unforgettable stay in Valley of Sabbia with holiday letting, either short term or seasonal rental. All ads are verified, travel with confidence, your holidays in Valley of Sabbia are here!
£295* week
book direct with the owners
from £42/Night*
See the rates
Up to-60%
Depending on the season
74 requests already received
Advert 26856
Display the map
Residence Vico Flat-Apartments 700 Sq.ft. in a residence. panoramic, not overlooked, swimming pool, garden/park viewFlat-Apartments Rental in Idro #26856 >Swimming poolParkingBaby chair
Interior facilities
- 1 Bathroom
- 1 Shower room
- Living room 215 Sq.ft.
- Typical style kitchen
- Dining room
- Mountain holidays
- Ideal for family and retired people
- Children welcome
- Setting : Lakeside, Suitable for a relaxing break
- Ski slopes at 15.5mi.
- Fishing
View the advert
3 bedrooms Gîte - Self Catering for rent for 8 people
£357* week
book direct with the owners
from £51/Night*
See the rates
Up to-69%
Depending on the season
141 requests already received
Advert 36475
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Park Alpini Stone-Built House 1,076 Sq.ft. in a private property. panoramic, mountain, swimming pool, garden/park viewGîte - Self Catering Rental in Idro #36475 >Swimming poolParkingDirect access to the beach
Interior facilities
- 1 Bathroom
- Living room
- 2 Double bed(s)
- 1 Sofa bed(s) 2 pers
- 1 Bunk beds
- 2 Possible extra bed(s)
- Mountain holidays
- Ideal for all age groups and family groups
- Children welcome
- Setting : Waterfront, Quiet
- Ski lifts at 15.5mi.
- Fishing
View the advert


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In the country 2.01 acre parcel in rural setting ready for your dream home. Build able tree lined lot. Call for help navigating the home buying and building process today.
Features Vacant Land
|1 Story SqFt Min: 1800
|2 Story SqFt Min: 2300
|2 Story 1st Flr SqFt Min: 1500
|Topography: Wooded
|Body of Water:
|Taxes: $683
|Tax Year: 2016
|Lot Size: irregular
|Est Acreage: 2.01
|School District: Whitewater
|High School:
|Middle School:
|Elementary School:
|Zoning: Residential
Estimate your monthly mortgage payment
How much can I borrow?
Speak with a mortgage consultant: 608-276-5210


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Website Design and style Company
There are various folks on world-wide-web who get in touch with by themselves as website designers and world wide web design providers. A lot of individuals get it done for entertaining, being a passion or part-time. Net structure companies full-time operations are method to go.. Website structure corporations executing it for residing will get your venture critically. Make certain your world-wide-web design and style business has done sufficient investigation on internet technologies Scott Keever Net Worth.
5 organization steps for finding the appropriate Website style and design corporation for you
one. Evaluate your preferences:
An expert Net style and design enterprise will function with you in lieu of in your case. It will make it easier to in defining your organization's specifications. Even now, you should have a essential idea of what your web web page can perform for you personally. Will it provide being an educational catalog? Will it seize new clientele? Will it strengthen relationship with your existing shoppers? Will it provide the demands of the staff?
You'll find couple preliminary necessities your firm should really aware of:
The supposed audience for the Internet web site
The meant target of your job
Predicted spending plan to the World-wide-web web-site
How the challenge will in good shape right into a bigger marketing/corporate plan
The individuals/departments within your organization dependable for that venture
These initial necessities will help you getting website structure company greatest fitted to your project. You should document them and use them for a foundation for assessing every internet style company. This document will serve as your benchmark to evaluate just about every net design business.
2. Listing Technology:
You can use many ways to go looking website design organization. You will find strengths and weaknesses on deciding upon a technique to find internet design and style organization. Blend of diverse ways might be your very best wager in hunt of internet style and design organization to your venture.
Competitors' web sites:
Try to look for internet web sites of corporations that you like, predominantly world-wide-web web pages of your competition. From time to time you've got to search for internet web sites apart from your business. Largely, web layout company consists of site credits (case in point: produced by 4MDESIGNERS). You could simply pay a visit to web style firm's world-wide-web web site and stick with it your assessment. When you simply cannot discover the credits on the web page, be at liberty to get hold of the group and request which World-wide-web structure company is responsible for the website web site? You'll be able to at least have some feedback about that world wide web style and design organization from the firm you can be contacting.


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CorporateCarOnline.com - 2013
Hackers stole and stored information online related to customers who used limousine and other ground transportation. The online information included plain text archives of credit card numbers, expiration dates, names, and addresses. Many of the customers were wealthy and used credit cards that would be attractive to identity thieves.


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My Nonprofit Reviews
Review for Anne Arundel County Literacy Council, Inc., Annapolis, MD, USA
The Anne Arundel Literacy Council has been a tremendous support to me as a new reading tutor.
When I need advice other tutors come to my aid and give me the benefit of their experience and give me ideas on how to proceed with my student. The training provided by the AALC has been wonderful.
You couldn't ask for better real-life instructors to help you through the process. You always have someone there. Other tutors really care about helping you and your student succeed.
More Feedback
If I had to make changes to this organization, I would...
I wish we had more frequent training sessions. They are great! Even if they were just occasional informal get-togethers, that would be helpful.
Would you volunteer for this group again?
For the time you spent, how much of an impact did you feel your work or activity had?
A lot
Did the organization use your time wisely?
Very Well
Would you recommend this group to a friend?
When was your last experience with this nonprofit?


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GraphicRiver Horse Mascot 3657133
Category: Stock Vector | Comment: ( 0 )
GraphicRiver Horse Mascot 3657133
Stock Vector \ Characters \ Animals
Sketch \ Speed \ Silhouette \ Running \ Rebellion \ Riding \ Sport \ Stallion \ West
Stock Vector - Horse Mascot 3657133 by seamartini @ GraphicRiver
Stock Vector \ Characters \ Animals
Graphics Files Included: JPG Image, Vector EPS; Layered: No; Minimum Adobe CS Version: CS.
Horse tattoo symbol for design isolated on white. Editable EPS8 (you can use any vector program) and JPEG (can edit in any graphic editor) files are included
Heart Tattoos; Set of Anchor Symbols; Set of Anchor Symbols; Horse Mascot; Horse Mascot; Snake Tattoos; Flower Symbols.
Keywords: animal, black, competition, design, domestic, horse, illustration, isolated, mammal, mane, mare, mustang, outline, purebred, race, racehorse, rebellion, riding, running, silhouette, sketch, speed, sport, stallion, strength, symbol, vector, west, wild.
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