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MAPkinase signalling is essential for cell growth, differentiation and cell physiology. G proteins and tyrosine kinase receptors each modulate MAPkinase signalling through distinct pathways. We report here that RGS14 is an integrator of G protein and MAPKinase signalling pathways. RGS14 contains a GPR/GoLoco (GL) domain that forms a stable complex with inactive Giα1/3-GDP, and a tandem (R1, R2) Ras binding domain (RBD). We find that RGS14 binds and regulates the subcellular localization and activities of H-Ras and Raf kinases in cells. Activated H-Ras binds RGS14 at the R1 RBD to form a stable complex at cell membranes. RGS14 also co-localizes with and forms a complex with Raf kinases in cells. The regulatory region of Raf-1 binds the RBD region of RGS14, and H-Ras and Raf each facilitate one another's binding to RGS14. RGS14 selectively inhibits PDGF-, but not EGF- or serum-stimulated Erk phosphorylation. This inhibition is dependent on H-Ras binding to RGS14 and is reversed by co-expression of Giα1, which binds and recruits RGS14 to the plasma membrane. Giα1 binding to RGS14 inhibits Raf binding, indicating that Giα1 and Raf binding to RGS14 are mutually exclusive. Taken together, these findings indicate that RGS14 is a newly appreciated integrator of G protein and Ras/Raf signalling pathways.
Heterotrimeric G proteins (G proteins) are molecular switches that regulate a broad range of signalling events important for many aspects of cell and organ physiology. The regulators of G protein signalling (RGS proteins) inactivate Gα-GTP subunits to negatively regulate G protein signalling. In conventional signalling models [1], G proteins link cell surface receptors of neurotransmitters and hormones to intracellular signalling events, and RGS proteins limit the lifetime of those signalling events. RGS proteins are defined by a shared 130 amino acid RGS domain which binds Gα-GTP to act as a GTPase activating protein (GAP) and inhibit G protein signalling [2–4]. Family members are classified into six or more distinct subgroups based on amino acid sequence identities and functional similarities. Many are relatively small (20–30 kDa) simple proteins with unremarkable features outside of the RGS domain, while others are larger (60–160 kDa) more "complex" proteins with additional domains that bind other protein partners to confer distinct signalling functions and/or act as novel integrators of G protein signalling.
RGS14 is a highly unusual complex family member that contains a canonical RGS domain, a tandem (R1 and R2) Ras/Rap binding domain (RBD) [5, 6], and a GoLoco/GPR (GL) motif. RGS14 was originally identified as a novel binding partner for the Ras-like GTPases, Rap1/2 [7]. The full-length sequence [8] encodes a ~62 kDa protein most closely related to RGS12 (85 kDa) and RGS10 (22 kDa). We and others show that RGS14 interacts selectively with the Gi/o subfamily of G proteins to regulate their guanine nucleotide binding/hydrolysis activity and signalling functions [9–13]. The RGS domain binds directly to activated Gi/oα-GTP to confer non-selective GAP activity towards Giα and Goα. By contrast, we find that the GoLoco/GPR (GL) motif binds specifically to inactive Giα1- GDP and Giα3-GDP, but not Giα2 or Goα, to act as an inhibitor of GDP dissociation (GDI) much like Gβγ subunits [10, 12, 13]. Furthermore, we have shown that RGS14 is actively recruited from the cytosol to the plasma membrane by inactive Giα1-GDP or Giα3-GDP [14]. This recruitment is mediated by the GL domain of RGS14, and Giα1/RGS14 form a stable complex at the plasma membrane in cells. Other known Gα/GL protein complexes are thought to be involved with unconventional G protein signalling pathways [15–17] that rely on the combined action of non-receptor guanine nucleotide exchange factors (GEFs) and GL proteins in place of Gβγ subunits to regulate and mediate Gα signalling. These pathways have been shown to be important for cell division, neuronal development and synaptic plasticity in both lower and higher eukaryote [16, 18]. Thus, RGS14 may serve new functions that can be attributed to the action of its poorly understood GL domain distinct from those contributed by its RGS GAP domain.
Both G proteins and tyrosine kinase growth factor receptors activate MAPkinase signalling by distinct pathways [19–21]. MAPkinase signalling is important for many aspects of cell physiology including cell growth, survival, and differentiation as well as neuronal development and synaptic plasticity [22]. Since RGS14 tightly binds inactive forms of Giα1/3 and also contains tandem (R1 and R2) Ras/Rap binding domains (RBD), we investigated whether RGS14 could integrate the actions of Giα, Ras and Raf kinases to regulate MAP kinase signalling. We report that RGS14 binds activated H-Ras and its effectors Raf-1, B-Raf and A-Raf to regulate PDGF-stimulated Erk phosphorylation, and that this activity of RGS14 is regulated by its interactions with Giα1. RGS14 is recruited by H-Ras to form a stable complex at the plasma membrane, and it binds both H-Ras and/or Raf kinases either alone or together in cells. Expression of RGS14 inhibits PDGF-stimulated Erk phosphorylation and this inhibition is dependent on H-Ras binding, but is reversed by Giα1 which also blocks Raf binding to RGS14. Our findings demonstrate that RGS14 serves as a novel scaffold to integrate Giα1 and Ras/Raf/ MAPkinase signalling events through the action of its GL domain.
The RGS14 cDNA used in this study was derived from rat (Genbank accession number of U92279) as previously described [10]. Truncated Flag-Raf-1 (ΔRaf-1) and 3xHA-B-Raf were kindly supplied by Dr. Haian Fu (Emory University). 3xHA-A-Raf cDNA was kindly supplied by Dr. Deborah Anderson (Saskatchewan Cancer Agency). HA-Raf-1 was kindly supplied by Dr. Ulf R. Rapp (Würzburg University). Glu–Glu epitope (EE) tagged recombinant Giα1/pcDNA3.1 plasmids, H-Ras 3xHA-tagged (N-terminus) plasmids and their constitutively active/inactive mutants were purchased from UMR cDNA Resource Center (Rolla, Missouri). The expression plasmids encoding RGS14 full-length, RGS14 deletion mutants coding for amino acids 1–213, 213–544, 213–490 and 444–544 cloned in-frame into the pcDNA3.1 (Invitrogen) were prepared as described previously [14]. Primer oligonucleotides encoding the 8 amino acid Flag tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) was used to generate N-terminally Flag-tagged RGS14 (Flag-RGS14). Site directed mutagenesis of RGS14 was performed by Quickchange Site Directed Mutagenesis kit (Stratagene). For the E92A, N93A double mutation placed into RGS14, the codon GAG was replaced by GCG, and the codon AAT was replaced by GCT; for the R333L mutation placed into RGS14, the codon AGA was replaced by CTT; for the H406A mutation, CAC were replaced by GCA. To express XFP-tagged Ric8A proteins in mammalian cells, the NheI/NotI YFP- and CFP-Ric8A DNA fragments described in Thomas, et al were subcloned into pcDNA3.1(+) (Invitrogen) [23].
Anti-Flag M2 antibody affinity gel was purchased from Sigma. Anti-Flag antibody, Alexa488-conjugated goat anti-rabbit and Alexa546-conjugated goat anti-mouse antibodies were purchased from Invitrogen. Anti-EE antibody was purchased from BD Biosciences. Monoclonal anti-HA horseradish peroxidase (HRP) conjugate antibody and monoclonal anti-HA TRITC (Rhodamine) conjugate antibody were purchased from Sigma. The following antibodies were purchased from Santa Cruz Biotechnology: anti-B-Raf (F-7) mouse monoclonal antibody (sc-5284), anti-Raf-1 (C-12) rabbit polyclonal antibody (sc-133), anti-Raf-1 (E-10) mouse monoclonal antibody (sc-7267), and anti-Giα1(R-4) mouse monoclonal antibody (sc-13533).
For cell imaging, HeLa cells were fixed for 10 min at room temperature with the following buffer: 20 mM PIPES pH 7.0, 1 mM MgCl2, 0.5 mM EGTA, 1 mM glutaraldehyde, 1 µg/ml aprotinin, 2 mM taxol, 0.1% Triton X-100, 2% paraformaldehyde. Fixed cells were subsequently blocked for 60 min at room temperature with PBS containing 10% goat serum and 1 mg/ml bovine serum albumin and incubated with a 1:1000 dilution of primary antibody, rabbit anti-Flag (Sigma), mouse anti-EE (BD Scientific), or Rhodamine-conjugated mouse anti-HA antibody (Sigma), for over night at 4°C. Cells were washed 3 times with PBS and then stained with 1:200 dilutions of Rhodamine-conjugated goat anti-mouse and FITC-conjugated goat anti-rabbit antibodies (Jackson Immuno-Research Laboratories) or Alexa553-conjugated goat anti-rabbit and Alexa633-conjugated goat anti-mouse antibodies (Invitrogen) for 1 h at room temperature. Cells were mounted with Vectashield mounting medium (Vector Laboratories). Images were collected on an Olympus IX51 inverted fluorescence microscope (Olympus) using a 100× oil immersion objective. Immunofluorescence analyses also were carried out using a LSM510 confocal laser scanning microscope (Zeiss). Images were acquired using an 63× oil immersion objective and processed using the Zeiss LSM image browser (version 2.801123) and Adobe Photoshop 7.0 (Adobe Systems).
HeLa cells were obtained from the American Type Culture Collection. Cell transfections were performed according to protocols described in our previous work [14]. RGS14 pull-down assays were performed as described [10, 14, 24] with modifications. Cells were transiently transfected with selected recombinant plasmids according to the methods described above. Transfected cells were lysed in buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM EGTA, 10 mM MgCl2, 50 µg/ml Aprotinin, 100 µg/ml Leupeptin, 1 µM phenylmethylsulfonyl fluoride (PMSF) and 1% TritonX-100. Each lysate was incubated with 10 µg of either full-length Trx-H6-RGS14 or His6-Giα purified proteins for 2 hours. The complexes were incubated an additional 1 h with 100 µl of Ni-NTA for 60 min. The Ni-NTA beads were centrifuged and then washed five times for Raf-1 and seven times (for B-Raf) each with 1 ml ice cold washing buffer. Protein complex were eluted into 100 µl Laemmli buffer. Samples were separated by 11% SDS-PAGE and transferred to nitrocellulose membranes (Millipore Corp., Bedford, MA) and immunoblotted with selected antibodies for visualization.
HeLa cells were seeded on 10 cm dishes and cDNA transfected using Lipofectamine 2000 (Invitrogen). For immunoprecipitation of expressed proteins, transfected cells were washed three times in ice cold PBS and lysed in buffer as described above in Ni-NTA pull-down assay. The lysate was cleared by centrifugation at 50,000 g for 30 min at 4°C. Protein concentrations were determined using the Bradford reagent (Bio-RAD). Lysates were then mixed with 50 µg Flag M2 gel (Sigma) directed against the Flag-tagged fusion protein. The mixed complexes were incubated for 2 h at 4°C with continuous rotation. Flag immunocomplexes were washed three times for Raf-1 and six times for B-Raf in ice-cold TBS buffer. Immunoprecipitates were suspended in a Laemmli sample buffer followed by boiling for 5 min. The protein samples were resolved by 11% SDS-PAGE, transferred to a nitrocellulous membrane (Bio-RAD) and probed with appropriate antibodies, e.g. HRP labeled monoclonal anti-Flag antibody (Sigma), HRP labeled monoclonal anti-HA antibody (Sigma), or polyclonal anti-Raf-1 antibody (Santa Cruz Biotechnology) followed by appropriate secondary antibody. Detection of immunolabelled proteins was accomplished by using the ECL-Plus chemiluminescent system (GE Healthcare).
HeLa cells were seeded on 12-well plates and cDNA transfected with Lipofectamine 2000 (Invitrogen) as described above. After 24 h, transfected cells were switched into serum free DMEM for 6 h. Cells then were stimulated with 25 ng/ml PDGF (Chemicon), 100 ng/ml EGF (Sigma) or fetal calf serum, FCS (20% final) for the times described in the text. The reactions were stopped by transferring cells to ice, and immediately lysed by removing media and the addition of 400 µl/well Laemmli sample buffer. Recovered samples were then boiled for 5 min. The sample mixture was electrophoresed on 11% SDS-PAGE and transferred to nitrocellulose membranes and probed for both active and total Erk using anti-phospho-p44/42 Erk polyclonal antibody (Cell Signalling) and anti-p44/42 Erk polyclonal antibody (Cell Signalling), respectively. Detection of immunolabelled proteins was accomplished using the ECL-Plus chemiluminescent system. Quantification of proteins was achieved by densitometry using a Scion Imaging system. Numbers derived using this analysis from multiple experiments were pooled and subjected to statistical analysis (paired t-test).
RGS14 was first identified as a binding partner for the Ras-like GTPase, Rap1/2 [7]. Ras and Rap each specifically bind RBD domains and RGS14 contains tandem (R1 and R2) RBDs [7, 25]. We tested if RGS14 could bind H-Ras. RGS14 (Flag-RGS14) was transfected together with different forms of Ras (HA-Ras) in HeLa cells. We observed that RGS14 bound to and co-immunoprecipitated with constitutively active (G/V mutant) H-Ras, but only weakly to wild type H-Ras and not at all to inactive (S/N mutant) H-Ras in HeLa cell lystes (Fig. 1A). We postulate that the weak binding of wild type H-Ras reflects a small population of active H-Ras-GTP present in cell lysates. We also examined the influence of H-Ras on RGS14 subcellular localization (Fig. 1B). When expressed alone in HeLa cells, RGS14-GFP localizes to the cytosol and H-Ras to membranes, in particular the plasma membrane. Active H-Ras(GV) localized most strongly to membranes, whereas inactive H-Ras(SN) failed to localize at membranes. When RGS14 is co-expressed with different forms of H-Ras, active H-Ras(GV) recruited RGS14 to the membrane. These findings strongly suggest that active H-Ras is a newly described binding partner for RGS14.
We next examined which region of RGS14 is responsible for binding H-Ras. RGS14 contains two contiguous Ras/Rap-binding domains (RBDs), R1 and R2 (Fig. 2A). Based on previous studies, RBD domains present in Raf kinases and other proteins serve as specific binding sites for activated Ras and Rap isoforms, and a conserved arginine residue within all RBDs is critical for this interaction [9, 26]. RGS14 contains an analogous arginine within the R1 RBD (R336) whereas a histidine occupies the analogous residue in the R2 RBD (H409) [9]. Disrupting R336 inhibits Rap2 binding whereas disrupting this H406 does not [9, 27, 28]. To investigate H-Ras interaction with RGS14, we introduced these same single substitution mutations (R333L or H406A) into the R1 and R2 RBD domains, respectively, of Flag-tagged RGS14 (Fig. 2A and 2B). When each of these RGS14 mutants was co-expressed with either wild type H-Ras, active H-Ras(GV) or inactive H-Ras(SN), we found that H-Ras was inhibited from binding to the R333L mutant but not the H406A mutant of RGS14 (Fig. 2B). This also was reflected in RGS14 subcellular localization (Fig. 2C). When co-expressed together in HeLa cells, the R333L mutant of RGS14 failed to co-localize with active H-Ras(GV) whereas the H406A mutant co-localized at membranes with active H-Ras(GV) in a pattern similar to wild type RGS14 (compare Fig. 2C to Fig. 1B). These findings indicate that active H-Ras(GV) binds the R1, but not the R2 RBD of RGS14.
The Raf kinases (Raf-1, B-Raf and A-Raf) are well described effectors of H-Ras. Therefore, we examined if RGS14 could also interact with one or more of the Raf kinases. Initial studies tested if purified recombinant His-RGS14 could bind any of the isoforms of Raf kinases (HA-tagged Raf-1 or B-Raf) when expressed in HeLa cell lysates. We found that expressed B-Raf and Raf-1 each bound purified His-RGS14 and were recovered by Ni-NTA resin (Fig. 3A). Similar results were observed when Flag-RGS14 was co-expressed with HA-B-Raf and HA-Raf-1 in HeLa cells. B-Raf and Raf-1 both were recovered following immuno-precipitation of RGS14 using anti-Flag resin (Fig. 3B), but the proteins did not bind non-specifically to the resin. RGS14-GFP also co-localized with either B-Raf or Raf-1 in both the cytosol and membranes of HeLa cells as visualized by confocal microscopy (Fig. 3C). We also found that RGS14 binds to and co-localizes with A-Raf in HeLa cells (Supplemental Figure 1). These findings suggest that Raf kinases are newly identified binding partners of RGS14, or components of RGS14-multi-protein complexes.
To elucidate the binding regions between Raf and RGS14, we generated four truncated forms of Flag-RGS14 (Fig. 4A) including: 1) the isolated RGS domain (amino acids 1–213), 2) the isolated RBD domains (213–490), 3) the RBD/GoLoco domains (213–544), or 4) the isolated GoLoco domain (444–544). Each of the truncated forms of RGS14 was co-expressed together with either HA-B-Raf or HARaf-1 in HeLa cells, and cell lysates were subjected to anti-flag immuno-precipitation (Fig. 4A). We found that only those fragments containing the RBD domains were able to bind either Raf-1 or B-Raf, but not those fragments containing only the RGS domain or the GPR/GoLoco domain, thereby demonstrating that the tandem RBD domain region is essential for Raf binding. In contrast to what was observed with H-Ras binding to RGS14, introduction of the R333L or the H406A single point mutations into the RBDs failed to alter Raf binding to RGS14 (data not shown).
Raf kinases share a conserved overall structural homology with three conserved domains designated as CR1, CR2 and CR3 (Fig. 4B). The CR1 region, located within the N-terminal region, contains a single RBD domain and a cysteine-rich zinc-binding domain (CRD). The CR2 region, located between CR1 and CR3, contains a serine/threonine rich domain, whereas the CR3, located in the C-terminal region, contains the serine/threonine (S/T) protein kinase catalytic domain. The CR1 and CR2 regions are important for the activation and regulation of Raf kinase activity and have been defined as essential sites for protein–protein interaction [29]. The N-terminal fragment region of Raf-1 (l-257) is able to homodimerize, whereas the C-terminal Raf fragment, BXB-Raf (l-25/306–648), does not [19]. We generated a truncated form of the Raf-1 (ΔRaf-1) missing the CR1 and CR2 regulatory regions but containing the CR3/kinase domain. When full-length Raf-1 and ΔRaf-1 were compared for their capacity to bind purified His-RGS14, we found that Raf-1, but not ΔRaf-1 bound RGS14, and was recovered using the Ni-NTA resin (Fig. 4B).
Several scaffolding proteins have been identified that are important for the spatiotemporal organization and context-specific signalling of the different MAPkinase signalling pathways [30]. One hallmark of scaffolds is their capacity to facilitate binding and assemble functionally related protein partners. Through the tandem RBD domains, RGS14 can bind active H-Ras and Raf kinase independently. We examined if Flag-RGS14 could behave as a scaffold to bind both HA-H-Ras(GV) and HA-Raf-1 simultaneously. All three proteins were co-expressed in HeLa cells and cell lysates were subjected to anti-Flag immunoprecipitation. We found that H-Ras and Raf-1 both co-precipitated with RGS14 (Fig. 5A). A complicating factor in these experiments is that active H-Ras(GV), Raf-1 and RGS14 each bind one another independently, so confirming that H-Ras(GV) and Raf-1 are binding to the same RGS14 molecule in cells is technically difficult. To address this question we expressed each protein (Flag-RGS14, H-Ras(GV) and Raf-1) independently in different sets of cells. Cell lysates of each protein were prepared separately (Fig. 5B), and the amounts of expressed protein were quantified. The cell lysates were then mixed in various combinations to control and equalize the amount of each protein present in the samples. Proteins were then subjected to anti-Flag immunoprecipitation and H-Ras and Raf-1 bound to recovered Flag-RGS14 were detected (Fig. 5B). We observed that detectable amounts of H-Ras and Raf-1 each bound RGS14 independently, as before. However, when lysates of each protein were all mixed together, we found that considerably more active H-Ras and Raf-1 bound to RGS14. These findings are consistent with the notion that RGS14 is a scaffold that assembles H-Ras and Raf-1 to form a stable RGS14/H-Ras/Raf-1 ternary complex.
Various extracellular factors activate H-Ras and Raf kinases to stimulate MAP kinase signalling pathways that culminate with the phosphorylation and activation of Erk1/2. These include, but are not limited to, various growth factors (e.g. PDGF and EGF) and other mitotic factors present in blood serum. Since RGS14 binds both H-Ras and Raf kinases, we examined if RGS14 can modulate H-Ras/Raf-mediated Erk phosphorylation. We observe that HeLa cells express cell surface receptors for PDGF, EGF and serum factors that, when activated, stimulate Erk phosphorylation (Fig. 6). When cells were transfected with an RGS14 construct, we find that RGS14 protein expression markedly inhibited PDGF-stimulated Erk phosphorylation (Fig. 6A). In contrast, RGS14 did not alter Erk phosphorylation stimulated by either EGF (Fig. 6B) or 20% serum (Fig. 6C). These findings suggest that RGS14 binding to H-Ras and Raf kinases, (as shown above), serves to inhibit PDGF-stimulated MAPkinase signalling.
We next examined the importance of H-Ras binding on RGS14 inhibition of PDGF signalling. As shown earlier (Fig. 2), the R333L mutation placed into the R1 RBD domain prevents H-Ras binding to RGS14. As shown in Figure 7A, RGS14 inhibits PDGF-stimulated Erk phosphorylation as before. However, in stark contrast, we observe that the R333L mutation of RGS14 failed to inhibit PDGF-mediated Erk phosphorylation in HeLa cells (Fig. 7A), indicating that RGS14 inhibition of PDGF signalling depends on H-Ras binding. We have previously shown that RGS14 directly binds inactive Giα1-GDP via its GPR/GoLoco domain, and that Giα1 recruits RGS14 to form a stable complex at the plasma membrane [14]. Therefore, we next examined the influence of Giα1 binding on RGS14 inhibition of PDGF signalling (Fig. 7B). We observed that co-expression of Giα1 reversed RGS14 inhibition of PDGF signalling, whereas Giα1 alone had no effect of PDGF-stimulated Erk phosphorylation. Taken together, these findings indicate that RGS14 inhibition of PDGF signalling depends on H-Ras binding and that this inhibition is reversed by Giα1 binding to RGS14.
We next sought to understand the basis for Giα1 reversal of RGS14 inhibition of PDGF signalling. HeLa cells were transfected with Flag-RGS14 alone or in the presence of Raf-1, Giα1 and/or H-Ras(GV) (Fig. 8). Cell lysates were subjected to anti-Flag immuno-precipitation and proteins bound to RGS14 were examined. As before, we observe that Raf-1, Giα1 and H-Ras(GV) each bind to RGS14 independently. However, in each case where Giα1 and Raf-1 are both present, Giα1 binds RGS14 and Raf-1 does not. In contrast, Giα1 binding does not affect H-Ras binding. This experiment was repeated in exactly the same way except with B-Raf in place of Raf-1, and similar results were obtained (data not shown). That is, Giα1 reduced the amount of B-Raf binding to RGS14. These findings suggest that Giα1 binding and Raf binding to RGS14 are mutually exclusive.
Since Giα1 inhibits Raf binding to RGS14 in cells, we examined Giα1 interactions with Raf kinases. Initial studies examined if Giα1 binds to Raf kinases. HeLa cell lysates expressing either Raf-1 or B-Raf were mixed with purified His6-Giα1. When Giα1 was recovered by Ni-NTA pull-down and examined for the presence of bound Raf, we found that neither Raf-1 nor B-Raf bound to purified Giα1 (Fig. 9A). However, we also observed that expression of Giα1 recruited either B-Raf or Raf-1 to the plasma membrane in HeLa cells (Fig. 9B). When co-expressed together with RGS14 and either B-Raf or Raf-1, Giα1 recruited both proteins to the plasma membrane (Fig. 9C). In parallel studies, we also found that Giα1 recruited A-Raf to the plasma membrane (Supplemental Figure 2). Together, these findings suggest that Giα1 does not directly regulate Raf activity, but somehow contributes to the co-ordinate translocation of the signalling complex to the plasma membrane.
RGS14 is a highly unusual signalling protein that contains two distinct Gα interaction sites (RGS domain and GL domain) and tandem (R1 and R2) RBD domains that are known in other proteins to bind Ras and Rap GTPases. Indeed, RGS14 was first identified as a novel Rap1/2 binding partner [7]. We and others have shown previously that the RGS domain of RGS14 is a promiscuous and nonselective GAP for active forms of Giα and Goα, and that the GL domain is a selective GDI for Giα, but not Goα [7, 10–12, 27]. We subsequently reported that the GL motif of RGS14 selectively binds inactive Giα1-GDP and Giα3-GDP, but not Giα2, and that both of these Giα isoforms strongly recruit RGS14 to the plasma membrane to form a tight complex with the GL domain to influence the dynamic subcellular localization of RGS14 [14]. Here we expand those findings to show that RGS14 binds activated H-Ras and its Raf kinase effectors (Raf-1, B-Raf and A-Raf) to modulate their signalling. H-Ras and Raf each bind RGS14 independently, but the two proteins appear to facilitate one another's binding to RGS14. Alternatively RGS14 may facilitate formation of the H-Ras/Raf complex which, in turn, binds more efficiently to RGS14. Active H-Ras selectively binds the R1 RBD domain of RGS14 and the Raf kinases also bind within the RBD region. We find that expression of RGS14 markedly inhibits PDGF-stimulated activation of Erk1/2 and that this inhibition is dependent on H-Ras binding but is reversed by co-expression of Giα1. In follow-up studies to understand this better, we found that Giα1 binding inhibited Raf binding to RGS14, suggesting that the binding of Giα1 and Raf are mutually exclusive. These findings demonstrate that RGS14 regulates the activity of multiple GTPases with distinct functions, and are consistent with the idea that RGS14 is a newly appreciated integrator of G protein (Giα) and Ras/Raf signalling.
RGS14 is one of only two known proteins (RGS12 the other) that contains three different binding domains for distinct GTPases, and it sits at the intersection of three families of signalling proteins that contain either an RGS domain, a GL domain(s) or an RBD domain(s). Recent studies indicate that proteins containing GL domains (GL proteins) modulate unconventional G protein signalling pathways and events that do not involve cell surface GPCRs [15, 17, 18]. Much of the previous work on RGS14 focuses on its presumed role as an RGS protein that modulates GPCR/G protein signalling. However, based on our findings here and our understanding of other proteins that contain GL domains, we propose that RGS14 is a newly appreciated integrator of unconventional G protein signalling and MAPkinase signalling. Our reported findings provide the framework of a model to describe how these functionally opposing domains work together to bind and modulate the functions of inactive and active Gα, and Ras/Raf/MAP kinase signalling.
Our proposed model highlights the GL domain as the first point of contact between Gα and RGS14, which differs from conventional models that highlight the RGS domain in this role [2, 4]. We envision RGS14 to function primarily as a GL protein that also contains an RGS domain. We have proposed that RGS14 is complexed with subsets of Giα1-GDP or Giα3-GDP via its GL domain and these complexes are devoid of Gβγ [14]. Our findings presented here are consistent with additional features of this model. We postulate that a non-receptor GEF such as Ric8A and/or possibly others [17, 31] stimulates nucleotide exchange and GTP binding to Giα which, in turn, promotes dissociation of RGS14 because the GL domain does not bind Gα-GTP. This is consistent with our recent observation that Ric8A interacts with RGS14 to promote Giα1 dissociation (C. Vellano et al, unpublished observation). Once free from Giα1, RGS14 is available to bind active H-Ras and Raf kinase to inhibit MAPKinase signalling, as we show here. In this model, the lifetime of the RGS14-Ras/Raf complex is limited by the RGS domain, which could restore Giα-GDP and promote reformation of Giα-GDP/GL-RGS14 complex, and consequential dissociation of Raf and H-Ras. Consistent with this idea, we find that Giα1 and Raf-1 binding to RGS14 is mutually exclusive. An arrangement that incorporates the RGS domain into the same protein restricts the RGS domain pairing to the Gα pre-selected by the GL domain (Giα1 or Giα3). This configuration imparts spatially restricted RGS/Gα selectivity and eliminates the necessity for intrinsic RGS/Gα selectivity. This is consistent with our earlier observation that the RGS domain is a non-selective GAP for Gi/oα [10]. We speculate that the RGS domain activity is regulated and unavailable as a GAP until the correct conformation is achieved. Consistent with this idea, we observe that both the GL domain [24] and the N-terminal region containing the RGS domain of RGS14 are phosphorylated (F.J. Shu, D.P. Cowan et al, unpublished observation), which could be important mechanisms for regulating these protein interactions.
Such a model exhibits many mechanistic parallels with established models of conventional GPCR/Gα signalling (1) except that a non-receptor GEF substitutes for a GPCR and the GL motif of RGS14 substitutes for Gβγ [23, 31, 32]. Of note, similar models have been proposed for proteins known to regulate cell division (but unrelated to MAPkinase signalling) in lower eukaryotes including worms and flies [32–34]. However, distinct GL and RGS proteins are thought to act in these systems whereas, in the case of RGS14 shown here, the RGS activity is already built into the GL protein and pre-positioned nearby to terminate RGS14 inhibition of Ras/Raf signalling. While some components of the working model are speculative, most are consistent with our observed findings and provide a framework for further experiments. Our previous work shows that RGS14 can modulate conventional GPCR/Gα (e.g. M2ChoR/Giα) signalling in membranes, and the model presented here does not rule out a parallel role for RGS14 in conventional GPCR/Gα signalling, as we have proposed [10, 35]. RGS14 may serve a (poorly understood) role in unconventional G protein signalling pathways involving non-receptor (GPCR) GEFs alone, or these in concerted action with GPCRs.
RGS14's closest relative, RGS12, also was shown to bind H-Ras and B-Raf to inhibit PDGF Erk phosphorylation, though no Giα regulation of this activity was reported. RGS12 inhibition of PDGF signalling may be mediated by direct binding to the PDGF receptor [36] suggesting that RGS14 and RGS12 serve distinct functions. Consistent with this idea, RGS14 and RGS12 share both overlapping and distinct sequence and predicted domains structures (63% identity; 70% similarity), and have very different patterns of subcellular distribution. RGS12 localizes to punctate structures in the nucleus and cytosol [37, 38], while RGS14 is distributed diffusely throughout the cytosol where it shuttles in/out of the nucleus and also accumulates at centrosomes and the plasma membrane with Giα1/3 [11, 28].
The exact mechanism by which RGS14 inhibits PDGF-stimulated MAPkinase signalling is unclear. We propose that RGS14 could serve as a molecular scaffold that sequesters active H-Ras and Raf from their signalling pathways to passively inhibit signalling. We favor this idea which is consistent with our proposed model. Alternatively, RGS14 could directly alter H-Ras or Raf-1 activity, perhaps by stimulating Ras GTPase activity and/or inhibiting Raf kinase activity. H-Ras binds specifically to the R1 RBD, and previous work has shown that the RBD domains of other proteins simply serve as binding sites for active Ras-GTP or Rap-GTP, but do not directly alter GTP binding to or GTP hydrolysis activity of these GTPases [5, 6]. Consistent with these reports, we previously showed the RGS14 failed to alter Rap GTP binding/GTPase activity [10, 24]. This would suggest that RGS14's effect on MAPkinase signalling is likely that of a passive scaffold that sequesters H-Ras and Raf. Alternatively RGS14 may serve to redirect Ras/Raf kinase activity away from MEK/Erk towards a different substrate and pathway. In this regard, RGS14 may act in a fashion similar to the well-described scaffold 14:3:3, which also binds Raf kinases to passively modulate their signalling activity. Further studies are required to determine if RGS14 redirects Raf kinase activity or if it competes with 14:3:3 for Raf binding.
Our previous work has shown that RGS14 is most highly expressed in neurons of adult brain [10], most notably within the hippocampus (Sarah E. Lee, et al, unpublished observation, and also http://www.brain-map.org). GL proteins studied thus far have been shown to be important for cell division, but also for neuronal development and synaptic plasticity in both lower and higher eukaryotes [16, 18]. Several studies have implicated a possible role for RGS14 in cell division [28, 39, 40]. However, since RGS14 is highly enriched in mitotic incompetent neurons of adult brain, it must serve functions distinct from (or in addition to) those relating to cell division. Within hippocampal neurons, MAPkinase signalling pathways play a crucial role in regulating various aspects of neuronal differentiation and synaptic plasticity [22]. It is possible that RGS14 serves an important, though as yet poorly defined role in regulating these processes. Other signalling proteins that share similar properties with RGS14 as scaffolds, GL proteins, and/or as regulators of MAPkinase signalling have been shown to play important roles in synaptic plasticity. For example, the mammalian partner of insceutable (mPins, aka LGN or AGS5) protein and RGS14 both contain GL/GPR motifs, are modulated by Ric8A [31], and are stabilized by Giα-GDP binding at the plasma membrane and centrosomes. In addition, mPins/LGN/AGS5 is reported to regulate opposing pulling forces during cell division of mitotic competent non-neuronal cells [41]. However, mPins/LGN/AGS5 also is expressed in mitotic incompetent adult hippocampal neurons (as is RGS14) and is enriched in synaptic membranes where it associates with PSD-95 and MAGUK scaffolding proteins in a Giα1-dependent manner to influence their trafficking and NMDA receptor surface expression [42]. Of note, we observe here (Fig. 9) that Giα1 does not bind Raf-1, yet is capable of recruiting this kinase to the plasma membrane along with RGS14, suggesting the involvement of a larger multi-protein signalling complex like PSD-95 or MAGUK. This also is consistent with a recent report showing that the FRMPD1 protein serves as a scaffold to regulate the subcellular localization and interactions of the GPR/GL-protein AGS3 and Giα3 [43]. Other scaffolding proteins play a key role in synaptic plasticity. Loss of Shank, a key scaffolding component of the postsynaptic density (PSD) of hippocampal neurons, results in mice that exhibit smaller dendritic spines, decreased synaptic transmission, but enhanced learning and memory [44]. Also, loss of the adaptor protein N-Shc/Shc-C, which directly links NMDA and BDNF-receptors to MAPkinase signalling in hippocampal neurons, yields mice that exhibit enhanced spatial learning and memory [45]. These reports support the idea that RGS14 may serve functions complimentary to those of other scaffolding proteins in neuronal synaptic plasticity.
We report that RGS14 binds activated H-Ras and Raf-1 kinases, both independently and together in cells. RGS14 inhibits PDGF stimulated MAPkinase activity in a H-Ras dependent manner. This inhibition of signaling and Raf-1 binding to RGS14 is reversed by Giα1, suggesting that Giα1 binding and Raf-1 binding to RGS14 are mutually exclusive. Giα1 expression in cells also recruits Raf-1 to the plasma membrane suggesting a larger Gi-regulated signalling complex. Together, our findings here suggest that RGS14 serves as a newly appreciated scaffold that integrate G protein and H-Ras/Raf/ MAP kinase signalling events.
*We thank Dr. Ulf R. Rapp (Wϋrzburg University), Dr. Haian Fu (Emory University) and Dr. Deborah Anderson (Saskatchewan Cancer Agency) for kindly sharing cDNA constructs used in these studies, as described in the Experimental Procedures section. We also thank Jon Waters for technical assistance in generating some mutant constructs. This work is supported by grants from the National Institutes of Health awarded to J.R.H. (R01NS37112 and R01NS049195).
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Need a substitute for wheat or gluten-based flours? Look no further! Shauna the Gluten-Free Girl provides an excellent tutorial: Just weight out the ingredients, then toss to mix.
These are just a collection of gluten-free flour mixes that I've liked using in baking and thickening sauces. Recipes can be completely gluten-free, including brownies, financiers, and cornbread.


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NLT Study Bible Personal Size TuTone Brown/Tan Leatherlike, Indexed
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Understand the Bible like never before with the NLT Study Bible. The product of over 40 Bible scholars and seven years of work, it's the most comprehensive study Bible ever created. The notes focus on bringing out the full meaning of the text, allowing the reader to understand the Bible more deeply than ever. Now available for the first time in a convenient personal size edition.
The New Living Translation is an authoritative Bible translation, rendered faithfully into today's English from the ancient texts by 90 leading Bible scholars. The NLT's scholarship and clarity breathe life into even the most difficult-to-understand Bible passages—but even more powerful are stories of how people's lives are changing as the words speak directly to their hearts. That's why we call it "The Truth Made Clear."
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The New Living Translation is an authoritative Bible translation, rendered faithfully into todays English from the ancient texts by 90 leading Bible scholars. The NLTs scholarship and clarity breathe life into even the most difficult-to-understand Bible passagesbut even more powerful are stories of how peoples lives are changing as the words speak directly to their hearts. Thats why we call it The Truth Made Clear.
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HEARTSRhode IslandAge: 55-65Gender: female5 Stars Out Of 5HelpfullMay 14, 2015HEARTSRhode IslandAge: 55-65Gender: femaleQuality: 5Value: 5Meets Expectations: 4This NLT helps me understand what is written in a clearer way. Sometime, Paul's writings can be confusing and this NLT helps with making the writing clearer. The study notes make me think about what is being said. Really like the extra information.
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First rejected icon for Bounce, our new local messaging app. We started this project a few months ago and along my graphic propositions was this icon that we decided not to feature.
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How did TMP get a G rating?
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October 9 2012, 12:07 AM
Lieutenant Commander
Re: How did TMP get a G rating?
mos6507 wrote:
Kirk also doesn't seem to grieve much when Ilia gets zapped by the probe. Decker does, of course, and doesn't like the cavalier way Kirk assigns Decker to work with the recreated Ilia-Probe as a sort of 'secret agent'. Kirk being somewhat empathetically challenged is very much a theme in TMP. I think they were going with him having a very selfish one-track mind to retake command of the Enterprise, so much so that it gave him a borderline personality disorder. When we get to Khan, Kirk behaves more like himself, but is just bored and brooding and not so much of a jerk.
That scene has always made me uncomfortable, particularly the way he says, "Carry out your assignment, Mr. Decker," as though there's no reason at all why Decker shouldn't be reluctant to interact with the Ilia-probe.
Later, after Spock was traumatized by his attempted mind-meld with V'Ger, Kirk stood at his bedside and demanded answers, even when it become apparent that Spock was too exhausted to even answer his questions. At the end of the scene, Kirk ordered that Spock be returned to duty, not taking McCoy or Chapel's feelings about his condition into consideration.
"I need Spock on the bridge."
Did he not think about Spock's fitness for duty? Did it occur to him that having a crew member working so soon after an injury could have placed the ship at risk? Spock had recovered, but there was no way to know that would happen when Kirk first discovered him floating back to the
, unconscious.
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TV Show (2011 - 2015)
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…but click Add to My Picks to add to your personal TV Schedule and LocateTV will tell you when Game of Thrones is next on TV.
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Peter Dinklage Tyrion Lannister
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- Season 5 Episode 10: Mother's Mercy
The High Sparrow and his militant followers appear to have made an indelible mark on King's Landing as Cersei finds herself seeking forgiveness. Meanwhile, in the North, Stannis embarks on a gruelling march, and Jon is challenged, and across the Narrow Sea, Daenerys is surrounded by strangers as the fifth chapter of the hit fantasy drama draws to a close. Starring Kit Harington, Emilia Clarke, Lena Headey and Stephen Dillane
- Season 5 Episode 9: The Dance of Dragons
In Westeros, Jon returns to the Wall from his controversial ranging mission, and Stannis faces a difficult decision. Meanwhile, in Braavos, Arya runs into a familiar face and Mace Tyrell visits the Iron Bank, and against her better judgement Daenerys hosts a traditional Meereenese celebration of athleticism. Fantasy drama, starring Kit Harington, Stephen Dillane, Maisie Williams and Emilia Clarke
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Arya makes progress in her training, Sansa confronts an old friend, Cersei faces a struggle and Jon embarks on a journey. Fantasy drama, starring Lena Headey
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Six guidelines for delivering praise effectively
Praising employees is a delicate business. Some experts advise you to praise every employee every day, while others argue that daily praise will undermine your management efforts.
Here are some issues to keep in mind when you praise:
- You may be subtly sabotaging employees' development. If employees become too dependent on your praise, they may stop taking risks and thinking for themselves, and start doing only what they know will earn them a compliment. Concentrate instead on sharing information about your employees' efforts to help them grow more independent.
- Are you listening as well as talking? Employees may have important things to tell you about their work, but if their only contact with you is the five minutes a day you spend praising them about something or other, you may never hear what they have to tell you. Instead of simply telling employees what a good job they're doing, take some time to ask them how they've succeeded in the job they're doing.
- Consistency is difficult. You can't see what every employee does every moment of the day. You may be praising something insignificant just because it caught your attention. If the employee realizes that your praise is irrelevant to his or her real efforts, cynicism may set in. Instead, explain to employees how their positive contributions affect the organization, and let them take pride in themselves without waiting for praise from you.
- Are you and the organization sending mixed messages? Employees who are stressed because they've got too much work and too little support won't suddenly feel better just because you spend five minutes a day praising them. Be honest with your people about the challenges facing all of you.
- Don't force it. If your attempts to praise are obviously just lip service, employees will feel manipulated and insulted. Give praise when it's deserved and appropriate, but don't look for excuses to hand it out. Your employees will recognize what you're doing.
- Is it personal? One size doesn't fit all when it comes to praise. If you use the same words and rewards to praise everyone, some people will feel that you're just repeating phrases you read in a management book. Get to know your people so you can honestly deliver the kind of praise they'll respond to.
Jules Ciotta is president of Motivation Communications Associates. He can be reached at (770) 457-4100 or email@example.com.
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Originally written on The Colts Authority | Last updated 11/5/11
I have a confession to make: I majored is in statistics. Dorky? I'll leave that to you. Nate sent me a link this morning to a research paper found in the Journal of Quantitative Analysis in Sports and I took the bait.Dr. Brian Skinner, a University of Minnesota physics research associate and Ph.D has taken a stab at explaining risk assessment and strategy in sports. His work is certainly heavy on theory but he brings it back to earth by explaining the use in common situations such as deciding between run, short passes and long passes: Equating Zrun and Zshort suggests that that team should run the ball whenever the number of yards to go is small enough that y 4.9N. In the intermediate range of y, such that 2.8N Got that? Maybe not so let me explain in more detail. The variable "y" is the number of yards needed. So Dr. Skinner says teams should run when they need less than 2.8 yards, throw between 2.8 and 4.9 yards and go deep otherwise. Seems intuititive but explains wh...
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text: THE WEB & CXXXVI - S.Q.U.E.A.K [DAST072] by THE WEB published on 2012/11/20 22:33:27 +0000 being human is overrated... track number three for "The Bunny Project" (work in progress). Music programmed by THE WEB; vocals written and performed by CXXXVI, http://soundcloud.com/the-web-cxxxvi THE WEB & CXXXVI - The Bunny Project EP (DAST72), download for free here: http://archive.org/details/DAST072_THE_WEB_and_CXXXVI_-_The_Bunny_Project_EP Lyrics (by CXXXVI) You woke up And realised you swapped bodies with your pet mouse Now you're stuck in a maze Doing tricks for cheese But it doesn't bother you You thought being human was overrated Besides you know the mice rule the Earth So it's good to have them on your side Yeah you sit back Stroke your whiskers And plan your next attack Yeah you're gonna Squeak Squeak 7 days of the week So if you see a mouse Crawling around your house Don't try to hit it with large mallet Even though it will reach a perfect pitch high C You gotta set it free. Yeah you're gonna Squeak Squeak 7 days of the week ENJOY WITH THE TRACK VIDEO: http://www.youtube.com/watch?v=j-QXeOTfIBA


text:
Blood: Plasma Pak (DOS)
Published by
Developed by
Critic Score
100 point score based on reviews from various critics.
User Score
5 point score based on user ratings.
German indexOn April 30, 1999, Blood Plasma Pak was put on the infamous German index by the BPjS. More information about what this means can be found in the game group.
ReferencesThe game contains a hidden level which is actually based upon Monolith's offices at Kirkland, Washington. The hall structures and layout are all to scale, and even the wall textures are the same. A a fat zombie wearing an apron inside can be found behind a door labeled "Management" in the main building.
Information also contributed by Maw and Xoleras
Contributed by Tomer Gabel (4361) on Aug 23, 2000. [revised by : Patrick Bregger (133164)]. -- edit trivia


text:
Reproducibility of swallow-induced cortical BOLD positive and negative fMRI activity.
ABSTRACT Functional MRI (fMRI) studies have demonstrated that a number of brain regions (cingulate, insula, prefrontal, and sensory/motor cortices) display blood oxygen level-dependent (BOLD) positive activity during swallow. Negative BOLD activations and reproducibility of these activations have not been systematically studied. The aim of our study was to investigate the reproducibility of swallow-related cortical positive and negative BOLD activity across different fMRI sessions. We studied 16 healthy volunteers utilizing an fMRI event-related analysis. Individual analysis using a general linear model was used to remove undesirable signal changes correlated with motion, white matter, and cerebrospinal fluid. The group analysis used a mixed-effects multilevel model to identify active cortical regions. The volume and magnitude of a BOLD signal within each cluster was compared between the two study sessions. All subjects showed significant clustered BOLD activity within the known areas of cortical swallowing network across both sessions. The cross-correlation coefficient of percent fMRI signal change and the number of activated voxels across both positive and negative BOLD networks were similar between the two studies (r ≥ 0.87, P < 0.0001). Swallow-associated negative BOLD activity was comparable to the well-defined "default-mode" network, and positive BOLD activity had noticeable overlap with the previously described "task-positive" network. Swallow activates two parallel cortical networks. These include a positive and a negative BOLD network, respectively, correlated and anticorrelated with swallow stimulus. Group cortical activity maps, as well as extent and amplitude of activity induced by volitional swallowing in the cortical swallowing network, are reproducible between study sessions.
Article: Compensatory recombination phenomena of neurological functions in central dysphagia patients[Show abstract] [Hide abstract]
ABSTRACT: We speculate that cortical reactions evoked by swallowing activity may be abnormal in patients with central infarction with dysphagia. The present study aimed to detect functional imaging features of cerebral cortex in central dysphagia patients by using blood oxygen level-dependent functional magnetic resonance imaging techniques. The results showed that when normal controls swallowed, primary motor cortex (BA4), insula (BA13), premotor cortex (BA6/8), supramarginal gyrus (BA40), and anterior cingulate cortex (BA24/32) were activated, and that the size of the activated areas were larger in the left hemisphere compared with the right. In recurrent cerebral infarction patients with central dysphagia, BA4, BA13, BA40 and BA6/8 areas were activated, while the degree of activation in BA24/32 was decreased. Additionally, more areas were activated, including posterior cingulate cortex (BA23/31), visual association cortex (BA18/19), primary auditory cortex (BA41) and parahippocampal cortex (BA36). Somatosensory association cortex (BA7) and left cerebellum in patients with recurrent cerebral infarction with central dysphagia were also activated. Experimental findings suggest that the cerebral cortex has obvious hemisphere lateralization in response to swallowing, and patients with recurrent cerebral infarction with central dysphagia show compensatory recombination phenomena of neurological functions. In rehabilitative treatment, using the favorite food of patients can stimulate swallowing through visual, auditory, and other nerve conduction pathways, thus promoting compensatory recombination of the central cortex functions.Neural Regeneration Research 03/2015; 10(3):490-7. DOI:10.4103/1673-5374.153701 · 0.23 Impact Factor
Article: Esophageal Reflexes Modulate Fronto-Parietal Response in Neonates: Novel Application of Concurrent NIRS and Provocative Esophageal Manometry.[Show abstract] [Hide abstract]
ABSTRACT: Background and Objectives: Central and peripheral neural regulation of swallowing and aero-digestive reflexes is unclear in human neonates. Functional near infrared spectroscopy (NIRS) is a noninvasive method to measure changes in oxy-hemoglobin (HbO) and deoxy-hemoglobin (HbD). Pharyngo-esophageal manometry permits evaluation of aero-digestive reflexes. Modalities were combined to investigate feasibility and to test neonatal fronto-parietal cortical changes during pharyngo-esophageal (visceral) stimulation and/or swallowing. Methods: Ten neonates (45.6±3.0wks PMA, 4.1±0.5kg) underwent novel pharyngo-esophageal manometry concurrent with NIRS. To examine esophagus-brain interactions, we analyzed cortical hemodynamic response (HDR) latency and durations during aerodigestive provocation and esophageal reflexes. Data are presented as mean ± SE or %. Results: HDR rates were 8.84 times more likely with basal spontaneous deglutition compared to sham stimuli (P=0.004). Of 182 visceral stimuli, 95% were analyzable for esophageal responses, 38% for HDR, and 36% for both. Of analyzable HDR (N=70): a) HbO concentration (µmol/L) baseline 1.5±0.7 vs. 3.7±0.7 post- stimulus was significant (P=0.02) b) HbD concentration (µmol/L) between baseline 0.1±0.4 vs. post-stimulus -0.5±0.4 was not significant (P=0.73). c) Hemispheric lateralization was 21% left only, 29% right only, and 50% bilateral. During concurrent esophageal and NIRS responses (N=66): a) Peristaltic reflexes were present in 74% and HDR in 61%. b) HDR was 4.75 times more likely with deglutition reflex vs. secondary peristaltic reflex (P=0.016). Conclusions: Concurrent NIRS with visceral stimulation is feasible in neonates, and fronto-parietal cortical activation is recognized. Deglutition contrasting with secondary peristalsis is related to cortical activation; thus, implicating higher hierarchical aerodigestive protective functional neural networks.AJP Gastrointestinal and Liver Physiology 05/2014; 307(1). DOI:10.1152/ajpgi.00350.2013 · 3.74 Impact Factor
- Clinical Gastroenterology and Hepatology 09/2014; 12(12). DOI:10.1016/j.cgh.2014.08.031 · 6.53 Impact Factor


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Business briefs: July 19
Wall Street mixed after earnings reports
NEW YORK - Wall Street closed out an impressive week with a mixed performance Friday after disappointing high-tech earnings punctured some of investors' enthusiasm over better-than-expected bank earnings reports.
Dodson Pest Control relocates office
LYNCHBURG, VA. - Dodson Pest Control has relocated its Knoxville office to 501 Dutch Valley Drive. The company had previously been at 3537 Papermill Road. Dodson expanded its services into the Knoxville area in 2003 when it acquired the assets of Volunteer Pest Control. Volunteer was in business in the Knoxville area for 14 years before the business was sold to Dodson Pest Control.
Established in Lynchburg in 1944, Dodson is currently one of the largest family-owned and operated pest management companies in the country, with 34 offices throughout Virginia, West Virginia, North Carolina, South Carolina and Tennessee.
Green Bankshares 2Q profit falls 79 percent
Green Bankshares Inc. reported Friday that its second-quarter net income plunged 79 percent to $1.46 million, or $0.11 per diluted share, from $7.08 million, or $0.62 per diluted share in the same period a year ago.
The drop reflects a sharp increase in the bank's provision for bad loans and an increase of 1,562,000 shares because of the acquisition of Civitas BankGroup in the second quarter of 2007.
The slumping residential real estate market hurt the company's loan portfolio in the quarter ended June 30.
In response, the parent company of GreenBank increased its loan loss provision to $11 million in the second quarter, up from $888,000 in the first quarter of 2008, Chairman and CEO Stan Puckett said in a statement.
Puckett said that the current residential real estate market is challenging "and it is unclear when this situation will improve."
Green Bankshares has 64 branches across East and Middle Tennessee, and one branch each in Bristol, Va., and Hot Springs, N.C.
Feds eye more Bear Stearns charges
NEW YORK - Prosecutors say they expect to bring more criminal charges against two former Bear Stearns hedge fund managers already charged with lying to investors about the collapse of the subprime mortgage market.
At a hearing in Brooklyn federal court on Friday, prosecutor Patrick Sinclair said Ralph Cioffi and Matthew Tannin probably would get hit with additional charges in the fall, though he declined to specify what kind.
Both men have pleaded not guilty to conspiracy and fraud charges and were released on bond. They could face up to 20 years in prison.
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well as the schizophrenia.
CitationsSadock BJ, Sadock VA (2007). Schizophrenia. In
Kaplan and Sadock's Synopsis of Psychiatry: Behavioral Sciences/Clinical Psychiatry, 10th ed.,
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If your faucet handles have seen better days, you can bring your faucet back to life with replacement handles. Replacing faucet handles is an easy project that will restore your faucet to like new condition. With brand specific fit handles, you can be sure that your new handles are an exact match to the original style and fit perfectly.
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Story of Survival
"Redeemed" by Dese'Rae L. Stage
(Paul G. Quinnett Lived Experience Writing Contest)
I tried to kill myself in the summer of 2006. I was 23. I spent a month deeply entrenched in an exhaustive cycle of breaking up and getting back together with Emma, the girl I'd loved for three years...
read the full story here
Latest Articles
|New Data Issued by the CDC Releases 2011 Suicide Statistics Showing Continued Rise in Suicide Rate 6/25/14
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|JUDY SHOULTS 2013 MHC Shining Star Award Recipient
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|Preventing Suicide: A Global Imperative, Executive Summary 2013
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The La Crosse Area Suicide Prevention Initiative is committed to promoting awareness that suicide is a public health problem that is preventable.
To educate
Educate the community that suicide is a preventable public health problem. We are able to educate through:
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We support current efforts to create and establish legislation for mental health insurance parity and suicide prevention.


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These Simple Truths: Deluxe Edition CDThese Simple Truths: Deluxe Edition has all the songs you love from their first album, plus one BRAND NEW song entitled "Know That You Are Loved." It also has two songs recorded live at the Lealand House Sessions.
Live Like That CD
Sidewalk Prophets released their sophomore album, Live Like That on March 27, 2012. With the help of their fans, Sidewalk... Learn More
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This CD+ DVD combination pack features Francesca Battistelli's 11-track album, Christmas, produced by Ian Eskelin, on CD as well as a DVD of the album performed live in Nashville, TN with additional bouns... Learn More
Zealand Worship - The EP Digital Album
PLEASE NOTE: This is a digital EP - you will not receive a physical CD with this purchase. The full digital EP will be available for download immediately following checkout.<... Learn More
Something Different
Preorder the new Sidewalk Prophets album, Something Different, today and you'll receive it on the album release date of August 28th, 2015!
Buy two copies and you'll receive... Learn More
The Wonderlands Surf Experience
The Wonderlands Surf Experience is the ultimate package for any Jon Foreman fan and features a once in a lifetime opportunity to hit the waves with Jon.
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The oceans and surfing have been a source of inspiration in Jon's life and music.
Upon the release date of the last The Wonderlands EP, Dawn, on October 23rd, 2015, you... Learn More
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Jon expresses himself not only through his music but through his artwork.
Upon the release date of the last The Wonderlands EP, Dawn, on October 23rd, 2015, you will... Learn More
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Coffee fueled the creation of The Wonderlands and fuels this collection.
Upon the release date of the last The Wonderlands EP, Dawn, on October 23rd, 2015, you... Learn More
The Wonderlands EP Box Set
Get Jon's The Wonderlands Series in a collectible wood box set! This box set also includes 4 postcards featuring the cover art from each EP.
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The Wonderlands Digital Set
The Wonderlands EPs:
- The Wonderlands: Sunlight EP - release date 5.26.15
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492 Views 1 Reply Latest reply: Oct 24, 2011 4:13 AM by UK-Max
UK-Max Oct 24, 2011 4:13 AM
Re: MacBook Pro Stolen: can apple block my device? in response to Finaport Solved
I'm afraid that there is nothing to be done from Apple's side.
Most importantly, you must focus on any sensitive information that you have stored on your Macbook. whether secured or insecure (in fact you have to consider it insecure from now). Particularly bank or financial information, and then consider passwords etc that could be used.


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I am a very inquisitive person. I love to learn and to travel, and I am lucky to have been able to do both. I hope to continue learning and exploring in the future.
I like figuring out how things work. I often find myself wondering how things became what they are today. This may be why I decided to be a history major.
I love spending time in nature, whether it be camping, or hiking, or exploring national parks. At the same time, I also like exploring the city, and getting to know new and interesting places.
I have a drive to create. I try to use my creative instincts both in work and in play. I especially love to write, and to work on films.
In my free time, among other things I like to play videogames, watch movies, and read. I like to spend time with others who share my interests, and I am always looking for new things to enjoy. I care a lot about my friends, and try to reach out to them as much as possible.


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Endowed Lectures
Endowed Lectures
That support is greatly appreciated as it contributes to the continuing education of the department's faculty, fellows, residents and students.
In addition to the traditional Chairman's Derby Lecture presented the week of the world-famous Kentucky Derby, the University of Louisville Department of Medicine hosts seven other "named" lectures.
Those lectures include:
John Walker Moore, M.D., was born in McConnellsville, South Carolina, and earned a BS degree at North Carolina's Davidson College in 1906. He began medical school at the University of North Carolina at Chapel Hill and earned his MD from the University of Pennsylvania in 1912.
After an internship at Episcopal Hospital in Philadelphia, he came to the University of Louisville as an instructor of pathology and bacteriology in 1915.
In 1917, he joined the U.S. Army as a captain and served overseas for 18 months with the American Expeditionary Force as Officer in Charge of Laboratories at the hospital center in Nantes, France.
Dr. Moore returned to Louisville in 1923 and was named chairman of the University of Louisville Department of Medicine as well as Staff Executive of Louisville General Hospital.
When Dean Stuart Graves resigned his post in 1928, Dr. Moore served in the interim administrative committee until his eventual appointment as dean at the end of that year and continued in those posts until 1949.
During his administration, the School of Medicine survived the Great Depression and the 1937 flood. It also added an annex at First and Chestnut streets, offered accelerated training during World War II, and in 1948 became the first unit of the University to receive regular state appropriations.
Dr. Moore retired as dean in 1949 and was the Alben W. Barkley Professor of Medicine until his death in 1952.
He is known for his work in cardiography, basal metabolism, blood chemistry as well as a dye-dilution method for determining cardiac output which he developed with J. Murray Kinsman.
Throughout his more than 50-year career, David H. Neustadt, M.D., FACP, MACR, has made a lasting mark in the field of rheumatology.
Specializing in arthritis and rheumatic diseases, Dr. Neustadt received one of the first fellowships in rheumatic diseases from the National Institute of Arthritis and Metabolic Diseases.
Dr. Neustadt's areas of special interest include intra-articular injection therapy, corticosteroids in rheumatoid arthritis, shoulder disorders and spondylitis. He has authored more than 150 scientific publications, numerous chapters in rheumatologic texts and three medical textbooks, and given more than 150 presentations in the United States, Europe and South America.
As a leader in his field, Dr. Neustadt has been the recipient of numerous awards. He is a Fellow of the American College of Physicians and the American College of Rheumatology (ACR), and in 1992 was named a Master of the ACR. In 1997, Dr. Neustadt was honored by the ACR as the recipient of the Distinguished Rheumatologist Award in recognition of outstanding achievements in the field of rheumatology.
He is on the board of directors of the Kentucky Chapter of the Arthritis Foundation, the Lupus Foundation of America and is a member of the American Association for the Advancement of Science, the American Medical Writers Association, and the American Medical Authors Association.
Dr. Neustadt continues to serve as clinical professor of medicine at the University of Louisville School of Medicine and also served as chief of the Division of Rheumatology. He continues to practice the specialty of rheumatology and teach fellows, medical house staff and medical students at U of L.
To honor Dr. Neustadt's commitment to rheumatic diseases, the David H. Neustadt, M.D., Fund for Rheumatology was created in the University of Louisville School of Medicine. This endowment benefits the Division of Rheumatology and supports fellowships and training, research opportunities, and special lectures and noted guest speakers.
Sponsored by the George W. Pedigo Fellowship Fund, the Pedigo Lecture was established in 1988 by Louisville natives Mr. and Mrs. Robert Hook.
The first annual George W. Pedigo Lecture was held on September 8, 1988. Dr. Pedigo chose his friend Dr. Edward W. Hook as the Visiting professor to present the first of the annual lectures.
George W. Pedigo Jr., M.D., FACP, was a native of Glasgow, Kentucky and he received his medical degree from the University of Louisville in 1938.
He joined the faculty of the University of Louisville in 1946, following training in Internal Medicine and service in the U.S. Army Medical Corps.
Dr. Pedigo was a former Governor of the American College of Physicians and Life Member of the University of Louisville Board of Overseers.
He was promoted to Emeritus Professor of Medicine in March 1981.
Dr. Pedigo received Kentucky's first Laureate award and was a made a Master in 1991. He passed away in July 1999.
He is remembered as an avid supporter of the Department of Medicine and an outstanding physician and academician.
The Theodore Segal Lecture fund was established December 7, 1982, in memory of an outstanding member of the academic community and a humanitarian.
Dr. Segal received his dental degree from the University of Louisville in 1954 and his orthodontic training at Tufts University in 1956.
He joined the staff of the University of Louisville Dental School in 1969 on a part-time basis, where he was one of the two founders of the new Department of Orthodontics. He maintained his faculty association until his death.
A veteran of World War II, Dr. Segal was a member of many dental and orthodontic associations and held offices in the Louisville and Kentucky Orthodontic Associations. He was also a member of the Kentucky Health Systems Agency and an active member of Alpha Omega professional fraternity.
Dr. Segal contributed to the education of many generations of orthodontists. Ted, as his colleagues and students knew him, always gave unselfishly of this time and talents.
His friendly disposition and willingness to help were almost proverbial, even though never imposed on anybody.
He died in 1982 of cancer and his family bequeathed this lectureship in Medical Oncology in his honor.
The Charles C. Smith Jr. Annual Lectureship in Medicine was established in 1987 in honor of Dr. Smith and as a memorial to his dear friend and patient, Armor Hugh Platt Taylor.
Mr. Taylor was fascinated by Dr. Smith's bedside teaching of tutorial students. Mr. Taylor's wife, Dr. Letitia S. Kimsey, a former medical microbiology teacher, endowed this lectureship.
Charles C. Smith Jr., M.D., FACP, grew up in the Appalachian community of Fonde, Ky. He attended Georgetown College and graduated summa cum laude.
In 1955, he received his medical degree from the University of Louisville where he was elected to Alpha Omega Alpha Honor Medical Society.
He completed a rotating internship at University Hospital and Hillman Clinic in Birmingham, Ala., then served as a U.S. Air Force flight surgeon for three years before completing his Internal Medicine residency at the University of Louisville Hospitals. He served as Chief Resident from 1960-61.
Dr. Smith was known as a consummate diagnostician, spending four decades taking exceptional care of his patients. Since 1961, he has served as clinical faculty at the University of Louisville, rising from Instructor to Clinical Professor of Medicine in 1985.
He welcomed medical students into his practice and always emphasized the importance of getting patients' medical histories. "The history is everything," he asserts. "A patient will usually tell you everything you need to make the diagnosis."
Dr. Smith was named one of the nation's "Best Doctors" in 1980. He was known as the "doctor to the doctors" for many years before retiring from private practice in 2001.
He has been involved with many organizations, including serving terms as president for the Innominate Society for the Study of Medical History, the Louisville Society of Internists, the Jefferson County Medical Society, and the Kentucky Medical Society (KMA), as well as Governor of the American College of Physicians.
Since 2006, he has served as Editor of Vital Signs, the Greater Louisville Medical Society's patient education quarterly publication.
Dr. Smith received the Outstanding Alumnus Award from Georgetown College in 1973, Alumni Service Award from U of L in 1987, Physician Laureate Award (Kentucky Chapter) from the American College of Physicians in 1991, Distinguished Alumnus Award from U of L School of Medicine in 1993, Excellence in Primary Care Award at the Caritas Foundation Doctors' Ball in 2004, Distinguished Service Award from the Kentucky Medical Association in 2008, and was named 2009 Ephraim McDowell Physician of the Year.
Dr. Smith continues to serve the community by making laundry runs for Hospital Hospitality House, serving as a key contact on KMA's National Legislative Activities Committee, serving on the Board of the Family Health Centers, developing a St. Catharine College health sciences satellite training center at the restored U.S. Marine Hospital to create "better health and better jobs in the Portland community" and supporting Red Bird Mission's health and wellness outreach in eastern Kentucky.
He and his wife, Rosemary together they have raised four children.
It is in loving memory of Nancy Middleton Smith that this lectureship is established by her grandson and University of Louisville School of Medicine alumnus Smith H. Gibson, M.D.
Nancy Middleton Smith was born in Evarts, Kentucky on January 12, 1874. She was a daughter of Judge James H. Middleton and Elizabeth Wynn Middleton. She married N.B. Smith on January 28, 1895. The eldest child of this marriage was Bell Smith Gibson, mother of Smith H. Gibson.
Smith, though possessing limited formal schooling herself, was exceedingly bright intellectually, encouraging her children and grandchildren to become educated to their fullest capacity.
She would be highly pleased that a grandson and two great grandsons have graduated with honors from the University of Louisville School of Medicine and a great granddaughter from the University of Louisville Louis D. Brandeis School of Law. Numerous other progeny have distinguished themselves as students elsewhere.
She was noted for her qualities of warmth and generosity. She ministered to the sick, consoled the bereaved, fed the poor, encouraged the downtrodden, and inspired the young, all the while committing her deep devotion to a large mountain family. A devout Baptist, she was truly in the ways of God.
Smith suffered periodically in her last years from an undefined cardiopulmonary disease and succumbed to acute pulmonary edema on August 19, 1927, at 53 years of age.
Beverly Todd Towery, M.D., was born in 1915 and thus was just the right age to be a part of the dramatic advances in medicine during and after World War II.
He loved the clinical aspect of his profession and he gloried in teaching.
He was at his best when doing either one, whether it was as an Army doctor during the invasion of Italy or on rounds in a teaching hospital, with students at his side.
He grew up in southern Kentucky and went to Western Kentucky State College where he received a bachelor's degree in 1936. He went to Vanderbilt University's School of Medicine and graduated first in his class.
Dr. Towery interned at Vanderbilt Hospital and spent a year as a resident in pathology at Mallory Institute in Boston.
For the following three years he was in uniform in the combat zones of Italy and France and emerged from the war a Major.
A civilian again, he became a senior resident in medicine at Massachusetts General Hospital where he had the opportunity to work with Fuller Albright, which led to a lifelong preoccupation with endocrinology.
Subsequently, he spent two years as a fellow at Thorndike Memorial Laboratory at Boston City Hospital, working with Robert Williams.
Dr. Towery returned to Vanderbilt, a Markle Scholar and the first chief of a newly established Endocrine Division. In July 1956, he became professor and chairman of the Department of Medicine at the University of Louisville.
With his arrival, the students and house staff saw a dramatic surge in the quantity and quality of bedside teaching, not only by Dr. Towery himself, but by newly-motivated faculty members.
Away from the demands of medicine he found relaxation in cabinetmaking – furniture for the suburban home he shared with his wife, Jane, and their three children, Lynn, Todd and Anne – or diving and tennis, two sports in which he excelled.
In 1970, Dr. Towery asked for release as chairman of the department in order to become chief of the Section of Endocrinology.
In the next few years he was able to devote full attention to his favorite medical activity, bedside teaching.
In 1974, he was struck by viral encephalitis. He never fully recovered and was unable to resume his brilliant career in teaching; he passed in June 1981.
In an attempt to honor the memory of this teacher, clinician, friend and gentleman, his colleagues in the medical community have established this lectureship.


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I paid $229 for my annual pass. I also spend approximately $20 to $30 on food and another $10 on a pin each time I go (Im being conservative with my approximations). I go about twice a month.
If Disney stopped selling their APs, Id go once a year. Maybe twice. Theyre losing about $840 ($35 x 24) a year from me. Without an AP Id be spending $120 a year, at most. [/QUOTE]
This may be true for you but for most people I would think that if they were to only visit Disneyland once a year that they would save up and make it a big trip with more than $25 spent on food and a single pin purchased. You may not spend $840. on food and extras on your once a year trip but most people who go once a year go to the Blue Bayou at $50.+ per person and buy several pins, a shirt, maybe a figurine or a watch, etc. So for the average person they might lose a few hundred, but I am guessing that would be made up by the many new APs they would sell because the Park is so pleasant and uncrowded. When I took a break from going many, many times per year and went twice a year each of those trips was much more costly than my weekly visits had been. Just my opinion.
So would you say that, as an AP holder, you should go MORE and spend less?
-JackDoc Brown had 2 Deloreans!
Personally I don't like to see things change too much but if they do, then I like to see it done with the Disney quality that everyone expects. However, I have never forgotten the arguments my brother and I used to get into when we were little. He hated going to Disneyland because he said that they never changed and that it bored him to death. Imagine if everyone else felt the same way. So as much as I hate to see change, I think it is necessary, for better or for worse.
I agree the park has to evolve and change with the times. The trouble is, it's hard to find execs who have the kind of vision Walt did. In my opinion, Walt was an artist first, who became an executive in order to turn his dreams into reality. Most executives these days went in to undergrad college knowing they were going for an MBA in order to turn a buck.
I'm glad that people like Lasseter are around who are motivated to keep the park a magical place. Without people like that, you definitely have just another theme park.
And I agree, the only way to vote is with your dollars.
As I wrote in another thread: change should be for the better. Not just for the bigger profit. Not just for the fad of the month.
Sorry, but I don't think Tarzan's Treehouse is bad show. Few people know who the Swiss Family Robinson is any more, despite the DVD release, and those who do know don't care.
Tarzan has been a part of our popular culture for 75 years and continues to be so because of the Disney film. The redo of the treehouse is great, and makes it more relevant to those who walk through it.
The new and changing effects are also more interesting to those under the age of 20!
Each attraction he created was different. Besides Dumbo/Casey Junior and Alice/Tea Cups, each ride at DL is very unique from each other.
Why not keep it that way instead of changing it because Pirates is the 'thing' right now. It won't be in 5 years, if not less.
And since they have all this money secured for the Island, why can't Disney actually use the Imagineers they have to create something totally new and exciting - not just Pirates...again.
And a Caribbean Island has palm trees on it, so, if it goes through, I am assuming they will have some palm trees. And palm trees would look really weird next to the Mark Twain and RoA.
The Island is perfect right now - in a sense. They just need to open back up the fort, install some really nice and creative playgrounds, create larger caves, add in nice and clean bathrooms, and have a nice eatery of some sort. That's all.
Personally myself, I don't know why Disney is wasting so much money on the Island. Not many go to it and there are many other areas in the park - Tomorrowland anyone? - that need more help than TSI. Oh, and what about DCA?
I hate coporate business sometimes...
Love your thread by the way...
You made excellent points.
Last edited by A Change In Me; 10-05-2006 at 05:11 PM. Reason: Misspelled "two"
This isn't saying that today's executives do not share some parts of this vison or passion, but this passion has to be there in order for any changes to be made successfully in this particular theme park.
My guess is, you never saw the Swiss Family Treehouse. If you did, then you certainly didn't see a great deal-- your imagination wasn't engaged.
I am glad I got to experience DL when it was great. I'm sorry for those who get to experience it after your generation gets through with it.
"Remember dreams come true", I would like to believe it is more than just a commercial tagline.
I really resonate with what DisneyIpresume just stated in his post! Reread it!
Well said!I think the main difference between Walt Disney and the latest round of executives is that Walt truly enjoyed the park, the rides, and everything else about it. Why else would he have put an apartment for himself inside the park? I think it is very difficult to find executives who have this vision, if that's what you want to call it; I think of this as more of a passion. Just think: Would Michael Eisner or Robert Iger ever move into an apartment in the middle of Disneyland, or even want to?
This isn't saying that today's executives do not share some parts of this vison or passion, but this passion has to be there in order for any changes to be made successfully in this particular theme park.
Last edited by CAspace; 10-05-2006 at 04:44 PM.