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We examined the activation of the p38 mitogen-activated protein kinase (p38-MAPK) pathway by the G protein–coupled receptor agonists, endothelin-1 and phenylephrine in primary cultures of cardiac myocytes from neonatal rat hearts. Both agonists increased the phosphorylation (activation) of p38-MAPK by ∼12-fold. A p38-MAPK substrate, MAPK-activated protein kinase 2 (MAPKAPK2), was activated approximately fourfold and 10 μM SB203580, a p38-MAPK inhibitor, abolished this activation. Phosphorylation of the MAPKAPK2 substrate, heat shock protein 25/27, was also increased. Using selective inhibitors, activation of the p38-MAPK pathway by endothelin-1 was shown to involve protein kinase C but not Gi/Go nor the extracellularly responsive kinase (ERK) pathway. SB203580 failed to inhibit the morphological changes associated with cardiac myocyte hypertrophy induced by endothelin-1 or phenylephrine between 4 and 24 h. However, it decreased the myofibrillar organization and cell profile at 48 h. In contrast, inhibition of the ERK cascade with PD98059 prevented the increase in myofibrillar organization but not cell profile. These data are not consistent with a role for the p38-MAPK pathway in the immediate induction of the morphological changes of hypertrophy but suggest that it may be necessary over a longer period to maintain the response.
The p38-MAPKs, extracellularly responsive kinases (ERKs)1 and stress-activated protein kinases/c-Jun NH2-terminal kinases (SAPKs/JNKs) constitute the three best-characterized subgroups of the mitogen-activated protein kinase (MAPK) superfamily (reviewed by Kyriakis and Avruch, 1996a, 1996b; Lee and Young, 1996; Cohen, 1997). MAPKs are activated by the dual phosphorylation of a Tyr and a Thr residue within a Thr-Xaa-Tyr motif and the identity of Xaa assists in the classification of subgroup membership. In the p38-MAPKs, Xaa is a Gly residue. Several isoforms of p38-MAPKs have been characterized. The original p38-MAPK (now sometimes called p38-MAPKα; Han et al., 1993, 1994; Freshney et al., 1994; Lee et al., 1994; Rouse et al., 1994; Zervos et al., 1995) exists as two alternatively spliced isoforms (Lee et al., 1994). Subsequently, the β1/β2 (Jiang et al., 1996; Kumar et al., 1997), γ (Li et al., 1996; Mertens et al., 1996), and δ (Goedert et al., 1997; Jiang et al., 1997) isoforms were identified. In many cells, p38-MAPKs are activated by a variety of cellular stresses including inflammatory cytokines, endotoxins, UV irradiation and hyperosmotic shock (reviewed by Lee and Young, 1996; Cohen, 1997). One substrate of p38-MAPKα and p38-MAPKβ is MAPK-activated protein kinase 2 (MAPKAPK2; Goedert et al., 1997; Kumar et al., 1997), an enzyme that phosphorylates the small heat shock protein 25/27 (HSP25/27; Rouse et al., 1994). In unstressed cells, HSP25/27 exists predominantly in an unphosphorylated state as high molecular mass aggregates (Lavoie et al., 1995). Phosphorylation leads to disaggregation (Lavoie et al., 1995) and is associated with protection of the actin cytoskeleton (Guay et al., 1997; Huot et al., 1997). Several other substrates of the p38-MAPK(s) have been identified (reviewed by Cohen, 1997).
In the intact heart, p38-MAPK is activated by a variety of stresses including ischemia and ischemia/reperfusion (Bogoyevitch et al., 1996; Yin et al., 1997), oxidative stress (Clerk et al., 1998), and hypertensive perfusion pressure (Clerk et al., 1998). The biological functions of the p38-MAPKs in the heart are not understood, although a recent report using an inhibitor (SB203580; Lee et al., 1994; Cuenda et al., 1995) that is selective for the α and β1 isoforms of p38-MAPK (Goedert et al., 1997; Kumar et al., 1997) suggests that their activation may protect the heart against ischemia (ischemic preconditioning; Weinbrenner et al., 1997). p38-MAPK(s) has also been implicated in hypertrophy of ventricular myocytes (Zechner et al., 1997). This is an important adaptational response to a demand for increased contractile power and is seen in a number of clinical situations in man. Transient transfection of primary cultures of neonatal rat ventricular myocytes with an expression plasmid encoding constitutively activated mutants of MKK6 or MKK3 (the MAPK kinases (MKKs) responsible for the activation of p38-MAPK) induces the transcriptional and morphological changes associated with the development of the hypertrophic phenotype (Zechner et al., 1997; Wang et al., 1998). SB203580 inhibited these changes (Zechner et al., 1997). In addition, SB203580 inhibited the hypertrophic response induced by phenylephrine (PE; Zechner et al., 1997), a powerfully hypertrophic α1-adrenergic agonist (Simpson, 1985; Lee et al., 1988; Iwaki et al., 1990; Knowlton et al., 1991).
G protein–coupled receptors (GPCRs) of the Gq/11 class participate in the cellular responses to α1-adrenergic agonists such as PE and endothelin-1 (ET-1; reviewed by Sugden and Clerk, 1997). Like the α1-adrenergic agonists, ET-1 is powerfully hypertrophic (Shubeita et al., 1990; Ito et al., 1991) and both α1-adrenergic agonism and ET-1 have been implicated in ischemic preconditioning (Banerjee et al., 1993; Wang et al., 1996). ET-1 and PE were first shown to activate the ERKs in cardiac myocytes (Bogoyevitch et al., 1993, 1994; Clerk et al., 1994) and this was the first MAPK cascade to be implicated in cardiac hypertrophy (Thorburn et al., 1994a; Gillespie-Brown et al., 1995). Subsequently, SAPKs/JNKs were shown to be activated in cardiac myocytes by ET-1 and PE (Bogoyevitch et al., 1995a; Ramirez et al., 1997), and these MAPKs have also been implicated in cardiac hypertrophy (Bogoyevitch et al., 1996; Ramirez et al., 1997). Although p38-MAPK has been implicated in PE-induced hypertrophy, there are only very limited data pertaining to the activation of p38-MAPKs in cardiac myocytes by PE (Zechner et al., 1997) and no data for ET-1 or other powerfully hypertrophic agonists such as PMA.
Here, we have characterized extensively the activation of the p38-MAPK pathway by the GPCR agonists, ET-1 and PE, and compared these responses with those to hyperosmotic stress and PMA. We examined the phosphorylation of p38-MAPK, activation of its substrate MAPKAPK2, and phosphorylation of HSP25/27 in primary cultures of ventricular myocytes isolated from neonatal rat hearts. Using SB203580, we also assessed the role of the p38-MAPK pathway in the development of the morphological changes associated with the hypertrophic response.
Materials And Methods
Laminin, microcystin LR, myelin basic protein (MBP), pertussis toxin (PTX), sorbitol, ET-1, PE, PMA, protein A–Sepharose and protease inhibitors were from Sigma Chemical Co. (Poole, Dorset, UK). PD98059 and GF109203X were from Calbiochem (Beeston, Nottinghamshire, UK) and were prepared as 25 mM and 10 mM stock solutions in DMSO. SB203580 was a gift from Dr. John C. Lee (SmithKline Beecham, King of Prussia, PA) and a 10 mM stock solution was prepared in DMSO. Lab-Tek tissue culture chamber slides were from Life Technologies (Paisley, Scotland, UK). Other tissue culture materials were from previously described sources (Bogoyevitch et al., 1995b). Prestained protein molecular mass markers, ECL Western blotting reagents, autoradiography film and streptavidi–Texas Red were from Amersham International (Amersham, Buckinghamshire, UK). γ-[32P]ATP was from NEN Life Sciences (Brussels, Belgium). SDS-PAGE reagents, IEF reagents, and Bradford (Bradford, 1976) protein assay reagent were from Bio-Rad (Hemel Hempstead, Hertfordshire, UK). Nitrocellulose was from Schleicher & Schuell and supplied by Anderman & Co. (Kingston-upon-Thames, Surrey, UK). cAMP-dependent protein kinase inhibitory peptide (sequence TTYADFIASGRTGRRNAIHD) was from Bachem (Saffron Walden, Essex, UK). The MAPKAPK2 substrate peptide (KKLNRTLSVA) was synthesized by Severn Biotech (Kidderminster, Worcestershire, UK). The antibody to HSP25/27 (sc-1049) was from Santa Cruz Biotechnology Inc. and supplied by Autogen Bioclear (Devizes, Wiltshire, UK). The antibody to the dually phosphorylated form of p38-MAPK was from New England Biolabs (UK) Ltd. (Hitchin, Herts., UK). A mouse monoclonal antibody to β-myosin heavy chain (β-MHC) was from Novocastra (Newcastle-upon-Tyne, Tyne and Wear, UK). Secondary antibodies (horseradish peroxidase– linked, FITC and biotinylated anti–mouse and anti–rabbit IgG antibodies) and fluorescent mounting medium were from Dako (High Wycombe, Buckinghamshire, UK).
Primary Culture of Neonatal Ventricular Myocytes and Sample Preparation
Myocytes were dissociated from the ventricles of neonatal Sprague-Dawley rat hearts by a previously described adaptation (Bogoyevitch et al., 1995b) of the method of Iwaki et al., (Iwaki et al., 1990). To deplete the myocytes of fibroblasts, the cells were preplated in DMEM/M199 (4:1 vol/ vol) supplemented with 10% (vol/vol) horse serum, 5% (vol/vol) fetal calf serum and 100 U/ml each of penicillin and streptomycin. The (nonadherent) myocytes were then plated on 60- or 35-mm culture dishes precoated with 1% (wt/vol) gelatin at a density of 1.4 × 103 cells/mm2. Myocytes were confluent and beating within 18 h. The cells were incubated for 24 h in serum-free medium in the absence or presence (in some experiments) of 1 μM PMA or 30 ng/ml PTX, and then exposed to agonists (0.5 M sorbitol, ET-1, PE or 1 μM PMA) in serum-free medium. For inhibitor studies, the cells were preincubated with inhibitor for 15 min before stimulation with agonist + inhibitor. For phospho–p38-MAPK immunoblot analysis and fast protein liquid chromatography (FPLC), myocytes (60-mm dishes) were washed in ice-cold Ca2+/Mg2+-free Dulbecco's phosphate-buffered saline (PBS) and scraped into 150 μl/dish extraction buffer A (20 mM β-glycerophosphate, pH 7.5, 50 mM NaF, 2 μM microcystin LR, 2 mM EDTA, 0.2 mM Na3VO4, 10 mM benzamidine, 200 μM leupeptin, 10 μM trans-epoxy succinyl-l-leucylamido-(4-guanidino)butane, 5 mM DTT, 300 μM PMSF, 1% (vol/vol) Triton X-100). The samples were centrifuged (10,000 g, 5 min, 4°C). For Western blotting, supernatants were boiled with 0.33 vol of sample buffer (0.33 M Tris-HCl pH 6.8, 10% (wt/vol) SDS, 13% (vol/vol) glycerol, 133 mM DTT, 0.2 mg/ml bromophenol blue). For FPLC, supernatants were re-centrifuged (10,000 g, 5 min, 4°C) before loading onto the column. Protein concentrations were determined using the Bradford method (Bradford, 1976).
Phospho-p38-MAPK Immunoblot Analysis
Myocyte extracts (25 μg protein) were immunoblotted for phospho-p38-MAPK according to the manufacturer's instructions. The bands were detected using enhanced chemiluminescence (ECL) with exposure to Hyperfilm MP. Blots were quantified by laser scanning densitometry.
Fast Protein Liquid Chromatography (FPLC) of ERKs and MAPKAPK2
ERKs (four 60-mm dishes/column) were separated on a Mono Q HR5/5 column equilibrated with 50 mM Tris-HCl, pH 7.3, 2 mM EDTA, 2 mM EGTA, 0.1% (vol/vol) 2-mercaptoethanol, 5% (vol/vol) glycerol, 0.03% (vol/vol) Brij-35, 0.3 mM Na3VO4, 1 mM benzamidine, and 4 μg/ml leupeptin. After a 5-ml isocratic wash, ERKs were eluted using a linear NaCl gradient (20 ml, 0–0.33 M NaCl) at a flow rate of 1 ml/min with collection of 0.5-ml fractions. They were assayed by the incorporation of 32P from γ-[32P]ATP into MBP by the direct method as previously described (Bogoyevitch et al., 1993, 1994), except that in some experiments the assay mix included 0.1% (vol/vol) DMSO or 10 μM SB203580 (final concentrations).
MAPKAPK2 (two 60-mm dishes/column) was partially purified by FPLC on a Mono S HR5/5 column equilibrated with 25 mM β-glycerophosphate pH 7.3, 2 mM EDTA, 5% (vol/vol) glycerol, 0.1% (vol/vol) Triton X-100 and 0.5 mM DTT. After a 5-ml isocratic wash, MAPKAPK2 was eluted using a linear NaCl gradient (15 ml, 0–0.3 M NaCl) at a flow rate of 1 ml/min with collection of 1 ml fractions. MAPKAPK2 was assayed by the incorporation of 32P from γ-[32P]ATP into KKLNRTLSVA peptide substrate (Rouse et al., 1994) as previously described (Bogoyevitch et al., 1996), except that the incubation time was decreased to 20 min. Total MAPKAPK2 activities were determined by integration of the areas under the FPLC peaks.
Isoelectric Focusing (IEF) and Immunoblotting of HSP25/27
Myocytes (in 35-mm dishes) were scraped into 100 μl/dish of extraction buffer B (12.5 mM Tris-HCl, pH 7.5, 2.5 mM EGTA, 1 mM EDTA, 100 mM NaF, 5 mM DTT, 0.3 mM PMSF, 0.12 mM pepstatin A, 0.2 mM leupeptin, 10 μM trans-epoxy succinyl-l-leucylamido-(4-guanidino)butane), and centrifuged (10,000 g, 5 min, 4°C). Supernatants were mixed with an equal volume of 8 M urea, 4% (wt/vol) Biolyte 3-10, 1% (vol/vol) Triton X-100. Samples (2 μl, 2–3 μg protein) were separated by IEF on 5% polyacrylamide gels containing 4 M urea, 0.2% (wt/vol) CHAPS, 5% (vol/vol) glycerol, 2% (wt/ vol) Biolyte 3-10 at 100 V (15 min), and finally 450 V (1 h). Proteins were transferred to nitrocellulose and probed with HSP25/27 antibodies (1/100 dilution). Blots were processed as previously described (Clerk et al., 1994).
Immunocytochemical Staining
Cells were plated in 8-well Lab-Tek tissue culture chamber slides precoated with laminin (20 μg/ml in PBS) and 1% (wt/vol) gelatin at a density of 5 × 104 cells/well (∼600 cells/mm2) and cultured for 18 h. Serum was withdrawn for 24 h before the experiments. Cells were pretreated (15 min) with 0.1% (vol/vol) DMSO or inhibitors and then left unstimulated or exposed to agonists in serum-free medium containing 0.1% (vol/vol) DMSO or inhibitors. After 4, 8, 24, or 48 h, the medium was removed and the cells washed with ice-cold PBS (3 × 0.5 ml). The cells were fixed for 10 min with 4% (wt/vol) formaldehyde, permeabilized with 0.3% (vol/vol) Triton X-100 in PBS and nonspecific antibody binding blocked with 1% (wt/vol) BSA in PBS containing 0.3% (vol/vol) Triton X-100. The cells were immunostained with an antibody to β-MHC (1/40 dilution, 1 h, 37°C), using a biotinylated anti–mouse IgG secondary antibody (1/200 dilution, 30 min, 37°C) and streptavidin–Texas Red (1/200 dilution, 15 min, 37°C). All antibody dilutions were in PBS and wells were washed with PBS (3 × 0.5 ml) between each incubation. Slides were mounted and examined using a Zeiss Axioskop microscope with a 100× oil immersion objective. Cells were photographed using Tmax 400 film with a set exposure time (30 s).
Phosphorylation of p38-MAPK
To assess the activation of p38-MAPK in cardiac myocytes, we used an antibody selective for the dually phosphorylated (activated) form of the p38-MAPKs for Western blot analysis. Hyperosmotic shock (0.5 M sorbitol) stimulated extensive phosphorylation of p38-MAPK (∼25-fold relative to controls; Fig. 1,A, top). This response was rapid and maximal within 5 min (Fig. 1,B). ET-1 (100 nM) and PE (100 μM) also stimulated a rapid but less extensive phosphorylation of p38-MAPK (∼12-fold) which was sustained up to at least 20 min (Fig. 1,A, center and B). The phosphorylation of p38-MAPK in response to PMA (1 μM) was rapid but was considerably less than with the other agonists (Fig. 1,A, lower and B). We directly compared the relative phosphorylation of p38-MAPK induced by the four agonists after 5 min (Fig. 2, A and B). Taking the response to 0.5 M sorbitol as 100%, the relative levels of phosphorylation induced by ET-1 (100 nM), PE (100 μM), and PMA (1 μM) were 52, 40, and 13%, respectively.
The EC50 for stimulation of many biological processes in cardiac myocytes by ET-1 is in the nM range (reviewed by Rubanyi and Polokoff, 1994; Sugden and Bogoyevitch, 1996). Consistent with this, the EC50 for ET-1 stimulation of p38-MAPK phosphorylation was 0.46 ± 0.01 nM (mean ± SEM of three independent experiments; Fig. 3, A and B). In two independent experiments, the ETA receptor antagonist, BQ123 (10 μM, 15 min pretreatment), inhibited the phosphorylation of p38-MAPK induced by ET-1 (100 nM, 5 min) as assessed by immunoblotting (results not shown). Quantitative analysis by laser scanning densitometry indicated that this inhibition was 71 ± 17% (mean ± SD).
Activation of MAPKAPK2
MAPKAPK2 is a substrate for p38-MAPK (Rouse et al., 1994). In addition to being an important effector in its own right, the stimulation of MAPKAPK2 is a useful indication of p38-MAPK activation. We measured MAPKAPK2 activity in cardiac myocytes after partial purification by Mono S FPLC. Sorbitol (0.5 M) stimulated a very rapid increase in MAPKAPK2 activity that was essentially complete within 5 min and was sustained for up to 30 min (Fig. 4,A). ET-1 (100 nM), PE (100 μM), and PMA (1 μM) stimulated modest but sustained increases in MAPKAPK2 activity (Fig. 4,A). The potency of these agonists was compared after 30 min incubation (Fig. 4,B). Sorbitol stimulated an 8.6-fold increase over control values, whereas the stimulation by ET-1, PE and PMA were 5.0-, 3.9-, and 2.8-fold, respectively. Consistent with activation of MAPKAPK2 by p38-MAPK, SB203580, a selective inhibitor of p38-MAPK/p38-MAPKβ (Goedert et al., 1997; Kumar et al., 1997), essentially completely inhibited the activation of MAPKAPK2 by all four agonists (Fig. 4 B).
MAPKAPK2 was initially identified as a substrate for the ERKs (Stokoe et al., 1992), which are strongly activated in cardiac myocytes by PMA, ET-1 and PE (Bogoyevitch et al., 1993, 1994; Clerk et al., 1994). To confirm that ERKs are not inhibited by SB203580 and the inhibition of MAPKAPK2 activity by this drug reflects its inhibitory effects on p38-MAPK, we purified ERK1 and ERK2 by Mono Q FPLC from myocytes exposed to PMA (1 μM, 5 min) and assayed activity in the presence of 0.1% (vol/vol) DMSO or 10 μM SB203580. There was no inhibition of either ERK1 or ERK2 by SB203580 (Fig. 4 C), indicating that p38-MAPK activity is essential for MAPKAPK2 activation in neonatal ventricular myocytes.
Mechanisms of Activation of the p38-MAPK Pathway
Inhibition by PTX.
ET-1, in addition to stimulating Gq/11-dependent pathways, also couples to Gi in cardiac myocytes (Hilal-Dandan et al., 1994). We investigated the potential involvement of Gi in the activation of the p38-MAPK pathway. Pretreatment of myocytes with 30 ng/ml PTX for 24 h had no effect on the basal levels of phosphorylated p38-MAPK, or on the phosphorylation of p38-MAPK induced by 100 nM ET-1 (Fig. 5,A) or 0.5 M sorbitol (Fig. 5,B). Similarly, neither the basal levels of MAPKAPK2 activity nor the activation of MAPKAPK2 by ET-1 (Fig. 5,C) or sorbitol (Fig. 5 D) was affected by PTX pretreatment. In separate experiments, this regime of PTX pretreatment inhibited Raf activation by ET-1 (Chiloeches, A., and P.H. Sugden, unpublished observations) indicating the efficacy of this protocol. These data demonstrate that ET-1 stimulation of the p38-MAPK pathway is not mediated through PTX-sensitive G proteins (i.e., Gi or Go).
Involvement of PKC.
Preexposure of myocytes to 1 μM PMA for 24 h essentially completely downregulates the classical and novel PKC isoforms (Clerk et al., 1995). This protocol increased the basal levels of phosphorylated p38-MAPK (Fig. 5,A) and MAPKAPK2 activity (Fig. 5,C) by two- to threefold. Phosphorylation of p38-MAPK induced by 100 nM ET-1 was inhibited by PMA pretreatment (Fig. 5,A). To evaluate the degree of inhibition, the basal levels of phosphorylated p38-MAPK or MAPKAPK2 activity were subtracted from values obtained in the presence of agonist. The inhibition of ET-1 stimulated p38-MAPK phosphorylation by PMA pretreatment was 79 ± 8% (mean ± SEM for three independent experiments). In contrast, the phosphorylation of p38-MAPK by 0.5 M sorbitol was not inhibited by PMA pretreatment (Fig. 5,B). Activation of MAPKAPK2 by ET-1 was inhibited (47 ± 7%, mean ± SEM for four independent experiments) by PMA pretreatment (Fig. 5,C), whereas the activation of MAPKAPK2 by sorbitol was unaffected (Fig. 5 D).
We also assessed the effects of a PKC-selective inhibitor, GF109203X (Toullec et al., 1991). This is a more selective PKC inhibitor than staurosporine and, unlike Ro318220 (Beltman et al., 1996), does not strongly activate the SAPKs/JNKs in cardiac myocytes (Clerk, A., and P.H. Sugden, unpublished observations). GF109203X (10 μM) increased the basal levels of phosphorylated p38-MAPK (Fig. 5,A), although there was no effect on basal MAPKAPK2 activity (Fig. 5,B). GF109203X (10 μM) had no effect on the phosphorylation of p38-MAPK (Fig. 5,B) or the activation of MAPKAPK2 (Fig. 5,D) by sorbitol, but inhibited the ET-1–stimulated phosphorylation of p38-MAPK (Fig. 5,A) and activation of MAPKAPK2 (Fig. 5,C) by 63 ± 8% and 64 ± 7%, respectively (means ± SEM for three or four independent experiments). A lower concentration of GF109203X (1 μM) had no effect on ET-1 stimulation of either p38-MAPK phosphorylation or MAPKAPK2 activation (results not shown). Published IC50 values for inhibition of partially purified PKC by GF109203X are ∼10 nM (Toullec et al., 1991), considerably less than we observed here. Therefore, using Mono Q FPLC to separate the MAPKs, we assessed the effects of GF109203X on the activation of ERKs in cardiac myocytes by ET-1, a response that is accepted to be predominantly PKC-dependent (Bogoyevitch et al., 1994). ET-1 stimulated two peaks of activity eluting at ∼0.20 M and 0.25 M NaCl (ERK2 and ERK1, respectively; Fig. 6,A). At 1 μM, GF109203X had no effect on the activation of ERK2, but inhibited the activation of ERK1 by ∼30% (Fig. 6,B). At 10 μM, activation of both ERK1 and ERK2 were significantly inhibited (Fig. 6 B). This indicates that the IC50 for the inhibition of PKC-dependent processes by GF109203X in cardiac myocytes is considerably greater than for purified PKC.
Involvement of the ERK Cascade.
PD98059 inhibits the activation of MEK1 and, to a lesser extent, MEK2 (Alessi et al., 1995; Dudley et al., 1995), both of which are involved in ERK activation. We examined the effect of this drug on the activation of the p38-MAPK pathway to determine whether the same MKKs are involved in the activation of ERKs and p38-MAPK in cardiac myocytes. PD98059 (50 μM) increased basal levels of phosphorylated p38-MAPK (Fig. 5,A) and MAPKAPK2 activity (Fig. 5,C). The phosphorylation of p38-MAPK and activation of MAPKAPK2 induced by 0.5 M sorbitol were unaffected by 50 μM PD98059 (Fig. 5, B and D). In contrast, 50 μM PD98059 inhibited the ET-1 (100 nM) stimulated phosphorylation of p38-MAPK (Fig. 5,A) and activation of MAPKAPK2 (Fig. 5,C) by 50 ± 3% and 67 ± 3% (means ± SEM for four independent experiments), respectively. In two separate experiments, a lower concentration of PD98059 (10 μM) gave no significant inhibition of MAPKAPK2 activation by ET-1 (results not shown). However, the activation of ERK1 and ERK2 by ET-1 was significantly inhibited (>80%) by 10 μM or 50 μM PD98059 (Fig. 6 C). These data indicate that although both ERKs and p38-MAPK may be similarly PKC dependent, the pathways diverge at a level before the MKKs. The results also suggest that whereas ERK activation is probably mediated through MEK1 in these cells (the IC50 for MEK1 inhibition by PD98059 is 10 μM; Alessi et al., 1995), activation of p38-MAPK may be mediated through MEK2 (the IC50 for MEK2 inhibition is 50 μM; Alessi et al., 1995) or another MKK which is sensitive to inhibition by PD98059.
Phosphorylation of HSP25/27.
In certain cell types, phosphorylation of HSP25/27 is associated with cytoprotection and stabilization of the actin cytoskeleton (Guay et al., 1997; Huot et al., 1997). HSP25/27 is a known substrate for MAPKAPK2 (Rouse et al., 1994). Therefore, we assessed the phosphorylation state of HSP25/27 in cells exposed to 100 nM ET-1 using IEF followed by immunoblotting. An increase in the phosphorylation of HSP25/27 increases the negative charge of the protein that allows it to migrate more quickly to the anode during the IEF stage. The individual phosphorylated forms of HSP25/27 are then specifically identified by immunoblotting. Up to three forms of HSP25/27 were detected using this method. In unstimulated cells, HSP25/27 was detected predominantly as bands 1 and 2, the most positively charged (least phosphorylated) forms migrating closest to the cathode (Fig. 7, top). In myocytes subjected to cellular stress (0.5 M sorbitol, 30 min), HSP25/27 was detected principally as bands 2 and 3, the more negatively charged forms. ET-1 stimulated an increase in the levels of band 3 from ∼5–8 min with a corresponding decrease in the levels of band 1, indicating an increase in the phosphorylation state of HSP25/27. This change was maximal at ∼30 min, and was sustained at least up to 1 h. The increase in phosphorylation of HSP25/27 induced by ET-1 was comparable to that induced by 0.5 M sorbitol. SB203580 completely inhibited this response (results not shown), consistent with the stimulation of HSP25/27 phosphorylation by ET-1 being mediated by p38-MAPK/ MAPKAPK2.
Effects of p38-MAPK Inhibition on Cardiac Myocyte Hypertrophy/Cell Survival.
We investigated the effects of p38-MAPK inhibition on the morphological changes induced in cardiac myocytes by ET-1 and PE over a period of 4–48 h. We compared the effects of SB203580 with those of PD98059, which inhibits the ERK cascade but also partially inhibits the p38-MAPK pathway (Fig. 6,C). Myocytes were cultured in chamber slides and either not stimulated or exposed to agonists in the absence or presence of inhibitors. The myofilaments were immunostained with an antibody to β-MHC. Control cells, cultured in serum-free medium for up to 24 h, were irregular and the myofilaments were disorganized (Figs. 8,A, 9,A, and 10,A). Myocytes exposed to 50 μM PD98059 (e.g., Figs. 9,D and 10,D) or SB203580 (e.g., Figs. 9,G and 10 G) appeared similar to the control cells at all time points studied.
From as early as 4 h, 40 μM PE (Fig. 8,B) or 40 nM ET-1 (Fig. 8,C) stimulated an increase in cell profile and myofibrillar organization relative to the controls (Fig. 8,A). By 8 h, cells exposed to either PE (Fig. 9,B) or ET-1 (Fig. 9,C) exhibited the typical morphology associated with cardiac hypertrophy. The cells were larger, with intense β-MHC staining and clear cross-striations. In myocytes exposed to either agonist in the presence of 50 μM PD98059, the myofibrillar organization was disrupted although there was no apparent decrease in the intensity of β-MHC immunostaining (Fig. 9, E and F). In contrast, 10 μM SB203580 had no effect on the morphology of myocytes stimulated with PE (Fig. 9,H) or ET-1 (Fig. 9 I).
After 24 h, control myocytes maintained in serum-free medium appeared smaller than those at 4 or 8 h, with reduced β-MHC immunostaining (Fig. 10,A), whereas cells exposed to PE (Fig. 10,B) or ET-1 (Fig. 10,C) exhibited a similar morphology to those at 8 h (Fig. 9, B and C, respectively). In myocytes exposed to either agonist in the presence of 50 μM PD98059 for 24 h, the myofibrillar organization was clearly disrupted although there was no apparent decrease in the intensity of β-MHC immunostaining or in cell profile (Fig. 9, E and F). SB203580 (10 μM) had no effect on any aspect of the morphological changes induced in myocytes by PE (Fig. 10,H) or ET-1 (Fig. 10 I) after 24 h.
In addition to inhibiting ERK activation (Fig. 6), 50 μM PD98059 also inhibits the activation of p38-MAPK and MAPKAPK2 by ET-1 (Fig. 5). However, 10 μM PD98059 (which does not significantly inhibit MAPKAPK2 activation, but does inhibit ERK activation [Fig. 6]) also disrupted the myofibrillar organization induced by ET-1 or PE for 8 and 24 h (results not shown). Furthermore, 10 μM SB203580 together with 10 μM (or 50 μM) PD98059 had no additional effect on the morphology of myocytes exposed to ET-1 or PE for 8 or 24 h (results not shown). These results indicate that the effect of PD98059 to disrupt myocyte myofibrillar organization is not attributable its inhibition of the p38-MAPK pathway.
After 48 h in serum-free medium, there were considerably fewer myocytes in the control wells and there was a substantial amount of cell debris (e.g., Fig. 11,A, arrowhead). The remaining myocytes were small, with very little β-MHC staining (Fig. 11,A). The number of myocytes in wells treated with PE or ET-1 appeared similar to the other time points studied (results not shown), suggesting that agonist stimulation promotes survival of cardiac myocytes. Myocytes exposed to PE for 48 h exhibited two patterns of morphology. Approximately 50% of the cells were large and clearly striated with very bright immunostaining for β-MHC (Fig. 11,B). The remaining cells were weakly stained for β-MHC, but were still striated (Fig. 11,C). Myocytes exposed to PE in the presence of PD98059 appeared weakly stained and the myofilaments were not organized (Fig. 11,D). In marked contrast to the results obtained up to 24 h, myocytes exposed to PE in the presence of SB203580 for 48 h were very small with minimal β-MHC immunostaining and no myofibrillar organization (Fig. 11 E). Similar results were obtained with ET-1 (results not shown). These data are not consistent with a role for the p38-MAPK pathway in stimulating any of the morphological changes induced by PE or ET-1. Rather, this pathway may be important over a longer period in maintaining the hypertrophic response. However, inhibition of the ERK cascade by PD98059 clearly disrupts the myofibrillar organization induced by PE or ET-1.
Activation of the p38-MAPK Pathway by GPCR Agonists
The p38-MAPK cascade is activated in many cell types in response to cellular stresses including hyperosmotic shock, UV irradiation and exposure to proinflammatory cytokines (reviewed by Kyriakis and Avruch, 1996a, 1996b; Lee and Young, 1996; Cohen, 1997). There are very few reports of activation of the p38-MAPK pathway by GPCR agonists. p38-MAPK is activated in platelets by thrombin (Kramer et al., 1995) and mediates thrombin stimulation of phospholipase A2 activity (Kramer et al., 1996). The chemotactic peptide N-formyl-Met-Leu-Phe also activates p38-MAPK and MAPKAPK2 in neutrophils (Krump et al., 1997). Cotransfection of m1 and m2 muscarinic acetylcholine receptors with an epitope-tagged p38-MAPK has shown that these receptors can activate the pathway (Yamauchi et al., 1997), but since the activation was only approximately twofold, it is difficult to assess its significance. A twofold activation of epitope-tagged p38-MAPK is also seen after β-adrenergic stimulation (Yamauchi et al., 1997). In similar experiments, transfection of a modified ETB receptor activates ERKs, SAPKs/JNKs and p38-MAPKs (Aquilla et al., 1996). In a limited study, PE has been shown to increase the phosphorylation of p38-MAPK twofold in cardiac myocytes after 30 min (Zechner et al., 1997), but this was not studied in detail and the activation of downstream events was not studied. We have previously demonstrated that p38-MAPK is activated in the perfused rat heart by pathophysiological stresses (ischaemia and ischaemia/reperfusion [Bogoyevitch et al., 1996], reactive oxygen species [Clerk et al., 1998] and hypertensive perfusion [Clerk et al., 1998]).
In this study, we have clearly demonstrated that, in addition to cellular stress (hyperosmotic shock), the GPCR agonists ET-1 and PE activate the p38-MAPK pathway in primary cultures of neonatal rat ventricular myocytes. Both agonists induced an ∼12-fold increase in the phosphorylation (activation) of p38-MAPK (Fig. 2) and a four- to fivefold increase in the activity of the p38-MAPK substrate, MAPKAPK2 (Fig. 4). This represents a significant activation of the pathway and is ∼50% of the response induced by a severe cellular stress, 0.5 M sorbitol (Figs. 2 and 4). The phosphorylation motif is similar in all the p38-MAPKs and the antibody to the dually phosphorylated form recognizes all isoforms. Thus, from these experiments, it is not possible to determine whether any specific p38-MAPK isoforms were activated by the different agonists. However, activation of MAPKAPK2 was completely inhibited by SB203580, which strongly inhibits p38-MAPK(α) and p38-MAPKβ1 and is a moderate inhibitor of p38-MAPKβ2 (Kumar et al., 1997), indicating that at least one of these isoforms is activated in cardiac myocytes. Furthermore, although p38-MAPK(α) is easily detected on immunoblots of myocyte extracts, we have yet to detect any of the other isoforms (Clerk, A., and P.H. Sugden, unpublished data).
From the results presented here, it is now apparent that ET-1 and PE can activate all three of the best-characterized MAPK cascades in the heart. The ERK cascade was first shown to be strongly activated by ET-1 (Bogoyevitch et al., 1993, 1994) and, to a lesser extent by PE (Clerk et al., 1994). ET-1 was then demonstrated to activate the SAPKs/ JNKs (approximately fivefold) whereas PE induced only an approximately twofold activation of this pathway (Bogoyevitch et al., 1995a). This twofold activation of SAPKs/JNKs by PE was subsequently confirmed by others (Ramirez et al., 1997). In contrast to the ERKs and SAPKs/ JNKs, the activation of the p38-MAPK pathway by PE is comparable to that by ET-1 (Figs. 2 and 4).
Mechanism of Activation of the p38-MAPK Pathway by ET-1
The ETA receptor mediates most of the effects of ET-1 in cardiac myocytes (reviewed by Sugden and Bogoyevitch, 1996). ET-1–stimulated phosphorylation of p38-MAPK was inhibited by BQ123, a selective ETA receptor antagonist (see Results), indicating that activation of both pathways is mediated through the same receptor. Consistent with this, the EC50 for p38-MAPK phosphorylation (0.49 nM, Fig. 3) by ET-1 in cardiac myocytes is similar to that for ERK activation (0.2 nM) (Bogoyevitch et al., 1993). Studies of COS cells transfected with ETB receptors have also indicated that a single receptor subtype can stimulate activation of ERKs, SAPKs/JNKs, and p38-MAPKs (Aquilla et al., 1996), although whether this is through a single G protein subtype remains to be determined. In our study, ET-1–stimulated phosphorylation of p38-MAPK and activation of MAPKAPK2 was not affected by pretreatment with PTX (Fig. 5, A and C), indicating that activation of this pathway in cardiac myocytes is not mediated through Gi/Go. We have previously shown that PTX inhibits ET-1–induced activation of Raf (Bogoyevitch et al., 1995c), the MKK kinase for the ERK cascade (reviewed by Sugden and Clerk, 1997). This suggests that activation of the ERK and p38-MAPK pathways by ET-1 require activation of different G proteins. As expected, the activation of the p38-MAPK pathway by 0.5 M sorbitol was unaffected by PTX pretreatment (Fig. 5, B and D).
Phosphorylation of p38-MAPK and activation of MAPKAPK2 by ET-1 appeared to be dependent on PMA-sensitive isoforms of PKC since downregulation of these isoforms or inhibition of PKC activity with GF109203X significantly attenuated this response (Fig. 5, A and C). This PKC-dependency was comparable to that of ERK, which is also inhibited by PKC downregulation (Bogoyevitch et al., 1994) and is inhibited by similar concentrations of GF109203X (Fig. 6,B). Activation of the p38-MAPK pathway by hyperosmotic shock (0.5 M sorbitol) was unaffected by either of these treatments (Fig. 5, B and D), indicating that the data do not reflect a nonspecific inhibitory effect of either PMA pretreatment or of GF109203X on the p38-MAPK pathway. Although the activation of p38-MAPK appears to be PKC-dependent, PMA activation of PKC only stimulated approximately a threefold increase in p38-MAPK phosphorylation and in MAPKAPK2 activity. These data suggest that although activation of the p38-MAPK pathway by ET-1 may be PKC-dependent, other factors are necessary for full activation. Activation of PKC is mediated through Gq-stimulation of phospholipase Cβ (reviewed in Fields and Casey, 1997; Sugden and Clerk, 1997), suggesting that the ERK and p38-MAPK pathways require Gq for full activation by ET-1.
MAPKAPK2 is phosphorylated on multiple Ser/Thr residues and these phosphorylations regulate its activity (Ben-Levy et al., 1995). Although MAPKAPK2 is now generally accepted to be a substrate for p38-MAPK (Rouse et al., 1994; Ben-Levy et al., 1995), it was originally identified as an ERK substrate (Stokoe et al., 1992) and at least one site (Thr25) may be preferentially phosphorylated by ERKs (Ben-Levy et al., 1995). Whereas phosphorylation of Thr25 does not activate MAPKAPK2 on its own, it may modulate the activation by the other phosphorylation sites (Ben-Levy et al., 1995). PD98059 is a selective inhibitor for MEK1 and, to a lesser extent, MEK2 (Alessi et al., 1995; Dudley et al., 1995), although we are unaware of any testing of PD98059 against other MKKs. PD98059 (50 μM) partially inhibited the activation of MAPKAPK2 by ET-1 (Fig. 5,C), but also partially inhibited p38-MAPK phosphorylation (Fig. 5,A). At this concentration of PD98059, activation of ERKs by ET-1 was essentially completely inhibited and a lower concentration (10 μM), which did not significantly inhibit MAPKAPK2 activation (results not shown), almost completely inhibited ERK activation (Fig. 6,C). These data indicate that ERK activation by ET-1 does not contribute to the activation of MAPKAPK2, and provide circumstantial evidence that PD98059 inhibits the activation of the MKK(s) of the p38-MAPK pathway. In addition, we have shown that SB203580 does not inhibit ERK activity (Fig. 4,C), but completely abolishes the activation of MAPKAPK2 by all agonists studied (Fig. 4 B). Although SB203580 does inhibit some isoforms of SAPKs/JNKs in cardiac myocytes (Clerk and Sugden, 1998), there is no evidence currently that these MAPKs phosphorylate and activate MAPKAPK2. Therefore, the data are consistent with phosphorylation of MAPKAPK2 by p38-MAPK being essential for activation.
Role of p38-MAPKs in Hypertrophy or Cell Survival?
p38-MAPKs have been implicated in the development of the hypertrophic phenotype in primary cultures of ventricular myocytes. Transfection of constitutively activated MKK6 (Zechner et al., 1997; Wang et al., 1998) or MKK3 (Wang et al., 1998) induces transcriptional and morphological changes associated with the hypertrophic response, indicating that activation of the p38-MAPK pathway has the potential to promote hypertrophy. However, the situation may be more complex as cotransfection of either MKK3 or MKK6 with p38-MAPK(α) or p38-MAPKβ suggest that p38-MAPK(α) promotes apoptosis, whereas p38-MAPKβ induces hypertrophy (Wang et al., 1998). Such transfection experiments are not unequivocal since (a) there may be cross-talk between the MAPK pathways, and (b) whereas activation of a single (overexpressed) pathway may induce a particular response, the physiological relevance of such a response cannot be assessed. Furthermore, in these studies, the myocytes are cultured in serum-free conditions for up to 70 h before analysis. Beyond 24 h, as discussed below, it becomes questionable whether studies of this nature are studies of hypertrophy or of myocyte survival.
The hypertrophic response induced by a 48 h exposure to PE is inhibited by SB203580 (Zechner et al., 1997). Here, we have shown that the morphological changes associated with cardiac myocyte hypertrophy induced by ET-1 or PE were apparent from as early as 4 h after stimulation (Fig. 8, B and C), and that between 8 h (Fig. 9, B and C) and 24 h (Fig. 10, B and C), the cells exhibited the mature hypertrophic phenotype. We also demonstrated that SB203580 had no effect on myocyte morphology over this time period (Fig. 9, G–I and 10, G–I), strongly indicating that the p38-MAPK pathway is not necessary for the induction of the morphological changes associated with cardiac hypertrophy. After 48 h, myocytes exposed to PE (Fig. 11, B and C) or ET-1 (results not shown) maintained their myofibrillar organization and cell profile. However, myocytes exposed to PE or ET-1 in the presence of SB203580 were small, with minimal β-MHC staining and no myofibrillar organization (Fig. 11,E and results not shown). These data at 48 h are essentially similar to those of Zechner et al. (1997) who only studied the effects at this time point, but in view of the absence of any effect of this inhibitor up to 24 h, suggest that the p38-MAPK pathway may be necessary to maintain the cells during hypertrophy rather than have a direct effect in stimulating the hypertrophic response. Consistent with a role for the p38-MAPK pathway in cytoprotection, activation of MAPKAPK2 by ET-1 induced the sustained phosphorylation of HSP25/27 (Fig. 7). Such phosphorylation of HSP25/ 27 is associated with cytoprotection in other cell types, and is particularly important in the maintenance of the actin cytoskeleton. Even though the activation of the p38-MAPK pathway by ET-1 was only ∼50% of that by 0.5 M sorbitol (Figs. 2 and 4), the extent of phosphorylation of HSP25/27 induced by either agonist was similar (Fig. 7) and may confer equivalent degrees of cytoprotection. Experiments by others have implicated the p38-MAPK pathway in ischemic preconditioning of the heart, whereby a short period of ischemia protects the heart against a subsequent more prolonged insult (reviewed by Sumeray and Yellon, 1997). Perfusion with SB203580 attenuates this preconditioning (Weinbrenner et al., 1997) suggesting that p38-MAPK, which is activated during ischemia (Bogoyevitch et al., 1996; Yin et al., 1997) plays an essential role in the preconditioning response. Ischemic preconditioning can be mimicked by perfusion with α1-adrenergic agonists (Banerjee et al., 1993), ET-1 (Wang et al., 1996), and in the rat heart, activation of PKC may be involved (Speechly-Dick et al., 1994; Li and Kloner, 1995; Mitchell et al., 1995). Our results are entirely consistent with a role for p38-MAPK in ischemic preconditioning. Moreover, since activation of the p38-MAPK pathway by GPCR agonists in cardiac myocytes is attenuated by inhibition of PKC, these data could explain the apparently disparate results implicating both PKC and p38-MAPK in ischemic preconditioning.
The Role of the MAPKs in Cardiac Hypertrophy
The ERKs, SAPKs/JNKs, and p38-MAPKs have all been implicated in cardiac hypertrophy, although the exact roles of each of these pathways has yet to be determined. Activation of the ERK cascade by transfection of myocytes with constitutively activated components of the pathway is not sufficient to induce the myofibrillar organization associated with hypertrophy (Thorburn et al., 1994a, 60b; Gillespie-Brown et al., 1995). However, in this study, we have shown that PD98059, which inhibits the ERK pathway and partially inhibits the p38-MAPK pathway, selectively inhibits the myofibrillar organization induced by PE or ET-1 (Fig. 9, E and F), suggesting that the activation of ERKs is essential for this response. However, it remains possible that the effects of PD98059 may be mediated through another, as yet unidentified mechanism. PD98059 does not inhibit the transcriptional changes associated with PE-induced cardiac myocyte hypertrophy (Post et al., 1996), although activation of the ERK cascade in transfection experiments can induce these changes (Gillespie-Brown et al., 1995). Such apparently conflicting data are probably indicative of the activation of multiple signaling pathways by physiological agonists. For example, whilst ERK activity may be necessary for myofibrillar organization, other signaling pathways may be necessary for the full response. Indeed, our data are fully supportive of hypertrophy being an integrated response to multiple signaling pathways, where ERKs may be involved in myofibrillar organization and p38-MAPK may be important in potentiating the hypertrophic response. It remains probable that both (and other) pathways are necessary for the full range of transcriptional and morphological changes associated with cardiac hypertrophy.
We thank R.J.A. Taylor for the preparation of the myocyte cultures.
This work was supported by grants from the British Heart Foundation, the Clinical Research Committee of the Royal Brompton Hospital, and the European Union BIOMED2 program.
Abbreviations used in this paper
extracellularly responsive kinase
fast protein liquid chromatography
G protein–coupled receptor
heat shock protein
mitogen-activated protein kinase
MAPK kinase
MAPK-activated protein kinase 2
MAPK/ERK kinase
myelin basic protein
- β-MHC
β-myosin heavy chain
Ca2+/Mg2+-free Dulbecco's phosphate-buffered saline
pertussis toxin
stress-activated protein kinase/c-Jun NH2-terminal kinase
Author notes
Address correspondence to Angela Clerk, Division of Biomedical Sciences, Imperial College School of Medicine, Charing Cross Campus, Fulham Palace Road, London W6 8RF, United Kingdom. Tel.: 011-44-181-846-7046. Fax: 011-44-181-846-7099. E-mail: firstname.lastname@example.com


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With more and more consumers upgrading to smartphones, Mastercard looks to change the way consumers view m-commerce with their new iPhone application 'Priceless Picks'. The Priceless Picks application lets you not only share your favorite places and those sensational deals you find with others, but also provides visibility to all the great things around you that others have tagged through utilization of your phones GPS technology. They bucket picks into 5 main categories: Priceless, Dining, Shopping, Entertainment and Other. In addition, the application offers a filter feature which lets you take customization a step further to ensure the content you are receiving is relevant to what you are looking for.
MasterCard Priceless Picks iPhone app from Kelly McCormick on Vimeo.
So this begs the question. How and when will retailers utilize this tool to engage their consumers?
Contributed by Michael Glunk

subset: commoncrawl


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Print News!
An email subject line can ensure viewers actually open the email, read it and act upon it. So, how do you create a compelling email subject line? Here's how.
How can businesses and individuals best reach out to consumers during the new spring season? Luckily, we have five super springtime marketing ideas to get you started.
The podcast audience is growing, so businesses are jumping on board with their marketing. We've put together a list of big benefits of launching a business podcast.
How do you plan on using Valentine's Day in your marketing strategy? If you're not sure yet, here are some ideas for how to show marketing love this Valentine's Day.
There's nothing like great content marketing, and blogging is the place to start. Today, we're going to talk about why you should be blogging, and some easy tricks to get going.
With a little effort there are ways to keep your current clients happy, satisfied and coming back for more. Check out these six tips for better customer retention.
There's a way to make copywriting a priority and save money at the same time—boost your own expertise. Here are sure-fire ways to improve your copywriting skills.
When it comes to content marketing for your business, an eBook is a great step. If you're not sure where to start, here are some big ideas for your next business eBook.
If you want to make sure 2024 is successful, there are a few things to put in place now. We've compiled a list of a few ways to start off a new year for marketing.
There are a few things you can do this year to make sure your business resolutions last throughout 2024! Here are seven steps to make (and keep) New Year's resolutions.


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Surgical hair restoration through the use of FUT is advantageous for patients who wish to cover a greater area of hair loss, as this procedure enables the surgeon to transplant a significant number of hair follicles in a single session. The FUT procedure will leave a scar where the incision was made at the donor area, but this incision is made in such a way that it is covered by existing hair and therefore remains unseen following this treatment for hair loss.
Month: August 2015
What Is The Cost Of The Hair Transplantation Process?
There are many factors that must be considered in determining the cost of hair transplantation. The extent of the hair loss and the needs of the client are major considerations including the number of grafts required and the technique utilized whether it is strip (FUT), FUE or Scalp Micropigmentation.
The price for your hair transplant procedure will also vary by country and location. These factors can be easily addressed through the consultation process available at a MAXiM hair transplant center. During the initial consultation, our doctors can discuss primary and supplemental treatment options while taking the goals of the patient into careful consideration.
At MAXiM, a hair transplant and other hair restoration procedures are very affordable. We accept cash, bank checks, wire transfers and credit cards. In the U.S., patient financing is available through third party patient financing companies.


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Classic Freight Transport is powered by 2014 or newer automatic Volvo's and five standard freightliners that are regularly inspected and serviced. Our high-rise tractors are fully loaded with air-conditioning, fridges, microwaves and comfortable living spaces. We offer 24-hour road-side assistance and designated staff on call at all times to communicate and help guide your journey.
We provide new hire orientation, training and everything you need to get started; chains, straps, ladder, shovel, etc. Our in-house cargo securement program has recently been visited by provincial and federal officials and both agree that when it comes to safety training in the industry, Classic Freight Transport is among the best.
We recently invested in ultra –light tarps that are extremely versatile, durable and easy to set up. Our flatbed division operates through North America, having a current passport or being in the process of obtaining one is recommended. Our operations team works hard to accommodate your schedule because we understand that home time is just as important!
that focuses on relationships and exceeding expectations


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Libraries at University of Nebraska-Lincoln
Date of this Version
Summer 9-30-2013
This paper discusses censorship and the challenges of Library services delivery in Nigeria. Librarians, through their professional associations, have long been committed to the social justice principle of free access to information. External censorship challenges to library collections threaten this principle overtly. However, censorship can also occur in libraries in various covert and often unconscious ways. This paper also raises concerns about current librarian practices and library censorship processes which can effectively censor library collections from within. In managing the library and its services, the librarian should promote inclusion and should not discriminate against any library user regardless of sex, ethnic group, nationality, social condition, religion or political opinions. It concludes by calling librarians to provide the highest level of service through courteous, prompt, adequate, skillful, accurate and unbiased responses to all requests for assistance to the user.


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If your dog is always waiting in the bathroom, you're probably contemplating the reasons and how you can do to fix it. This article will provide the possible causes and the ways you can do about them.
Then, why is my dog have to wait outside of the bathroom? The reasons your dog is waiting outside the bathroom is that it suffers from separation anxiety or it is a bit protective and has learned that it is rewarded for its behavior or is it a behavior that is done in its natural way.
There are a variety possibilities for causes to be considered, it might be beneficial to think about what could make each one more likely. When you have an concept of the root reason, it will be much easier to address it.
Why your dog guards you in the bathroom
Below are some possible causes , and what might make each than likely to constitute the primary motive.
You are reading: Why Does My Dog Wait Outside The Bathroom?
Separation anxiety
It could be because there is a sense of separation anxiety. This is especially likely if it exhibits symptoms of anxiety while going to the bathroom, and when it is anxious when you leave the house or getting ready to go to go to bed in the evening. The signs of anxiety might include crying or pacing.
The reason could be that it's trying to protect itself. This is especially likely if it is known to be extremely cautious in the presence of animals or other people who are present. It's also more likely to occur if it is not to exhibit any sign of stress and isn't waiting for anything from you.
Encouraging the behavior
It is possible that it has realized that it earns benefits for waiting out of of the bathroom. If you are prone to giving it something like more attention, toys or even treats, while it's waiting outside the bathroom, it'll likely perform the same thing more often.
Read more : How Long After Applying Frontline, Can I Pet My Dog?
Animals evolved to be pack animals and it's normal for them to want to be with other members of their group. The reason your dog is following your bathroom to go might be due to the fact that it wants to be near you, so it feels like it's part of the group and is more secure. This is especially true in the event that it tends to follow you around to other places after being taken care of and fed.
Things to consider
Here are some suggestions to take into consideration when trying to determine the reason behind why your dog is doing this.
What else happened when it first started doing it
If your dog was not always stay outside of the bathroom It is helpful to look at what else went on at the time it began to do it. It may be that it was an incident that triggered it.
If it began to do it in a sudden manner and suddenly, it could be because of factors like discovering that the behavior will be reward-based or some other reason that caused it to become anxious at times, for instance, if you adopted a pet.
What is different when your dog waits outside the bathroom
If it doesn't always stay outside in the restroom, it might be beneficial to think about what's different about when it does not perform it. For instance, if it doesn't do it after it is fed or exercised It could be that it does so when the waiting is for the things it needs.
The body language it shows
It is also helpful to think about the body language it displays when it does this.
If it displays symptoms of anxiety, like crying, appearing smaller or pacing around it is more likely to be experiencing it because of something that makes it scared or anxious like separation anxiety.
While if it's calm, while waiting at the outside of the toilet, it'll be more likely to does so naturally since it is more comfortable in your presence or is trying to protect itself.
What to do about your dog waiting outside the bathroom
Below are a few alternatives you can choose from in order to convince the dog off.
Avoid encouraging it
Read more : Why Does My Brothers Dog Look Like Frank Gallagher
Like we said there is a possibility that it's the case that it's been taught that certain behaviors are reward. It is better in rewarding the behavior when it doesn't go to the bathroom with you and avoid rewarding the behavior when it is rewarded.
Limit reasons why it might be anxious
It is also helpful to narrow down the causes for anxiety. A few things to consider include ensuring it is fed, active and that it is able to go outside to pee prior to going away for a time.
Positive reinforcement training
Positive reinforcement training involves encouraging the behaviors that you want to see by rewarding them and discouraging unwanted behaviors by not rewarding them. In order to encourage your dog to not follow you to the bathroom , you might make it appear as if you're heading to the bathroom, and give it a treat when it does not follow you.
This post could include affiliate hyperlinks. Pet Dog Owner could receive a payment from the businesses mentioned in this article. It has no impact on the cost that you pay. We would be grateful for any assistance.
Should I stop my dog from waiting outside the bathroom?
The first step in stopping your dog from snoozing in the bathroom is to figure out whether there is a problem initially. If it's not bothering your dog and you're both calm, then I see no issue with your dog standing outside the bathroom.
But, you must to understand why your dog is doing this, in the event that there's an ulterior motive behind this type of behavior. Your dog might need assistance, particularly when he's experiencing anxiety or is unhappy due to reasons. It is possible to consider the following questions:
Has my dog suddenly started waiting outside the bathroom?
An uneasy dog is a miserable dog , and as dog owners we would like to ensure that our dogs are happy. If your dog is suddenly beginning to stand outside the bathroom after you enter with his tail curled in between the legs of his paws, be looking for indications of anxiety. Maybe, your dog has become anxious and requires help in managing separation anxiety.
It's cute that your dog waits outside the bathroom door to greet you However, it could also be a sign of a bigger problem. If you notice other indications that indicate separation anxiety, do not hesitate to consult your vet as it's a very common issue.
Source: https://petstutorial.com
Category: DOGS


text: A federal bankruptcy judge has approved an agreement allowing former Farmland Industries Chief Executive Harry Cleberg to file $7.4-Million in claims against Farmland. Under the agreement, Cleberg will be treated like any other claimants in the Chapter 11 proceedings against Farmland. Cleberg claims the bankrupt company owes him deferred compensation and retirement benefits. Representatives of Farmland's unsecured creditors and bondholders had opposed allowing Cleberg to become part of the bankruptcy proceedings. They still are pressing ahead with an investigation of whether Cleberg and other Farmland executives were in breach of their duties for the financial shipwreck at Farmland.


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Travel Fun Fact
The Eastern Bearded Dragon uses camouflage or inflates itself when threatened. Their beards protrude from their necks when puffed up and they will also open their mouths wide, exposing the bright yellow lining. The dragons typically grow to about 55cm. Water dragons are found in eastern Australia and southern New Guinea. We encountered this dragon at the Lone Pine Koala Sanctuary. Read more about our visit there in our post, Campervan Camping and Road Tripping from Brisbane to Melbourne.


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← Go back
Overall, very well. Very warm, engaging and welcoming demeanor, all of which are of great importance in creating a safe space. The use of the DSM as a brief "counter example" of what we do in CIT was excellent reinforcement that CIT is not therapy and is focused on strength not weakness. Referred back to the need to create/maintain a safe space when setting up/explaining RW/MD exercise. Good affirmation of participant feedback, which is another way of creating a safe space (ensuring people feel their input in welcome and valued).
Allowed PPt to guide the presentation of skills and concepts. Largely went to participants first when PPt introduced new concept.
Very well. A few refinements needed for "hand rubbing" activity: We are not necessarily looking for pleasant or neutral sensations during this activity, just sensations (some might be unpleasant, like the tensing of shoulders or the burn in the triceps). Also, we are not looking only to increase sensation vocabulary but sensation awareness.
Did well referring back to Tracking and Resourcing when explaining Grounding, and including how natural it is for us to engage in various grounding practices even unconsciously and how often we do it. Provided good reminder that one of the benefits of Grounding (as well and Tracking and Resourcing) is that, when needed, we can do it any time in our minds and do it surreptitiously, without anyone even knowing we're doing it.
Provided an excellent setup in Skill 1 for use of RW and MD through the rest of the course, including the behaviors needed for active listening.
Using the DSM as an example of what CIT is not was excellent. Could provide a bit more detail here, e.g., DSM focused on detecting and defining the symptoms of mental/emotional illness and dysfunction to "fix" people, while CIT is focused on defining, detecting and developing skills that are natural to us all and promote wellbeing and flourishing...but overall this was a great way to start out.
Of note for next time, it's important that the whole program be set up/overviewed during Skill 1 when presenting Slide 1. It needn't involve great detail; just the three Series and the uniqueness of CIT's systems skills in Series III (better to not include so much detail about the Resilient Zone in the opening slides).
Remember to set up Accountability Partner practice when setting up MD exercise.
Remember to have participants get up and move and try out different positions when introducing Grounding.


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- 6000-6500k color temperature
The high quality crystal chandelier always make you proud of having such chandelier which provide brilliant shining and elegance even throughout the home.Our first product on this review comes with 3 circle rings with many top grade K9 crystals with 27.7,19.7 and 11.8 inches diameter to enlighten your place perfectly.
You can be carefree about the required specific ceiling to hang them as this can fit any ceiling as high, low and slanted sloped. This has build in LED light source which is extremely energy saving even 3 years warranty for every part of this take rick of your investment.Shading of cool white light won't hurt your eyes which works with 110-220 volt.
Personalized shape and height of each circle offer convenience and different look of your place. Thought this has no dimmable feature while this have 6000K to 6500 K color temperature to provide classy and luxurious lighting through this lighting fixture. Metal wire and frame with outstanding light source goes rounds of the years without damage with such beautiful light.
Why we picked it
- Energy sufficient
- Suitable for any ceiling
- 3 years warranty for parts
- Shameless match with your room style
- Classic and modern design
- Wonderful for the price
2. Round Crystal Chandelier – Chrome Round Crystal Chandelier Ceiling Fixture Light
Key Feature
- Dimmable with dimmed switch and bulb
- Works with 4 E12 base bulb
- Fixture and shade have chrome color
- 17 inches diameter chandelier
- 60 W for per bulb
- Shade comes with metal and crystal blending
Most of the crystal won't come with a multiple finish where you can't purchase one with your required finish with an exact lighting fixture. But this lighting fixture has position 2nd on this list of best crystal chandelier offers four finishes such as black, chrome, oil rubbed bronze and antique copper as you can find best match of finish according to your home need.
Featuring this glam lighting with glass crystal with exceptional arrangement to allow more illumination with perfect decoration.Along with 4 light bulbs which have E12/14 with 60W for each bulb produce right amount of lighting for 200-360 sq. Ft large room or space through 17 in diameter and 20 inch height.And the chain comes with 39 inches which can be shortened to remove small pieces.
The unit have metal ceiling canopy with 5.1 x 1 inch dimension which hold the amazing lighting fixture which have metal and crystal combines shade.With dimmable switch and bulb you can set this cozy and mode lighting atmosphere.
Why we picked it
- Source of comfortable lighting
- Adjustable length
- Suggested for 200-360 square
- High quality materials
- Provides great brightness
- Elegant enough to match any room decoration
3. Contemporary Crystal Chandelier – Pendant Ceiling Chandelier Lighting
Key Feature
- Halogen E12 bulbs included
- Have 2700k color temperature
- Blending of classic and contemporary materials
- Works with 8 lights
- Sturdy metal lamp holder
- Equipped with premium K9 crystal
Meelighting always trustworthy lighting fixture manufacturer and here the another example of this which have 10 years warranty feature which is not possible for others.Because of their incredible sturdy metal construction and top quality K9 material they have such confident to ensure your investment is safe.
The modern contemporary design of this lighting offer a great match for dining room, living room, porch, hotel and more this will be eye-catching centerpiece with 3 tier pendent lights which have E12 base with only halogen. Luckily this comes with halogen bulbs as a gift. Moreover, 19.7 diameter and 13.4 inch height have wide coverage with 2700K color temperature and 60W for each bulb. Dimmable capacity is available when it set with dimmer switch and bulb.
Black matte finish add versatility and sophistication to blend any style and background of home. Instruction manuals for correct assembly 48 inch long chain allow to hand this fixture according to your desired length. Plus this fixture has instant on feature to brighten up your place.
Why we picked it
- Highly versatile
- Offer beautiful lighting
- Worth of price
- Surprising warranty
- Height adjustability
- Beautiful and long-lasting
4. Crystal Chandelier For Bedroom – Gris Modern Ceiling Light Flush Mount Ceiling Light
Key Feature
- Has 38 triangle cord crystal bar
- 308 octagonal crystal beading
- 51 splendid crystal balls
- Required G9 cool or warm white bulbs
- Real crystals decorated
- Has warranty feature and manual instruction
The Size of any crystal chandelier is an important factor dependent the overall measurement of placing area. While this comes with 15.7 inch wide,9.56 inches height and 15.7 inch length for 5 m2 area to provide light intensity actually what you expect from your chandelier.
More interestingly this equipped with three different crystal type to makes this piece more elegant and amazing touch for your place.There are 51 splendid crystal balls for providing more well-organized while 38 triangle cord crystal bar attached to their upper portion which create more variable light with 308 octagonal crystal beading for amazing glittering and translucently.
Again it has a sturdy and sleek modern plate with clear texture and silver finish which is made from stainless steel as anti corrosion and rustproof. It requires G9 8 bulbs with 60W while these bulbs won't pack with this product. Moreover, used sparkling crystal have glass touch as these are real not acrylic. While warranty included all service for this unit.
Why we picked it
- Appealing design
- Mirror chroming base
- Worth of your investment
- Ensure amazing illumination
- Blend any sense of style
- Lightweight and flimsy
5. Mini Crystal Chandelier – Mini Crystal Chandeliers Acrylic White Chandelier Light
Key Feature
- Fast assembly and installation
- Ideal for 150-200 sq. ft. place
- Included required installation tools and manual
- Use polycarbonate crystal
- Need 4 bulbs with E12 base
- Can be height adjusted
In case you are finding a crystal chandelier which is more from acrylic than you are in right position as this made from poly carbonate material which is used for sunglasses or window but spread good amount of lighting like glass made one but not easy to break them and lightweight for fast and safe portable without compromising performance and looking of lighting.
The dimension of this light is highlight its comparably mini size such as 15 inch diameter with 17 inch adjustable chain. To produce the right amount of brightness with 4 bulbs with E12 base require 40 watts for each of them. It comes with all necessary hardware which are needed to faster assembly and installation.This could be a good option for 150-200 sq. ft.
The lamp comes with hardwired, so you need not do many things as you need to unfold the arms and attach the bread with the main unit and go for installing them properly.Through manual instructions your installation is very effortless. So this modern acrylic light is an ideal piece for dramatic statement and classy touch with glass stem and metal frame and arm.
Why we picked it
- Luxurious appearance
- Glow of vintage lighting
- Lightweight yet sturdy
- Build to last
- Dimmable comparability
- Hardwired
6. French Empire Crystal Chandelier – French Empire Crystal Chandelier Chandeliers Light
Key Feature
- Good sizing for standard place
- Free shipping
- European traditional lighting
- Classy design
- Easy to assemble
- Quality glass crystal
If you want to bring a lighting piece which have blending of traditional glow with accent looking this french crystal chandelier won't disappoint you. This comes with plastic and real glass blending such as large and small sizes crystals round the beads are plastic while the small dangle and larger top one is glass crystal.So whatever is that after assemble and lighten up this have dramatic glow with beautiful shimmers.
For best result you need to choose smaller option than 40W at 25W with E12 3 bulbs which provide good sparkle for your place. Once you hung this on your place you will forget the assembling time which sometimes ca be lengthening because this comes with vogue manual instruction, but it will be easy it is for to start the task after good inspect of the pieces carefully.
A chandelier is worth when it was capable to shade good lights with authentic look. While this have amazing lighting from every angle. You can place this fixture flat and vaulted ceiling without no issue. Overall we can suggest you as a beautiful fixture which worth of money.
Why we picked it
- Seems expensive
- Eye catching for any place
- Versatile applications
- Decent prize
- Very bright
- Timeless elegance
7. Vintage Crystal Chandelier – LED Light Crystal Pendant Lighting Ceiling Light
Key Feature
- Wide bulb compatibility
- Varying heights
- Used high quality crystal material
- Suitable for modern home design
- Classic finish for more elegance
- Impressive glow comes with 8 bulbs
Here is the stylish lighting with vintage inspired design for best match while you have modern contemporary and traditional theme to decor your home or informal office andmore. It comes with 30.7 x 21.6 inches with durable metal construction and height adjustability chain can fix 0-39 inches. This beautiful and impressive looking chandelier have a black finish to meet rustic, vintage and classic requirements.
To provide great illumination it has 8 heads which have E12 base and takes 40W for each bulb among incandescent, energy saving, halogen and move which you prefer most. Clear K9 crystal which is more gorgeous than you imagine or looking in picture.
Mid century character is more eye catchy in these days to create trendy look while this pieces have sparky rainbow which can go for kitchen, foyer, hallway, living room and bedroom through its ambivalence and appearance even whoever looked at once in light up with love this. The only drawback of this product is it have not dimmable capacity.
Why we picked it
- Add brightness with comfortable lights
- Fashionable and elegance outside
- Mid century arrangements
- Great for the price
- Idea for home restaurant and informal office
- Good coverage
8. Antique Crystal Chandelier – Light Antique Bronze
Key Feature
- Single bulb lighting fixture
- Beautiful antique finish
- Simple to install and assemble
- Wide applications
- Long-lasting metal and crystal craft
- Simple yet decorative
Mesmerizing decoration of this fixture will force you to purchase and hang them in your place. The durable construction and incredible performance of this antique crystal chandelier confirm with UL listed and energy star certification. This antique finish and transparent shade offers best match for your home whichever you picked.
With 11x11x 15 inches dimension and unique craftsmanship this provides a hundred possibility of positioning them. This works with 1 light bulb which required 60w to make perfect brightness with complacent and lightweight design. This originated from China to allow more overflow of style of creativity and crafty. As this comes with hardwired you can assemble them faster.
It will surely satisfy with their reasonable price and extraordinary design and lighting. While the small size fit any small corridor staircase or room to brighten up thought it has excessive darkness. Utmost ambience and luxurious lighting comes with reflection of top quality crystal. Through a dimmer switch you can enjoy soft lighting.
Why we picked it
- Good choice for small place
- Soft lighting
- Absolute for any decor
- Transparent and neutral color
- UL listed
- Energy Star certification
9. Gold Crystal Chandelier – Antilisha Gold Crystal Chandelier Light
Key Feature
- Use boutique selected crystal
- Durable stainless steel frame
- Gold painted beautiful chandelier
- Dimmable bulb included
- Cake tired pattern
- Clear tassel raindrop crystal
For great look in larger solace round chandelier unbeatable ans Antilisha presenting a beautiful chandelier with good coverage from 17.7 inches diameter with height quality stainless steel frame which have up to 85 percent crystal attached.To make a great blending of shadow and great lighting this has different arrangement of frame and glass. Don't be doubtful about this sturdy construction and performance while this have 5 years warranty to use them care freely.
Convenience installation is another feature of this fixture which have video and clear manual instruction. Ships with 12 E12 base bulbs which works with 40W.This fits any ceiling from slanted to high correctly with out affect of lighting. Use dimmer switch and bulbs to set as dimmed lighting source.
Is more ornamented chandelier is attractive? This piece make you if you have this thinking till now. As this have cake tired pattern and with simple clear tassel rain drop crystal to fit any popular style included mid century, post-modern, luxes, Paris, French empire and more throughout the art deco style. This comes from 5.5" round canopy with 59 inch adjustable height chain to hang this per your desirable length.
Why we picked it
- Instant on feature
- 5 years warranty
- Touch of art deco
- Fits most of the types of ceiling
- Unique and gorgeous
- Adjustable length chain
10. Globe Crystal Chandelier – Bronze Chandeliers Globe Crystal Chandelier Light
Key Feature
- Height adjustable
- Suggested for 120-160 sq. Ft space
- Can be hung for any ceiling
- Ideal for foyer, restaurant, staircase and more
- Need 100 watts to light up
- Compatible with any standard bulb with E12 base
This beautiful chandelier build with metal frame with globe design quite different in look from a casual crystal chandelier. It has two finish as black and antique which are considered the most neutral finish for these type of crystal chandelier. The compact size of this has 7.5 x 15.7 x 4.72 inch round chandelier have good coverage as 60-120 sq. ft.
If there is a curious mind to bring industrial look in your café or home staircase, villa or dining room this could be lightened up with this UL listed lighting.You can choose this for farmhouse style through a vast option of light bulbs among LED, CFL, halogen and more with one E26 base which takes 100W and 120 volts.Visual appearance and mode with cheers with dimmable compatibility with regarding switch and bulbs.
Whether this seems small one for large space but adequate for highlight any small are like kitchen island and dine take. This offer comfortable lighting without hurting your eyes. Using of 39.37 adjustable chain you can hand this on appropriate position which is needed per your cheating type and application.
Why we picked it
- UL listed
- Farmhouse inspired design
- Easy to put together and hang
- Hardwired
- Can be dimmable
- Two finish available
11. Outdoor Crystal Chandelier – Chandelier Solar Multicolor Clear Crystal Wind Chimes
Key Feature
- Made with acrylic and metal
- Elegant looking
- Reflection of sun
- Super easy to hang
- Accent lighting
- Suitable for garden, patio, lounge and more
Lighting of outdoor have several benefits but when it is not accomplishes with electricity even solar power undoubtedly it is more fun than others. This chandelier is designed for outdoor applications such as garden, foyer and lounge for spreading glow with multicolor crystals. This color will change automatically with consistence timing.In dark night or going sun down this will start its magical work.
You will get unlimited compliments from your neighbors when you hand this on your garden. It comes with 6 inches diameter and 19 inches height while this acrylic jewel comes in different length around the chime for more dashing and charming look. This had metal construction with beautiful two shaped acrylic with craftsmanship silver frame best crystal chandeliers.
More interestingly this is a combo pack as chandelier for night while source of musical peaceful sound maker as wind chimes. Through a shepherd hook you can easily install this anywhere in your garden which capable to shimmer for outdoor accent pieces by reflecting the sunlight. To enlighten your backyard this is best adding such friendly budget which goes long times except any damage or gull.
Why we picked it
- Solar chandelier
- Changing color
- Properly illuminate outdoor living place
- Gorgeous and very functional
- Create musical note
- Small but effective
12. White Crystal Chandeliers – Surpars House White Feather Crystal Chandelier 4-Light
Key Feature
- Adjustable cord length
- Easy to assemble and hang
- Real glass crystal
- White feather shade
- Works with E12 base bulbs
Our best crystal chandeliers list won't be delicate except we are not reviewing such a wonderful lighting.You have already been viewing the picture which comes with white feather shade as well as two shaped crystal for the purpose of decoration, the snow-white feather allow mid century arrangement in your pace undoubtedly here the reason to considered it as the most gorgeous and glamorous lighting fixture.
The lamp use E12 base 4 incandescent bulls to product good numbers of lighting. For any standard size room 59 inches height and 19.68 inches diameter is practical enough through candelabra. To create warm feeling and elegant looking this has versatile placement while its living room, girls bedroom or make up room which match and decoration and background which are already existence.
Sturdy painted base and metal wire provide best support when hung this from ceiling and utmost matching for modern home through unique ambivalence while it has not dimmable capacity with 120 watts for best lighting. This requires manual instruction to faster assembly and hang as this comes with half finished.
Why we picked it
- Source of warm lighting
- Unique and elegant
- Crustal are quite sparkly
- Wonderful to the price
- Glamour of mid century
- Wide application
13. Traditional Crystal Chandelier – Grey Glass Candle Crystal Chandelier
Key Feature
- Glossy chrome painted finish
- 21 inc diameter chandelier
- Equipped with K9 crystals
- Adjustable height
- Blending of modern and contemporary style
- Operate with E12 bulbs
Elegance and classic design blending is the charming feature of this traditional crystal chandelier. The elaborate decor must come with this lustrous lamp piece. This is a beautiful chandelier with gray crystals arrangements with gray glass shade to give which have versatile fit for any decoration of bedroom kitchen living shop hotel and restaurant.
It follows European style which able to utmost fit in style modern to traditional with quality illumination through 21 diameter and 6 lightbulbs which works with E12 base of any standard types of bulbs. This has sturdy build with chrome paint for durability and eye catchy appearance.Furthermore, the height adjustability from 0-39.37 inches makes chances of best height for your demand.
Other than this, it has strong export package and free damage replacement as this manufactured from professional lights factory. This uplight has comfortable light which won't hurt eyesight but provide the right amount of lighting for 30-40 room size.Candle shape light allow great featuring of mid century with art deco style for keeping home decor personalized and unique.
Why we picked it
- Impressive appearance
- Sturdy construction
- Comes with 3 years warranty
- Easy assembly
- Features with flush mount
- European style
14. Best Crystal Chandeliers Lamp – Semi Flush Mount Ceiling Light Fixture
Key Feature
- Use 24 sparky crystal
- Require flush mount
- Antique black finish
- Crystal attached by metal twist
- Handmade antique piece
- Excessive robust metal shade
As lighting fixture decoration pieces crystal is always praiseworthy while if you have to choose metal frame for durability and versatile option for this is a popular pick.If you really want something new in your place you can look this one. This comes with 24 sparky glass to offering shining effect instantly.
With the 12 cm round ceiling canopy it requires semi flush mount while you did not need to more drilling of your ceiling. To give different and ambulance lighting for home remodeling or other purpose this could be reliable in foyer, bathroom and hallways. Also, black finish for this antique lighting gives sophistication with E26 base varieties of bulbs.
The handmade frame with 18 cm width is specially designed for creating crafted double twisting design around the crystals which make shadow on the ceiling which is incredible to make something different in your home. A single bulb with 60W offer dazzling bright whether you are trying to bring modern or antique look in bar, office balcony and more. glass chandeliers.
Why we picked it
- Flavor of farmhouse theme
- Good for modern home lighting
- Rustic look match any style
- Suitable for both residence and premise
- Comfortable lighting
- Accent addition for room decor
15. Industrial Crystal Chandelier – Industrial Crystal Pendant Ceiling Light Fixture
Key Feature
- Recommended only for flat ceiling
- Can be dimmable
- Three layer metal frame design
- High quality crystal incorporate
- Included installation hardware and manual
Sparking lights comes from 108 crystal strips and one vintage filament light bulb for best result to light up your selected area. Moreover, 3 layers of inner metal ensure utmost sparkle from every angle of your lamp. Durability comes with their construction of 3 layer and mesh frames which also a successful attempt from manufacturer to bring industrial vibe even in modern decoration.
Ceiling light fixture with dimmer option always makes you more comfortable with new lighting option when you will have use dimmable switch and bulb. To ensure comparable small coverage is comes with 15 inc Dia x 23.5 inch height for kitchen island, commercial location living room and more. This light have 4,75 inch ceiling canopy which suitable for flat positing.
Oil rubber bronze finish offers this hanging piece classic and timeless look which it's you can think best match for your place and in which decoration. But certainly this crystal chandelier is an elegance option for any place.
Why we picked it
- Industrial vibe
- Romantic lighting
- Wide location to place
- Removal mesh shade
- Quick and easy installation
- Oil rubber bronze finish
Frequently Asked Question Of Best Crystal Chandeliers
What are crystal chandelier made of?
Basically crystal chandelier made from glass are which different from the usually used for windows and eyeglasses as this have lead oxide to make is more sparkle and brilliant. While acrylic are made with poly carbonate which offer super cutability and lightweight.
How do you clean crystal chandeliers?
Cleaning of crystal chandelier depends on how often you're cleaning and which method cleaning is choosing to apply.You can clean with crystal on with available cleaning product of market with spraying bottle and clean perfectly, or you can go with crystal off procedure with you need to apart the crystal and other parts of the chandelier to clean them overly. We suggested to this method when your chandelier have worsened dirt or lost its glam.
What style are crystal chandeliers best for living room?
Various style of chandelier makes it is a more popular decorative fixture from informal office to your bathroom. Waterfall or raindrop, big and spiraling are most attractive for your living room which capable to provide elegance with flair of enough bright.
What is the difference between Royal cut and elegant cut crystal?
A great combination of high quality lead free machine cut which have machine polished to give it authentic desirable showmanship while the invisible coating ensure easy cleaning and more shining defined royal cut of crystal.On the contrary Elegant
cut crystal have higher end cut with most faceted which is the reason of greater shine and sparkle. So only cut makes difference between Royal cut and elegant cut crystal.
Are crystal chandelier outdated?
No, crystal chandelier is still popular like it was before as timeless but modern chandeliers are more updated with power source and different style of pick. These have a dramatic look for bedroom, living room, bathroom and so on from traditional era to modern. Moderately several of them is had theme base design like farm house mid century or Bohemian which can blend decoration and being a focal point of your entire house.
See More: Best Chandeliers Reviews
For more spark and shine anyone cant deny installation of best crystal chandelier no matter it is home or commercial place restaurant or bar setting this can easily being a centerpiece once you select them perfectly actually which material shade and lighting type you need.
Choosing the right chandelier depends on the position of placement with its size if you wanted to hang on a large room you can purchase a larger. On the other hand for smaller room standard size won't disappoint you with noble design and style.
Here the large list with different dimension layer, colors of crystal chandeliers from Amazon with reasonable prize and good rating which are helpful to update your place lighting with this glamorous and functional options.


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Download the School calendar 2023-2024 (Official) |
Haiti - Agriculture : Rebuilding the agricultural sector 24/06/2011 13:20:36 "Ensuring technical cooperation has been the core of IICA's work for over 70 years," declared Victor Mr. Villalobos. "Today's challenges in agriculture development calls for increased investment, innovation and risk management [...] Governments must find ways to promote sustainable development in order to help raise the rural poor out of poverty. IICA pledges its full commitment to supporting its member countries in this critical endeavor." During the session, Alfredo Mena, IICA's Representative in Haiti, shed light on some challenges facing the sector. He said, "Helping to strengthen the Ministry of Agriculture to implement policy and plans is paramount [...] Without this component, it will be increasingly difficult to put in place systems that allow for increased investment which will ultimately create jobs for rural people and feed Haiti, while protecting it's natural resources and environment." Mena also cited examples of horizontal cooperation, current projects in negotiation and provided key highlights of the 2010-2014 Haiti Country Strategy. He reiterated the focus of IICA activities. "Ensuring food security remains a top priority for us [...] We've just begun the 4th year of the Pro-Huerta Project, a family agriculture program which has benefited over 15,000 families to date [...] Beneficiaries also include many other families who have received funds from PROHUERTA from Spain and the International Fund for Agriculture Development (IFAD)" adding that "The IICA Office in Haiti works in coordination with the Ministry of Agriculture and natural Resource (MARNDR) and have implemented 17 priority projects with the donor community..." The Haiti Country Strategy, a road map to assist the Ministry of Agriculture in rebuilding Haiti's agricultural sector, aims to increase national production to support food security; support animal and plant health programs; expand reforestation, promote agribusiness and the private sector; provide technical assistance to the Ministry of Agriculture, particularly with the new government; preserve natural resources and reinforce cooperation and between committed organizations and governments. "The Haiti Country Strategy serves as an important guide on how IICA can best help to meet country level needs to build a sustainable agriculture," concluded Villalobos. See also : https://www.haitilibre.com/en/news-2287-haiti-argentina-food-self-sufficiency-soon-a-reality-in-haiti.html PI/ HaitiLibre
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Maladroit | ||
Studio album by Weezer | ||
Released | May 14, 2002 | |
Professional reviews | ||
Metascore | 72 |
Maladroit Reviewer: Jon Horowitz (Nude as the News) Publishing date: Unlisted |
No rating given |
The first listen to Weezer's latest offering, Maladroit, is tough to swallow. The milkshake-thick riffs were edgier than those found on the band's previous releases. The vocals were delivered with harsh condescending tones. This was a recipe for a crunchier, rougher Weezer.
But I recall the unappetizing first bites of Pinkerton and the Green Album, the band's third and untitled LP. With Pinkerton, I expected another "Surf Wax America" or "Undone -- The Sweater Song," and was initially lost in Rivers Cuomo's hang-ups and the band's supposed less-than-radio-friendly sound. As I digested Pinkerton, though, I realized it had an inviting dark side, with more artistic integrity than the Blue Album, the band's first and also untitled LP. Same thing happened with The Green Album. I expected Pinkerton-esque longings for lesbians and distant Asian women, but instead got a bubblegum beach song in "Island in the Sun," "Peter Gunn"-meets-drug-culture on "Hash Pipe" and a barrage of other wonderful ditties. Within hours of listening to the album, I was jonesing for more.
So I gave Maladroit the second chance it deserves (and in a much more conducive listening environment – on the open road). I quickly discovered Weezer sounding more raw than ever –- a fresh batch of bold, intense nerd rock delivered well before The Green Album even had a chance to reach its expiration date.
Maladroit combines the best parts of the three previous Weezer albums: creamy guitar riffs, addictive beats, staple "hoo hoo's" and the brilliant mystique of Cuomo's lyrics. Consider the band a modern, refined version of Kiss – the musical geeks behind the makeup.
"Dope Nose" is Weezer's most infectious tune since "Hash Pipe" or "Buddy Holly." It's got that "I can't get this song out of my head" quality. Fortunately, you won't want it out of your head. Also like "Hash Pipe," "Love Explosion" continues the subtle thievery of riffs and melodies. At times, the song bears a resemblance to "The Loco-Motion." Frightening as this seems, Weezer's ability to mate cheesy pop with crunchy punk rock gives birth to the band's signature fresh sound.
"American Gigolo," the album's opening track, is tightly crafted musically, and is even better crafted lyrically. Like Pinkerton, Maladroit delves into Cuomo's psyche, but he only reveals snippets of (if any) personal info. And he scribes these lyrics in some universal code all his own -– simple and concise, yet vague and mysterious –- and always ready to be sung along to. "If you hate this / No, I can't blame you / Because I'm hurtin' / So I'll flame you." Sure, you can venture at what inner demons possessed Cuomo to write those words (a stab at critics or former fans, perhaps), but it's just as easy for Jane Listener to use this song as a response to a fight with her boyfriend.
The only disagreement I have with Maladroit, as with previous Weezer LPs, is the album's length -- clocking in at just over a half hour. But if the band continues to crank out an LP per year, a half hour might be all they'll need.
So while the first slurp of Maladroit's extra-thick pop-punk offerings gave me a brainfreeze, I couldn't help but thirst for seconds, and thirds, and…
— Jon Horowitz


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Lights...Camera...Action... - My Bonkers Brain
A backstage pass to the world of #fashionheaven
Take a peek behind the scenes to see how some of the features on emmaheaven.com are created and brought together every month.
"And now we invite you backstage to see this month's shoot: My Bonkers Brain."
Producer: Emma Heaven
Location: Studio Heaven
Photography & Post Production: Adrian Heaven
Makeup created and applied by: Beth Jones facebook.com/bethjbeauty Instagram: @bethjbeauty
Video filmed by: Jack Heaven
Video edited by: Adrian Heaven
Social media clips & photos: Cath Esaw
Music: Hourglass - Squeeze
Thank you to everyone on #TheDreamTeam for creating this issue's shoot and video.
Your creativity and commitment never ceases to amaze me!
Tags: Lights camera action


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A lot of the fringe players got opportunities and did well for Arsenal against Dundalk on Thursday night, with a few catching Mikel Arteta's eye.
Mikel Arteta rotated heavily for Arsenal's match against Dundalk on Thursday, bringing in almost a full team of new players compared to Leicester City lineup. Only Granit Xhaka kept his place in the side, with even goalkeeper Bernd Leno dropping out.
For a couple of the younger players, this was a rare chance to impress. Joe Willock and Reiss Nelson were making their first starts of the season outside of the League Cup. Arteta liked what he saw from both of them.
"[Joe and Reiss] looked good tonight and always looked like threats," Arteta said. "They looked like they could do something different in the game to change it.
"They were good around the box, they were creative, they had courage to make the right decision in the right areas. I'm pleased with both of them."
Nicolas Pepe hasn't been quite so much on the fringes, starting three of the last four games. But this was still a chance for him to make his case ahead of the weekend's game against Manchester United. He did so with a nice goal on his right foot.
Again, Arteta had praise for another one of Thursday's starters, as well as some constructive criticism.
"I think he's so willing to do well and show everybody – first of all, his team-mates and myself – and also the fans that he is a player that can make the difference every week," Arteta said of Pepe.
"It's about finding the consistency. Not through the games, but during the games as well to be most instinctive and to be able to do the right things in many, many occasions where he has to make the right decisions on the pitch.
"[The goal is] nothing to do with me, that! That was his action and he scored a magnificent goal."
As for whether the performances will put any of Thursday's group in contention to play against United, Arteta suggested he makes the decisions on a case-by-case basis, not using team results.
"It's not because we lose or win, it's what they bring to the team, how they play, and what they have shown.
"The results at the end are the consequences of many things but individually how they do things is what determines whether they have the opportunity to play or not."