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Previous studies have suggested that G protein coupling, phospholipase C activation, phosphoinositide hydrolysis, and protein kinase C activation may be required for α1B-adrenergic receptor regulation, particularly for their endocytosis into intracellular vesicles. Accordingly, the internalization and down-regulation properties of mutated receptors with defects in G protein coupling and second messenger generation were investigated. The Δ12 and Δ5 receptors, previously shown to be defective in G protein coupling, exhibited greater agonist-induced losses of cell surface accessibility assessed by radioligand binding to intact cells on ice than for the wild-type receptor; however, these receptors were completely defective in endocytosis into intracellular vesicles assessed by sucrose density gradient centrifugation. These receptors also did not undergo down-regulation with long-term agonist exposure as did the wild-type receptor; instead, a prominent up-regulation was observed. The Y348A receptor, previously shown to be defective in phosphoinositide hydrolysis and endocytosis was also defective in down-regulation but did not exhibit significant up-regulation. In contrast, a receptor construct with amino acid residues 246 to 261 deleted (Δ[246–261]) was also defective in stimulation of phosphoinositide hydrolysis but exhibited internalization and down-regulation properties essentially identical to those for the wild-type receptor. Together, these results suggest that stimulation of phosphoinositide hydrolysis by α1B-adrenergic receptors is not required for their endocytosis or down-regulation but that similar and overlapping receptor structural domains are involved in mediating these processes.
The effects of G protein-coupled receptors (GPCRs) are mediated by activation of specific G proteins and their downstream effector enzymes and second messenger pathways. Epinephrine binding to α1B-adrenergic receptors (α1BARs) activates members of the Gq/11 family of G proteins, which stimulate phospholipase C (PLC) and phosphoinositide (PI) hydrolysis, eliciting cellular responses by activation of protein kinase C (PKC) and regulation of intracellular Ca2+ (Graham et al., 1996; Zhong and Minneman, 1999). Agonist binding to GPCRs also initiates desensitization, a series of adaptive changes that decrease responsiveness of the receptor to subsequent or continued agonist exposure (Perkins et al., 1991). Receptor-specific changes occurring during desensitization include a rapid uncoupling of the receptor from G protein activation; a rapid internalization of receptors into compartments no longer accessible to ligands at the cell surface, due to receptor "sequestration" within the plasma membrane and/or "endocytosis" into intracellular vesicles; and a slower decrease in the total number of receptor binding sites, referred to as down-regulation and generally thought to result from receptor degradation (Ferguson et al., 1996; Bohm et al., 1997; Krupnick and Benovic, 1998). Although the cellular and molecular mechanisms involved in desensitization are best characterized for the Gs-coupled β2-adrenergic receptor (β2AR), similar changes have been shown to occur for Gq-coupled α1BARs (Cotecchia and Mhaouty-Kodja, 1999;Garcı́a-Sáinz et al., 2000; Toews, 2000).
The extent to which G protein coupling, effector enzyme activation, and second messenger generation and action contribute to GPCR internalization remains a question of interest. In the case of the β2AR, it has been known for many years that agonist-induced internalization can occur independently of G protein coupling, cAMP formation, and activation of the cAMP-dependent protein kinase, because β2AR internalization occurs normally in S49 lymphoma cell variants that lack Gs or functional coupling between β2ARs, Gs, and adenylyl cyclase (Clark et al., 1985; Mahan et al., 1985). Instead, considerable evidence indicates that internalization of β2ARs is mediated by second messenger-independent phosphorylation of agonist-occupied receptors by members of the GPCR kinase (GRK) family and their subsequent translocation to clathrin-coated pits mediated by binding of the adaptor protein β-arrestin to the phosphorylated receptor (Ferguson et al., 1996; Krupnick and Benovic, 1998). Recent studies have shown that GRKs are also able to phosphorylate and desensitize α1BARs and that β-arrestin can modulate α1BAR desensitzation and internalization (Diviani et al., 1996; Mhaouty-Kodja et al., 1999). However, we and others have shown that direct activation of PKC with the second messenger analog phorbol-12-myristate-13-acetate or related compounds can induce or promote α1BAR internalization, at least under some conditions (Cowlen and Toews, 1988; Fonseca et al., 1995; Zhu et al., 1996). Furthermore, both PLC inhibitors and PKC inhibitors have been reported to inhibit agonist-induced internalization of α1BARs (Fonseca et al., 1995; Zhu et al., 1996; Awaji et al., 1998), suggesting that G protein coupling, effector enzyme activation, and second messenger-regulated kinases may play a more important role in internalization for α1BARs than for β2ARs.
We have presented evidence that sequestration of receptors within the plasma membrane (defined as a loss of cell surface accessibility of radioligand binding sites) and endocytosis within intracellular vesicles (defined as a shift of receptors from the plasma membrane fraction to a light vesicle fraction with sucrose density gradient centrifugation) are two distinct steps or components of receptor internalization for α1BARs (Cowlen and Toews, 1988; Wang et al., 1997; Toews, 2000). Two specific aspects of those studies prompted us to further investigate the possible requirement of G protein coupling and downstream signaling events specifically in the endocytosis step of α1BAR internalization. First, we showed that agonist plus the PKC activator phorbol-12-myristate-13-acetate could induce endocytosis of α1BARs in DDT1 MF-2 cells, a cell line in which treatment with agonist alone induced sequestration without endocytosis (Cowlen and Toews, 1988). Second, in a subsequent study aimed at investigating the role of the NPIIY sequence in the seventh transmembrane domain of the α1BAR as an internalization signal, we found that the Y348A-mutated α1BAR was essentially completely defective in stimulation of PI hydrolysis and in agonist-induced endocytosis, although agonist-induced sequestration of the mutated receptors within the plasma membrane was similar to that for the wild-type receptor (Wang et al., 1997).
To investigate whether the defect in G protein coupling and PI hydrolysis of the Y348A receptor was responsible for its failure to endocytose and to further investigate the requirement of G protein coupling for other aspects of receptor function and regulation, we have now investigated the binding, functional, and regulatory properties of three additional mutated α1BARs that are defective in stimulation of PI hydrolysis. The results suggest that G protein coupling is not required for α1BAR endocytosis or down-regulation but that the receptor determinants involved in G protein coupling are very similar to, yet distinct from, those required for endocytosis and down-regulation.
Experimental Procedures
Cell culture medium, serum, trypsin, G418, and LipofectAMINE reagent were from Invitrogen (Carlsbad, CA). The Muta-Gene In vitro Mutagenesis kit was obtained from Bio-Rad (Hercules, CA); other enzymes were from New England BioLabs (Beverly, MA). [3H]Prazosin was from PerkinElmer Life Sciences (Boston, MA) and [3H]inositol was from Amersham Pharmacia Biotech (Piscataway, NJ). Epinephrine, phentolamine, sucrose, and other biochemicals were from Sigma Chemical (St. Louis, MO).
Mutagenesis, Transfections, and Cell Culture.
The preparation of the stably transfected CHO cells expressing the wild-type and Y348A α1BARs used in these studies has been described in detail previously (Wang et al., 1997). To generate the Δ[246–261] receptor construct, the cDNA encoding the α1BAR inserted between theHindIII and XbaI sites of phage M13 mp18 was used as the substrate for oligonucleotide-directed mutagenesis with the Bio-Rad Muta-Gene M13 kit as in our previous studies (Wang et al., 1997, 2000). The mutagenic primer for the Δ[246–261] receptor construct was as follows, where the carat represents the site where the deletion was introduced: GTCATGAAGGAGATG ∧ GAGGACACCCTCAGC, deleting residues 246 through 261 (Fig. 1). After confirmation of the mutation by DNA sequencing, the mutated α1BAR cDNA was cut from M13 mp18 by usingHindIII/XbaI and subcloned into the expression vector pRC/CMV, followed by DNA sequencing to reconfirm the mutation. The mutated α1BAR plasmid was then stably transfected into CHO-K1 Chinese hamster ovary cells by using LipofectAMINE, and clones resistant to 400 μg/ml G418 were isolated and screened for α1BAR expression, as in our previous studies (Wang et al., 1997, 2000).
The pCMV plasmids encoding the Δ12 and Δ5 receptors, whose preparation has been previously described (Wu et al., 1995), were kindly provided by Dr. Dianqing Wu (University of Connecticut, Farmington, CT). These plasmids were transfected into CHO-K1 cells together with the pMAMneo plasmid encoding neomycin resistance by using LipofectAMINE, and clones resistant to G418 (400 μg/ml) were isolated and screened for α1BAR expression. Sequencing of Δ12 confirmed the correct deletion of the YIV sequence at residues 227 to 229. However, sequencing of the Δ5 plasmid indicated the presence of additional changes beyond those originally described (Wu et al., 1995), as described under Results and in Fig. 1.
Cells were maintained in monolayer culture in Ham's F12 medium supplemented with 10% fetal bovine serum and 200 μg/ml G418 at 37°C in a humidified incubator with a 5% CO2atmosphere. Cells from confluent flasks were trypsinized and plated in culture dishes at 3000 to 5000 cells/cm2. Cells were typically used for experiments on the 4th day of culture.
Binding and Functional Assays.
Binding and functional assays were conducted as described in greater detail for previous studies (Wang et al., 1997, 2000), with minor modifications. For membrane binding assays, cells grown on 100- or 150-mm dishes were lysed by scraping in hypotonic buffer and membranes were isolated by centrifugation. The membrane pellets were resuspended in binding buffer (20 mM Tris, pH 7.4, 2 mM MgCl2, 140 mM NaCl) and aliquots were incubated with [3H]prazosin in binding buffer for 60 min at 37°C; for these studies 100 μM phentolamine was used to define nonspecific binding. The reactions were stopped by filtration over Whatman GF/B glass fiber filters (Whatman, Maidstone, UK) followed by washing and quantitation by liquid scintillation counting. For saturation assays, six to seven different concentrations of [3H]prazosin were used. For competition binding assays, [3H]prazosin was used at approximately 300 pM and the concentration of epinephrine as competing ligand was varied; this high concentration of [3H]prazosin was used to avoid binding more than 10% of the radioligand for those clones with high levels of receptor expression.
For PI hydrolysis assays, cells grown on 35-mm dishes were prelabeled overnight with 2 μCi/ml [3H]inositol and then stimulated for 20 min with various concentrations of epinephrine in medium containing 10 mM LiCl. Labeled compounds were then extracted from the cells and the aqueous and organic phases were separated. Inositol phosphates in the aqueous phase were isolated by chromatography on Dowex 1-X8 (formate form) columns and quantitated by liquid scintillation counting. Data are presented as the percentage of conversion of [3H]inositol phospholipids to [3H]inositol phosphates.
Internalization and Down-Regulation Assays.
Assays of receptor internalization and down-regulation were all conducted essentially identically to our previous studies (Wang et al., 1997,2000). Assays of radioligand binding to intact cells on ice, referred to as "ice binding assays", were used to assess decreases in cell surface accessibility of receptors, referred to as "loss of ice binding". Cells grown on 35-mm dishes were incubated for 30 min in the absence or presence of 10 μM epinephrine to induce receptor internalization. Cells were quickly washed and then incubated on ice for 4 h with 1.8 nM [3H]prazosin in serum-free medium; nonspecific binding was defined as that occurring in the presence of 10 μM phentolamine. Cells were then rapidly washed in medium containing 10 μM phentolamine and dissolved in 1 ml of 0.2 N NaOH. Radioactivity associated with the dissolved cells was assessed by liquid scintillation counting.
For sucrose density gradient centrifugation assays of receptor endocytosis, cells grown on 100-mm dishes were incubated for 30 min in the absence or presence of 10 μM epinephrine to induce internalization. Cells were washed and then lysed by scraping in ice-cold hypotonic buffer. The lysate was layered on top of a discontinuous sucrose density gradient consisting of 1.7 ml 15% sucrose (w/v), 5.0 ml of 30% sucrose, and 2.5 ml of 60% sucrose. Samples were centrifuged at 28,000 rpm for 60 min at 4°C in an SW41 rotor in a Beckman L8–70 refrigerated ultracentrifuge. Fractions were then collected from the top of the tubes, and binding of [3H]prazosin (1.4 nM) to the membranes in each fraction was determined essentially as described above.
For down-regulation assays, cells grown on 100-mm dishes were incubated in the absence or presence of 10 μM epinephrine for 24 h and then washed and lysed. Membranes were isolated by centrifugation, and binding of [3H]prazosin (3.7 nM) to the isolated membranes was then determined as described above.
Data Analysis.
Nonlinear regression analyses of saturation and competition binding assay and dose-response curve data were performed with GraphPad Prism (GraphPad Software, San Diego, CA). Values for all parameters for all mutations are the averages from multiple experiments, with duplicate or triplicate determinations in each experiment, including assays with at least two different clones and performed on at least two different days. Data are presented as the means ± S.E.M. (n = x, y), where x indicates the total number of determinations and y represents the number of different clones tested.
Mutated Receptor Constructs.
The G protein-coupling properties of two mutated α1BAR constructs with deletions at the amino-terminal end of the third intracellular loop (Fig. 1) were reported previously (Wu et al., 1995). The mutation referred to as Δ12 (deleting three amino acids, residues 227–229) was shown to be defective in coupling to all members of the Gq/11family of G proteins (Gq, G11, G14, and G16), whereas the mutation referred to as Δ5 (deleting 35 amino acids, residues 227–261, including the residues deleted in Δ12) was defective in coupling to Gq, G11, and G14 but retained a low level of coupling to G16. We confirmed the deletion of three amino acids (residues 227–229) in the Δ12 plasmid cDNA by sequencing. Our sequencing of the Δ5 plasmid indicated that 35 amino acids (residues 227–261) were deleted, as originally described; however, our sequencing indicated the insertion of an Asn and a Ser residue at the carboxyl-terminal end of this deletion, as indicated in Fig. 1. Whether these two residues were present in the receptors in the previous study is unknown. We generated an additional related α1BAR mutant, referred to as Δ[246–261], in which only the carboxyl-terminal 16 amino acids (residues 246–261) of the originally reported Δ5 deletion were deleted. Each of these constructs was stably transfected in CHO cells, and the binding, functional and regulatory properties of multiple clones of each construct were investigated and compared with those for the wild-type α1BAR.
Functional Properties of Mutated Receptors.
Epinephrine stimulated PI hydrolysis in cells expressing the wild-type receptor by approximately 4-fold, with half-maximal stimulation at approximately 200 nM (Fig. 2). The EC50 value for the wild-type receptor from this set of experiments is higher than the values of 31 and 43 nM reported in our two previous studies (Wang et al., 1997, 2000). The reason for this lower potency compared with our earlier studies is not completely clear; however, it is likely to result from differences in "coupling efficiency" or "receptor reserve" between the cells used in the earlier studies and those in the current studies, because the affinity of the receptor for epinephrine in competition binding assays has remained essentially constant in all of these studies (Table1). The Δ12 and Δ5 receptors expressed in CHO cells appeared to be completely defective in epinephrine stimulation of PI hydrolysis, similar to the previously reported results with these receptors in transiently transfected COS-7 cells (Wu et al., 1995). The newly generated Δ[246–261] receptor was similarly defective in stimulation of PI hydrolysis. The averages ± S.E.M. of the fold stimulation values with 10 μM epinephrine from the individual PI hydrolysis experiments were 5.08 ± 2.01 for wild-type, 0.85 ± 0.12 for Δ12, 1.14 ± 0.19 for Δ5, and 1.20 ± 0.13 for Δ[246–261]; these values were significantly different from 1.0 (p < 0.05, analysis of variance followed by Dunnett's multiple comparison test) only for the wild-type receptor.
Binding Properties of Mutated Receptors.
Clones with high-level expression were obtained for all of the mutated receptors. Receptor expression levels and antagonist binding properties were tested in saturation binding assays with the antagonist radioligand [3H]prazosin (Table 1).B max values ranged from 0.1 to nearly 10 pmol/mg of membrane protein in the clones analyzed, similar to the range of expression levels obtained for the wild-type receptor. The affinities of all three of the mutated receptors for [3H]prazosin were 2- to 3-fold lower than that of the wild-type receptor. These results are similar to the decrease in antagonist radioligand affinity observed for the F303G and F303N α1BARs that are uncoupled from G protein activation due to mutations in transmembrane domain VI (Chen et al., 2000); however, they are in contrast to our previously characterized Y348A-mutated α1BAR, which had [3H]prazosin binding affinity identical to the wild-type receptor (Wang et al., 1997).
Agonist binding properties were assessed in assays of epinephrine competition for [3H]prazosin binding (Table 1). The Δ12 and Δ5 receptors both exhibited markedly higher affinities for epinephrine than did the wild-type receptor, similar to the results with the Y348A-mutated α1BAR (Wang et al., 1997) and with other α1BARs with defects in G protein coupling and PI hydrolysis due to mutations in transmembrane domain VI (Chen et al., 2000) or in the DRY motif in the second intracellular loop (Scheer et al., 1996). In the previous study with transient transfection in COS-7 cells, the Δ5 receptor exhibited higher affinity than the wild-type receptor for the agonist norepinephrine, but the affinity of the Δ12 receptor for norepinephrine was the same as for the wild-type receptor (Wu et al., 1995). In contrast to the results with the other mutated receptors, the Δ[246–261] receptor exhibited an affinity for epinephrine that was slightly lower than that of the wild-type receptor, suggesting that the nature of the coupling defect for this receptor may be different from that for the other mutated receptors. Together, these binding and functional assays indicate that the failure of these mutated receptors to stimulate PI hydrolysis is not due to a failure of the receptors to be expressed or an inability to bind agonist.
Assays of Agonist-Induced Changes in Cell Surface Accessibility of Receptor Binding Sites.
Assays of [3H]prazosin binding to intact cells on ice were used to assess agonist-induced changes in the cell surface accessibility of the various receptor constructs (Fig.3, top). Pretreatment of cells expressing the wild-type receptor for 30 min with 10 μM epinephrine led to a 26 ± 2% decrease in [3H]prazosin binding. Agonist pretreatment of cells expressing the Δ12 and Δ5 receptors led to 80 ± 3% and 53 ± 6% decreases in [3H]prazosin binding to intact cells on ice, respectively, decreases that were greater than that seen for the wild-type receptor assayed under the same conditions. In contrast to the results with the Δ12 and Δ5 receptors, the agonist-induced decrease in [3H]prazosin binding to intact cells on ice for cells expressing the Δ[246–261] receptor was 24 ± 5%, essentially identical to that for the wild-type receptor.
The loss of ice binding was further investigated in saturation binding assays with intact cells on ice for the Δ12, Δ[246–261], and wild-type receptors (Table 2). Agonist pretreatment induced both a decrease inB max and a decrease in affinity for the Δ12 receptor, similar to our previous results with the Y348A receptor (Wang et al., 1997). However, in contrast to the previous results with the Y348A receptor, the decrease inB max observed in the saturation assays with the Δ12 receptor was about twice as great as for the wild-type receptor, similar to the data in Fig. 3. The values for the loss of ice binding observed in the assays with a single concentration of [3H]prazosin that are shown in Fig. 3 are more similar to the decreases in B maxobserved in the saturation assays in Table 2 for the current experiments than in the previous study, because a much higher and nearly saturating concentration of [3H]prazosin was used in the single concentration assays in the current studies. In contrast to the results with the Δ12 receptor, the loss of ice binding for the Δ[246–261] receptor was due to a decrease inB max with no change in affinity, and the decrease in B max for the Δ[246–261] receptor was similar to that for the wild-type receptor (Table 2).
One possibility for the apparent decreases in affinity seen for the Δ12 receptor in this study and for the Y348A receptor in our previous study (Wang et al., 1997) is incomplete removal of the pretreatment agonist during the washes before the binding assay. Low concentrations of retained agonist from the pretreatment would be more likely to interfere with the subsequent binding assay for these receptors because they have significantly higher affinity for epinephrine compared with the wild-type receptor and the Δ[246–261] receptor, and this is in fact the pattern of effects that is observed. In experiments with the Δ12 receptor pretreated with 0.1 μM rather than 10 μM epinephrine to lessen the possible contribution of retained agonist, the decrease in affinity after epinephrine pretreatment was smaller but still present; thus, retained agonist likely contributes to the observed decrease in affinity, but additional changes in receptor binding properties induced by epinephrine pretreatment may also be involved.
Sucrose Density Gradient Centrifugation Assays of Agonist-Induced Endocytosis.
The agonist-induced endocytosis of the various receptor constructs was assessed by sucrose density gradient centrifugation to separate plasma membrane receptors from those in intracellular endocytotic vesicles (Fig. 3, middle). Pretreatment of cells expressing the wild-type receptor with the agonist epinephrine (10 μM) for 30 min induced a shift of 24% of the receptors initially in the plasma membrane fraction to the light vesicle fraction, indicating that for these receptors, endocytosis into intracellular vesicles is sufficient to account for essentially all of the loss of cell surface-accessible receptors detected in the ice binding assays. In contrast, pretreatment of cells expressing the Δ12 and Δ5 receptors with epinephrine did not induce a shift of receptors from the plasma membrane fraction to the light vesicle fraction. This lack of agonist-induced endocytosis is similar to the results obtained previously for the Y348A α1BAR (Wang et al., 1997). However, pretreatment of cells expressing the Δ[246–261] receptor with epinephrine induced a shift of plasma membrane receptors to the light vesicle fraction that was similar in magnitude to the shift for the wild-type receptor. Together with the results from the cell surface accessibility assays mentioned above, these results indicate that agonist exposure leads to sequestration of the Δ12 and Δ5 receptors within the plasma membrane but not to their endocytosis into intracellular vesicles, similar to the results reported previously for the Y348A α1BAR. In contrast, the Δ[246–261] receptor, in spite of its defect in coupling to PI hydrolysis, undergoes both sequestration and endocytosis similar to the wild-type receptor.
Agonist-Induced Changes in Receptor Expression.
Cells expressing each of the receptor constructs were pretreated with10 μM epinephrine for 24 h to assess their ability to undergo agonist-induced down-regulation (Fig. 3, bottom). Under these conditions, the wild-type receptor was down-regulated by 39 ± 2%. For cells expressing the Δ12 and Δ5 receptors, 24-h pretreatment with epinephrine did not induce down-regulation but led instead to a marked up-regulation of the total number of [3H]prazosin binding sites. For the Δ12 receptor, the value for epinephrine-pretreated cells was 245 ± 29% of the value for control cells (145% up-regulation) and for the Δ5 receptor the value for epinephrine-pretreated cells was 255 ± 16% of the value for control cells (155% up-regulation). In contrast, agonist pretreatment led to 46 ± 2% down-regulation of the Δ[246–261] receptor, similar to results with the wild-type receptor. The down-regulation properties of the Y348A α1BAR were also investigated, because these assays were not included in our original studies of this receptor (Wang et al., 1997). The Y348A receptor did not undergo either down-regulation or up-regulation with agonist pretreatment; the value for binding to membranes from Y348A cells pretreated with10 μM epinephrine for 24 h was 107 ± 6% of the value for control cells (n = 8, 8).
The goal of these studies was to investigate the hypothesis that G protein coupling and subsequent PKC activation are not required for α1BAR sequestration within the plasma membrane but are required for endocytosis into intracellular vesicles and for the subsequent down-regulation of these receptors. Three of the four mutated α1BARs that were defective in stimulation of PI hydrolysis were also defective in agonist-induced endocytosis and down-regulation (Δ12, Δ5, and Y348A), suggesting a relationship between PI hydrolysis and endocytosis and down-regulation. However, the ability of the fourth mutated receptor (Δ[246–261]) to undergo normal endocytosis and down-regulation, despite an apparently complete lack of PI hydrolysis, strongly suggests that PI hydrolysis is not required for either process. Recent studies with other Gq-coupled receptors have also provided evidence that Gq coupling and PI hydrolysis are not required for receptor internalization. These include studies with mutated receptors for AT1 angiotensin receptors (Hunyady et al., 1994), ETA endothelin receptors (Bhowmick et al., 1998), and pituitary adenylyl cyclase-activating protein type 1 receptors (Lyu et al., 2000) as well as studies demonstrating thyrotropin-releasing hormone receptor internalization in cells lacking Gq and G11(Yu and Hinkle, 1999). The data presented here appear to conflict with other evidence that the PKC inhibitor staurosporine is effective at inhibiting α1BAR internalization (Fonseca et al., 1995; Zhu et al., 1996; Awaji et al., 1998). One possible explanation for this discrepancy is that the effects of staurosporine on internalization are due to inhibition of a kinase other than PKC. Alternatively, it is possible that ongoing PKC activity is required but that further activation of PKC by the specific receptor being internalized is not required.
Our conclusion that PI hydrolysis is not required for internalization is based on the properties of the Δ[246–261] receptor. However, we cannot rule out the possibility that an undetectably low level of PI hydrolysis occurs with the Δ[246–261] receptor and that this very low level of second messenger generation is sufficient to promote receptor internalization. A previous study using confocal microscopy to monitor internalization of green fluorescent protein-tagged α1BARs stably expressed in αT3 pituitary gonadotroph cells showed that receptor internalization was blocked by the PLC inhibitor U73122, suggesting that PLC activation is required for α1BAR internalization (Awaji et al., 1998). We attempted to use U73122 as a second approach to investigate the role of PI hydrolysis in α1BAR internalization. However, we discovered that U73122 inhibits ligand binding to the α1BAR at similar concentrations to those that inhibit PLC activation (unpublished data), preventing its straightforward use in these studies. The contribution of receptor blockade to the effects of U73122 will be particularly prominent when high concentrations of U73122 are used together with relatively low concentrations of α1BAR agonist, such as the combination of 10 μM U73122 with 100 nM norepinephrine used in the previous study (Awaji et al., 1998). Thus, it is possible that the inhibition of α1BAR internalization observed in their studies resulted from U73122 blockade of receptors rather than from PLC inhibition. More detailed studies of the multiple and complex effects of U73122 and related compounds on PLC activation, receptor binding, and receptor internalization assessed by various assays are in progress.
Although our results suggest that G protein coupling and PI hydrolysis are not required for endocytosis, the observation that three of the four mutated receptors that were unable to stimulate PI hydrolysis were also defective in endocytosis suggests that highly similar and overlapping receptor domains are required for G protein activation and for endocytosis. The Δ[246–261] mutation differentiates between these two responses, blocking PI hydrolysis but not endocytosis. Thus, amino acid residues 246 to 261 of the α1BAR are apparently critical for proper functional interaction of the receptor with G proteins but not for its interaction with the cellular machinery involved in receptor endocytosis. In contrast, residues 227 to 229 are important for both processes. Whether these residues are directly involved in G protein coupling and endocytosis or whether their mutation alters the structure of other regions of the receptor remains to be determined. Studies of additional mutations, including smaller deletions as well as substitution mutations, will be required to more precisely identify the roles of this region of the receptor and of individual amino acid residues in both G protein coupling and endocytosis. Several recent studies with other receptors have succeeded in separating the structural domains involved in internalization from those involved in G protein activation for various Gq-coupled receptors (Hunyady et al., 1994;Bhowmick et al., 1998; Lyu et al., 2000). Of greatest interest in relation to our data is a recent study of the regulatory properties of the constitutively active D142A-mutated α1BAR, which was able to activate Gq and PI hydrolysis but was defective in both agonist-induced receptor phosphorylation and internalization (Mhaouty-Kodja et al., 1999). This mutation also separates G protein activation from receptor internalization, but with the "opposite" phenotype from our Δ[246–261] mutation, which is defective in G protein activation but able to undergo normal internalization. Further studies based on this interesting pair of mutated α1BARs could yield important information on the distinct structural domains and conformations required for interaction with G proteins versus those required for interaction with the cellular endocytosis machinery.
The results presented here provide further evidence that the process that we call sequestration does not require coupling to PI hydrolysis, because it occurred for all three mutated receptors studied here to an extent equal to or greater than that for the wild-type receptor, similar to previous results with the Y348A receptor (Wang et al., 1997). Phosphorylation of GPCRs by GRKs requires only agonist occupancy of receptors, but not second messenger generation, and GRK-mediated phosphorylation and subsequent binding of β-arrestins have been implicated in the pathway for internalization of many GPCRs (Krupnick and Benovic, 1998; Ferguson, 2001), including α1BARs (Diviani et al., 1996, 1997). GRK-mediated phosphorylation of α1BARs followed by β-arrestin-mediated targeting of the phosphorylated receptors to an inaccessible subdomain within the plasma membrane is thus a likely mechanism to account for the process that we call sequestration, although this remains to be demonstrated. Whether this "compartment of sequestration" is an intermediate compartment on the pathway to endocytosis in clathrin-coated pits, vesicles, and endosomes, or whether it represents an alternate pathway to remove receptors from accessibility to ligands at the cell surface, remains to be determined. Identification of this compartment may also help to explain the greater extent of sequestration observed with the Δ12 and Δ5 receptors than with the wild-type and other mutated receptors. The Δ12, Δ5, and Y348A mutated α1BARs, which traffic to this compartment but do not undergo endocytosis, provide powerful tools for assessing the nature of this compartment and the molecular mechanisms involved in receptor transport to and from this compartment.
The three mutated receptors that were defective in endocytosis were also defective in down-regulation. The Y348 receptor did not down-regulate in response to agonist, whereas the Δ12 and Δ5 receptors showed a marked up-regulation in response to agonist. The Δ[246–261] receptor, which endocytosed normally, also down-regulated normally. If down-regulation is mediated by receptor protein degradation in lysosomes after receptor endocytosis, still the most widely accepted mechanism (Tsao et al., 2001), then it would be expected that defects in endocytosis would correlate with defects in down-regulation, as observed with these mutated receptors. However, our recent studies of α1BARs with carboxyl-terminal tail mutations showed that down-regulation can occur even for receptors that are completely defective in rapid receptor sequestration and endocytosis, raising questions about the lysosomal degradation hypothesis for down-regulation of these receptors (Wang et al., 2000). Down-regulation without endocytosis has been reported for other receptors as well (Tsao et al., 2001). Studies are in progress to further characterize the prominent up-regulation observed with the Δ12 and Δ5 receptors and to identify the mechanisms involved; potential mechanisms include the nuclear factor-κB-mediated mechanism recently proposed for up-regulation of serotonin receptors (Cowen et al., 1997) and the ligand-mediated stabilization of "inherent instability" that has been shown to occur for both constitutively active and inactive mutations of adrenergic and other receptors (Gether et al., 1997; Alewijnse et al., 2000; Wilson and Limbird, 2000).
The three mutated receptors that were defective in endocytosis also exhibited affinities for the agonist epinephrine that were 8- to 40-fold higher than that for the wild-type receptor (Table 1; Wang et al., 1997), similar to results with several other α1BAR mutations that inhibit coupling to PI hydrolysis (Scheer et al., 1996; Chen et al., 2000). In contrast, the Δ[246–261] receptor exhibited an affinity for epinephrine slightly lower than the wild-type receptor. Thus, although it does not stimulate PI hydrolysis, the Δ[246–261] receptor is otherwise more similar to the wild-type receptor than to the other three mutated receptors in terms of agonist binding affinity as well as internalization and down-regulation properties. These results suggest that the nature of the coupling defect for the Δ[246–261] receptor may be different from that for the other mutated receptors and that further studies of the Δ[246–261] receptor could provide new insights into G protein coupling for α1BARs.
In summary, we have generated a new mutated α1BAR that is defective in stimulation of PI hydrolysis but exhibits binding and regulatory properties different from those of three other α1BARs with defects in PI hydrolysis that have been described previously. Together, our studies of these mutated receptors suggest that at least four responses to agonist occupancy of α1BARs can occur without detectable stimulation of PI hydrolysis, namely, sequestration, endocytosis, down-regulation, and a novel up-regulation. Occupancy of the ligand binding site of the receptor by agonist and the resulting conformational changes alone may be sufficient to mediate these responses; alternatively, it is possible that one or more of these changes are mediated by as-yet-unidentified G protein-independent signaling pathways, several of which have been recently identified for other GPCRs (Heuss and Gerber, 2000). These mutated receptors provide a powerful set of tools for more detailed investigation of the specific receptor domains and conformations mediating both receptor signaling and the multiple adaptive responses that occur after agonist binding to these receptors.
We thank Dr. Dianqing Wu (University of Connecticut) for providing the Δ12 and Δ5 mutated receptor constructs, and Drs. David Bylund (University of Nebraska Medical Center) and Susanna Cottechia (Institut de Pharmacologie et de Toxicologie, Facultéde Médecine, Lausanne, Switzerland) for helpful discussions.
- G protein-coupled receptor
- α1BAR
- α1B-adrenergic receptor
- phospholipase C
- phosphoinositide
- protein kinase C
- β2AR
- β2-adrenergic receptor
- G protein-coupled receptor kinase
- Chinese hamster ovary
- Received July 17, 2001.
- Accepted September 21, 2001.
- The American Society for Pharmacology and Experimental Therapeutics


text:
Finding it a symbol of racism and hate, Gov. Tom Wolf ordered a Confederate flag removed from a Flag Day exhibit in the state Capitol after the chairwoman of the black legislators' caucus took it down but event organizers put it back up.
Wolf took action Wednesday after Capitol police had been summoned to retrieve the flag and return it to the display.
The reproduction of an 1863 Confederate battle flag was part of a historical society's collection of flags that have flown over what is now the U.S., including from territories and the colonial period.
Rep. Vanessa Lowery Brown, a Philadelphia Democrat, said she was astonished to see the flag as she walked through the Capitol's east wing on Tuesday.
"I just did what I thought was right and I took the flag down," said Brown, who heads the Pennsylvania Legislative Black Caucus.
She carried it into the chambers and gave it to the House speaker.
More @ ABC

subset: commoncrawl


text:
Please learn it. It'll save you so much time on the phone.
Alpha, Bravo, Charlie, Delta, Echo, Foxtrot, Golf, Hotel, India, Juliet, Kilo, Lima, Mike, November, Oscar, Papa, Quebec, Romeo, Sierra, Tango, Uniform, Victor, Whiskey, X-Ray, Yankee, and Zulu.
Ever tried to spell something out and the other person mixes around letters?
S—Not F, S! S as in Sam!
That doesn't work.
Sierra, Echo, November, Delta, November, Uniform, Delta, Echo, Sierra.
Works like a charm.
EDIT: For those that don't get it, what does it spell?


text:
I went to Mexico thinking this would be the worst of the 4 hotels we were due to stay in, and it ended up being the best by far. I've stayed in a couple of these in the UK and they've been awful but I loved it!
We arrived at 6.50am after an overnight bus expecting just to drop our bags but they had a room ready and let us check in for no extra charge which was excellent.
The room was HUGE! Two big double beds, sofa, desk area and the shower was probably the biggest I have ever been in, bigger than a double. There was also a safe, coffee percolator, aircon and mini balcony - none of which you will get in the UK version of an Express.
Rooms black out completely at night and the only noise transference was from noisy guests in the corridor. Great night's sleep.
Breakfast was good for a buffet though I have had better (they are a bit slow refilling the hot food when it's gone and it goes cold very quickly). That's pretty much the only fault I could find. You may perhaps want to be a bit nearer town but it's an easy and very safe 10-15min walk (this is obviously a middle class residential area) and a very easy walk to the 1st class bus station.
My biggest annoyance was probably the other guests - it caters to a lot of tour groups who all breakfast at the same time so you are sometimes forced out to the patio.
I'd definitely stay here again
- Official Description (provided by the hotel):
- The Holiday Inn Express Centro Historico Oaxaca is conveniently located in the Historical Center of the city just a short walk from promenade which leads to the town square (zócalo). The beauty of this award winning hotel is complemented with an art gallery of original oil paintings from the most renowned Oaxacan artists. All our rooms and suites feature remote-controlled air conditioning, hair dryer, coffeemaker, iron and ironing board, telephone with two lines and data port. Express Breakfast (American Breakfast Buffet), business center, heated outdoor swimming pool, indoor parking on premise and special events hall with capacity for up to 170 persons are additional facilities and services offered by the hotel. The Holiday Inn Express Oaxaca represents a style of service and hospitality that is traditional to the State of Oaxaca. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with IHG, Expedia, Hotels.com, Travelocity, Hotwire, Cancelon, HotelQuickly and getaroom.com so you can book your Holiday Inn Express Centro Historico Oaxaca reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Holiday Inn Express Centro Historico Oaxaca Hotel Oaxaca
- Holiday Inn Express Oaxaca
- Holiday Inn Oaxaca


text:
|( 7 upvotes )
|...aaand, I imagine you have a nice a**s.
|Quite a-ite, seven. You have provided entertainment in many forms for this old fossil for a rather long time now. I have always appreciated the creative side of that mind of yours. You offer a remarkable challenge to keep up.
|Gaaaaaaaay!Sorry...that was a typo...I dropped the "r".
|I....I....I think I love you, Steven.
|You may be old, Gaz and have older attitudes but from what I have gathered from your chats and comments, you're a class act to everyone...no matter what. :)
|Ralf (not an acronym). Sorry, I'm old, and apparently have a weak stomach.
|I'll be looking for a picture of an Asian, transgendered, Jewish single, homeless, disabled mother with a Downs child, next...and that still won't cover everything...Prejudice is wrong in whatever form it takes...
|You forgot "transgender"
|( 30 upvotes )
|I'll Try !
|very nice Gary
sooooooooooooooooo good
|And now, now I get to play
|Had to come to meetings with this mind set in order to contend with idiotic investors and megalomaniacal corporate honchos.
|Y'know, Bandit, its funny (not haha funny), I've been turning things upside down and backward in the workplace for over 40 years.
|Gary always sheds a different light on a situation.
|'Works'? it's all play, Connie, a-l-l-l-l play. Thnx guys.
|It's funny how the mind works. I'm not talking about the poster I'm talking about you. another 5 er
|Thank you, Mr. Gary. Much appreciated
|( 10 upvotes )
|as Alex would say "a 5er right right"
|Geez steve, 20 shares in like...now? Whatchoo got, man?
|orange you glad I loved it?
|( 7 upvotes )
|And that'd be the house I'd love to see!!!
|...Especially the elaborate Halloween-themed murder-suicide crime-scene displays put-up on some peoples lawns!... Oh-wait, that's just on mine... Just a display, yeah.
|Black, lots of black! and purple! Spiders, webs, fake blood! Not only time to freshen up my décor, but stock up for pranks! I luuuuuv Halloween! 5 goth lions!
|( 9 upvotes )
|Gimme time....
|Nick, while you take the time to leave a gamut of witty remarks on various posters, how 'bout grabbing some pics and try a hand at yer own, hmmmm?
|How 'bout some nice, relaxing milk? Does a body goooooooood....
|i invited him, he should be a good opponent
|Thanx Missy.
|A Mr. Gary tribute...impressive 'cern! Congrats! And 5Ler Mr. Gary.
|He most certainly would. Thnx JayDub
|Nice one, Gary! I wish cern would join the poster evolution. He would certainly add a new dimension to it. 5.
Misc info


text: Just wanted to share my birthday gift from my "special someone". A new wallet! It's my first Dior anything, and I've been eyeing it for a while. I think it's an older model (if anyone knows the name, let me know!), but I love it none the less! Yay for me! It "almost" makes getting older a bit more bearable!


text:
Let's start stickering. Buy 4, save 25%. Buy 10, save 50%.
Oil on Canvas. 28″ × 20″ SOLD
landscape painting oil original country nature sheep animals the lake disrtict cumbria uk
Incredibly beautiful. You are truly the best painter on RB. I am in awe of your talent Joe.
Ah thanks so much Lyn. Regards Joe
Wonderful work Joe! Instant fav!
Many thanks for your comment. Regards Joe
fantastic work Joe Beautiful Scene
Glad you like it Maggie, thank you. RegardsJoe
Beautiful work Joe! This is great :-)))R
Thank you very much. Regards Joe
You are the master of landscape Joe! i am always amazed at the amount of detail and how you capture the light…and the sheep are painted perfectly.
Thanks for your great words Joe, means alot. Regards Joe
Beautiful composition and details, it is a lovely location and your work has done it justice!!
I appreciate that Shaun, thank you. Regards Joe
Stunning work!
Thank you. Regards Joe
Great work you are very clever
Thanks so much Julie. Regards Joe
Superb pastel colours and stunning detail, you have captured the light of the area perfectly.
Much appreciated David. Regards Joe
Awesome ! Another winner Joe.
Thank you Stephen. Regards Joe
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text:
MOVIE SYNOPSIS [ General Audiences ] With the film direction of Cyprien Vial, Harmandeep Palminder leads the cast of this film of France, with script in English (see subtitles), whose presentation to the public was scheduled for 2014. You can watch Bébé tigre online on TV cable or theaters with original audio in English at different dates. This film premiered in theaters in 2014.
Rating: 5 by 16 users./
Runtime: 87 minutes
Harmandeep Palminder, Vikram Sharma, Elisabeth Lando, Bilal Baggad, Billel Brima, Amandeep Singh, Karim Leklou, Aurore Broutin, Gérard Zingg, Marie Berto, Lauriane Escaffre, Arsh Kumar, Sukh Palminder, Mahipal Singh, Navpreet Singh and Agnès Tassel.
87-minute film (full movie) directed by Cyprien Vial.
The original audio of Bébé tigre has dialogues in Dolby Digital, it was produced in France.
It premiered in cinemas or television in 2014.
Screenplay written by Marie Amachoukeli-Barsacq, Cyprien Vial and Céline Sciamma.
Pierre Cottereau.
Isabelle Madelaine and Emilie Tisné.
This film was produced and / or financed by Centre National de la Cinématographie (CNC), Fonds Images de la Diversité, Sofica Manon 4, Ciclic - Région Centre, Dharamsala, Darius Films, Ciné , Procirep, Angoa-Agicoa, Canal and Région Ile-de-France.
Haut et Court.
Without commercial breaks, the full movie Bébé tigre has a duration of 87 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in EnglishDolby Digital in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, Bébé tigre in FULLTV Movies.


text:
Community Partner Sign Up
Community Partner Application (Open registration)
Presenting Sponsor
|AMITA Health – will be at the market weekly providing various health screenings and great freebie stuff! "Like" them on Facebook!
|BMO Harris – staff from the Kimball/Dundee branch will be on hand at the information booth helping people use their Link and credit/debit cards out at the market. Plus they will be offering their double value offer when using your Link Card. For more info click HERE. "Like" them on Facebook!
|Davey Tree Expert Company – "Ask an Arborist" – providing weekly expert tree service information and answering your questions at the market from bushes to trees. "Like" them on Facebook!
|Waste Management – Providing clean restrooms and hand washing station at the market, as well as trash and recycling receptacles. "Like" them on Facebook!
|Chiro One Wellness Centers – Giving free spinal screenings and information with great customer care! "Like" them on Facebook!
|Shared Harvest Food Cooperative – a local foods grocery store in the making – they will be on hand with information and how to buy shares! "Like" them on Facebook!
|Ziegler's Ace Hardware – a local Downtown Elgin hardware store for one's garden needs and DIY projects. "Like" them on Facebook!
|Boys & Girls Club – A positive place for at risk youth grades 1st-12th to participate in activities with mentors.
|CCI Greenheart – Since 1985, CGI Greenheart has offered cultural exchange programs in the U.S. that connects American with international students. As the leading nonprofit cultural exchange sponsor that blends service learning and volunteerism into all their programs, they are effectively creating a community of compassionate and conscious global citizens. CCI Greenheart is a division of the global nonprofit, Greenheart International.
|City of Elgin Parks & Recreation – Fitness center located in Downtown Elgin containing two indoor pools, meeting spaces, childcare, indoor track and pavilion, a gym and much more!
|Community Crisis Center – Their mission is to provide Safety, hope, advocacy, respite and education for those impacted by crisis and violence in the Fox Valley Region and Illinois. Will be on hand with informational materials about the Crisis Center and its programs.
|Congresswoman Tammy Duckworth (Field Office) – Congresswoman Tammy Duckworth serves the residents of the 8th Congressional District of Illinois. Residents of the 8th District will have the opportunity to share their questions, comments and concerns with the Congresswoman's staff.
|Ecker Center – Founded in 1955, the Ecker Center is a non-profit outpatient community mental health center. The Ecker Center provides comprehensive mental health services for adults as well as 24/7/365 psychiatric in-person emergency evaluation and assistance for people of all ages including children. Their clients are primarily very low income people. They will be promoting their largest fundraiser of the year, the Festival of Trees, which is annual event in November.
|Elgin Bicycle & Pedestrian Advisory Committee – Promote safe and comfortable accommodations for cycling and walking, and improved access to transit in Elgin. Education, promotions, advice recycling cycling, walking & transit in Elgin.
|Elgin Climate Change Organization/Sierra Club – will be on hand with interesting information on advocacy for the environment and sustainability relating to Sierra Club and climate change.
|Elgin Area History Museum – is a non-profit organization whose purpose is to preserve and interpret Elgin's history by providing educational opportunities for residents of the city and surrounding communities. Old Main is listed in the National Register of Historic Places. Through permanent and changing exhibits, the Museum brings to life Elgin's rich heritage.
|Elgin Public Museum – of Natural History and Anthropology serves the public by enhancing knowledge of the natural sciences and cultures, past and present, through the use of exhibits and interactive experiences.
|Gail Borden Public Library – The library serves 134,000 residents in Elgin, South Elgin and portions of Hoffman Estates, Streamwood and Bartlett. In June they will be marketing their summer reading program, Read to the Rhythm and their showing of "Project 2-3-1". Visit their website to learn more happenings at your local library!
|Girl Scouts of Northern IL – Girl Scouts of Northern Illinois (GSNI) serves over 17,000 girls and 4,000 adults in 16 counties in northern Illinois. Their mission is to build girls of courage, confidence, and character, who make the world a better place. Through a variety of leadership-oriented and girl-led programming, girls participate in age-appropriate activities in the areas of Healthy Living, STEM (Science, Technology, Engineering and Math), Financial Literacy, Outdoor and Environmental Awareness, and Community Service.
|Hemmens Cultural Center – Elgin's premier arts and entertainment venue is nestled on the banks of the Fox River in Elgin's Civic Center, and is at the heart of Elgin's growing arts community. This intimate 1,200 seat theatre is a favorite venue for seeing stars up close. With an expansive Exhibition Hall and ample Lobby space The Hemmens offers a unique style for almost any occasion; including weddings on the stage. Watch this summer for Elgin Summer Theatre's production of "Next to Normal".
|Judson University World Leaders Forum – The World Leaders Forum is an event that raises money for scholarships and an endowment fund to start and entrepreneur program at Judson University. The University hosts world leaders like Condoleeza Rice and George W. Bush to inspire the Elgin community and Judson students, alike.
|Literacy Connection – is a community-based affiliate of ProLiteracy. Their trained volunteers provide free, accessible, and customized one-on-one tutoring. Their mission is to help adults reach their literacy goals. They also provide English conversation groups, Family Literacy, and small group tutoring. They serve learners in 16 northwest suburban Chicago communities. Games, activities, contests, etc. to raise awareness about literacy and the need for tutors in the Kane County area will be at their booth. Tote bags will occasionally be offered, as well as other items such as bookmarks, pencils, etc.
|Side Street Studio Arts – was founded as an outlet for the Elgin arts scene. Side Street's mission is to provide an accessible space encouraging all aspects of the artistic process from creation to curation. We are pleased to announce that Side Street Studio Arts will lead the DNA's Harvest Market "Rainbowland – An Adventure at the Market" for kids. Rainbowland will run from 12pm-2pm on market days, June through the beginning of August.
|University of Illinois Extension – Kane County Master Gardener Program – Master Gardeners help others "learn to grow" by answering gardening questions, identifying insect, disease and other plant problems, distributing fact sheets and bulletins, presenting garden lectures and demonstrations, and supporting community projects. Master Gardeners will answer gardening questions and distribute horticultural information.
|YWCA Elgin – Dedicated to eliminating racism, empowering women and promoting peace, justice, freedom and dignity for all.


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|Professional Automotive Repair Technical Discussions for August 31, 2007
International Automotive Technicians' Network (iATN) - Members Only
|(Also Available: All, Asian, Chrysler, European, Ford, GM.)
|Vehicle Owners: If you are seeking an automotive repair professional to service your vehicle, visit the iATN Repair Shop Finder.
Open Help Requests for European
|Help Request
|2000 Audi S4 2.7L 1st gear VIN: WAUDD68D6Y
Hi Audi tech's this S4 has a whine in first gear and cannot really hear in second but it still sounds like a main shaft bearing. Just wondering before I tear down if anyone has seen this only in 1st? No metal in oil. We have delt with shift issues but no noise...
|2000 Jaguar XKR 4.0L Right Rear 1/4 window VIN: SAJJA42B8Y
Hello Group, The right rear quarter window will not move on this convertible. The noises that it makes sounds like a typical window regulator which I have ordered and have here. I have not done any disassembly on the vehicle yet because I thought it would be ...
|2004 Land Rover Range Rover HSE 4.4L radio VIN: SALMF11464
This nightmare has inoperative FM radio, making the aftermarket XM satellite radio inop. I recently removed the glove box to replace the blower motor regulator. after returning car to customer they called back and stated the FM and the XM would not play. AM ra...
|1986 Mercedes-Benz 300E 3.0L a/c vents VIN: WDBEA30046
Good morning everyone I have an 86 m/b 300e that came in for no air from the center ducts. It comes out good through the outer ducts and the defrost. I suspected a vacuum leak so I checked the source at the intake and the various vacuum hoses under the hood an...
|2000 Mercedes-Benz ML320 3.2L Hesitation, WONT TAKE THE GAS VIN: 4JGAB54E1Y
Why did I take a Benz in for service !!! Here goes... This vehicle is starving for fuel, worse on cold start yet idles good. Upon accel falls on its face... sometimes if gas pedal is slowly pushed rpm's will come up to the rev limiter and run good. Fuel presur...
|2003 Mercedes-Benz C230 Kompressor 1.8L Coolant Warning VIN: WDBRN40J93
We have a C230 that will start normally and show "no malfunction" on the driver's information panel. After about 30-45 secs a "coolant- visit workshop" warning comes on. The coolant level sensor has been replaced, the coolant has been flushed, the coolant temp...
|1999 Volkswagen Beetle GL 1.8L Poor Idle Quality, Cuts out, Stall, MIL Lamp on VIN: 3VWCD21C6X
Hello Everyone. I have a 99 New beetle that likes to die intermittently. This problem happens with no warning or symptons. The customer is a little old lady,and she drives this car like one. She can get it to malfunction at every stop sign. When I drive it try... Trouble codes: P1127 (Boost pressure control exceeded)
|2000 Volkswagen Beetle GL 2.0L MIL Lamp on VIN: WVWZZZ9CZY
Carried out tests according to trouble tree, and come up blank. Cleared MIL, after 3 days MIL back on, same code. Vehicle has been to agents on numerous occasions apparently for same problem. I have noticed in past posts that MAF failure seems to be a common p... Trouble codes: 16556 (P0172 System too rich)
|1998 Volvo S70 2.4L engaged all the time VIN: YV1LS5570W
looking for volve tech's that may have seen this concern.starter will not stop running unless i remove the relay.also the headlamps and heated seats and def switch and the blinkers all work with the key out of the ign.unless i pull the 60 amp fuse that goes to...
|2002 Volvo V70 X/C 2.4L accessories VIN: YV1SZ58D92
We have purchased a new CEM and have been unable to load it with VIDA. VIDA cannot communicate with the vehicle with new CEM in place even after entering VIN in manually it will not recognize the new CEM to this vehicle. If you command VIDA to read the vehicle...
Fixes & Summaries for European
|2003 BMW 325Ci 2.5L Compressor VIN: WBABS33492
I want to thank everybody for their help. I feel kind of dumb. As it turns out the ground wire on the compressor was loose. I could not see it until I removed the compressor. I was ready to purchase the BMW support for my Auto Enginuity if I needed to. For s...
|1992 Jaguar XJ6 4.0L Electronic VIN: SAJK1743NC
The jag is fixed Thanks for all the help. Yes it was the darlington transistor. I took the blower motor out and replaced the transistor and everything went to working fine. There is still a door problem but a no go from owner. A big thanks to Donald Blanchard ...
|1993 Jaguar XJ6 4.0L battery VIN: SAJHW1747P
We found the left front seat lumbar inflater switch jamed and holding the switch in the "on" position.
|1994 Jaguar XJ6 4.0L No Start VIN: SAJHX1741R
The problem was water in the fuel. I had checked or replaced everything on the ignition side and I checked all the specs, grounds and voltages for the fuel system right at the ECM connectors and found nothing out of line. With nothing else to check I figured i...
|1988 Mercedes-Benz 560SEL 5.6L Poor Idle Quality, Stall VIN: WBDCA39DOJ
well checked all the basics,..all good,..replaced crank sensor,..and checked all the other items as suggested,...car ran great for like 3 weeks and started all over again,..so the customer has the car and we will continue to try and diagnose the problem,...but...
|1988 Mercedes-Benz 560SL 5.6L Cuts out, Stall VIN: WDBBA48D1J
Signal from throttle position sensor was normal, but on suggestion, plugged in new sensor and car did not die for 2 days, running about 2 hours each time. Properly installed sensor 8/30, and car ran all day yesterday and no problems in 2 hrs this morning, so ...
|1983 Porsche 944 2.5L diff noise VIN: 1GWNT308T9
This 944 is not fix yet,I found the noise come from the torque tube,will replace the torque tube and also will check out the clutch system.Thank you for all replies,will post the fix latter.
|1995 Volkswagen Jetta GL 2.0L Excessive Emissions VIN: 3VWRC81H6S
|1998 Volkswagen Jetta GL 2.0L Poor Idle Quality VIN: 3VWRB81H9W
Once again, thanks to all who replied. The common concern seemed to be with grounds. One frequent reply was about the ground from batt to trans bell housing. If this is left off, and tried starting, it would damage the computer, even if later reconnected pr...
|2000 Volkswagen Beetle GLS 2.0L Hesitation, Stall VIN: 3VWCA21C8Y
Thanks to John [...] The Fix... Replaced the 3rd MAF, ran throttle adaptation, after dissconnecting the battery & touching cables together to drain completly. (The MAF was bad but car WOULD NOT perform untill battery was disconnected & cables connected & dra...
|2002 Volkswagen Jetta GLS 2.0L A/C Compressor VIN: 3VWSB69M12
I would like to take the time to thank you for all of your help!! It turned out that the shop was checking compressor voltage at the A/C compressor after installing the cooling fan control module and was receiving no voltage. When I arrived to recheck the vehi...
|1998 Volvo V70 2.4L MIL Lamp on
Mass air flow good - No Vac leak - problem still persists - Customer has asked for black tape to cover light. Thanks to everyone.


text:
Currency Displayed in
Gateway to multimedia
For those who want a larger display and simple controls, JVC's KW-V11 DVD receivers supplies a vibrant 6.2" touchscreen that lets you control and see info from your music and videos. Keys on each side of the screen allow you to access Source, Eject, Volume, and other frequently used functions, and you can customize the screen to display your five favorite sources.
You can pop in CDs, DVDs, and USB drives loaded with digital music for hours of tunes for the road. Plug your iPod, iPhone, or flash drive into the front USB port for even more music on the go. A big thumbs up — iPhone users even get to enjoy Pandora® Internet Radio using the receiver's display and controls.
Video on the go
You can watch your favorite DVDs on the built-in screen whenever you're parked. This JVC includes a rear A/V input for a portable video player or video game console, along with a rear input for a handy backup camera. Once installed, the JVC's screen will show the camera feed when your vehicle is in reverse — a great feature, especially if you drive a larger vehicle.
Control your music
When your iPod's connected, you can view album art and browse through your playlists with a few swipes of your finger. Of course, the KW-V11 plays all your CDs, DVDs, and recordable discs, plus you can plug a thumb drive loaded with hours of MP3, WMA, and WAV music into the receiver's USB input.
The KW-V11's touchscreen controls let you easily navigate to your sources and set up your system. When your music is playing, you can use its Monitor Touch Control to quickly swipe basic controls such as skipping tracks and cranking the volume.
Apps for iPhone 4 or 4S
Got an iPhone 4 or 4S? Using JVC's App Link Mode, you'll get basic control over audio apps like Tunein Radio, including functions like play/pause or track up/down, and you can also view traffic info from Inrix, Cobra iRadar, and other driving apps. For powerful navigation, download the MotionX-GPS Drive app (subscription required) and you'll be able to see maps and live traffic reports on the receiver's screen. You'll still need to access your iPhone with this app, so consider adding an iPhone mount for easy use.
Making it all sound great
Select one of the eleven preset EQ settings, or you can use the 5-band parametric equalizer to adjust the tone for your separate audio sources. The KW-V11 also provides three pairs of RCA preamp outputs that'll send a clean, high-voltage signal to any outboard amps in your system. You can even adjust the crossover points for the speakers and a connected subwoofer.
Want to keep your steering wheel controls?
In most cars, you'll need an adapter to retain those controls with this car stereo. Connect a few wires, then program the adapter for your particular car and radio, and you'll retain the convenience of your steering wheel controls. You'll see the adapters that work with your vehicle when you add this receiver to your cart.
iPod/smartphone note: If you're going to use your iPod or smartphone with this receiver, be sure to check "Details" for compatibility information.
Product Highlights:
General Features:
DVD/CD player with AM/FM RDS tuner
built-in amplifier (22 watts RMS/50 peak x 4 channels)
6.2" widescreen touchscreen with Monitor Touch Control
App Link Mode for control of music, navigation, and other apps with iPhone 4/4S
Pandora control with select iPhones
USB 1A connection provides extra power for charging devices
fits double-DIN (4" tall) dash openings
optional remote control
Audio/Visual Features:
plays CDs, DVDs, and USB memory devices (see "Details" for full capabilities)
11 EQ preset sound modes and 5-band parametric EQ
high- and low-pass filters
built-in iPod, iPhone, and iPad control (see Details tab for model compatibility)
inputs: front USB input, rear A/V input, rear-view camera input
outputs: video (RCA) output, 6-channel preamp outputs (4-volt front, rear, sub)
This product hasn't received any reviews yet. Be the first to review this product!
All prices are in USD.


text:
Summer Tyme (AKA Summer) is a dark-haired American beauty from Schenectady. She first showed up in 2002's Sorority Babes 2, getting nasty alongside sorority sisters like Shelbee Myne. It was enough to get her noticed and get fans wanting more, though she would only shoot a handful of movies before disappearing from the circuit around 2005. Measuring a gorgeous 34A-26-32, this beautiful and talented slut will remain a part of our collective fantasies forever.
She showed up in Gang Bang Squad, getting her interracial freak on with four Black studs, including Shane Diesel. Summer starts with blowjobs all around, and then the studs take turns smashing her doggystyle. She takes her time, focusing on just a guy or two at once and making sure everyone gets what they came for. The action ramps up as it moves along, and Summer is eventually getting her asshole stretched and taking double penetration. The cumshots are so heavy, she can't even open her eyes.
Summer really seems to have a thing for taking it up the ass, as she does anal in most of her movies. Her blowjobs are incredible, too, and definitely deserving of more camera time. To this end, she starred in Oral Fantasies 1, a flick that showcased her oral abilities. Summer Tyme seems to be done with the porno business, but perverts are happy to discover her work and become new fans.


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Imagine life as one big spinning wheel, the outer skirts can be a dangerous, chaotic and dizzy, at any moment we risk falling off completely. As we migrate toward the centre we find the balance, calm and harmony that has always been there within us. A peaceful resting place where everything is just as its meant to be. This is called contentment.
Throughout life, I think we will always tether between the inner and outer skirts of the spinning wheel. Whatever point we are at and whatever we are gripping onto just to stay on the wheel there will always be important and fundamental lessons to being human. The only way to move towards the centre is to open our hearts and minds, let go of what is pushing us outwards and live moment to moment, as we silence the monkey mind we are open to hear, acknowledge and understand what the universe is telling us.
The goal is to find our centre of gravity, understand our place, our existence, our purpose. Go with the natural flow and allow the miracle of life to dazzle you and take you higher than you have ever been before.
2016 taught me a lesson or two, I believe we can read information and comprehend the concepts but we will not fully understand what it really means until we learn through first hand experience. These are things I thought I understood until my path lead me to learn them for myself.
- Read those good vibe books you have been wanting to get to.
- Learn to be alone without being lonely.
- Experience life in all of its dualities, You cant stop the waves but you can learn to surf.
- Practice gratitude.
- Yoga Every day.
- Meditate, even if its only 5 minutes.
- Give love without expectations or conditions.
- Get out in nature, feel her communion and experience her wonder.
- Failure is success delayed, don't get too caught up in the chatter.
- Understand that your ego is a fictitious character made up by your mind. Be humble. there is no room for hierarchy.
I'll be honest, I sometimes forget the very same lessons I have learned, as mentioned before, we are human. The important thing is that we are always reaching for the centre of the spinning wheel. We will revisit the same lessons over and over and we will always be encountering new ones along the way. Life is a journey and my intention is to be present for every second of it. There should be no wishing to reach the destination, because then what? you've made it? all dressed up and no where to go from there…so its time to embrace the now.
My recommended feel good books to bring you into the now.
Namaste and welcome 2017
Experience life in all of its dualities here
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Staying physically active is the best way to stay physically and mentally fit after a limb loss. Most sports have some kind of adaptation that allows players of all capabilities to participate. For today's blog post, JP&O chose to focus on some of the most popular competitive sports among individuals with prosthetic devices or wheelchairs.
Volleyball can either be played sitting or standing. In sitting volleyball, players must keep at least one buttock in contact with the floor whenever they make contact with the volleyball. In a standing volleyball match, athletes have a choice to play with or without their prostheses. Athletes with amputations, spinal cord injuries, cerebral palsy, brain injuries, and strokes are eligible to compete.
Tennis is one of the fastest growing wheelchair sports in the world. It can be integrated easily because the same court, rackets and ball are used. It is a popular sport for many amputees because upper and lower limb amputees, as well as individuals using walkers and wheelchairs, can participate. The game rules are the same as able-bodied tennis, with the only exception being that a wheelchair player can allow the ball to bounce twice.
Basketball is the most popular sport for individuals with limited mobility. Most of the major rules remain the same with a few exceptions. The player's wheelchair is considered part of the player and general rules of contact and fouls apply. There are also rules about dribbling and how many pushes a player may use without being called for a traveling violation.
The rules for swimming are adapted to accommodate those with limited mobility. The starting positions vary and there are adaptations allowed for visually impaired swimmers. Swimmers may start the race standing on a platform and diving in, sitting on the platform, or starting in the water.
Sports are a great way to stay active and socialize with new people. They have also been found to increase confidence in those who participate. At JP&O, we can help you find the best device to help your performance and keep you in the game!Tags: limited mobility, physical activity, wheelchair sports


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Speaking with an elderly parent whom you believe needs care or assistance can be very difficult, especially if they are resistant to the idea. They may feel strong and healthy enough to care for themselves, or they may simply not be comfortable with the thought of having a stranger caring for them. If they are developing any level of dementia, they may not be able to rationally see their own need. When approached about this topic, many seniors fear they are losing personal control and independence. Adding to this emotional upset is the complex, new relationship forming between parent and child. Their rolls are shifting and now the adult child must make decisions for their parent. This new identity structure can feel strange and uncomfortable for both, and may add more friction to the situation.
Strategies for Communicating with Parents
Consider the following strategies as you plan for a conversation with your parent about their care needs:
1. Listen and learn why they are resistant. Avoid interrupting them — give them plenty of time to express their feelings so that you can understand why they do not want or think they need help. It could be something expected, such as a fear of the cost involved. Or, they may be apprehensive because they think the goal is to place them in a nursing home instead of simply arranging for a few days of in-home assistance each week.
2. Sometimes resistance and inability to think clearly is effected by illness, especially if they are experiencing dementia or Alzheimer's Disease, or if they have had recent surgery or a stay in the hospital. Depression, which is very common among seniors, can cloud their thinking. Just being diagnosed with an illness can also lower an elderly person's capacity to think and reason clearly. In cases like these, it is very important to express yourself and your concerns in a very calm manner. Avoid forcing answers or decisions. Assure them of your concern; perhaps that you may, unfortunately, not have the time to care for them as much as you would like, and that having additional help would be a great support for you as well as for them. Express that you want to continue enjoying your relationship with them as their adult child, and not become their caregiver.
3. Try to keep the conversation positive, focusing on the benefits. Encourage them about how great it would be to have a companion in their home to enjoy conversation and prepare home cooked meals, especially if they have been living alone. Because home care aides can usually provide transportation, discuss the fun of getting out in the car to run errands, trips to the store, hair appointments, even visiting with friends. Perhaps your parent has difficulty with bathing or dressing; describe the comfort and assurance they will feel having assistance with these basics of daily living. Many home care companies, or individuals who are caregivers, are willing to work with the family and give the relationship a test run for a short amount of time. Often, when a good match is found between caregiver and senior, they become more willing for and interested in continued care.
4. Consider including a professional in the conversation. Often seniors will listen to their physician before they will take the advice of their own children. Also, research "geriatric care managers" in your area — most will come to the home and talk about the many options and benefits of in-home care.
For more information, or to take a senior care assessment, go to CareFamily.com.


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GraphicRiver Ship Crew Member with Binoculars 7957437
Category: Stock Vector | Comment: ( 0 )
GraphicRiver Ship Crew Member with Binoculars 7957437
Stock Vector \ Characters \ People
Transportation \ Travel \ Transport \ Transfer \ Scarf \ Ship \ Travelling \ Vessel
Stock Vector - Ship Crew Member with Binoculars 7957437 by interactimages @ GraphicRiver
Stock Vector \ Characters \ People
Graphics Files Included: JPG Image, Vector EPS; Layered: Yes; Minimum Adobe CS Version: CS.
Illustration of a ship crew with a telescope on a white background
Happy Family Jumping in the Street; Female Superhero Near a Fullmoon; Kids Forming School Letters; Ballerina Dancing on a Stage; Elephant, Lion and Bear Near a Tree; Rat Sitting Above Cheese with an Empty Banner; Hen at the Farm with a Rainbow and Sign.
Keywords: attendant, background, boat, cartoon, crew, employee, exploration, explore, explorer, exploring, female, girl, human, isolated, lady, person, scarf, ship, transfer, transport, transportation, travel, travelling, vessel, white, woman, wood, wooden, worker, working.


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GOLDEN VALLEY, Minn.- Ringing in the New Year often brings with it the promise of new beginnings and the burden of self-improvement. Fueled by the nostalgia of the holidays, armed with a year's worth of regrets, almost half of Americans are expected to make New Year's resolutions. Dan Ament, Financial Advisor with Morgan Stanley joins us to discuss financial resolutions to make in the new year.
Financial Resolutions for the New Year
Track your finances: The first step toward financial improvement is to get the lay of the land. A minority of Americans have a budget, according to survey data from the National Foundation for Credit Counseling. Consider utilizing one of the many tools available to help you track your income and expenses to better identify opportunities to save more and / or reduce debt. Resources to research may include www.Quicken.com, www.Mint.com or one of the many personal finance apps available to track your income & expenses.
Automate where possible: One of the most easily avoidable mistakes that people make in regards to their finances is missing due dates. Often due to pure forgetfulness, tardiness can have serious ramifications on your financial life – such as missed credit card payments fostering credit score damage. Set up recurring monthly payments from a checking account to pay off your credit card each month in full. If you are a responsible user of credit cards, consider automating other monthly recurring payments on your credit card. If you research a good card, you will earn rewards while paying it off each month.
Build an emergency fund: More than half of American's do not have a rainy day fund, according to the Financial Industry Regulatory Authority's National Financial Capability Study. Building up some cash reserves as a safety net to cushion unforeseen expenses or job loss should be high on your list.
Reduce debt / Save More: U.S. consumers have about $1 Trillion in revolving debt. REDUCE DEBT - High cost consumer debt can become black hole that is difficult to crawl out of financially. Add to that the risk of increasing payments with higher interest rates and it becomes an even greater challenge. Create a plan to prioritize and start by paying down higher rate debts you are carrying. Accomplishing this will allow you to SAVE MORE - 1 in 4 people nearing retirement age have absolutely no money saved up, according to the Federal Reserve. Well, why don't we take some steps to change that in 2016? Retirement obviously isn't your only savings need. You also need to save for college, weddings, vacations, etc.
Monitor and improve your credit score: The first step in the credit-improvement process should be to get your free credit report and review it for errors. Once you've confirmed that everything is accurate, you can begin shoring up the weak points in your score. The grades on the Credit Analysis section of your WalletHub account will tell you what those weaknesses are. The real cost of bad credit? It is estimated that the annual difference in cost between good and bad credit is roughly $650 in credit card payments, $1,400 on your auto loan and $2,300 on a mortgage.
Sources and excerpts:
Visit www.annualcreditreport.com to request a copy of your report from the three agencies.


text: Robert Myers wrote: > Is anyone aware of a source of such wiring which might be willing to > sell the stuff in smaller quantities? You can get it from Digikey, my favorite mail order electronics store. (www.digikey.com) If you enter "teflon wire" in the parts search portion of the main page, it should bring up several hits. If you then click on "hookup wire", you'll get exactly what you're looking for. (Well, at least white and black) -Chris


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Bright, stylish, beautiful, fashionable and creative pendant. This lamp comes in Bel Occhio Collection. Lamps from this collection are distinguished creative design. It can be continuously adjusted with the touch of a hand. Provides both focused and ambient illumination. The high-quality materials, this lamp will serve you for many years. This lamp is available in two colors: white and orange.
- Pendant
- Contemporary Style
- Bel Occhio Collection
- Brend: Pablo Design
- Clear
- White or Orange Diffuser
- Acrylic sphere
- Steel canopy
- Fabric cord
- Full-range dimmer
- Voltage: 120/220V
- (1) White BT15 - med base (75W)
- UL Approved components, CE
- Fixture Height: 15.5 inches
- Fixture Width: 16 inches
- Fixture Depth: 16 inches
- Chain or Rod Length: 96 inches
- Canopy Height: 1 inches
- Canopy Width: 4.5 inches
- Canopy Depth: 4.5 inches
- Wire Length: 96 inches
- Assembled Weight: 13 lbs
Pablo Designs is absolutely innovative designer and manufacturer of home lighting. Pablo Designs was formed in 1993 by Venezuelan born industrial designer Pablo Pardo and has built its studio in San Francisco. The award winning designs successfully combine great functional capabilities with sleek and ultra-modern design to provide your home with the best sources of light. At ModernManhattan.com you will find Table Lamps, Wall Lamps, Pendants and Floor Lamps.
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Ceiling + Pendant Lights
Ideal supplement over a dining room table or bar, ceiling lights are perfect to brighten living rooms, hallways and bedrooms. Our great selection of pendants in range of colors can provide you with direct lighting and make a dramatic statement in your home. Made of different materials and with various shapes and forms, they serve as a modern embodiment of classic chandeliers. From modern to traditional and casual ceiling fixtures, our lighting is what you are looking for to enliven your home!
When choosing a contemporary style you achieve shiny and airy chic with glass and stainless steel surfaces. By choosing traditional and transitional design you get the space full of rich colors like bronze or gold and rustic romance. Whether you want to use singular flush mount or three or five-light candelabra you are sure to find a great assortment here.


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Logo design: Plans, schematic drawings, diagrams, curves, outlines ; One line or one band ; Broken lines or bands (except A 26.11.13)
The European Union Trademark for this logo was filed on 24.07.1998 for goods and services in class(es) 5, 16, 31, 41 and 42 at the European Union Intellectual Property Office (EUIPO). The current status of the trademark No. 000887075 is 'registered'.


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9 Fairfax + Buckeye Articles | Page:
St. Luke's garners national spotlight
Preservation magazine
Friday, April 28, 2017
The National Trust for Historic Preservation shines a light on the stunning resurrection of St. Luke's in Buckeye. From Katherine Flynn for Preservation magazine:
St. Luke's was vacant for a total of eight years, and things weren't looking good; it was the target of vandals and copper wire prospectors, and moisture damage severely eroded the plaster on the building's interior. In 2006, the community development organization
Cleveland Neighborhood Progress
(CNP) came up with what some may have called a far-fetched plan to put it back into use.
"Frankly, a lot of folks in Buckeye had just gotten used to this kind of cultural erasure happening," says Wayne Mortensen, the director of design and development at the organization. "When businesses or institutions shut down, the buildings would fall into disrepair and they would just eventually be torn down.
"So the ability to bring that [St. Luke's] back," he says, "was a pretty big shot in the arm for a neighborhood that was just assuming that it would be lost."
Read the whole story
Brandon Chrostowski named CNN Hero
Saturday, March 19, 2016
Local hero
Brandon Chrostowski
got some national recognition this week. Per CNN:
"Foodies savor the French cuisine at Edwins, an upscale restaurant that's earned a reputation as one of Cleveland's finest eateries. But this high-end establishment provides far more than a good meal. It's staffed almost entirely by people who were once incarcerated.
By day, ex-offenders learn the fundamentals of the culinary arts industry. By night, they put their skills to work."
Get the whole story
Interest free loans for Mt. Pleasant residents
Hebrew Free Loan Association
Wednesday, March 02, 2016
The Saint Luke's Foundation recently awarded a grant to the Hebrew Free Loan Association (HFLA) of Northeast Ohio to provide interest free loans of up to $5,000 to the residents in the Mt. Pleasant neighborhood who are in need of home repair or funds to purchase side yards.
Qualifications include income that will allow repayment of the loan, a co-signer and quotes from an insured and bonded contractor. Loan checks will be written directly to the service provider. Perfect repayment of this loan will allow applicants to qualify for future interest free loans.
For additional information
click here
or call 216-378-9042.
Calabrese advocates for transit funding at Statehouse
Greater Cleveland Regional Transit Authority
Wednesday, February 17, 2016
Joe Calabrese, CEO and General Manager of the Greater Cleveland Regional Transit Authority (RTA) advocated for more transit funding in testimony on Feb. 16 at the Statehouse.
"RTA is the largest public transportation agency in Ohio. My employees, who reside in 16 of Ohio counties, serve approximately 50 million customers each year in Cuyahoga and several neighboring counties," said Calabrese in his address.
"Public transit in Ohio is a $900 million industry that supports many manufacturers, suppliers and jobs.
Public transit gets workers to work, students to school, connects important destinations, drives economic development and provides mobility to many Ohioans who have no other mobility option due to economic realities or disabilities."
Read his comments in their entirety
Travel + Leisure readers rank Cleveland one of America's best food cities
Thursday, March 26, 2015
"The rust belt city offers some old-fashioned, even old-world, charms. Readers ranked it at No. 5 for its rich food halls, like
West Side Market
—with spices, baked goods and delis—which dates back to 1912, when it catered primarily to the city's immigrants."
Read the full story
city club ceo asks: can cleveland overcome its race problem?
Wednesday, February 25, 2015
"As chief executive of the City Club of Cleveland—a 102-year-old institution created to foster dialogue about local, national and international issues—I often find myself in the midst of conversations about the city. So when I—a white guy—am in a meeting about policing or witnessing the inability of some white people here to understand why Tamir's death catalyzed such vocal and visible protests, I remember what a divided city this really is."
Read the full story
healthline cited as model of bold, beautiful bus rapid transit
City Lab
Wednesday, January 28, 2015
"Unlike standard bus stops, which are situated along the curb on opposite sides of a street, BRT stations function better in the center. There are practical reasons for this design, the biggest being that it's cheaper to build one nice station per stop than two. But it all comes back to presenting BRT more as a train platform than a bus stand (below, a rendering of a central station in Vientiane, Laos, and a finished one on the HealthLine BRT in Cleveland)."
Read the full story
mayor jackson announces plans to introduce $100m bond to city council
The City of Cleveland
Wednesday, December 03, 2014
Cleveland Mayor Frank Jackson announced this week that on
Monday, December 8th,
his administration will be "introducing to Cleveland City Council an ordinance for a $100 million bond as an investment in the future of the City of Cleveland."
The bond, the city stated in a press release, will be used to invest in four areas: roads and bridges, the city's emergency response fleet, city-owned facilities and new neighborhood projects.
According to the release, the Jackson administration believes it can stimulate investment in neighborhoods beyond those usually cited as examples of success -- such as Ohio City and University Circle -- and especially areas that have not alrady seen recent private development.
"The final $25 million will go towards new city neighborhood projects and investments outside of the city's central business district," the press release states. "Previous public investments in city neighborhoods have resulted in positive private investment occurring; however, some neighborhoods, in spite of public investments, have not seen equal private investment."
"The City of Cleveland believes that, through focused neighborhood planning and continued investment in the city infrastructure and facilities, the $100 million bond issue will provide essential public capital intended to leverage private investment in neighborhoods where it has been lacking," the press release continues.
Mayor Jackson is quoted as saying: "With this investment, we will not only have better roads, bridges, facilities, vehicles, and recreation centers, but it will also stimulate economic growth, and promote more private investments in the city's neighborhoods that have the greatest challenges to private investments."
To read the complete press release, click
discover cleveland's neighborhoods through cle city life tours
Cleveland Neighborhood Progress
Thursday, November 20, 2014
Cleveland Neighborhood Progress has announced that it will be hosting two CLE City Life tours on Saturday, November 29th and Saturday, December 27th.
"Cleveland Neighborhood Progress is pleased to offer citywide bus tours to introduce (or re-introduce) you to some of the coolest and most unique places to live in Northeast Ohio," the website states. "Join us and see why Tremont and Ohio City receive so much publicity. We'll show you why University Circle is considered the most intellectual square mile in the nation. And you'll understand why demand is so high for Downtown living options. All this and more!"
The cost is $12. You can register
9 Fairfax + Buckeye Articles | Page:
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