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PPARa-dependent induction of the energy homeostasis-regulatingnuclear receptor NR1i3 (CAR) in rat hepatocytes: Potential rolein starvation adaptationNadine Wienekea, Karen I. Hirsch-Ernstb, Manuela Kunaa, Sander Kerstenc,Gerhard P. Pu ¨schela,*aUniversita ¨t Potsdam, Institut fu ¨r Erna ¨hrungswissenschaft, Biochemie der Erna ¨hrung, Arthur-Scheunert-Allee 114-116, D-14558 Nuthetal, GermanybGeorg-August-Universita ¨t Go ¨ttingen, Institut fu ¨r Pharmakologie und Toxikologie, Abteilung Molekulare Pharmakologie,Robert-Koch-Strasse 40, D-37075 Go ¨ttingen, GermanycNutrition, Metabolism and Genomics Group and Nutrigenomis Consortium, Wageningen University, P.O. Box 8129,6700 EV Wageningen, The NetherlandsReceived 10 October 2007; accepted 5 November 2007Available online 20 November 2007Edited by Laszlo NagyAbstractpivotal relevance for animals. Recent evidence suggests aninvolvement of the nuclear receptor NR1i3 (CAR). Fasting in-duces CAR by largely unknown mechanisms and CAR-deficientmice are defective in fasting adaptation. In rat hepatocytes CARwas induced by WY14643, a PPARa-agonist. A DR1 motif inthe CAR promoter was necessary and sufficient for this control.The PPARa-dependent increase in CAR potentiated the pheno-barbital-induced transcription of the prototypical CAR-depen-dent gene CYP2B1. Since free fatty acids are natural ligandsfor PPARa, a fasting-induced increase in free fatty acids mightinduce CAR. In accordance with this hypothesis, CAR inductionby fasting was abrogated in PPARa-deficient mice.? ? 2007 Federation of European Biochemical Societies. Publishedby Elsevier B.V. All rights reserved.A tight hormonal control of energy homeostasis is ofKeywords: Phenobarbital-responsive enhancer module;Nuclear receptor protein; Constitutive androstane receptor;CYP2B1 promoter; Thyroid hormone; Xenobiotic metabolism1. IntroductionA stringent control of energy homeostasis is of pivotal rele-vance in animals and humans. Excess in either energy expendi-ture or intake in the long run leads to diseases like cachexia orobesity with metabolic syndrome and type 2 diabetes, respec-tively [1,2]. Therefore, complex control mechanisms haveevolved that adjust food intake to the actual demands. In addi-tion, a healthy organism can adapt energy expenditure to vari-ations in fuel supply over a wide range. Thus, resting energyexpenditure increases when food supply is ample whereas itdecreases upon starvation. To achieve this high level of flexibil-ity metabolic pathways that influence energy expenditure areunder tight hormonal control. In this respect, thyroid hor-mones are of outstanding importance. The abundance of theactive form of thyroid hormone, T3, is controlled in a dualway: Synthesis and liberation of both T3 and the less activeT4 from the thyroid gland is stimulated by TSH, whose releaseis controlled by the hypothalamus-pituitary axis and is inhib-ited by thyroid hormones in a negative feedback inhibitionloop. Apart from this, regulated processing and eliminationof thyroid hormones provides an additional level of control.The majority of T3 in the circulation is derived from deiodin-ation of the outer ring of its precursor T4 in the liver. Modifi-cation of thyroid hormones by enzymes of the xenobioticmetabolism (sulfotransferases, UDP-glucuronosyltransferases,multi drug resistance associated proteins) prevents this activa-tion step and in addition leads to the inactivation and elimina-tion of T3 [3,4]. The transcription of genes coding for proteinsinvolved in the inactivation of thyroid hormone is under thecontrol of the nuclear receptor NR1i3 (CAR, constitutiveandrostane receptor). In particular, induction of hepatic xeno-biotic-metabolizing and thyroid hormone-metabolizing en-zymes by phenobarbital is mediated by CAR. Recently, ithas been shown that CAR and as a consequence thyroid hor-mone-inactivating enzymes were also induced by starvation [5].In addition, mice lacking the CAR gene showed a severely im-paired fasting tolerance. These results clearly indicate thatCAR is a central player in the adaptation of energy homeosta-sis to reduced fuel supply. The mechanisms by which fastinginduces CAR expression are, however, largely unknown. A re-cent publication provides evidence that a fasting-induced in-crease in plasma adrenalin leads to a cAMP-mediatedinduction of PGC1a which in turn enhances the HNF4a-dependent transcription of the CAR gene [6]. CAR in cooper-ation with PGC1a then enhances the transcription of CARtarget genes. The mechanism proposed in this study can, how-ever, explain only part of the fasting-induced CAR expressionsince fasting-dependent CAR induction was retained to a cer-tain extent in HNF4a-deficient mice. In addition, excessivelyhigh adrenaline concentrations (100 lM) that never can beachieved in vivo were employed in this study to evoke the ob-served effects in cell culture experiments. Therefore, we soughtfor an alternative mechanism by which starvation can lead toan induction of CAR. A hallmark of fasting adaptation isthe increase in plasma free fatty acids. Free fatty acids areligands for the nuclear receptor PPARa [7]. We therefore ana-lyzed whether PPARa activation enhanced CAR expressionand modulated the expression of a CAR target gene. It was*Corresponding author. Fax: +49 33200 88 541.E-mail address: email@example.com (G.P. Pu ¨schel).0014-5793/$32.00 ? 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.doi:10.1016/j.febslet.2007.11.011FEBS Letters 581 (2007) 5617–5626
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found that the PPARa agonist WY14643 induced CARexpression and potentiatedinduction of the typical CAR target gene CYP2B1 in primarycultures of rat hepatocytes or in vivo after treatment of ratswith Phenobarbital and WY14643. In line with our hypothesis,fasting-dependent induction of CAR expression was impairedin PPARa-null mice.thephenobarbital-dependent2. Materials and Methods2.1. MaterialsAll chemicals were purchased from commercial sources indicatedthroughout the text. Oligonucleotides were custom-synthesized byMWG Biotech AG (Ebersberg, Germany) and Sigma/Genosys (Stein-heim, Germany). The pGL3 constructs pGL3C2B1, pGL3CD2 andpGL3CD8 were kindly provided by Bauer [8].2.2. AnimalsMale Wistar rats (200–300 g) were purchased from Charles River(Sulzfeld, Germany) and kept on a 12 h day/night rhythm (light from06:00 to18:00 h) with free access to water and the standard rat diet1320 (altromin, Gesellschaft fu ¨r Tierhaltung, Lage, Germany).Male pure-bred Sv129 and PPARa-null mice on a Sv129 back-ground were used at 3–5 months of age (Jackson Laboratories, BarHarbor, ME). Animals were kept on a 12 h day/night cycle with freeaccess to normal laboratory chow (RMH-B diet, Arie Blok animalfeed, Woerden, the Netherlands).Treatment of the animals followed the German and the NetherlandsLaw on the Protection of Animals and was performed with permissionof the state animal welfare committees.2.3. Animal experimentsFor three consecutive days male Wistar rats (3 in each group) wereinjected either vehicle (saline) or 3.13 mg PB/kg BW. The last day thePB was administered alone or together with a single dose of 50 mg WY14643/kg BW or vehicle (equal parts of DMSO and saline). The fol-lowing day livers were removed from anesthetized rats (ether). Liverswere homogenized in 3 ml KBS buffer (10 mM Na2HPO4, 10 mMNaH2PO4, 110 mM KCl, pH 7.4)/g liver, containing 5 mM EDTA,50 mM NaF and 200 lM pefabloc and 10 lg/ml trypsin inhibitor.Microsomes were prepared immediately by differential centrifugationand the CYP2B-catalyzed O-depentylation of 7-pentoxyresorufin wasmeasured fluorometrically (see below). Fed mice were killed at theend of the dark cycle. Fasting was started at the onset of the light cyclefor the times indicated. Mice were killed by cervical dislocation. Liverswere isolated and frozen immediately for later RNA quantification.2.4. Hepatocyte preparation and cultureRat hepatocytes were isolated as described previously [9]. Hepato-cytes (1.2 · 105 cells/cm2) were plated on plastic tissue culture plates(Sarstedt, Nu ¨rnbrecht, Germany) in Williams E medium containing0.5 nM insulin, 100 nM dexamethasone (all Sigma–Aldrich, Deisenho-fen, Germany), 10 mg/ml penicillin/streptomycin (Biochrom, Berlin,Germany) and for the first 4 h of culture 4% newborn calf serum(v/v) (Biochrom, Berlin, Germany). Culture was then continued in ser-um free medium at 37 ?C in a humidified atmosphere of 5% CO2and95% air. Medium was supplemented with ITS (insulin 5 mg/L, transfer-rin 5 mg/L, sodium selenite 5 lg/L) (Sigma–Aldrich, Deisenhofen, Ger-many) as noted. The medium was replaced daily. At the time indicated1 mM phenobarbital and/or 10 lM WY 14643 or the solvent 0.1%DMSO were added and the culture was continued for a total of72 h. Hepatocytes were washed twice with PBS (137 mM NaCl,2.7 mM KCl, 1.5 mM KH2PO4, Na2HPO4Æ 2H2O, pH 7.3), shock fro-zen in N2and stored at ?70 ?C for subsequent analysis.2.5. Generation of the CAR promoter constructs and PPARa expressionplasmidsFragments containing 4.4 kb, 2.6 kb or 1.9 kb of the CAR promoterwere generated from rat genomic DNA by PCR using the primerslisted in Table 1, then blunt-end ligated into pJet and subcloned inthe right direction into pGL3basic (Promega, Mannheim, Germany).Starting from a vector containing the 1.9 kb promoter fragment addi-tional shorter fragments were generated using unique restriction sitesor PCR-based site-directed mutagenesis. The translated region of therat PPARa gene was amplified by PCR from hepatocyte cDNA withproofreading thermostable polymerase. The PCR-fragment was clonedinto the expression vector pcDNA3.2.6. Cell transfection and luciferase reporter gene assayPrimary adherent rat hepatocyte cultures, plated onto plastic 3.5 cmdiameter tissue culture plates (0.7 · 106cells/plate), were transientlytransfected immediately after seeding with 1 lg of each pGL3-derivedplasmid using a modified calcium phosphate transfection protocoldescribed previously [10]. Afterwards the hepatocytes were treated asdescribed above. At the end of the experiment at 72 h of cell culturehepatocytes were lysed in 100 ll of luciferase cell culture lysis reagent(Promega) and firefly luciferase activity was measured in 25 ll of celllysate using the Fluostar Optima (BMG Labtech, Offenburg,Germany). HEK293-cells were transiently transfected with the pcDNAexpression vector constructs as described previously [11].2.7. Microsome preparationHepatocytes were scraped into 100 ll KBS buffer (10 mM Na2H-PO4, 10 mM NaH2PO4, 110 mM KCl, pH 7.4) per 3.5 cm diameterplate, containing 5 mM EDTA, 50 mM NaF and 200 lM pefabloc,10 lg/ml leupeptin and 10 lg/ml trypsin inhibitor. Suspensions werethen sonified. The cell homogenates were centrifuged for 10 min at2500 · g and the supernatants were centrifuged further for 120 minat 36700 · g. The pellets were then resuspended in 100 mM potassiumphosphate buffer pH 7.4, containing 1 mM EDTA, 5 mM NaF and20% glycerol and shock frozen in N2and stored at ?70 ?C. Prior tofurther use protein content was determined [12].2.8. Cell lysate preparationHepatocytes were scraped into 100 ll lysis buffer per 3.5 cm diameterplate (20 mM Tris/Hcl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Tri-ton X-100, 2.5 mM Na4P2O7, b-glycerol phosphate 50 mM NaF,200 lM pefabloc, 10 lg/ml leupeptin and 10 lg/ml trypsin inhibitor).Suspensions were then sonified. The cell homogenates were centrifugedfor 15 min at 10000 · g. The collected supernatant was stored at?20 ?C. Prior to further use protein content was determined [12].Table 1Sequences of the primers used to generate reporter gene constructsPrimerSequencerCARprom_R+rCARprom_F1.9+rCARprom_F2.6+rCARprom_F4.4rCARprom_DDR1d_RrCARprom_DDR1d_FrCARprom_DDR1c_RrCARprom_DDR1c_FrCARprom_DDR1p_RrCARprom_DDR1p_F50-CAGGAATTAGGGATTGTGGTCCTC-3050-CAGAGTGATGAAGACAGAGCTTGG-3050-CGCAGTAGCAAGCAAACTAGAGTAG-3050-CAGAGTGATGAAGACAGAGCTTGG-3050-GCCGCCcgatcgGTTAAGCCGTGGCGGCTCGATC-3050-GCCGCCcgatcgGAACTGCTGATTTCCAGAC-3050-GCCGCCcgatcgCACACTCAGTGGCTCATTGG-3050-GCCGCCcgatcgGTGGATTCCAGGGGTCAAAG-3050-GCGGCGcgatcgCTGTCTGATATTCTGGGGCTAGAG -3050-GCGGCGcgatcgCTGTGGGTACTAGGTGTACACATT-305618N. Wieneke et al. / FEBS Letters 581 (2007) 5617–5626
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2.9. Enzyme activity assayPentoxyresorufin O-dealkylation activity was measured by directfluorometric detection of resorufin formation [13]. Briefly, dealkylationactivity of 5 lg of protein was measured in 200 ll of a 50 mM potas-sium phosphate buffer pH 7.4, containing 4.5 lM 7-pentoxyresorufin,65 lM NADP+, 165 lM glucose-6-phosphate, 165 lM MgCl2Æ 6 H2Oand 0.4 U glucose-6-phosphate dehydrogenase (all Sigma–Aldrich,Deisenhofen, Germany) over a period of 20 min. The reaction ratewas determined by external calibration with solutions of known res-orufin concentrations.2.10. Immunoblotting proteinHepatic microsomes, cell lysates (CYP2B, 50 lg of protein) or nucle-ar extracts (30 lg of protein) were subjected to SDS–PAGE. Afterelectrotransfer onto polyvinylidene difluoride membrane (Roth,Karlsruhe, Germany), blots were blocked with Tris-buffered salinecontaining 0.10% Tween and 5% dry milk for 1 h at room temperatureand incubated overnight at 4 ?C with a mouse monoclonal antibodyagainst rat liver CYP2B (Oxford Biomedical Research, Oxford MI,USA), a rabbit polyclonal antibody against mouse CAR (Axxora Deu-tschland GmbH, Gru ¨nberg, Germany) or a rabbit polyclonal antibodyagainst PPARa (Santa Cruz Biotechnology Inc., Santa Cruz, CA,USA). Immune complexes were detected using horseradish peroxi-dase-linked goat anti-mouse IgG or goat anti-rabbit IgG accordingto the ECL Plus Western blotting kit (Amersham Pharmacia Biotech.).Immunoreactive bands were visualized and quantified by using theimaging system QuantityOne (Biorad).2.11. RNA preparation and Northern blot analysisTotal RNA was isolated from hepatocyte cultures by guanidiniumthiocyanate phenol extraction, [14] subjected to electrophoresis onformaldehyde-agarose gels, and subsequently transferred to Hybond-N nylon membranes as described previously [15]. RNA blots werehybridized to the oligonucleotide probe 50-GGTTGGTAGCCGG-TGTGA-30specific for the rat CYP2B1 gene (bases 49–66 of exon 7region; Genebank L00318) which had been 50end-labeled by T4-poly-nucleotide kinase utilizing [c-32P] ATP [16] RNA expression was quan-tified by an imaging system (Fujix BAS 1500 Bio-Imaging Analyzer,Fujix, Tokio, Japan). Control hybridizations were performed using aGAPDH antisense oligonucleotide probe [17].2.12. Real-time RT-PCRRNA was isolated using the SV total RNA kit (Promega). To gen-erate standards for CAR and b-actin RT-PCR using the oligonucleo-tides listed below was carried out with hepatocyte cDNA as templateand the resulting fragments were cloned blunt end into pJET orpUC57 (both MBI-Fermentas). Hot start real-time PCR for the quan-tification of each transcript was carried out in a reaction mixture of15 lL of iQ SYBR Green Supermix (Bio-Rad, Mu ¨nchen) and 5 lLof cDNA. PCR was performed with an initial enzyme activation stepat 95 ?C for 3 min. Followed by 40 cycles of denaturation at 95 ?Cfor 30 s, annealing at 63 ?C for 30 s and extension at 72 ?C for 1 minin a real-time DNA thermal cycler (iCyclerTM, Bio-Rad). Thefollowing oligonucleotides where used as forward and reverseprimersrespectively:50-TCTCACTCAACACTACGTTC-3050-CTGGAAAAGGATCCAAGCCTGGG-30for CAR [18] and 50-CCCTAAGGCCAACCGTGAAAAGATG-30and 50-AGGTCCCG-GCCAGCCAGGTCCAG-30for b-actin. The initial copy number ofthe amplified cDNA was quantitated by determining the number of cy-cles required to raise the fluorescence signal above a defined thresholdduring the logarithmic phase of amplification. Using the respectivestandard curves the copy numbers were calculated. The results areshown relatively to the control level after normalization to that ofb-actin.and2.13. Electrophoretic mobility shift assayThe electrophoretic mobility shift assay was performed essentially asdescribed previously [8]. Nuclear extracts were prepared from hepato-cytes which were kept in culture for 72 h as described above. Alterna-tively, nuclear extracts were prepared from HEK293-cells that hadbeen transiently transfected with either pcDNA3 or pcDNA3-ratP-PARa. A double stranded oligonucleotide probe was generated byhybridizing50-GAGTGTGTATAAAGGTCAGAGAACAACCTGand50-GGCCCCTGCAGGTTGTTCTCTGACCTTTATAwhichcontain the sequence of the central DR1-motif of the rat CAR pro-moter (bold). The 50-overhangs were filled with a Klenow reactionmix containing a[32P]dCTP. Binding of 5 lg nuclear extract proteinto 0.2 lCi (0.5 pmol) probe was performed in presence of 2 lg of unre-lated DNA (pGL-basic) to avoid unspecific binding. Binding was com-peted with a about 100-fold excess of unlabelled probe or a 5-foldmolar excess of plasmids containing the wild-type or mutated 1.9 kbpromoter fragments as indicated.3. Results and discussion3.1. PPARa-dependent induction of CAR in the adaptiveresponse to fastingAs reported previously by others, CAR mRNA was inducedabout 4-fold in livers of rats starved for 24 h (see supplemen-tary material). Similarly, CAR mRNA was induced about2,5-fold after a 12 h to 24 h fast in wild-type Sv129 mice(Fig. 1). Since, among other mechanisms, the activation ofthe nuclear receptor PPARa during caloric restriction regu-lates the adaptive response of the energy metabolism [19,20],it was hypothesized that PPARa might also play a role inthe fasting-dependent induction of CAR. In line with thishypothesis the fasting-dependent induction of CAR mRNAobserved in wild-type mice was significantly reduced inPPARa-deficient mice, indicating that a functional PPARa isessential for the induction of CAR by fasting. Further support-ing this hypothesis, CAR mRNA was also induced about 1.5-fold by intraperitoneal injection of the PPARa-specific agonistWY14643 in rats (Fig. 2A).3.2. Potentiation of the induction of the CAR target geneCYP2B1 by PPARa agonistsTo analyze whether the PPARa-dependent increase in CARwas functionally relevant the impact of a PPARa agonist on aprototypic CAR-dependent gene whose transcription, unlikethat of for instance UGT1A1, was not affected by the PPARaagonist itself was analyzed. Rat CYP2B1 appeared to fulfillthese requirements. CYP2B1 activity was almost not detect-able in livers of control rats (Fig. 2B). Activity was inducedby a low dose of phenobarbital (3.13 mg/kg body weight) toabout 6 lU/mg. This induction was enhanced about 4-foldFig. 1. Attenuation of fasting-dependent CAR induction in PPARa-deficient mice. Control or PPARa-deficient mice were starved for thetimes indicated. CAR mRNA levels were determined by qPCR in liversof these animals. Statistics: Student's t-test; (a) significantly differentfrom control mice, P < 0.05, n = 5.N. Wieneke et al. / FEBS Letters 581 (2007) 5617–56265619
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by the simultaneous application of the PPARa agonistWY14643 which induced CAR mRNA (Fig. 2A) but did notaffect CYP2B1 activity by itself (Fig. 2B). Phenobarbital didnot alter CAR expression significantly (Fig. 2A).To further analyze the PPARa agonist-dependent increaseof CYP2B1 primary cultures of rat hepatocytes were incubatedwith the PPARa-selective agonist WY14643 for 24 h and withphenobarbital for 48 h as indicated (Fig. 3). Like in the liver ofPPARa agonist treated or fasted rats CAR mRNA was in-creased about 3-fold in hepatocytes treated with the PPARaagonist. In hepatocyte cultures kept in the presence of WY14643 plus phenobarbital CAR mRNA-level was elevatedabout 10-fold over the basal level. Apparently, phenobarbitalpotentiated the PPARa-dependent CAR mRNA induction;yet phenobarbital by itself did not significantly augment theCAR mRNA levels. The induction of CAR by WY 14643but not the potentiation of this induction by phenobarbitalwas also seen on the protein level by Western blot analysis(Fig. 3, bottom). The CAR target gene CYP2B1 was almostnot detectable in untreated rat hepatocytes, neither by mea-surement of the enzyme activity nor by Western blot analysisor on the mRNA-level by Northern blot analysis (Fig. 4). Asseen in vivo, CYP2B1 was induced by treatment of hepatocyteswith phenobarbital for 48 h. This induction was enhancedabout 2-fold by the simultaneous presence of the PPARa ago-nist for 24 h. However, the PPARa agonist by itself did not in-duce CYP2B1, clearly indicating that the PPARa effect onCYP2B expression was indirect, and most probably via theinduction of CAR.To further corroborate the hypothesis that CAR-inductionis a prerequisite for the potentiation of phenobarbital-inducedCYP2B activity by the PPARa agonist, the transcription of aluciferase reporter gene construct under the control of a frag-ment of the CYP2B1 promoter was analyzed in primary hepa-tocyte cultures. In accordance with the protein and mRNAdata, transcription of the reporter gene containing part ofthe wild-type CYP2B1 promoter (pGL3C2B1) was increasedby phenobarbital (Fig. 5). This increase was further enhancedby the simultaneous presence of the PPARa agonist, which, atvariance with the protein and mRNA data, also by itself in-creased the transcription of the reporter gene. This discrepancymight be attributable to additional posttranscriptional controlFig. 2. PPARa-dependent induction of CAR an potentiation of phenobarbital-induced CYP2B expression in rats in vivo. Male wistar rats (200–300 g) were injected vehicle (saline) or 3.13 mg PB/kg BW for three consecutive days. The last day the PB was administered either alone or togetherwith a single dose of 50 mg WY 14643/kg BW or vehicle (a mixture of equal volumes of DMSO and saline). The following day livers were removedfrom anesthetized rats (ether). RNA or microsomes were isolated. CAR mRNA was quantified by real-time PCR as described in the methods section,CYP2B activity was determined as described in legend to Fig. 2. Values are means ± S.E.M. of n P 3 independent experiments. Statistics: Student'st-test for paired samples: (a) significantly higher than control, P < 0.05 and (b) significantly higher than phenobarbital-stimulated, P < 0.05.5620N. Wieneke et al. / FEBS Letters 581 (2007) 5617–5626
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mechanisms by which phenobarbital could affect CYP2B1mRNA stability or translation. Evidence that CYP2B1 mRNAabundance is regulated on the posttranscriptional level wasrecently provided [21]. The synthesis of functional CYP2Bprotein also depends on the adequate supply with hemewhose synthesis is controlled by amino levulinate synthase 1(ALAS-1) an enzyme that is induced by phenobarbital in aCAR-independent manner [22]. To distinguish a possibleCAR-independent PPARa effect on the CYP2B1 promoterfrom an effect that is dependent on a prior induction ofCAR, additional reporter gene constructs were employed: Ina reporter gene construct lacking the phenobarbital-responsiveenhancer module (PBREM, nucleotides ?2626 to ?2156),which contains the presumed CAR binding site, phenobarbi-tal, as expected, did no longer increase the reporter gene activ-ity (pGL3CD8), whereas the induction of the reporter gene bythe WY14643 was preserved in this construct (Fig. 5). By con-trast, a reporter gene construct lacking a 178 bp fragment inthe proximal promoter region (nucleotides ?353 to ?176,pGL3CD2) was no longer directly activated by the PPARaagonist, however, it retained the full response to phenobarbitaland displayed a similar potentiation of the phenobarbital-dependent induction by WY14643 as the wild-type promoter(Fig. 5). Thus, this construct behaved as expected for an idealprobe that is not directly activated by PPARa but senses thePPARa-dependent induction of CAR. PPARa may stimulateCYP2B1 promoter activity directly as well as indirectly via amechanism involving CAR. These two effects are mediatedby distinct regions within the CYP2B1 promoter. These datafurthermore suggest that the induction of CAR by the PPARaagonist might be relevant for the control of the transcription ofother CAR-dependent genes.3.3. Characterization of the CAR promoterTo gain further insight into the mechanisms by which thePPARa agonist increased CAR mRNA, reporter gene con-structs were generated that express the luciferase gene underthe control of the rat CAR promoter. Luciferase transcriptionwas increased by the PPARa agonist WY14643 in constructscontaining 4.4, 2.6 or 1.9 kb of the rat CAR promoter whereasluciferase transcription was not affected by the PPARa agonistin the control vector (Fig. 6). Phenobarbital did not influencethe basal transcription rate in any of the constructs. At varianceto what was seen on the mRNA level (Fig. 3), phenobarbitalFig. 3. Induction by WY14643 of CAR in rat hepatocytes. Primarycultures of rat hepatocytes were cultured as described in materials andmethods. Phenobarbital and the PPARa agonist WY14643 were addedas indicated after 24 h and 48 h of culture, respectively. After a totalculture period of 72 h hepatocytes were harvested, mRNA wasextracted by affinity chromatography and quantified by real-timeRT-PCR. The copy number was normalized to the copy number ofGAPDH-mRNA. Values are means ± S.E.M. of n P 3 independentexperiments. Statistics: Student's t-test for paired samples: (a) signif-icantly higher than control, P < 0.05 and (b) significantly higher thanphenobarbital-stimulated, P < 0.05. CAR protein was detected bywestern blot with a polyclonal antibody against a conserved peptidebetween mouse and rat CAR. A representative blot of three similarindependent experiments is shown.Fig. 4. Potentiation by WY14643 of the phenobarbital-dependent induction of CYP 2B1. Rat hepatocytes were cultured as described in the methodssection. Phenobarbital and the PPARa agonist WY14643 were added as indicated after 24 h and 48 h of culture, respectively. After a total cultureperiod of 72 h hepatocytes were harvested. Cyp2B activity and protein were determined in a microsomal fraction by a pentoxyresorufin-depentylatonassay and Western blot, respectively. mRNA was determined by Northern blot. Band intensities were quantified densitometrically. Values aremeans ± S.E.M. of n P 3 independent experiments. Statistics: Student's t-test for paired samples: (a) significantly higher than control, P < 0.05 and(b) significantly higher than phenobarbital-stimulated, P < 0.05.N. Wieneke et al. / FEBS Letters 581 (2007) 5617–56265621
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also did not further augment the PPARa agonist-dependent in-crease in CAR transcription, indicating that the phenobarbital-dependent potentiation of the PPARa-elicited increase inmRNA occurred on a posttranscriptional level possibly by sta-bilizing CAR mRNA. In this context it is worth noting, thatphenobarbital has been shown to unspecifically stabilizemRNAs [23]. However, the phenobarbital-dependent increaseof CAR mRNA was not reflected on the protein level (Fig. 3,bottom). On the protein level, phenobarbital rather reducedthe PPARa-induced CAR expression slightly. The pattern ofCAR induction on the protein level thus is in better agreementwith the CAR promoter studies than the induction on themRNA level.Computer analysis of the 1.9 kb CAR promoter fragmentidentified three DR1 (direct repeats separated by 1 nucleotide)motifs which are potential PPARa binding sites (Fig. 7A).Two of them, the proximal and the central one, are highly con-served across species rat, mouse and man. To elucidate whichof the three DR1 motifs is necessary for the PPARa-dependentinduction of CAR additional truncation mutants were gener-ated containing either none, one, two or all three of the DR1motifs. As expected, luciferase transcription from a reportergene construct containing none of the DR1 motifs did not dif-fer from the control vector and was not induced by the PPARaagonist (not shown). Surprisingly, a 1 kb promoter fragment,which contained the proximal conserved DR1 motif, but wastruncated at a unique PstI site immediately proximal of thecentral DR1 motif also did not respond to the PPARa agonistwith an increase in transcription rate (Fig. 7B). By contrast,transcription of a 1.2 kb construct that was truncated at aBstXI site about 180 bp upstream of the central DR1 motifand about 400 bp downstream of the distal DR1 motif(Fig. 7B) was enhanced by the PPARa agonist to a similar ex-tent as the 1.9 kb construct, revealing that the region contain-ing the central, conserved DR1 motif appears to be sufficientand necessary to confer the PPARa-dependent induction ofCAR gene transcription, whereas the distal DR1 motif, whichis only present in the rat promoter but is lacking in the humanand mouse promoter appeared to be dispensable.To exclude that essential promoter sequences apart from theDR1 motif were lost by the consecutive shortening of the pro-moter, additional constructs were generated in which one ofthe DR1 motifs was deleted selectively within the 1.9 kb pro-moter fragment by site directed mutagenesis. Deletion of theproximal or the distal DR1-motif did not affect the PPARa-dependent activation of the reporter gene transcription whiledeletion of the central DR1 motif completely abrogatedthe PPARa-dependent reporter gene induction (Fig. 7C). Alabeled oligonucleotide probe comprising the central DR1 motifplus 12–13 nucleotides up- and downstream bound specificallyto nuclear extracts of hepatocytes (Fig. 7D). The binding wascompletely abolished by a 100-fold excess of an unlabeledoligonucleotide competitor. Specific binding of proteins tothe labelled oligonucleotide probe was also reduced if a 5-foldmolar excess of a plasmid containing the 1.9 kb promoter frag-ment with the three DR1 motifs was included into the bindingreaction. Similarly binding was reduced when the plasmidswhich contain a 1.9 kb promoter fragment lacking either theproximal or distal DR1 motif were added to the bindingreaction. By contrast, a plasmid containing a 1.9 kb promoterconstruct lacking the central DR1 motif reduced binding onlyto a similar extent as a similar amount of vector DNA withoutthe promoter region and thus apparently did not compete spe-cifically with the binding of the oligonucleotide probe furthersupporting the relevance of the central DR1 motif. In orderto corroborate that the bands observed in hepatocyte nuclearFig. 5. CAR-dependent potentiation of the phenobarbital-induced expression of a reporter gene under the control of the CYP2B1 promoter byWY14643. Hepatocytes were cultured as described in the methods section. Immediately after seeding they were transfected with the reporter geneconstructs by a modified calcium phosphate method. Phenobarbital and the PPARa agonist WY14643 were added as indicated after 24 h and 48 h ofculture, respectively. After a total culture period of 72 h hepatocytes were harvested. Luciferase activity was determined in cell lysates. Values aremeans ± S.E.M. of n P 3 independent experiments. Statistics: Student's t-test for paired samples: (a) significantly higher than control, P < 0.05 and(b) significantly higher than phenobarbital-stimulated, P < 0.05.5622N. Wieneke et al. / FEBS Letters 581 (2007) 5617–5626
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extracts really represent PPARa-binding to the probe, addi-tional experiments were performed with HEK293 cells inwhich rPPARa had been transiently overexpressed (Fig. 7E).Western blot analysis of nuclear extracts of non-transfectedHEK293 cells revealed a weak band at about 50 kDa whichwas markedly enhanced in nuclear extracts of cells transientlytransfected with an expression vector containing the translatedregion of a PPARa cDNA. In an EMSA, a single shift bandwas observed with nuclear extracts of HEK293 transfectedwith the control vector. This band was much more pronouncedin cells transfected with a vector containing the translated re-gion of the rat PPARa gene. It migrated at a position similarto the upper band observed with hepatocyte nuclear extracts.Taken together the data indicate that the central DR1 motifis necessary and probably sufficient to confer the PPARa-dependent activation of the CAR promoter.3.4. Physiological significance of PPARa-dependent CARinductionRecently it was reported that the fasting-induced increase inCAR expression might depend on an adrenaline and cAMP-dependent induction of PGC1a, which binds together withHNF4a to a conserved sequence element in the proximalCAR promoter. However, although the fasting-dependentinduction of CAR target genes was abolished in livers of ani-mals lacking HNF4a the induction of CAR itself was blunted,yet not abolished [6]. In our current study with rat hepatocytes,in contrast to PPARa agonists neither adrenaline nor anothercAMP-elevating hormone, glucagon, increased CAR mRNAor reporter gene activity under control of a 1.9 kb promoterfragment that contained all elements that had been identifiedby Ding et al. to be relevant for cAMP-dependent inductionin mice (not shown). Species differences and differences in theprotocol for hepatocyte isolation and cultivation might explainpart of this discrepancy; in addition the majority of the exper-iments performed by Ding et al. were carried out in tumor cellsrather than hepatocytes. Although this previous study clearlyshowed that HNF4a is essential for basal CAR-expressionand modulation of HNF4a activity contributed to the fastingadaptation of CAR expression, current data in rat hepatocytestogether with the experiments in PPARa-deficient mice implythat a PPARa-dependent increase in CAR expression isneeded to get a full response to starvation. Possibly PPARaactivation and the fasting-dependent increase in cAMP act inparallel.A glucocorticoid response element has previously beendescribed in the far upstream region of the human CARpromoter [24]. Glucocorticoids have been shown to increaseCAR expression in human hepatocytes [25] and to potentiatethe induction of CAR-dependent genes including CYP2Bin human, mouse and rat hepatocytes [26]. Since glucocorti-coid levels are increased during fasting, this might be anadditional mechanism by which fasting enhances CAR expres-sion.In accordance with our current finding, various conditionsthat lead to an increase in plasma free fatty acid concentrationshave been shown to increase CAR mRNA in mice. Thus apartfrom fasting an increase in free fatty acids in db/db mice led toa CAR induction [27]. In addition, induction of a number ofCAR-dependent genes has been shown to be increased in insu-lin-resistant mouse models with increased levels of plasma freefatty acids [28].Fig. 6. Activation by WY14643 of the transcription of a reporter gene construct under the control of rat CAR promoter fragments. Hepatocyteswere cultured as described in the methods section. Immediately after seeding they were transfected with the reporter gene constructs by a modifiedcalcium phosphate method. Phenobarbital and the PPARa agonist WY14643 were added as indicated after 24 h and 48 h of culture, respectively.After a total culture period of 72 h hepatocytes were harvested. Luciferase activity was determined in cell lysates. Values are means ± S.E.M. ofn P 3 independent experiments normalized to control activity. Statistics: Student's t-test for paired samples: (a) significantly higher than control,P < 0.05.N. Wieneke et al. / FEBS Letters 581 (2007) 5617–56265623
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Fig. 7. Identification of a DR1 motif in the rat CAR promoter as potential target for the PPARa-dependent induction of CAR. (A) Positions of theputative DR1 motifs in the CAR gene promoter are shown underlined and bold. A PstI site immediately proximal to the central DR1 motif that wasused to generate the contruct pGL3rCAR1.0 is shown in bold italic. (B) Hepatocytes were cultured as described in the methods section, except, thatinsulin concentration was reduced to 0.5 nM after the initial adherence phase. Immediately after seeding they were transfected with the reporter geneconstructs by a modified calcium phosphate method. Phenobarbital and the PPARa agonist WY14643 were added as indicated after 24 h and 48 h ofculture, respectively. After a total culture period of 72 h hepatocytes were harvested. Luciferase activity was determined in cell lysates. (C)Hepatocytes were cultured as in B, however, they were transfected with reporter gene constructs in which one of each of the DR1 motifs had beeneliminated selectively. Values are means ± S.E.M. of n P 3 independent experiments normalized to control activity. Statistics: Student's t-test forpaired samples: (a) significantly higher than control, P < 0.05. (D) Hepatocyte nuclear extracts (5 lg protein) were incubated with a labelled probecomprising the central DR1 motif plus 12–13 nucleotides up- and downstream. Binding was competed with an 100-fold excess of unlabelled probe(competitor) or a 5-fold molar excess of plasmids containing no promoter fragment (pGL-basic), a 1.9 kb promoter fragment with all three DR1motifs (CAR 1.9 kb) or promoter fragments in which either the proximal (D-DR1 prox), central (D-DR1 cent) or distal (D-DR1 dist) DR1 motif wasdeleted as indicated. The intensity of the two shift bands that are marked by the arrowheads were quantified densitometrically. A representative ofthree independent experiments is shown. (E) Nuclear extracts (5 lg protein) of HEK293 cells transiently transfected with either pcDNA3 (pcDNAcontrol) or pcDNA3 containing the translated region of the rat PPARa gene (pcDNA-rPPARa) were incubated with a labelled probe comprising thecentral DR1 motif plus 12–13 nucleotides up- and downstream. Binding was competed with an 100-fold excess of unlabelled probe. PPARaexpression was monitored by Western blot analysis.5624N. Wieneke et al. / FEBS Letters 581 (2007) 5617–5626
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In conclusion, the newly discovered PPARa-dependentinduction of CAR via a conserved DR1 motif appears to beof high physiological significance both in the control of energyhomeostasis and xenobiotic metabolism.Acknowledgements: The excellent technical assistance of Elvira Stein-meyer, Ines Kahnt, Sonja Blume, Christoph Schmitz-Salue and Gud-run Ru ¨dell is gratefully acknowledged. This work was supported bythe Deutsche Forschungsgemeinschaft through the Sonderforschungs-bereich 366, Teilprojekt A16 and Sonderforschungsbereich 402, Teil-projekt A2 and the Grant Pu 100-3.Appendix A. Supplementary dataSupplementary data associated with this article can befound, in the online version, at doi:10.1016/j.febslet.2007.11.011.References[1] Tisdale, M.J. (2005) Molecular pathways leading to cancercachexia. Physiology (Bethesda) 20, 340–348.[2] Kahn, S.E., Hull, R.L. and Utzschneider, K.M. (2006) Mecha-nisms linking obesity to insulin resistance and type 2 diabetes.Nature 444, 840–846.[3] Mol, J.A. and Visser, T.J. (1985) Rapid and selective inner ringdeiodination of thyroxine sulfate by rat liver deiodinase. Endo-crinology 117, 8–12.[4] Qatanani, M., Zhang, J. and Moore, D.D. (2005) Role of theconstitutive androstane receptor in xenobiotic-induced thyroidhormone metabolism. Endocrinology 146, 995–1002.[5] Maglich, J.M., Watson, J., McMillen, P.J., Goodwin, B., Willson,T.M. and Moore, J.T. (2004) The nuclear receptor CAR is aregulator of thyroid hormone metabolism during caloric restric-tion. J. Biol. Chem. 279, 19832–19838.[6] Ding, X., Lichti, K., Kim, I., Gonzalez, F.J. and Staudinger, J.L.(2006) Regulation of constitutive androstane receptor and itstarget genes by fasting, cAMP, hepatocyte nuclear factor alpha,and the coactivator peroxisome proliferator-activated receptorgamma coactivator-1alpha. J. Biol. Chem. 281, 26540–26551.[7] Nakamura, M.T., Cheon, Y., Li, Y. and Nara, T.Y. (2004)Mechanisms of regulation of gene expression by fatty acids.Lipids 39, 1077–1083.[8] Bauer, D., Wolfram, N., Kahl, G.F. and Hirsch-Ernst, K.I. (2004)Transcriptional regulation of CYP2B1 induction in primary rathepatocyte cultures: repression by epidermal growth factor ismediated via a distal enhancer region. Mol. Pharmacol. 65, 172–180.[9] Pu ¨schel, G.P., Kirchner, C., Schro ¨der, A. and Jungermann, K.(1993) Glycogenolytic and antiglycogenolytic prostaglandin E2actions in rat hepatocytes are mediated via different signallingpathways. Eur. J. Biochem. 218, 1083–1089.[10] Gaunitz, F., Papke, M. and Gebhardt, R. (1996) Transienttransfection of primary cultured hepatocytes using CaPO4/DNAprecipitation. Biotechniques 20, 826–830, 832.[11] Neuschafer-Rube, F., Engemaier, E., Koch, S., Boer, U. andPuschel, G.P. (2003) Identification by site-directed mutagenesis ofamino acids contributing to ligand-binding specificity or signaltransduction properties of the human FP prostanoid receptor.Biochem. J. 371, 443–449.[12] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.(1951) Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193, 265–275.[13] Lubet, R.A., Mayer, R.T., Cameron, J.W., Nims, R.W., Burke,M.D., Wolff, T. and Guengerich, F.P. (1985) Dealkylation ofpentoxyresorufin: a rapid and sensitive assay for measuringinduction of cytochrome(s) P-450 by phenobarbital and otherxenobiotics in the rat. Arch. Biochem. Biophys. 238, 43–48.[14] Chomczynski, P. and Sacchi, N. (1987) Single-step method ofRNA isolation by acid guanidinium thiocyanate-phenol-chloro-form extraction. Anal. Biochem. 162, 156–159.[15] Aubrecht, J., Kahl, G.F. and Hohne, M.W. (1993) Regulation offibronectin mRNA expression in primary hepatocytes in responseto EGF and phenobarbital. Biochem. Biophys. Res. Commun.190, 1023–1028.[16] Omiecinski, C.J., Walz Jr., F.G. and Vlasuk, G.P. (1985)Phenobarbital induction of rat liver cytochromes P-450b andP-450e. Quantitation of specific RNAs by hybridization tosynthetic oligodeoxyribonucleotide probes. J. Biol. Chem. 260,3247–3250.[17] Hirsch-Ernst, K.I., Schlaefer, K., Bauer, D., Heder, A.F. andKahl,G.F. (2001)RepressionCYP2B1 mRNA induction by reactive oxygen species in primaryrat hepatocyte cultures. Mol. Pharmacol. 59, 1402–1409.[18] Yoshinari, K., Sueyoshi, T., Moore, R. and Negishi, M. (2001)Nuclear receptor CAR as a regulatory factor for the sexuallydimorphic induction of CYB2B1 gene by phenobarbital in ratlivers. Mol. Pharmacol. 59, 278–284.[19] Kersten, S., Seydoux, J., Peters, J.M., Gonzalez, F.J., Desvergne,B. and Wahli, W. (1999) Peroxisome proliferator-activatedreceptor alpha mediates the adaptive response to fasting. J. Clin.Invest. 103, 1489–1498.[20] Kroetz, D.L., Yook, P., Costet, P., Bianchi, P. and Pineau, T.(1998) Peroxisome proliferator-activated receptor alpha controlsthe hepatic CYP4A induction adaptive response to starvation anddiabetes. J. Biol. Chem. 273, 31581–31589.[21] Truong, N.T., Moncion, A., Barouki, R., Beaune, P. and deWaziers, I. (2005) Regulatory sequence responsible for insulindestabilization of cytochrome P450 2B1 (CYP2B1) mRNA.Biochem. J. 388, 227–235.[22] Kakizaki, S., Yamamoto, Y., Ueda, A., Moore, R., Sueyoshi, T.and Negishi, M. (2003) Phenobarbital induction of drug/steroid-metabolizing enzymes and nuclear receptor CAR. Biochim.Biophys. Acta-General Subjects 1619, 239–242.[23] Matsumura, S. and Omura, T. (1973) Stability of messengerRNA's for microsomal NADPH-cytochrome c reductase andcytochrome b 5 in the livers of normal and phenobarbital-treatedrats. J. Biochem. (Tokyo) 73, 407–416.[24] Pascussi, J.M., Busson-Le Coniat, M., Maurel, P. and Vilarem,M.J. (2003) Transcriptional analysis of the orphan nuclearreceptor constitutive androstane receptor (NR1I3) gene promoter:Identification of a distal glucocorticoid response element. Mol.Endocrinol. 17, 42–55.[25] Pascussi, J.M., Gerbal-Chaloin, S., Fabre, J.M., Maurel, P. andVilarem, M.J. (2000) Dexamethasone enhances constitutiveandrostane receptor expression in human hepatocytes: conse-quences on cytochrome P450 gene regulation. Mol. Pharmacol.58, 1441–1450.[26] Kocarek, T.A., Schuetz, E.G. and Guzelian, P.S. (1994) Biphasicregulation of cytochrome P450 2B1/2 mRNA expression byof phenobarbital-dependentFig. 7 (continued)N. Wieneke et al. / FEBS Letters 581 (2007) 5617–56265625
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dexamethasone in primary cultures of adult rat hepatocytesmaintained on matrigel. Biochem. Pharmacol. 48, 1815–1822.[27] Yoshinari, K., Takagi, S., Sugatani, J. and Miwa, M. (2006)Changes in the expression of cytochromes P450 and nuclearreceptors in the liver of genetically diabetic db/db mice. Biol.Pharm. Bull. 29, 1634–1638.[28] Pass, G.J., Becker, W., Kluge, R., Linnartz, K., Plum, L., Giesen,K. and Joost, H.G. (2002) Effect of hyperinsulinemia and type 2diabetes-like hyperglycemia on expression of hepatic cytochromep450 and glutathione s-transferase isoforms in a New Zealandobese-derived mouse backcross population. J. Pharmacol. Exp.Ther. 302, 442–450.5626N. Wieneke et al. / FEBS Letters 581 (2007) 5617–5626


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Wheeler retires after long career as president of Lowe's track
Updated: May 23, 2008, 3:26 PM ETAssociated Press
CONCORD, N.C. -- Humpy Wheeler announced his retirement as president and general manager of Lowe's Motor Speedway on Wednesday, ending a 33-year career as one of NASCAR's top promoters.
Wheeler will step down after Sunday's Coca-Cola 600 at the track near Charlotte. No replacement was selected for the 69-year-old Wheeler, who plans to write a book and host a television show.
Wheeler said he first considered retirement about a year ago, although he sidestepped a question on whether ...
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text: Sue and Art were great hosts at their B&B. From a clean room to unbelievable breakfasts and sharing their knowledge and love of the area. We used this as our base as we explored the Erie Canal trail by bike and we're also amazed at the quality of restaurants in the area. Jim and Laura


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April 5, 2011
Spring Practice Photo Feature #2
Spring practice will culminate in 10 days with the Spring Game on April 15. The new Golden Hurricane coaching staff has been focused on improving all areas of the team, and the players are progressing with each and every practice. This second ITS photo feature from spring drills focuses on the five TU quarterbacks.
• LINK: 2011 Spring Practice Photo Feature #2
Copyright 2011 by InsideTulsaSports.com. Reproduction of these photos in any form is strictly prohibited without the expressed written consent of InsideTulsaSports.com.
• Spring Practice Notebook #3
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• Spring Scrimmage #2: First Impressions
• Spring Practice Notebook #2
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• Recruits on hand to watch TU scrimmage
• Spring Practice Notebook #1
• Spring Practice Photo Feature #1
• Spring practice gets underway at TU
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5 Parks near LaBelle, Florida
Port LaBelle, Florida 2.21 miles NE
Palmdale, Florida 15.4 miles NE
Aqua Isles Boulevard, LaBelle, FL 33935, USA LaBelle, Florida 33935 (map) | (863) 675-2331
Sites: 300 | Latest Rate: $50.00 | 1 Reviews
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Dec 2015 - $50.00
We didn't do the daily rate, stayed a month for $700.
Beautiful, clean, friendly park but waaaaay overpriced.
Mostly permanent mobiles and "florida homes" with about 15% RV's.
Right on the river (from Lake O all the way to the Gulf at Ft. Meyers), with its own launch ramp and docks.
Joined some friends during our winter in "the south", just to check it out.
The entire place revolves around their "activities".....ain't my thing.
Management is stiff, formal, and unyielding, but "inmates" are very nice.
If you have the money and get excited over shuffleboard, this is the place. - MajorOz
LaBelle, Florida (map) | (863) 675-3641
Sites: 120 | Latest Rate: $32.00 | 1 Reviews
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Mar 2011 - $32.00
This is an older campground that boasts mature pine and palm trees and "grass" between sites. Quite a variation in site dimensions but mostly comfortable for spacing. Grass is not watered any more so is rather brown and sparse in the winter months. Nice paved streets and concrete parking slabs with side extension for table and chairs. Picnic tables provided. Perhaps 50% Permanent sites. Very friendly residents. Morning breakfast in Chiki-Hut by volunteers. Some activities. Shuffleboard, Horseshoe pits seldom used but in nice shape. Hot tub at 102, Pool at 80. Monthly rate about $650. Walking distance to Groceries. Closest spots a block from street making for rather quiet surroundings. - easydoesit
LaBelle, Florida (map) | (863) 674-0085
Sites: 306 | Latest Rate: $100.00 | 29 Reviews
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Feb 2015 - $100.00
This is where all the rich people go. Very friendly resort. First day we were made to feel very welcome and old friends. These people have all the toys. Remote control race cars and a scale race track with grandstand. Scale model remote control trains that cover about an acre. Remote control sail boat races. Remote control race boats. Drones. Tricked out golf carts. Bentleys, Vipers, SSRs, etc. Casitas. Impeccable landscaping. Pool is 90+ degrees in February. Socials almost every day of the week. - Royce Holm
Jan 2015 - $60.00
We stayed at Riverbend for six weeks from late December to early February. We were is site 204 which is considered a wood-site lot with slightly lower rate. This is really nice resort with a very friendly atmosphere and lots of activities. While we were there we made use of the pickle-ball courts, pool and hot tub, watched some remote car races, participated in the twice weekly exercise class and took advantage of the breakfast and lunch offerings. The park is spacious with wide paved roads and large concrete sites. We drive a 44 ft motorhome and tow a car. East to navigate and park. Resort staff is friendly and helpful. Our site had lawn table and chairs for our use but some rental sites do not. Lovely landscaping between most sites which are spacious and lots of grass in the resort. Cable is ok. We did not use the resort Wifi as they require you install a modem which they supply, so we went with our hotspot which worked well. Good satellite reception. There is a dog run next to the pickle-ball courts but we did not often go there. From our experience, this is the nicest resort we have visited and hope to return next year for a couple of months. - LvR
Jan 2015 - $90.00
We stayed at RiverBend Luxury Motorcoach Resort for a week over the New Year. Each lot is about 50 x 100-feet and has full hookups with cable TV, and telephone and DCL connections. Although WiFi doesn't reach the individual lots, it is available in all the facilities or you can sign up for the DSL service. Our Verizon MiFi worked great. RiverBend is an ownership resort with many lots available to rent. Nearly all have extensive landscaping. The resort facilities are simply outstanding. They have a beautiful clubhouse with bar and restaurant right on the Caloosahatchee River, boat docks, a nature center, store, large heated pool, good sized laundry, and many organized community activities. There is an extensive model railroad layout, dirt racetrack for electric or gas-powered cars, and four lakes. Every person we encountered at RiverBend were friendly and happy to be there. We definitely will return next year. - CNRUSA
LaBelle, Florida (map) | (863) 675-0334
Sites: 40 | Latest Rate: $23.00 | 1 Reviews
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Mar 2011 - $23.00
Riverside Retreat has a campground/RV park open to all. Also hosting a camp for churches, organizations, schools, etc. the campground/RV park is separate from that camp area. Situated on the Caloosahatchee River, Riverside Retreat's motto is, "It's the Nature of the Place." It is a quiet park in a natural setting. You may spy gopher tortoises, bobcats, or armadillos on any of the hiking paths. Canoe or kayak the Caloosahatchee. The roads and sites are not paved, but it is a full hook-up campground with a friendly staff. We highly recommend this little gem of a campground. We visit every winter. - Dawn1955
LaBelle, Florida (map) | (863) 675-6888
Sites: 461 | Latest Rate: $55.00 | 19 Reviews
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Aug 2015 - $55.00
Loved this place. We want to go back this winter as soon as we sell our Florida house in Ft. Pierce. Nice spacious parking spots. Lots of permanent homes, but also lots of transients like us. Many activities, meals with other guests. Just impressed us to no end! - Genielady
Mar 2015 - $30.00
We had a wonderful stay at Whisper Creek. We arrived on a Saturday evening after dark and were taken to our spot and assisted in backing in, so everything was to our liking. The pool was warm and inviting. The bathrooms as clean as could be. The staff friendly and helpful. I searched the internet extensively to find a quiet campground with warm temps for our first ever month long vacation. We stayed here for 2 weeks, and were sad to leave. This is the campground we hope to someday call our winter home. - R-D
Mar 2015 - $26.00
We were looking for a place for our first "snowbird" experience. After checking reviews for about 50 RV resorts/ campgrounds and then visiting 10 in the greater Fort Myers area we chose Whisper Creek for our stay January- April of 2015. The rate reflects that 3 month stay.
We couldn't have been happier with our choice. The sites are huge ( we were in the Glenn), staff is very accommodating and helpful, plenty of activities, everyone very friendly and welcoming. We plan to return for the winter. - Oma&Opa


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Salsa Zucchini Recipe
5 1
It took Teri Wolfson just three ingredients to create this tasty and super easy side dish. The reader from Knoxville, Tennessee writes, "It's great on the grill but the best part is there's no clean up."
- 2 small zucchini, cut into 1/2-inch slices
- 1 cup salsa
- 1/2 cup shredded Monterey Jack cheese
- Place zucchini on a double thickness of heavy-duty foil (about 18 in. x 12 in.). Top with salsa. Fold foil around zucchini and seal tightly.
- Grill, covered, over medium heat for 25-30 minutes or until zucchini is crisp-tender. Open foil carefully to allow steam to escape. Transfer zucchini to a serving bowl; sprinkle with cheese. Yield: 4 servings.
Originally published as Salsa Zucchini in Simple & Delicious July/August 2007, p43
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Seattle Utilities eBilling
View and pay your Seattle City Light and Seattle Public Utilities bills online. Our system is completely paperless and there is no additional charge to view and pay your bills.
Once you've signed up for eBilling:
- You'll be notified by email when your new bill is available
- You'll have the option to set up automatic payments
- You'll be able to view up to two years of bills and payment history
- You'll be able to add additional utility accounts
To sign up you'll need your SPU or SCL account number and ZIP code, which you can find at the top of your bill.


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Music Production & Engineering Courses
This course is a survey of the history and techniques used in producing surround audio. The focus is split between understanding surround tools (microphones, consoles and monitoring), surround acquisition (recording techniques), and presentation (mixing and authoring) in the context of audio for disc, video, games and broadcast. The course includes practical work to reinforce theoretical and technical learning. It addresses the aesthetic considerations of surround production relating to varying end uses, and examines the elements of music that invite surround treatments based on the compositional ideas and production values.


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1168 posts tagged with love.
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Was he interested or not?
An older guy flirted with me at work. I finally got a chance to give him my number but it's been awhile and he hasn't called. I'm VERY confused. Details inside. [more inside]
How do I know whether it's worth giving my relationship a shot?
I'm so confused about my relationship that I've taken to MeFi to ask for advice – any thoughts would be hugely appreciated. My boyfriend of almost one year is 29; I'm 34. He's kind, caring and all-round deeply lovely, we get on absolutely brilliantly, have loads in common and I feel incredibly comfortable and happy around him. Life is sweet. Except two not-so-tiny things: he's scared of commitment and doesn't ever want to talk about my ticking biological clock. How much time do I give this? [more inside]
The Cycle Continues
Are my kids forever damaged? Can I teach them to show love? [more inside]
How to love a fearful-avoidant partner
I've seen these questions about how to change a fearful-avoidant attachment style, but I can't find any information on how to help a partner who is fearful-avoidant feel loved and secure. [more inside]
Give me your best NON-RELIGIOUS wedding readings
I'm looking for a short reading for our wedding that we can ask an athiest friend of ours to read. Other portions of the ceremony will contain religious elements, but we don't want to ask him to read anything that might make him feel uncomfortable or awkward. [more inside]
Help me love
Guys, I've gotten off track. I've lost my way. And I'm worried that I won't get to be in love again because it scares me. [more inside]
Modern Dating/Online Matching 101, please?
For a fiction project, I need to come to a better understanding about how modern dating works, particularly with regard to Tinder. From my perspective (I'm older, I've been married a long time and I have absolutely no experience with the modern dating scene), I wonder why women might be using Tinder. Is it just for hooking up? Or do woman (or men) look to Tinder to try to meet someone for actual dating and long-term relationship? [more inside]
In love with best friend for about a year but he's taken. Now what?
About a year ago I realised I loved a close friend of about a decade. Now what? [more inside]
Movies about fanatics
What are some more movies about lives being consumed by objects or crafts? [more inside]
Realistic love songs
I love love and I hate most love songs. I feel that real love is incredibly beautiful in it's complexity yet knowledge of it's frailty. I want songs that express this sentiment without being cynical.
My boyfriend "forgot" me and let his ex stay during the holiday with him
Please help me get over the bitterness towards my boyfriend's actions. I am turning into a person I truly dislike, but I can't get over myself and all the bad feelings this story brought up. I have been with my man for almost three years in a LDR. He is great in so many ways and I do love him. However, I have lost my trust and confidence in him that he will be a considerate and respectful man, and a partner to count on in the future. [more inside]
Websites about love and self-worth/knowledge/respect/etc
I really like this website and I was wondering if you can recommend similar ones. [more inside]
Please Help Me Think Of 40 Love Songs and How Should I Package Them?
Mr. Westridge's 40th birthday is coming up. I have decided to give him 40 of something every day for the forty days leading up to his actual birthday. One thing I want to give him is a collection of 40 love songs but I'm running out of ideas for songs. Also what would be the best way to gather all these songs together and present them? [more inside]
I think I've fallen out of love with my husband. What now?
More detail below the cut, but mostly I'm seeking some experience and wisdom from the hivemind on the more existential aspects of this realization, and maybe a reality check if needed. Thanks. [more inside]
More songs for baby video, like M. Ward's "One Hundred Million Years"
I'm looking for more songs similar to M. Ward's "One Hundred Million Years" for a 20-minute baby photo/video compilation. I have found plenty of kids' songs and songs written about having children. I'm looking for more indie songs that echo the sentiment, "And this love, this love between you and I, Is older than that burning ball of fire up in the sky." Songs by indie singers about endless love, happiness, joy, the beauty of life, etc. would fit the bill. [more inside]
When a man tells you "You are the one" does he really mean it?
How soon do you realize that the person you are with is the one you are marring? I am sure everyone is different and have a different opinion about this but, I just started to seeing this guy(we both are in 40s), we are doing great, we've been together every day since the day we met. I'm at his house every night and waking up with him every morning. So the things are more than great. But the bottom line is, I only have known him for 2 months. Yes we know alot about each other, he's trying to learn about me fastest he can. I see that every day. but 2 month is still 2 month. [more inside]
Only a loser would settle for me
I can't imagine someone choosing to date me when there are people out there who are so much more successful, competent, fit, daring, worldly, and self-assured. So if someone likes me, I assume they can't do better than me, and that's a turnoff, because I'm not so great, so THEY must not be so great either. How can I get out of this mental trap? [more inside]
Is it unfair to tell my colleague I'm in love with her?
I'm in love with my female colleague. We went on a date in 2014 and left it as friends. Now I'm thinking of finding a new job - should I tell her why or is this unfairly burdening someone else with my problem? [more inside]
Who am I and how do I learn to love her?
After tons (and maybe too much self-reflection) I've come to realize all my problems boil down to the fact that I hate myself. Help? [more inside]
Passion ambivalence - committed to relationship when in love with third?
What is more important: having stability with your partner, whom you love (but possibly non-amorous) and whose company you enjoy or the being-in-love with a third person with whom you feel alive, passionate and excited [more inside]
Can this 'bond' turn into a relationship?
I fell for a girl...we keep a really nice and friendly connection going on between us, but nothing more happens. She knows how I feel but keeps rejecting me. She have her reasons to do so though. [more inside]
Navigating a 10 Year Age Gap While Dating
Yesterday an incredible guy messaged me on Match and I can't stop looking at his profile because he seems like such a great fit for me. The only catch is that he's 10-11 years older than I am (though still in his 30s) and that's making me very leery. Am I right to be or am I being silly? [more inside]
Single parent dating: I want to start meeting nice men again
A long time has passed since I posted here, and a lot has happened in that long time. Quick update is that now I am legally divorced. I also got laid off last year and thankfully was able to find another great job quickly. Moved to NYC, which is a fantastic city and I went to school here so have a lot more friends here than when I was in Boston. Anyway, so I want to date. I don't know if I want to get into a serious relationship just yet, but I just want to share my life with someone and do fun things on weekends. Where do I meet responsible, loving, educated men who would want to date me? I haven't seen anyone in two years (since separation/divorce)… I think I should change that… [more inside]
Powering through when it feels hopeless
I am having the worst holiday season of my life. Help me cope. [more inside]
Romantic Triple Threat! How to move forward?
I've fallen for my coworker-friend-roommate I've known for a year-and-a-half, and I need advice on how to escalate without it all blowing up in my face. We've roomed together (just the two of us) for six weeks, but I've been away for half that time. Also, we get along well and flirt occasionally, but I'm the worst at reading signs. [more inside]
Am I looking a gifthorse in the mouth?
I found the greatest guy when I wasn't even looking. But I'm not too long out of a relationship (10 months) where I think I really loved him (despite current man looking so much better on paper), intimacy is taking a while to grow and I just feel a bit wary and not sure I'm into him some of the time. It's been 3/4 months of dating but I'd now say we're firmly headed to relationship territory. But I don't want to stay and waste this person's time if he's not the one for me. I also don't want to throw away something which could be really special, and this person is special, but maybe I know that on paper but don't really appreciate it which doesn't seem like a good start to a relationship. [more inside]
Please help me decide if I should move back to NYC from SF
Can't decide if I should move back to NYC from SF. Please help me find some new ways to think about the problem. [more inside]
"No touching!" Physical touch for the less inclined?
You are not a touchy feely person - that is, you do not openly express emotion or affection through physical contact. How have you learned to give and share love with people in your life whose primary love language is physical touch? [more inside]
Should I stay or should I go.......again?
My on and off love affair is off again.....I think. We had a row.....made up.....next day she cancelled forthcoming date....not heard from her since. Why do I care what happens next? [more inside]
San Fransisco Corgi.
My girlfriend's birthday is next weekend, and I'd love to surprise her with an hour or two or seven of playing with a corgi in a park, somewhere in SF. [more inside]
Keeping love alive and well
What are some of the things you do, and your partner does for you, on a daily basis and perhaps as an occasional surprise that makes you feel loved and helps keep the romantic love in your relationship alive? Anything worthwhile takes some serious effort to maintain and nourish in the long term. I am very much interested in learning about the efforts healthy adults in healthy relationships have made that took their relationship to a higher level of love, maturity and intimacy. [more inside]
What percentage of trans individuals are gay/queer?
I am interested in learning to what extent individuals who identify as transexual also identify as queer or homosexual. Have there been any good studies of this? I googled but could not find much - please link me to any studies/findings about the issue.
I like a guy who has a girlfriend. What should I do?
I have a crush on a guy that I met in grad school several months ago. He's very nice, smart, and fairly handsome. He appeared a little awkward at first but once I got to know him, he's actually one of the few guys that I can have a long and meaningful conversation with. Although our conversations are mostly school-related, he seems to remember a lot of details from our convo, such as where I used to work a few years ago, what my goals are, etc. He recently joined the same student organization and volunteered at the same research lab with me. I'm very inexperienced in dating so I'm not sure if he's interested in me or not. However, my gut tells me that he might be interested because he often asks me to attend certain meetings or events together, he also occasionally calls me to ask questions (which he could simply ask through text or in person). I recently found out that he has a girlfriend and they both live together so I'm assuming his relationship is pretty serious. Therefore I'm trying to keep my distance from him and not cross the line. But the more I see him/talk to him, the more I like him and I'm not sure what to do. Please advise. Thank you!
Do I meet my catfish?
I am in Buenos Aires on holiday visiting a relative for two weeks, and this place is so full of emotion. [more inside]
Finally had the "marriage" talk with long term boyfriend
Do our expectations of when to get married one day match up? [more inside]
Help me help my 11 year old brother grow up into an awesome person!
What can I do as his sister to help my brother grow up to an awesome person that's hardworking, successful, happy and responsible (and morally good) person. Also one who's educated and has a good amount of fun, worldly experiences. Right now I'm very afraid of him growing up and being a bad person destined for because because of all of the current situations in his life including the way he's being parented. [more inside]
Should I end it with my great companion of a SO?
I'm unsatisfied in the passion and effort in my relationship but love the companionship. We are like best friends. However, it's been so long since I've seen makeup or hair down, or her really initiating to me. Tons of details inside... thanks for your help. My frustration has been building over a long time. [more inside]
It's been a year and he still won't sleep with me.
I am a female and my boyfriend and I have been seeing each other about once a week for over a year. I am 27 and he is 30. I know he isn't seeing anybody else, and that he likes me. Why have we still not slept together? [more inside]
Plan to move to across the country for love. How do I tell my parents?
Since having a manic episode about a year and a half ago, my parents started treating me like I was a minor again (I'm almost 30 now). How do I most respectfully tell them that I'm am going to move without turning it into "I'M NOT ASKING FOR PERMISSION, DAMMIT: I'M MOVING AND YOU CAN'T STOP ME"? Thanks, mefites. [more inside]
Please tell me how to get through this breakup with sanity intact
I went through a breakup this summer and the mental aftermath has put me in a very bad place. Please help me find peace and perspective. [more inside]
How do find a job or career where compassion, sensitivity are assets?
Are there jobs where having a *thin* skin is an asset instead of a liability? Where being loving, caring, emotional, sensitive are a good thing and you don't have to fight those tendencies, especially as a leader? [more inside]
Sharing relationship problems for advice with best friend
Is it okay to ever talk about relationship problems with your closest trusted friend(s) for advice or to vent? [more inside]
Relationship plagued by fear/anxiety or is it just bad fit?
How is it possible to feel anxious and questioning most of the time, but also feel genuine moments of excitement to see him and genuine calm/happiness to be in his presence? [more inside]
Why is it so hard to find a women interested in long-term relationship?
I'm a male in my late 20's who's having trouble finding a woman to settle down with. I keep getting attracted to/and attracting women who are either not interested in serious relationships (only casual/physical relationships) or have some self-esteem issues/other problems that makes them very unstable/emotionally fluctuating. I wouldn't describe myself as overly attractive (very average looks and slim build) or promiscuous, so I'm kind of puzzled by how and why these women feel attracted to me. [more inside]
Marriage Discussion in Long Term Relationship
How would you bring up discussion of marriage without scaring your partner or making them feel pressured? [more inside]
Dealing with a love Conundrum
Should I take the leap, and take the risk, or should I just forget about it and move on? [more inside]
Should I or should I not ? (date someone while not over someone else)
I am a man. I had an emotional affair with a friend - let's call him James - whom I was very much in (unrequited) love with, and recently decided to "break up". The long story is here. Then I decided to start dating and recently met this person, let's call him Fred, whom I like very much. The problem is that I am absolutely not over James, and we've even managed to re-establish contact, still very guarded at this point, although we have not yet met. So I find myself in a quandary and wondering whether it's fair to start something with Fred when my heart is really with James. I mean, I like Fred very much and could ever grow to love him. But I fear I may be just using them as a convenient toy while waiting for a James moment that will never come. And Fred is a nice enough person that I don't want to play with him. But then, I also need company and intimacy and I like having him around and he seems to feel the same about me, and is prepared to wait for as long as it takes for me to be truly over James. [more inside]
Is he a narcissist, or just very self-centred?
I've been dating a guy (30) for half a year, and though I am blinded by love, I'm starting to see signs that he could be narcissistic, but am not sure. [more inside]
How do i deal with my boyfriends distance and communication issues?
My boyfriend cancels so often on our dates, and we haven't had sex even though we have been together for half a year, which makes me think he could have a sexual problem or is afraid of getting close to me. Details inside. [more inside]
Stay for love or move for money?
I have a possible job opportunity (and similar ones in the future, if I don't take this one) at a prestigious institution in my field that would allow me to live a comfortable middle-class life, pay my debts, and raise a family or own a house someday. I love my current job, relationship, home, and community, but I'm stuck at a pay level that's barely adequate to cover my bills each month and that's unlikely to increase much over the course of my career. How do I proceed? [more inside]


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Legends of the High Seas by JonHodgsonWatch
©2007-2016 JonHodgson
(c)2007 Games Workshop Ltd, all rights reserved.
Cover to a new game from Warhammer Historical.
Corel Painter, Adobe Photyshop.
Cover to a new game from Warhammer Historical.
Corel Painter, Adobe Photyshop.
Oh WOW. This was so good I had to listen to my favorite swashbuckler music tracks. This rocks, completely and utterly.
"HAHA! also, your wife is now pregnant with my child!"
"I'm just that AWESOME!"
this pic is...AWESOME
"I'm just that AWESOME!"
this pic is...AWESOME
the movement is cool
great work
Don't mention it, pal!
Pirates kick major ass!! Awesome work!
this is terrific. I like how we can see the ship tip forward us and the actions that are happening on board. the contrast between the background and the two foreground characters are really nice also.
It's a very cool piece, you can almost smell the smoke!
great composition
great composition
sweeeeeeeeeet work!
Wow, brilliant scene
That is *awesome* The dynamics are great, the action is fantastic! Definitely a favorite!
Impressive - most impressive, but can you do Ninja?
arrrrrrr me matey! that be a fine painting you go there!
okay I did my pirate thing. I'm sure I won't be the only one
It looks fantastic!
okay I did my pirate thing. I'm sure I won't be the only one
It looks fantastic!
Heh heh, its a tough temptation to avoid! And hey its good for you, so why try!
more one great work!
cool work!
Arr, me matey!!! A bottle of rum for ye for such an impressive work! Avast!
Wow Jon! I totally dig pirates and you did a damn fine job here!
yay, pirates! what a dynamic piece, i just love it. i feel the colors shadows could've been a bit darker in some places to bring the whole piece together compositionally. the boots/hat/flag are a bit too dark compared to the rest of the painting, but otherwise this just rocks. wonderful characters


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University of Redlands > Academics >
School of Music >
Summer Camps >
Summer Piano Camp >
Courses and Activities
General Courses
Applied Skills
Musical Explorations
Apply Today
Students who join us get to play a part in fun and engaging group courses and activities. Students also have the option to choose a Musical Focus and Musical Exploration.
All students participate in the following:
Piano Ensemble
Performance Forum
Theory: "FUN"-damentals of Music
Activities Showcase
APPLIED SKILLS (choose one):
The following Applied Skills classes give the student a chance to apply his or her musical knowledge and understanding to the development of a specific skill that will enhance the student's pleasure and performance at the piano. No previous experience in these areas is necessary.
A. Adventures in Composition
The Composition class will explore fascinating aspects of music creation. The course includes experimentation with diverse sound sources, working with modern as well as traditional methods of notation, and exploring principles of form, tension/resolution, character and mood.
B. Accompanying – The Pianist as Partner
Students in this class will explore the skills needed to be a fine accompanist. They will have the unique opportunity of preparing music with another instrumentalist and/or vocalist.
C. Organ and Harpsichord for Pianists - Keyboards Across the Ages
In this class, students will explore the similarities and differences among piano, organ, clavichord and harpsichord. Each student will have a hands-on opportunity to perform on the mighty four- manual Casavant organ in Memorial Chapel, and get experience on a harpsichord as well.
D. Percussion Ensemble - Drumming
This class is a joyful way for students to develop rhythmic skills. Students not only will see, hear and handle professional orchestral percussion equipment such as drums, gongs, marimbas and maracas, they will also learn to play them through an exciting percussion ensemble experience.
E. Sight Reading for Fun
The sight-reading class will help students improve their ability to play new music for fun the first time they see it. The focus is on quick recognition of notes, rhythms, and musical patterns.
MUSICAL EXPLORATIONS (choose one):
The University of Redlands Piano camp offers an ever-changing array of musical experiences and explorations for students. This year, there are four explorations from which to choose.
A, Music and ART: "Drawing by Ear"
Students will uncover the common elements that shape music and art, specifically color, form, texture and rhythm. Using a variety of raw materials, and inspired by musical examples, each student will have a chance to create his or her own artwork. This summer students will have the opportunity of working with a tie-dye expert.
B. Music and DANCE: "How Music Makes Us Move"
Working with an experienced dance and music instructor, students will learn about, and kinesthetically experience, music's relation to dance. Music and movement go hand in hand.
C. Music and MULTI-MEDIA: "Listening with our Eyes!"
Students will create a "Nature Symphony" on film, juxtaposing nature scenes they videotape on location around campus with the recorded piano performances of other campers. Students will learn the basics of videography, including how to handle a camera, sound recording, and editing.
D. Our Sonic Universe: "Does Nature Sing?"
Unlike our eyes, the ears never close! In this class students will become discerning listeners in a rich sonic environment. Besides examining sources of sound, they will experiment with the acoustics of sound and discover what constitutes "musical" sound.
The Sustainable University of Redlands Farm (SURF) is a natural farm in its beginning stages, but already grows and sells fresh vegetables to Bon Appetit, the food management company on campus.
© 2016 University of Redlands. All Rights Reserved.


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Welcome to the sports seasons of the end of baseball and start of football and a column full of savory snack recipes excellent for creating a festive feeling for sport viewing.
Recipe ingredients are available at the supermarket and require little or no cooking.
Garlic-herb dip — Halve 3 heads garlic crosswise. Place in an oven-safe dish. Drizzle with olive oil, wrap in foil and roast at 400 degrees for 40 minutes. Cool slightly until easy to handle. Squeeze out the soft garlic pulp; mix with 1 cup mayonnaise, 1 teaspoon each lemon zest, lemon juice and parsley. Taste to determine if additional, salt, lemon or parsley is needed.
Hot crab dip — Mix 3 ounces cream cheese, 3/4 cup mayonnaise, 2 teaspoons lemon juice. Add hot sauce and chopped parsley to taste. Fold in 1/2 pound crabmeat. Spread in a baking dish, top with breadcrumbs or panko (Japanese breadcrumbs). Bake at 425 degrees until golden, about 45 minutes.
Blue cheese dip — Mix 4 ounces crumbled blue cheese, 1/2 cup each mayonnaise and sour cream, 1/4 cup buttermilk and some minced chives.
Buffalo dip — Make blue cheese dip adding 2 tablespoons hot sauce and 1/2 cup finely chopped celery, and replace chives with minced celery leaves.
Bacon-jalapeño dip — Mix 8 ounces cream cheese, 2 cups shredded cheddar cheese, 1/2 cup sour cream, 1/4 cup milk, 4 slices chopped cooked bacon, 1 tablespoon bacon dripping, 1 teaspoon white wine vinegar and 2 thinly sliced jalapeños. Spread in baking dish; top with buttered breadcrumbs/panko. Bake at 350 degrees until golden and bubbly, about 30 minutes.
Queso dip — Cook 1 tablespoon each butter and flour in a saucepan over medium heat, stirring 1 minute. Stir in 1/2 pound shredded cheddar cheese and 1/4 cup each cream cheese, milk and jarred salsa until smooth. Meaty variation, stir in 1/4 cooked beef or ground turkey.
Veggie dip — Combine 1/4 cup each finely chopped red and yellow bell pepper, celery, zucchini and green onions. Mix 8 ounces softened cream cheese with 2 tablespoons milk. Stir cheese mixture into the vegetables; season with salt and pepper.
Smoked salmon dip — Make veggie dip but omit zucchini; add 4 ounces chopped smoked salmon and 2 tablespoons each capers and finely chopped fresh dill.
Onion dip — Cook 2 thinly sliced onions in oil in a skillet over medium heat until golden, about 35 minutes. Cool, chop and mix with 1 cup sour cream, and 4 chopped green onions. Salt to taste.
Bacon-onion dip variation — Stir in 6 slices crumbled cooked bacon.
Pizza snack mix — Bake 1/4 pound sliced pepperoni at 350 for 15 minutes; drain on paper towels. Toss 2 cups each pita chips and cheese crackers, 2 tablespoons olive oil and 2 teaspoons each minced parsley, oregano and basil, or use pizza seasoning to replace the herbs.
Asian snack mix — Combine 1 cup each sesame sticks, wasabi peas, peanuts and crisp rice cereal. Toss with 1/2 stick melted butter, 1 tablespoon each soy sauce and sesame seeds, 2 tablespoons brown sugar, and 1/2 teaspoon each red pepper flakes and salt. Bake at 350 degrees, 20 minutes, stirring once. Remove from oven and toss with 5 cups vegetable chips.
Curry snack mix — Toss 4 cups crisp rice cereal, 2 cups puffed rice, 1 1/2 cups coconut flakes, 1/2 stick melted butter, 2 teaspoons brown sugar, 1 teaspoon curry powder, 1 large pinch cayenne and 1/2 teaspoon salt. Bake at 350 degrees for 20 minutes. Remove from oven and toss with 1 cup chopped dried mango.
Enjoy easy and tasty snacks with family and friends.


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Spring Break 2015: Missouri. In-district actions show local demand for Medicaid expansion.
Legislative inaction hasn't sat well for numerous Missourians across the state who have urged the Missouri legislature to expand Medicaid. Over Spring Break, community leaders have led several in-district events to raise awareness and urge legislators to close the coverage gap by expanding Medicaid this session.


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Graceful use of these wavy frond diamond designs with a rustic overtone.
Monday - Friday
8am - 5pm
927 S Homer St Seattle, Washington 98108
email (
Their selection of ceramic tiles is nothing short of remarkable, not to mention a knowledgeable staff that seems to always go above and beyond any reasonable expectations!
© 2016 StatusCeramics All Rights Reserved


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Irwin Naturals, Dr. Linus Pauling, Vitamin C, 1,000 mg, 90 Tablets
By Irwin Naturals58 Reviews
Expiration Date:?Jun 2017
Expiration Date
The length of time for the expiration date or "best used before" date depends on the type of product, as well as the brand.
Perishable items (such as flax oils or certain probiotics) generally have shorter expiration dates. Although our warehouse is fully air-conditioned, these more fragile items are put in cold storage (freezer or refrigeration unit) for maximum freshness.
Our receiving department does its best to verify and then enter the correct expiration dates for all incoming products. However, discrepancies do occur from time to time. This being said, the exceptionally high turnover at iHerb ensures that our inventory is among the freshest in the industry.
Shipping Weight:?0.53 lbs (0.24 kg)
Shipping Weight
The Shipping Weight includes the product, protective packaging material and the actual shipping box. In addition, the Shipping Weight may be adjusted for the Dimensional Weight (e.g. length, width & height) of a package. It is important to note that certain types of products (e.g. glass containers, liquids, fragile, refrigerated or ice packed) will often require protective packaging material. As such, these products will reflect a higher Shipping Weight compared to the unprotected product.
- Product Code: IRW-25317
- UPC Code: 710363253172
- Package Quantity: 90 Tablets
- Dimensions: 2.3 x 2.3 x 4.2 in, 0.45 lbs (0.2 kg)
Total: $32.16
Combo Discount: -$12.00
Together: $20.16
Product Overview
- Premium Vitamins
- Easy to Swallow Caplets
- With Bioflavonoid Complex
- Dietary Supplement
Vitamin C is an important antioxidant that neutralizes free radicals which cause cellular damage in the body and contribute to the aging process. Vitamin C also helps to fortify the immune system and is necessary for the formation of healthy bones and connective tissue.
Along with friend Albert Einstein, Linus Pauling is considered by many to be a leading scientific figure of the 20th century. Here are only a few of his accomplishments:
- The only person to have received two unshared Nobel prizes (1954 and 1962)
- One of the founders of both modern chemistry and molecular biology
- Co-founder of the Linus Pauling Institute of Science and Medicine
- Considered by many to be the "father" of vitamin research
- Published two of the first best selling nutritional books: "Vitamin C & The Common Cold" and "How to Live Longer & Feel Better"
In 1993, Dr. Pauling lent his name to support a ground breaking new line of vitamin products. Dr. Linus Pauling Premium Vitamins are formulated according to Dr. Pauling's scientific principles of nutrition.
Suggested Use
(Adult) Take one (1) tablet twice daily. Do not exceed two tablets per day.
Other Ingredients
Microcrystalline cellulose, stearic acid, methylcellulose, silicon dioxide and magnesium stearate.
Do not use if safety seal is broken. Check with your doctor before using this product if you are using medication or have any medical conditions. Do not use if you may become pregnant, are pregnant or nursing. Do not exceed recommended daily intake. Not intended for use by persons under 18. Keep out of reach of children. Store in a cool, dry place.
(State of California Prop 65) This product contains a chemical known to the State of California to cause birth defects or other reproductive harm.
|Supplement Facts
|Serving Size: 1 Tablet
|Servings Per Container: 90
|Amount per Serving
|% Daily Value
|Vitamin C (as Ascorbic Acid)
|1000 mg
|Citrus (Citrus spp.) Bioflavonoid powder (fruit)
|100 mg
|Life Extension Complex
Ginkgo powder (leaf), Garlic powder (bulb), Spirulina (Arthrospira platensis) powder, Activin Grape Seed extract, CoQ10
|50 mg
|†Daily Value not established
iHerb Customer Reviews
I have found this product one of the best Time Realese Vitamin C with a Bioflavonoid complex around. It is non irratating to the stomach as well. And keeps going all day. :)
Dr. Pauling did so much research on vitamin c; I'm pleased to see products with his name on them! Here is a very economical way to have a great Vitamin C product. This one is also very easy on my stomach, even though it isn't buffered.


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How Cold Reading Works
3 Apr 2014 - 6:16pm
183 reads
The scariest part of phoning a psychic hotline is giving up all information the clerk wants before she hands you off to the psychic. At Psychic Power Network a foreign-born clerk had to echo plus repeat each letter and numeral I gave her. "'S' as in snake," I said. "'F' because inside Frank,' she said. "No," I said, more boldly, "'S as inside snake. S because inside serpent. S as in Superman." I gave her all the information she asked for except my email. I was just taking up their provide of a 10-minute psychic reading for hot customers, just $1.95. To my surprise I received exactly which. Not each amateur ouija board, tarot card, plus astrology consumer realizes or comprehends the unseen dynamics of what they are doing... however the door is opened to demons in every instances. Demons do not care if a person participates from ignorance, curiosity, or full understanding- they are surprisingly legalistic. They can even claim a right to enter based on just being present plus suggesting nothing while others do what is incorrect! One thing to keep in your mind is that those with true psychic presents will likely not tell you anything that is not for a individual growth or for your own superior. You probably won't be able to recognize when you'll die, whenever the globe can end, or factors like that from a genuine psychic. This really is because telling we these points wouldn't become the right thing for we. Some details are not meant to be known. Even when a psychic sees this in a reading, it's not considered ethical to reveal certain items to a client which may harm them in certain means. In all instances, a succeeds
is generaly continual. It means for one query there is commonly one answer even if there are multiple psychic reading. You may discover that most psychics are open and honest plus convenient to get along with throughout a initially reading. You must also find out when the psychic needs anything of we during the reading. Some psychics can ask to see a pic of somebody which they need to focus on and other items. Pasqualina's website is based in France, because is the website of the notorious and openly fake "Gabriella" plus "Sara Freder." These are typically not women or folks. These are typically computers programmed to send we links to vague plus lengthy computerized "free readings" -- Pasqualina's "Free Personal Horoscope" is 3600 words; here, see mine -- that are mainly sales pitches for paid readings that are furthermore computerized. This gives you a tremendous joy, a fulfillment which you are related to existence, and existence cares for we. Once you're clean and well-defined, you are able to see tremendous love dropping on you from all dimensions.
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Bullhorn Back Office
iOS Universal Business
Make timesheets and expenses EASY with Bullhorn Back Office! Bullhorn Back Office was built by former Staffing executives to handle the inefficiencies associated with the collection of timesheets for Staffing and Recruiting Firms. Eliminate the paper timesheet process with Bullhorn Back Office.
Mobile applications include:
- Time and expense entry
- Notifications to agree to prior to timesheet submittal
- Multiple assignments per user
- Calendar to navigate to prior and future dates
- Multiple timesheet options
- Timesheet Note
- Manager approval
- Expense Attachments by photo upload
Web application includes:
- Invoice creation with timesheets attached
- Accounting integration
- Payroll integration
- Applicant tracking system integration
- Reporting
- Commission module
- Group timesheets and VMS import
- Email approvals
What's New
- Bug fix
- Enhanced expense tracking to support Reimbursable and Non-reimbursable expenses


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Stayed there in Mar 12 for one night. The room is spotless. There is a beautiful garden with a windmill. Breakfast is impeccable. I left my cloth there and they sent back to me within a week. Really appreciated. Will definitely stay there again.
Top-rated Attractions Nearby
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Centralizes developer build, deployment packaging, and server process management in a single tool.
Centralizes developer build, deployment packaging, and server process management in a single tool.
Manages builds
Local server
Building an application works by loading the capitol.json file from the project, as well as from any packages in node_modules, and then loading all tasks, and kicking off the tasks specified in the configuration to run in each of ss's build phases.
It can include configruations for any number of builds, but the common setup is a dev vs. prod build.
secret-service build {{dev/prod}}
To install a new dependency, use npm --save/--save-dev like usual, but after installing, use the following command to package the file:
secret-service modules package
This will create a .ZIP file in the modules/ directory of your package.
To explode a module, use the following command to check your compare your current node_modules files to the module dependencies:
secret-service modules explode
And to just run the check sequence:
secret-service modules check
Most of the specialized commands above really just kick off gulp tasks in a more pre-defined way, but you can start individual gulp tasks using:
secret-service app {{commands...}}
To see a list of available tasks and any associated help with the task, just do:
secret-service tasks
This is the primary configuration file of the Capitol framework, and for secret-service.
In a tasks subdirectory of your project, add a task file that matches the pattern "ss-*.js". In it, export a function with the signature:
module.exports = function(gulp, config) { gulp.task("my-task", function() { ... }); };
You will get a single gulp instance across all task files and modules. The configuration object will be a merged version of the capitol.json files found in the main project and dependencies.


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Williams-Sonoma Chocolate Croissants
Enjoying rich, flaky pastries hot from the oven couldn't be easier, thanks to this box of wonders. The croissant dough arrives pre-shaped and frozen. Simply thaw the pieces overnight, then pop them in the oven first thing in the morning for the ultimate breakfast treat.
To buy: $40 for 15 croissants, williams-sonoma.com.