text:
- Research
- Open Access
Akt1 sequentially phosphorylates p27kip1 within a conserved but non-canonical region
- Lucas P Nacusi1, 2 and
- Robert J Sheaff1, 2Email author
© Nacusi and Sheaff; licensee BioMed Central Ltd. 2006
- Received: 30 March 2006
- Accepted: 16 June 2006
- Published: 16 June 2006
p27kip1 (p27) is a multifunctional protein implicated in regulation of cell cycling, signal transduction, and adhesion. Its activity is controlled in part by Phosphatylinositol-3-Kinase (PI3K)/Akt1 signaling, and disruption of this regulatory connection has been identified in human breast cancers. The serine/threonine protein kinase Akt1 directly phosphorylates p27, so identifying the modified residue(s) is essential for understanding how it regulates p27 function. Various amino acids have been suggested as potential targets, but recent attention has focused on threonine 157 (T157) because it is located in a putative Akt1 consensus site. However, T157 is not evolutionarily conserved between mouse and human. We therefore re-evaluated Akt1 phosphorylation of p27 using purified proteins and in cells.
Here we show purified Akt1 phosphorylates human and mouse p27 equally well. Phospho-peptide mapping indicates Akt1 targets multiple sites conserved in both species, while phospho-amino acid analysis identifies the targeted residues as serine rather than threonine. P27 deletion mutants localized these sites to the N-terminus, which contains the major p27 phosphorylation site in cells (serine 10). P27 phosphorylated by Akt1 was detected by a phospho-S10 specific antibody, confirming this serine was targeted. Akt1 failed to phosphorylate p27S10A despite evidence of a second site from mapping experiments. This surprising result suggested S10 phosphorylation might be required for targeting the second site. We tested this idea by replacing S10 with threonine, which as expected led to the appearance of phospho-threonine. Phospho-serine was still present, however, confirming Akt1 sequentially targets multiple serines in this region. We took two approaches in an attempt to explain why different residues were previously implicated. A kinetic analysis revealed a putative Akt1 binding site in the C-terminus, which may explain why mutations in this region affect p27 phosphorylation. Furthermore, commercially available recombinant Akt1 preparations exhibit striking differences in substrate specificity and site selectivity. To confirm S10 is a relevant site, we first showed that full-length wild type Akt1 purified from mammalian cells phosphorylates both human and mouse p27 on S10. Finally, we found that in cultured cells under physiologically relevant conditions such as oxidative stress or growth factor deprivation, endogenous Akt1 causes p27 accumulation by phosphorylating S10.
Identifying where Akt1 phosphorylates p27 is essential for understanding its functional implications. We found that full-length wild type Akt1 – whether purified, transiently overexpressed in cells, or activated in response to cellular stress – phosphorylates p27 at S10, a noncanonical but evolutionarily conserved site known to regulate p27 activity and stability. Using recombinant Akt1 recapitulating this specificity, we showed modification of p27S10 also leads to phosphorylation of an adjacent serine. These results integrate PI3K/Akt1 signaling in response to stress with p27 regulation through its major phosphorylation site in cells, and thus identify new avenues for understanding p27 deregulation in human cancers.
- Wortmannin
- Growth Factor Deprivation
- Multiple Serine
- PDVF Membrane
- Full Length Wild Type
Information transmitted by signaling pathways determines whether a cell continues the proliferative cycle or adopts an alternative fate. This decision requires regulating Cyclin Dependent Kinases (CDKs), which are activated by phosphorylation and temporal association with a unique cyclin subunit (D, E or A type in G1/S phase) [reviewed in [1]]. Two families of CDK inhibitory proteins (CKIs) have been identified: INK4 proteins (p15, p16, p18 and p19) specifically inhibit cyclin D-CDK4/6, while Cip/Kip proteins (p21, p27, and p57) are considered more broad-spectrum inhibitors of cyclin D, E, and A CDK complexes [2, 3].
P27 regulation is particularly important because it functions as a tumor suppressor that is often disrupted in human cancers, usually by compromising its stability and/or location [4–7]. Consistent with this view mice without p27 develop pituitary tumors and display increased susceptibility to carcinogens [8–10]. Mice lacking a single copy of the p27 gene are still hypersensitive to carcinogens, illustrating the importance of precisely controlling its levels and activity [11, 12]. Despite these observations the role of p27 in tumorigenesis remains enigmatic due to its multifunctionality. Although first characterized as a CDK inhibitor that negatively regulates cell cycle progression [13–16], p27 also possesses CDK-independent functions such as inhibiting the adaptor protein GRB2 (to regulate signaling) or targeting RhoA (to regulate adhesion) [17–19]. Disrupting these p27 activities could also contribute to the disease state, especially given recent evidence cancer cells do not necessarily require hyperactive CDKs [20, 21].
The PI3K/Akt1 pathway plays a key role in cell survival and proliferation by modulating gene expression, protein synthesis, and cell cycle machinery [33]. PI3K regulates p27 function via a multi-pronged effort that includes altering p27 transcription, protein levels, and location [34, 35]. Akt1 contributes to this process by phosphorylating and inhibiting Forkhead transcription factors that promote p27 transcription [36]. In addition, Akt1 directly phosphorylates p27 in order to alter its levels and/or location in the cell [30, 37–42]. Thus, inappropriate PI3K/Akt1 activation could contribute to cellular transformation in part by deregulating p27 [35]. Identifying the p27 residue(s) phosphorylated by Akt1 is essential for determining their regulatory significance. Numerous sites have been proposed, but recent attention has focused on threonine 157 (T157). This residue is located in an Akt1 consensus sequence "RKRPAT157" within the p27 NLS. Recombinant Akt1 failed to phosphorylate a p27 mutant lacking this site, and in some breast cancer cell lines Akt1 hyperactivation altered p27 location [39–42].
Important p27 phosphorylation sites are usually conserved among species (e.g. T187 and S10), but T157 is an alanine in the mouse. This observation prompted us to re-evaluate the site(s) targeted by Akt1. We now show purified Akt1 phosphorylates mouse and human p27 equally well. Phospho-peptide and phospho-amino acid analysis revealed Akt1 modifies neighboring serine residues that are conserved among species. Analysis of p27 deletion mutants localized these residues to the N-terminus 1–86 amino acids, which contain the well known S10 phosphorylation site. We confirmed Akt1 phosphorylates S10 by western blot using an antibody specific for phospho-S10. Surprisingly, Akt1 failed to phosphorylate p27S10A despite mapping evidence indicating existence of an additional site. This suggested S10 phosphorylation might be a pre-requisite for its modification. We confirmed this idea using a p27 mutant containing threonine in place of S10, which now resulted in both radiolabeled threonine and serine. Our data indicate Akt1 sequentially phosphorylates multiple serine residues within a conserved but non-canonical region, one of which (S10) has already been identified as a major regulator of p27 function. These results were confirmed in cultured cells where co-expressing Akt1 with p27 leads to an increase in S10 phosphorylation. Finally, we show that stressing tissue culture cells with oxidative insult or growth factor deprivation causes an Akt-dependent increase in p27S10 phosphorylation.
Akt1 phosphorylates both human and mouse p27
Akt1 phosphorylates p27 at multiple serines
Tryptic peptides for Human p27Kip1
Akt1 phosphorylates serine 10
The predicted tryptic digest shows only one N-terminus peptide containing two serines (Table 1; amino acids 6–15). Our attention was immediately drawn to S10 – despite the fact it is not within an Akt1 consensus motif – because it is a well known, physiologically relevant p27 phosphorylation site. To determine if S10 is one of the sites targeted by Akt1 we utilized a commercially available antibody directed against p27 phosphorylated at this location. This antibody clearly recognizes p27 only after incubation with Akt1 in a kinase reaction (Figure 4A). In contrast, it failed to recognize p27S10A whether or not Akt1 was present. To further confirm that the S10 antibody is recognizing phosphorylated p27, products of the kinase reaction were incubated alone or in the presence of alkaline phosphatase (Figure 4B). As expected, the signal was significantly diminished after phosphate removal. Serine 10 and surrounding amino acids are conserved in both mouse and human p27 (Figure 1), and we confirmed Akt1 targets S10 in mouse p27 using the same methodology (Figure 4C; lanes 3 and 4). Furthermore, phospho-peptide maps of mouse p27 were identical to that of the human protein, confirming the same sites are phosphorylated in both species (Figure 4D).
P27S10 phosphorylation is required to target a second site
We directly tested this idea by taking advantage of the fact Akt1 is a serine/threonine kinase. If S10 alone is phosphorylated then converting it to threonine should only generate radiolabeled phospho-threonine. However, if an additional serine is also targeted both phospho-threonine and phospho-serine should be present. We generated a p27S10T mutant by site-directed mutagenesis. Akt1 still phosphorylated hp27S10T, albeit more weakly than wild type p27 (Figure 5B, compare lanes 1 and 3). Nevertheless, the amounts were sufficient for phospho-amino acid analysis. As shown previously Akt1 phosphorylated wild type p27 almost exclusively on serines (Figure 5C, third panel). In contrast, p27S10T clearly shows both phospho-serine and phospho-threonine (Figure 5C, bottom panel). These results provide further evidence Akt1 targets S10, and reveal existence of a second modified serine. Because p27S10A is not radiolabeled by Akt (see Figure 5A and B), we conclude that modification of the second site is dependent on first phosphorylating S10. This scenario could also explain why the second site appears to be phosphorylated less efficiently.
Kinetic analysis of p27 phosphorylation
Even though mutating T157 to alanine had no obvious effect on p27 phosphorylation, other residues in the C-terminus might still be involved in Akt1 binding. We addressed this question by determining if p27(87–198) can inhibit phosphorylation of the full length protein. Recombinant Akt1 was incubated with [32P]-γ-ATP and full length p27 in the presence of increasing C-terminus. Figure 6D shows phosphorylation of hwtp27 was diminished when p27(87–198) was present. Similar results were obtained when the outcome was measured using the phospho-specific S10 antibody (Figure 6E). Thus, mutations in the p27 C-terminus could conceivably compromise an Akt1 binding site and affect phosphorylation at N-terminal residues. Although we were unable to demonstrate this consequence when T157 was mutated in our system, alteration of this site might affect p27 phosphorylation under different reaction conditions.
Purified preparations of recombinant Akt1 show different specificities
Full length Akt1 from mammalian cells phosphorylates serine 10 in vitro
Stress activated Akt phosphorylates p27S10 in cultured cells
Akt is activated in response to a variety of different stimuli such as growth factors or cellular stress (50–53). Oxidative stress via exposure to copper was previously shown to activate Akt (50), so we examined whether treating cells with CuSO4 had an effect on p27 phosphorylation. Indeed, incubation of HEK293 cells overexpressing p27 and Akt1 with CuSO4 resulted in higher levels of p27S10 phosphorylation (Figure 9B, compare lanes 3 and 4). Total p27 levels also increased as expected given previous work showing S10 modification stabilizes p27 (28). Importantly, pre-treatment of cells with Akt inhibitor totally abolished S10 phosphorylation, indicating the CuSO4 effect was mediated by Akt1 (Figure 9B, lane 5). To determine if endogenous Akt targets p27S10 in response to stress, HEK293 cells were transfected with p27 alone and then treated with CuSO4. Exposure to CuSO4 again caused an increase in p27S10 phosphorylation (Figure 9C, compare lanes 2 and 3) which was abolished by pre-treating cells with wortmannin or Akt inhibitor (Figure 9C, compare lane 3 to lanes 4 and 5). As expected we observed increased levels of total p27 due to the increased stability of the S10 phosphorylated form (Figure 9C, middle panel).
To confirm that CuSO4 treatment was activating Akt1 via generation of reactive oxygen species (ROS), we repeated the experiment with H2O2 (51). H2O2 treatment of HEK293 cells overexpressing p27 with increased p27S10 phosphorylation, which was inhibited by pre-incubating cells with wortmannin or Akt inhibitor (Figure 9D). In marked contrast, mitogen stimulation also leads to increased Akt1 activation and p27S10 phosphorylation (Figure 9C). However, in this case it is not prevented by the Akt inhibitor or wortmannin. These results suggest Akt1 specifically phosphorylates p27 at S10 in response to cellular stress, while a different kinase carries out this modification in response to mitogens.
Previous analysis of p27S10 phosphorylation in cells revealed it increases in response to the stress of mitogen withdrawal. To determine if Akt1 is responsible we analyzed p27 phospho-S10 levels after serum starving Hela cells in the absence or presence of Akt inhibitor. Serum starvation increases p27S10 phosphorylation, and this effect is dramatically blocked by Akt inhibitor (Figure 9E). These results strongly suggest Akt is largely responsible for phosphorylating p27S10 in response to mitogen removal. In contrast, mitogen stimulation of serum-starved cells also leads to Akt1 activation, but as predicted above this does not correlate with nor lead to increased p27S10 phosphorylation (Figure 9F). These results suggest that although Akt1 is activated in response to a variety of stimuli, it specifically phosphorylates p27S10 when cells are exposed to cellular stresses such as reactive oxygen species or growth factor deprivation.
Previous work has clearly demonstrated that Akt1 phosphorylates p27 [30]. Due to the multifunctionality and complex regulation of p27, determining the physiological significance of this event would be greatly facilitated by precisely identifying the targeted residue(s). Akt1 is a serine/threonine kinase, so there are 27 potential targets in human p27 (18 serines and 9 threonines). Several residues throughout the polypeptide chain have been postulated as Akt1 targets (S10, T157, T187, T198, or combinations thereof), based mainly on deletion and mutation analysis using purified proteins and commercial recombinant Akt1 [30, 39–42]. This methodology has proven effective at identifying physiologically relevant phosphorylation sites in many different proteins, including p27. For instance, cyclin E-CDK2 phosphorylation of p27T187 was initially revealed by this approach [24]. However, its relative ease and simplicity compared to more labor intensive techniques like peptide mapping is tempered by potential indirect effects (e.g. mutating a binding rather than phosphorylation site).
Recent attention has focused on p27T157 because of its location within a putative RXRXXT157 Akt1 consensus site located within the p27 NLS (Figure 1) [39–42]. T157 is not present in mouse p27, however, which is curious given that both p27 and the PI3K/Akt1 pathway are highly conserved in the two species. This lack of conservation is in marked contrast to other physiologically relevant p27 phosphorylation sites such as T187 and S10. Nonetheless, there is precedence for nonconservation of sites with other Akt1 substrates. For example, Akt1 blocks apoptosis by phosphorylating human caspase-9 within the "RXRXXS196" consensus site even though S196 is not present in mouse and dog [44, 45].
To try and resolve these issues we re-examined p27 phosphorylation by Akt1. Our results show that Akt1 phosphorylates human p27, mouse p27, and human p27T157A equally well. Phospho-peptide mapping of p27 revealed three radiolabeled spots, two of which lie in a diagonal characteristic of the same peptide containing different amounts of phosphate [43]. The less intense third spot may represent a partial digestion product that can arise when two basic amino acids (e.g. positions 15 and 19 in p27) are in the same vicinity [43]. Alternatively, the third spot might represent a less efficiently targeted residue residing on a different peptide. Phospho-amino acid and deletion analysis revealed that phosphate is incorporated almost exclusively into N-terminus serines. Our attention was immediately drawn to S10 for three reasons: 1) It is located in the only N-terminus tryptic peptide containing multiple serines (Table 1); 2) It has been previously identified as a major site of p27 phosphorylation in cells [28]; and 3) S10 was previously suggested as a potential Akt1 phosphorylation site [30]. A commercially available phospho-S10 antibody specifically recognized both mouse and human p27 after pre-incubation with Akt1 and ATP, confirming at least one of the modified residues is S10. Because phospho-peptide mapping indicated a second serine is located on the same peptide, it could be either S7 or S12. Experiments are currently underway to precisely identify this site, which is complicated by its close proximity to S10 and the sequential nature of the reaction.
We generated hp27S10A with the expectation it would still be phosphorylated by Akt1 at the second site. However, the kinase completely failed to radiolabel this mutant, suggesting S10 modification is a pre-requisite for targeting the other residue. We attempted to mimic S10 phosphorylation by changing it to glutamic acid, but Akt1 did not label this mutant (data not shown). As an alternative approach we changed S10 to threonine. A kinase assay confirmed p27S10T is still phosphorylated, and phospho-amino acid analysis now revealed both radiolabeled threonine and serine. These results confirmed Akt1 phosphorylates S10, and showed its modification is required for targeting the second serine. Phospho-S10 could alter p27 structure to reveal the second site, perhaps involving the proline at position 11. Akt1's ability to sequentially target multiple residues in p27 raises the interesting possibility a similar mechanism occurs at the previously identified RXRXXS/T consensus site, which might have been overlooked by mutational analysis.
The region surrounding S10 is not the typical Akt1 consensus site (RXRXXS/T) originally characterized using a panel of purified peptides [46]. S10 is immediately adjacent to Pro11, and the peptide analysis suggested such a configuration is not targeted by Akt1 [46]. Obviously, however, the structure or recognition of an isolated peptide may be quite different within the context of a larger amino acid sequence. Thus, our results hint at the existence of a novel sequence motif that might be utilized in other Akt1 substrates. A blast search using a possible motif derived from the p27S10 region (RXSXXS/TPSXXR) identified several proteins involved in the PI3K/Akt1 pathway (e.g. Actin-binding LIM protein 1, PP2A, ABL1 and eIF4E binding protein). It will be of interest to determine if any of these potential candidates are phosphorylated by Akt1.
Why has there been so much confusion surrounding identity of the p27 residues(s) targeted by Akt1? One possible complication is pointed out by our observation that while Akt1 phosphorylates the N-terminus, it can also bind the C-terminus. Depending upon the experimental system and reaction conditions, mutations in this region could inhibit phosphorylation of the N-terminal residues. Such a situation is not unprecedented given that cyclin E-CDK2 binds p27 at the N-terminus region yet phosphorylates T187 [24]. Likewise, another candidate for S10 phosphorylation (hKIS) was originally identified by its affinity for the C-terminus portion of p27 [29]. The confusion could also be explained by the use of different recombinant Akt1 preparations, which we found display varying specificity towards potential substrates. These differences could arise due to a contaminating kinase, or more interestingly as a result of mutations introduced into Akt to facilitate its activation and/or purification. Regardless, our observations suggest caution is warranted when using recombinant Akt1 to characterize substrate specificity. For this reason and to confirm S10 is a relevant site, we analyzed p27 phosphorylation by full length wild type Akt1 in vitro and in cells. In both cases we found that Akt phosphorylates p27 at serine 10.
Many different stimuli activating Akt1 have been identified. We found that two well characterized types of cellular stress known to activate Akt1 – reactive oxygen species and growth factor withdrawal – also caused an increase in p27 phosphorylation at S10. Furthermore, pre-treating cells with small molecule inhibitors of Akt activation or activity blocked this stress dependent increase in S10 phosphorylation.
This effect appears to be quite specific, as mitogen stimulation also resulted in Akt1 activation but did not lead to an increase in S10 modification. Akt activation in response to stress is known to initiate a pro-survival response, and p27S10 phosphorylation/stabilization could play a role in this process by blocking cell cycle progression and/or modulating signal transduction pathways [18, 19]. These possibilities must be tempered by our observation that when targeting S10 Akt1 can also phosphorylate a neighboring serine, which could have unexpected consequences or alter regulatory events typically associated with S10 modification. Given our in vitro analysis suggesting Akt1 specificity can vary, it remains possible that Akt1 targets p27 residues other than/in addition to S10 in response to different physiological conditions. Regardless, our observation that Akt1 phosphorylates p27S10 in response to cellular stress has important implications for current efforts to understand how PI3K/Akt1 normally regulates p27, as well as how this process is disrupted in human cancers.
Akt1 phosphorylates p27 to regulate its activity, but the identity of targeted residue(s) remains controversial. Here we show that full-length wild type Akt1 – either purified or in cells – phosphorylates p27 at S10, a noncanonical but evolutionarily conserved site known to regulate p27 function. Using recombinant Akt1 that recapitulates this specificity, we showed modification of p27S10 also leads to phosphorylation of an adjacent serine. Although Akt1 is activated by many different stimuli, we found that it specifically phosphorylates p27S10 in response to cellular stress such as reactive oxygen species or growth factor deprivation. These results integrate PI3K/Akt1 signaling with p27 regulation through its major phosphorylation site in cells, and thus identify new avenues for understanding its deregulation in human cancers.
Protein expression constructs
P27, His-tagged Akt1, His-tagged cyclin E and CDK2 were cloned into the CS2+ mammalian expression vector in which protein production is driven by the CMV promoter [24]. HA-tagged Akt1 was a gift of Dr. Richard Nho. Tags are located at the N-terminus. For bacterial expression p27 was cloned into a Pet16B vector [24]. P27 mutants were generated using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and confirmed by DNA sequencing.
Recombinant proteins and protein purification
Purified GSK protein was purchased from Cell Signaling Technology. Purified recombinant Akt1 protein was obtained from Cell Signaling Technology (prep1:#7502; and prep3: #9274) and from Calbiochem (prep2). His-tagged p27 was purified from E. coli extracts by Ni2+-affinity chromatography. Briefly, the BL21 strain of E. coli was transformed with Pet16B expression vectors containing the cDNA of interest, grown to an OD of 0.6, and protein synthesis induced by 1 mM IPTG for 4 hours. Cells where lysed in non-denaturing buffer pH8 (50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole, 0.05% tween) and His-tagged proteins purified under native conditions according to the manufacturer's directions using Ni-NTA kit (QIAGEN). Purified proteins were dialyzed against 10 mM Tris pH8.0, 1 mM EDTA, 20 mM NaCl, 2 mM DTT and 10% glycerol for 16 hours. P27 concentration was determined by the Bradford method and comparison with a known standard (BSA; BioRad) by Coomassie staining. His-tagged cyclin E-CDK2, full length His-tagged Akt1 and full length HA-tagged Akt1 were purified after transient overexpression in HEK293 cells. Cells were harvested under native conditions in non-denaturing buffer pH8. Full length His-tagged Akt1 and His-tagged cyclin E-CDK2 were purified by incubating whole-cell extract with Ni2+-agarose beads (QIAGEN) for 1 hour at 4°C. Beads were washed 3 times with 1 ml of the above lysis buffer containing 20 mM Imidazole, then once with kinase reaction buffer (25 mM Tris pH7.5, 5 mM β-glycerophosphate, 2 mM DTT, 50 nM Na3VO3, 5 mM MgCl2, and 0.2 mg/ml BSA). Full length HA-Akt1 was purified by immunoprecipitation with an antibody against HA-Tag (Santa Cruz, Santa Cruz, CA) using protein A-agarose (Roche) as described previously [22]. The kinase reaction was performed as described below.
Kinase assays
Akt1 or cyclin E-CDK2 were incubated with purified His-tagged p27 in a final volume of 20 μL containing 20 μM non-labeled ATP (Sigma-Aldrich), 4 μCi [32P]-γ-ATP (ICM), and kinase reaction buffer (25 mM Tris pH7.5, 5 mM β-glycerophosphate, 2 mM DTT, 5 μM Na3VO3, 5 mM MgCl2, and 0.2 mg/ml BSA). Reactions were performed at 30°C for 60 min (unless otherwise indicated) and quenched by addition of SDS sampler buffer. Samples were heated at 90°C for 5 minutes, centrifuged at 13000 rpm for 3 minutes, and separated by SDS-PAGE. Proteins were then transferred to a PDVF membrane and incorporation of radiolabel visualized by autoradiography or phosphoimager.
Phospho-peptide and phospho-amino acid analysis
Phospho-peptide (PPA) and phospho-amino acid (PAA) analysis were performed as described by Boyle et al. [43]. Briefly, kinase products were separated by SDS-PAGE and transferred to PDVF membrane. Bands containing the radiolabeled protein were visualized by autoradiography. For PPA, proteins were eluted by soaking excised membrane in 0.5% polyvinylpyrrolidone (PVP-360; Sigma) plus 100 mM acetic acid at 37°C for 30 minutes to block nonspecific absorption of trypsin. The membrane was then washed with water and 50 mM ammonium bicarbonate. Radiolabeled proteins were digested by incubation with 10 μg of trypsin in 50 mM ammonium bicarbonate at 37°C for 2 hours, followed by a second incubation with an additional 10 μg of trypsin overnight at 37°C. Phospho-peptides were spotted on cellulose-TLC plates and electrophoresed at 1500 V for 25 minutes. After air drying the plates were placed in a chromatography tank containing isobutyric acid buffer (1250 ml isobutyric acid, 38 ml n-Butanol, 96 ml pyridine, 58 ml glacial acetic acid, 558 ml water). Ascending chromatography was performed for 12 hours perpendicular to the direction of electrophoresis. Peptide maps were visualized by exposing plates to the phosphoimager. To determine identity of phosphorylated amino acids (PAA), bands containing radiolabeled proteins were hydrolyzed with 6N HCl at 100°C for 1 hour followed by lyophilization with a vacuum centrifuge. Dry pellets were resuspended in pH1.9 buffer containing 1 μg of nonlabeled phospho-serine and phospho-threonine standards. Because Akt1 is a serine threonine protein kinase it was not necessary to look for phospho-tyrosine, which allowed us to run electrophoresis in one dimension. Samples were spotted on TLC plates and electrophoresed at 1500 V for 25 minutes. Phospholabeled amino acids were visualized by exposing plates to the phosphoimager. Standards were visualized by 0.25% ninhydrin in acetone spraying and 15 minute incubation at 65°C.
Cell culture
HEK293 and Human Diploid Fibroblasts (HDF) cells were obtained from ATCC and cultured in Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO) supplemented with 10% (v/v) fetal bovine serum (FBS) (Atlas Biologicals) at 37°C in 5% CO2. Hela cells were obtained from Dr. Anja Bielinsky (University of Minnesota) and cultured in DMEM (1 mg/ml glucose) (Cellgro) supplemented with 10% (v/v) FBS (Atlas Biologicals). Mitogen deprivation was performed by growing cells to confluency and removing FBS for 48 hours. Cells were then stimulated with 20% FBS for the indicated times. When treating cells with CuSO4, DMEM was removed, cells were washed with PBS and incubated for 2 hours in Hanks' balanced salt solution (HBSS) containing CuSO4. Where indicated, cells were pre-treated with 200 nM wortmannin (Calbiochem) or 1 μM Akt inhibitor VIII (Calbiochem #124108) for 30 minutes, washed with PBS, and then incubated in HBSS containing CuSO4 and inhibitors.
Transient transfections
Transfections were performed using the calcium-phosphate precipitation method as described previously [24]. Briefly, indicated amounts of plasmids containing the cDNA of interest were diluted in 500 μL calcium-phosphate solution, then added to 60 mm cell culture plates containing proliferating HEK293 cells in 3 mL of DMEM with 10% FBS. After 12 hours cells were washed with PBS and allowed to recover for 24 hours in DMEM plus 10% FBS prior to harvesting in indicated buffer. H2O2 was added to transfected cells for 2 hours prior to harvest. Where indicated, cells were pre-treated with 200 nM wortmannin (Calbiochem) or 1 μM Akt inhibitor VIII (Calbiochem #124108) for 30 minutes prior to H2O2 addition. In the case of CuSO4 treatment DMEM was removed, then cells were washed with PBS and incubated for 2 hours in Hanks' balanced salt solution (HBSS) containing CuSO4. Where indicated cells were pre-treated with 200 nM wortmannin (Calbiochem) or 1 μM Akt inhibitor VIII (Calbiochem #124108) for 30 minutes, washed with PBS, and then incubated in HBSS containing CuSO4 and inhibitors.
Western blotting
Purified proteins or whole cell extracts were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to PDVF membranes, and visualized by immunoblotting with indicated antibodies. Bound antibodies were detected using horseradish peroxidase-linked anti-mouse or anti-rabbit antibodies (Amersham Pharmacia Biotech), and visualized by enhanced chemiluminescence (Santa Cruz) followed by exposure to X-ray film (Kodak X-Omat AR). The antibodies employed are: anti-Akt1 and anti-phospho-AktS473 (Cell Signaling Technology); anti-p27 (Transduction Labs); anti-phospho-p27S10 and anti-HA-tag (Santa Cruz).
Alkaline phosphatase treatment
After the kinase reaction utilizing non-labeled ATP, phosphorylated p27 was incubated for 1 hour at 30°C in the presence of 40 units of calf intestinal alkaline phosphatase (Promega) in 20 μL of buffer containing 50 mM Tris-HCl (pH 8.0) and 1 mM MgCl2. The reaction was stopped by adding SDS-sample buffer and then subjected to SDS-PAGE, followed by western blot analysis of total and S10 phosphorylated p27.
We thank Dr. Lincoln R. Potter for expert assistance with PPA and PAA and Dr. Stephanie J. Moeller for technical assistance and critical review of the manuscript.
This work was supported by an Ellison Medical Foundation New Scholars Award in Aging to RJS (AG-NS-0140-01).
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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About the ShelterAnimal Harbor is a limited-admission animal shelter facility serving Franklin County, Tennessee. It is operated by members of The Franklin County Humane Society, a private non-profit 501(c)(3) organization. Animal Harbor opened its doors in February of 2003. It was the first animal shelter in Franklin County.
The shelter operates under a no-kill philosophy-- we do not euthanize pets for space. Once a dog or cat is admitted to Animal Harbor, it receives the best of care. All of our pets are inoculated, wormed, and spayed or neutered at the appropriate age.
Animal Harbor is located at 25 Walnut Grove Road-- at the corner of Highway 64 and Walnut Grove Road. This is nine miles south-west of downtown Winchester, Tennessee, just past Belvidere, Tennessee, going toward Huntland, Tennessee. Coming south just past Belvidere, watch for a favorite local landmark, the Swiss Pantry, and Animal Harbor will be one mile further, on the right. Click here for map.
Tuesday-Thursday: Noon — 5:00 PM
Friday: Noon — 6:00 PM
Saturday: 10:00 AM — 4:00 PM

subset: commoncrawl


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The already shortened Democrat convention in Charlotte just shrank a little bit more. Convention officials announced this morning that Obama's acceptance speech at Bank of America stadium is being scrapped and moved inside the vastly smaller convention hall. The stadium seating capacity was 75,000. The hall? About 20,000. Democrats are blaming the move on the "weather."
Forecasters are saying there is a 40% chance of rain tomorrow in Charlotte. Earlier this week, a convention spokesman said the speech would take place "rain or shine." Today, however, they site fears of lightening as the reason for the move. (Perhaps God is mad the Democrats took him out of their platform.)
Of course, this comes amid what had been building concern over whether the Democrats would be able to fill the large stadium. Convention staff had recently been aggressively offering tickets to anyone. There were also plans to bus in thousands of supporters from neighboring states to ensure a full stadium. Fortunately, the Democrats won't have to worry about that now.
Democrats had months ago taken the unprecedented step of canceling the first day of the convention, mostly due to problems with fundraising. A giant celebration at the massive Nascar track in town was also scrapped, replaced by a quaint "street-festival."
2012 is looking increasingly like the year of diminishing returns for Democrats.


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As your business partner we want to make your business banking easier. Our most common business lending products are overviewed below. We also believe you will find of value a visit to our
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Commercial Mortgage Loan
A loan used to finance the purchase of real estate (or construction). It appears on the business' balance sheet as land and improvements (buildings). A commercial mortgage is repaid from net income of the business on a defined amortization.
Term Loan
A secured loan used to finance fixed assets (other than real estate), such as equipment. The term of the loan depends on the life of the asset being financed and the cash flow of the business operation.
Revolving or Seasonal Line of Credit (LOC)
Generally, a credit arrangement for financing fluctuations in current assets. The need predominantly results from a sales volume that cannot be supported by the internally generated cash flow of the business or a seasonal need, like inventory for peak times in a landscaping business. Repayment is from conversion of non-cash assets to cash, such as inventory and accounts receivable.
Permanent Working Capital
A loan used to finance cash needs. This type of loan is used when the business operation's cash disbursements exceed its ability to generate cash internally. Repayment is from net profits.
If your business is expanding; needs 100% financing; or if you require a unique payment plan, leasing may be the option for you.
Click here for more information.
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A Letter of Credit is an instrument issued by a bank on behalf of a client authorizing an individual, or firm, to draw drafts on the bank, or on its account at one of its foreign correspondent banks, under certain conditions stipulated in the Letter of Credit.
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saddle tramp(redirected from a saddle-tramp)
saddle tramp
1. slang A cowboy, particularly one who lives a nomadic lifestyle. Primarily heard in US. You can't trust him—he's just a saddle tramp who roams from town to town!
2. slang One who rides on horseback. Primarily heard in US. A: "I hear hoofbeats." B: "Yes, there's a saddle tramp approaching in the distance."


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Five Common Pitfalls of Property Investors
Whether you're looking to invest in your first or fifth property, everyone can learn a thing or two from these some common investor mistakes.
1. Leasing serviced apartments
Serviced apartments are popular with investors at the moment. With strong demand from business travel customers, continued high growth levels in the sector are predicted.
However, this growth potential has to be weighed against a number of things:
- the necessity of spending a good deal of money on furnishing the apartment up front
- the hefty ongoing management fees involved
- and the inflexibility of having to lock in a long-term commercial management lease.
Which can make the property difficult to sell in the short term, if that's what you decide to do.
Note that banks also tend to be wary of serviced apartments, and will usually require that you have a larger percentage of deposit than for a traditional property purchase. Crunch your forecast numbers very carefully – and remember that if a deal looks too good to be true, it usually is.
2. Only viewing the property during the day
If possible, make sure you see the property at night as well as during the day. If you can't access the property itself in the evening, at least spend some time in the neighbourhood.
Noise from traffic and neighbours can differ greatly during the night. Is the street full of riotous party houses? Do trucks start rolling in during the early hours of the morning? You won't be able to tell from a daytime visit. Also consider whether the area feels safe after dark. Some areas may take on a very different atmosphere when the sun sets.
3. Over and under-capitalising on improvements
As a rule, you should spend no more than 10 per cent of a property's value when renovating, otherwise your rental gains will not justify the outlay on improvements. If you purchase an old property that needs a new kitchen, for example, make sure the cost of the kitchen will be under this threshold.
On the other hand, especially for older properties, if you don't invest in any improvements, you risk not keeping pace with modern living standards and the rent will remain low. Finding a balance between these two dangers is essential.
4. Buying a rental property with a friend or family member
Especially for first timers, sharing the costs of buying a rental property with family or friends is an increasingly popular option. There is nothing inherently wrong with doing this, provided you avoid the dangers involved.
No matter how close you are with your co-purchasers, make sure there is a clear written contract about how all costs are shared. These include not just the mortgage repayments, but also maintenance, agency management, body corporate fees, council rates and more.
If you have any doubts about a party's capacity or willingness to pay these, or if you detect differing visions for the management of the property or portfolio, rethink the wisdom of such an arrangement. Remember that you risk jeopardising relationships that are far more valuable than the potential financial gain.
5. Following the crowd
It's tempting to buy when the market is hot and certain areas are trending, but there's no harm in looking further afield. Do your own research and due diligence, and remember that by the time an area is publicly designated an investor 'hotspot', the growth potential has already been exceeded.
For more expert guidance on property investment and management, contact the investment professionals at K.G. Hurst today.
Image courtesy of Chris Sharp / FreeDigitalPhotos.net


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Hentai Picture: Hooded Stephanie moves her bootie up and down on Robbie's trouser snake!
Look at mock-obedient and courteous babes of Lazy Town with their cute legs apart to show their sweet sleek pussies and meaty warm stems slapping their bollocks over meaty yummy ass sphere. Sex-starved Lazy Town babe wants to feel hard black dick pushed al the way down into her butt snatch, and then blow it off till it shoots tons of cum into her mouth and on her face! Robbie Rotten is brimming with anxiety to get fucked in both her cum receptacles and when she gets it at last she squirts hard.
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I'm chuffed to let you know that ADOX is getting ready to ship ADOX CHS 100 II which they claim will be very close in characteristics to the original ADOX CHS 100 ART and will be available in the full range from 135, 120 to large format except 127 format. Because it is newly developed, it will benefit from new technologies to avoid problems with light piping (no more loading the film in the dark) and a new coating avoiding scratching the emulsion during development. See here for details.
On Monday 22 July, ADOX followed up on their Facebook page with the following statement:
Today CHS 100 II saw the dark of the coating alley. Now it has to settle and harden. After August 15th we can start cutting it up. We hope to keep "August" as a delivery date for the first rolls.


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Reviews & Commentary on Film Festival Screenings, Select New Releases and Contemporary Foreign Film.
Tuesday, May 7, 2013
Hot Docs 2013 Film Review - Rent A Family Inc.
Director Kaspar Astrup Schroder came up with the idea for Rent a Family Inc. on one of his many trips to Japan. He was looking through the classifieds and noticed the unusual items that were available for rent. The papers had dogs that could be rented for an hour to take them for a walk in the park. The paper also had a section for renting people. Intrigued Schroder contacted Ryuichi who owned a business I Want To Cheer You Up Ltd. The business is run though a web site and a cell phone. Ryuichi will rent himself out as a father, husband, boss, colleague or even get together a group to serve as one side of the family for a wedding.
Ryuichi is the father of two boys who does not have a great relationship with his family. He hardly speaks to his wife. They live in small cramped quarters outside of Toyko. His oldest son now sleeps with his mother in the bed that Ryuichi bought for himself so he could get a good nights sleep. Father's day goes by each year unnoticed while mother's day is a big deal in the household. His family had no idea about his business. His wife does not care what he does as long as he brings home money to support the family. The only affection that Ryuichi gets at home is from the small family dog Chappi.
Schroder interviews Ryuichi using a one camera shot with the questions coming from out of the frame. Ryuichi admits that he is lonely and has thoughts of suicide. Money is always an issue and if he dies his family would get to keep the home. His wife is interviewed in the same fashion using a Japanese translator. She recounts that the only way she knows that he has changed jobs is based on the new delivery company uniform that she finds in the wash. Schroder also injects Japanese shows into the film one of which has Ryuichi on as a guest, identity hidden to give details of his business. The shows are loud, extremely busy, visually stimulating and speak to the main social and cultural issues of the day.
The film recounts four I Want to Cheer You Up assignments. In the first Ryuichi plays the new husband of a woman that wants her ex to pay child support and give her control of the funds. Her ex had always refused to do so in the past but with Ryuichi playing the role of the new husband and pointing out that he has to take on responsibility for the children the ex agrees to let the client control the funds for the kids.
The most elaborate job is the assignment to provide the entire brides side of a wedding. Ryuichi plays an Uncle of the bride and a highlight of the film is the group going over their roles on the train ride to the wedding getting their names and back stories straight. The wedding goes off with a few hitches and mistakes but the troop are generally moved by the ceremony many crying at the event.
Rent a family Inc is a unique look into Japanese culture. The need for individuals to seek approval from others and an underlying tone that people are aware that something is not right with the situation but go along to get along. It's a film that I can recommend.
*** out of 4
Rent a Family Inc. | Kaspar Astrup Schroder | Denmark | 2013 | 77 Minutes.
2013 Hot Docs Film Festival.
Tags: Classifieds, Renting people, Stand Ins, Secret Job, Dysfunctional Family, suicide, Japanese culture, Japanese Television.
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The unit is open 24-hours and 7 days a week providing care to more than 20,000 patients each year. All emergency cases will be attended by our very experienced Medical Officer specially trained to handle trauma and emergency cases and a team of highly experienced nurses.
Referral to specialist is done immediately when necessary.
The unit is also equipped with a minor operation theatre for minor surgeries and other emergency procedures.
Quality Policy
At Lablink everyone of us from both the technical and administrative sections work in harmony in pursuing and meeting QUALITY STANDARDS in laboratory practice. We shall meet the needs and expectations of the medical practitioners who are our direct clients and in doing so our service will provide maximum benefits and satisfaction to the patients.
We take every opportunity given to us by our client doctors to make their already complex job easier, by providing accurate laboratory results and correct interpretations along with appropriate advice in the quickest possible "turn around" time. We work hard towards achieving this goal and helping our doctors to keep up with the continued advances in laboratory science. In providing prompt, cordial and cost effective service, patients' welfare is uppermost on the mind of our staff.
The laboratory is equipped with the latest state-of-art facilities providing services such as
- Clinical Biochemistry
- Hematology
- Blood Bank
- Microbiology/Urinology
- Immunology
- Hispathology/Cytology
Operating hours
Laboratory is open on Sunday/Public Holiday and after office hours. Overtime charge is charged on the tests done during these hours.
Please visit our website for further information.
Website : http://www.lablink.com.my
Email : firstname.lastname@example.com
The Diagnostic Imaging (Radiology) department is equipped with the latest state-of-the-art facilities which include:
1. General x-ray machines
2. Fluoroscopy machine for screening procedure & special investigations
3. Ultrasound Machine
4. 64-slice CT Scan
5. Mammogram machine
6. Magnetic Resonance Imaging (MRI)
7. Lithotripsy Machine
8. Fluoroscopy Machine
General Radiography
A basic radiological imaging procedure on body parts upon clinician request
A technique of using ultrasound modality to produce images within the body to diagnose and high frequency of sound wave is use instead of radiation.
Utrasound Examination
Types of ultrasound examination:
- Abdomen
- Pelvic region
- Liver, gall bladder and pancreas
- Thyroid
- Breast
Also known as magnetic resonance imaging, is a pain-free non-invasive test used to produce two dimensional images of body structures by using intense magnetic fields on to patient body.
Common MRI investigation
- Brain - Lesion , bleed or looking for early detection of infarct
- Extremities and joints.
- Spines and spinal cord
- MR angio.
How to get our service
Request for x-ray examination must be referred by consultants and general practitioner via
1. Request form
2. Appointment via phone (need referral letter upon arrival)
3. On line request (available within hospital premises)
4. Memo instruction by authorized qualified medical personnel.
CT Scanning
An x-ray procedure which combines many x-ray images with the aid of a computer to generate cross-sectional views and, if needed, three-dimensional images of the internal organs and structures of the body. A CT scan is used to define normal and abnormal structures in the body with high resolution images.
Our high end facility
64 Multislices CT ( MDCT):
What is an advantages of 64 multi slice CT or (64MDCT)
CT Scan is used to diagnose such as:
- Brain defect of bleed
- Bone defect
- Lung defect
- Bleed in other organs
- Lesion
- Vertebral defect
- CT Angiography (CTA)
- 3D reconstruction
- Dental MPR scan
What you need to do for CT examination.
- Patient must be in the department 15 minute earlier .
- Patient with medication must inform the radiographer on duty.
- Radio opaque should be removed
Note : Liquid diluted gastrografin (contrast media) to be taken orally 20-30 minutes before examination (as required).
An Imaging procedure for dynamic study which specialization examination area examined by doctors.
Fluorocopy (Special Examination)
Special examination is an imaging procedure involving difference of positive contrast (Barium sulphate solvent or organic iodine suspension). The contrast media is given to the patient through tube, injection or oral.
Types of examination & the contrast used
Barium swallow
Barium meal
Barium enema Dissolved Barium sulphate
2. Urinary System
IVU (Intravenous Urogram)
MCU (Mict. CystoUrethrogram) Organic iodine suspension
3. Nerve System
Myelogram - organic Iodnie Suspension
4. Vascular System
Arteriogram Organic Iodine suspension
5. Others
- HSG (Hysterosalphingogram)
- T-Tube
- Sialogram
- Sinogram
( Please inform the nurse or radiographer if you are allergic to any drugs before examination on during the booking of appointment ).
Our Cardiac Catheterisation Laboratory has the latest equipment with the state-of-the-art Technology. This facility is special X-ray equipment used to perform angiogram (special X- rays of the arteries).
The following services are available: - Coronary angiogram to assess the severity of the coronary heart disease and suitability for bypass surgery or coronary angioplasty
- Right and left heart catheterization to assess the disease of the heart valves, hole in the heart, etc.
- Permanent pacemaker implantation to treat life threatening abnormal heart beat.
- Coronary angioplasty or PCI—balloon dilation and stenting of the coronary arteries.
- Angiogram and angioplasty of the kidney arteries.
Cath. Lab Angiography
It is part of interventional radiography examination such as blood vessel in some organs and heart.
Some example that dedicated angio system can be use as following examination :
1. Coronary Angiogram
2. Aortogram
3. Interventional Radiology (IR) procedure
Dietitian, who is the nutrition and dietetic professional will be responsible to provide dietary consultation to all patients and plan menu for patients requiring specific nutritional intervention.
Service Provided
• Diet counselling for inpatient and outpatient
• Enteral feeding assessment and regimes for inpatient and outpatient
• Design patient meal plan for patient requiring individual diet modification
• Conduct group counselling or nutritional education to patient in group
We accept appointment via:
• Phone call
• Doctor referral
Cancellation should be notified earlier for rescheduling of your appointment
Dietitian's office is situated at the Angkasa Ward (Level 4) of KPJ Seremban Specialist Hospital.
Operating hours
Contact Dietitian
Phone: 06 767 7800 ext 1454
Email: firstname.lastname@example.com
Physiotherapy Services that specializes in Rehabilitation is one of the clinical support services available at KPJ Seremban Specialist Hospital.
Our Physiotherapist provides client-centred services that develop, maintain and restore people's maximum movement and functional ability. They can help patient at any stage of life, when movement and function are threatened by ageing, injury, diseases, disorders, conditions or environmental factors.
KPJ Seremban Physiotherapist helps patients in maximise their quality of life, looking at physical, psychological, emotional and social wellbeing. They work in the health spheres of promotion, prevention, treatment/intervention and rehabilitation.
In KPJ Seremban Specialist Hospital physiotherapist treat patients from all groups of ages. We help the patients to manage pain and recuperating during rehabilitation period using various types of methods to aid recovery. It takes a holistic approach that involves the patient directly in their own care.
We cater the needs of the Hospital's inpatients, as well as referrals from General Practitioners, various Consultants and other Physiotherapists. Interventions rendered from therapeutic exercise, manual therapy as well as thermal electrotherapeutic modalities base on the assessment outcomes and patients' needs.
Most conditions treated by our Physiotherapists require a short course of treatment, which after careful assessment, are tailored specifically to your individual physical capabilities and rate of progress.
Occupational Therapy
Occupational therapy is a client-centred health profession concerned with promoting health and wellbeing through occupation. The primary goal of occupational therapy is to enable people to participate in the activities of everyday life. Occupational therapists achieve this outcome by working with people and communities to enhance their ability to engage in the occupations they want to, need to, or are expected to do, or by modifying the occupation or the environment to better support their occupational engagement.
Occupational therapy is the art and science of restoring function and independence of an individual due to physical, social, mental or psychological disorders.
It aims at optimizing independence by regaining or maintaining functions so as to maximize independence in activities of daily living (ADL) which includes daily routine work and leisure activities. Our Occupational therapist believes that occupations (activities) describe who you are and how you feel about yourself. If you are unable to do the things you want, or need to do, to live and enjoy your life, your general well-being may be affected.
Occupational therapist at KPJ Seremban Specialist Hospital provide its services in the field of orthopaedic and hand injury hand, neuro-surgical neurology, paediatric and psychosocial areas e.g. CVA (stroke) head injuries, fractures, nerve and tendon injuries of hand, joint mobility sustaining in rheumatoid arthritis and others. Tailored made pressure garment for post burn cases as well as custom splinting service is also provided.
KPJ Seremban Physiotherapist and Occupational Therapist works in tandem towards restoring function and independence of the clients to lead a healthy and effective lifestyle.
Services Provided
Treatment is available for a wide range of conditions such as:
• Stroke cases
• Intensive care
• Musculoskeletal (bones, joints and soft tissues)
• Sports related injuries
• Pre and Post-Surgical care
• Acute/chronic Orthopaedic cases
• Cardiorespiratory condition/illness
• Neurological condition
• Paediatric (children)
• Geriatric (care of the elderly)
• Back and neck conditions
• Industrial injuries
• Post burn cases
• Woman Health and Pre/Post Natal
• Education and health promotion
At KPJ Seremban Specialist Hospital, the major emphasis of our practice is to educate patients regarding self-help, home treatment programme and to involve them closely in the management of their conditions.
We accept appointment via:
• Phone with Referral
• Walk in with Referral (subject on availability)
A dedicated Home Nursing Team offers Post-Operative Rehabilitation Care upon discharge. This ensures that correct care and necessary follow up rehabilitation work continues to take place at the comforts of our patients' home.
Operating Hours
Monday – Friday
For further information, kindly contact us at:
Email: email@example.com
Phone: +606 767 7800 ext 1540/1541
Our Mission
To ensure that all patients receive pharmaceutical products that are safe, efficacious and of quality as well as advice on their rational use. Objectives
1- To provide an efficient and courteous service to all our patients.
2- To dispense correct medications to all our patients as prescribed by doctors.
3- To purchase and maintain an adequate stock of drugs which is up-to-date and comprehensive.
4- To ensure that all our patients obtain prescribed medication within a suitable time period.
5- To provide drug information and professional advice to patients and healthcare professionals.
To Our Customers
All medications dispensed shall be labelled accordingly with the name of the medications. We strongly advice patients to:
1- Know the name of your medications
2- Inform your doctor about any other medications that you are currently taking if not prescribed by him/her
3- Inform your doctor if you are pregnant or breast feeding
4- Inform your doctor and/or pharmacist if you have experienced any allergic reactions with your medications.
Working Hours
We provide 24-hours comprehensive Pharmacy services to all inpatients and outpatients of our hospital which also complements our 24-hours Accident and Emergency Department.
Our outpatient services are available at:
1- Ground Floor Pharmacy - 8:30 AM - 9:00PM
General Advice On The Use And Handling Of Medicines
1- Do not exceed the dose prescribed by your doctor. In case of overdosage, seek medical attention at once.
2- If you develop an allergic or unusual reaction to any medicine, contact your doctor at once.
3- The medicine prescribed for you are specific to treat your present condition. Never give your medicine to anyone else.
4- To avoid mistakes, do not take medicines in the dark or mix different medicines in one container.
5- Keep all medicines out of the reach of children.
6- Store medicines away from heat and direct sunlight.
7- Medicines which are expired or no longer needed should be flushed down the toilet, unless otherwise directed.
8- The word "POISON" which may appear on your medicine packages means a controlled medicine under the Poisons Ordinance 1952.
We have a reputable team of Residents and Visiting Medical Consultants who are well qualified and experienced in their respective field.
They are strongly supported by a dedicated nursing team and paramedical staff in all the discipline of medicine to deliver personalised service.
The combined force provides specialised outpatient as well as inpatient services.
• Anaesthetist
• Cardiologist
• Dermatologist
• ENT, Head & Neck Surgeon
• General Surgeon
• General & Vascular Surgeon
• Physician & Neurologist
• Neuro Surgeon
• Obstetrics & Gynaecologist
• Orthopaedic
o Trauma & Sport Surgeon
o Spinal Surgeon
o Upper Limb Surgeon
• Opthamologist
• Paediatrician
• Paediatrician (Paediatric Infectious Diseases)
• General Physician
• Physician Nephrologist
• Physician Respiratory & Internal medicine
• Physician & Cardiologist
• Psychiatrist
• Urologist
• Cardiothoracic Surgeon


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5i's CEO, Ryan Modesto will be appearing on BNN Market Call on Thursday, May 24th, 2018. Set your PVR or tune in live at 12 PM to hear Ryan's latest top picks, how the past picks have performed, and him and his team's latest investing insights.
For readers who don't get a chance to call in, you can always utilize our Q&A section to ask questions you may have throughout the year. If you're not yet a member, you can get one free question credit by signing up for a free 30-day trial here (no credit card required).
Submit your questions when he's live by calling BNN's Market Call at 1-855-326-6266, emailing email@example.com, or submit your questions on Twitter by using @marketcall during the interview.


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Johnny Depp and Christian Bale have the finest sets of cheekbones in moving pictures today, and they sure get a workout in Michael Mann's Public Enemies. Bale's in particular are a wonder; thanks to the way they frame his expressionless lips he could be gnawing the inside of his mouth into corned beef. Not to be outdone, Depp loosens and contorts to him to fetching effect, especially when he holds a tommy gun or makes promises to Marion Cotillard that even Clark Gable and William Powell couldn't utter without making their mustaches wither.
Glowering in fantastic clothes -- that's all Mann gets out of Depp and Bale (like Clint Eastwood in the seventies and eighties, Bale's recent stint of non-acting has acquired a patina of respectability). While Mann is too obsessive about art direction and such to ever conform to hackdom, his scripts show a second-rate mind susceptible to the influence of pop psychology and mytho-poetic macho twaddle. But Mann's recent forays into genre pictures has flattened his ambitions. Collateral was surprisingly boring, Miami Vice redundant, but Public Enemies is his most anonymous work yet. For reasons I can't fully explain, Mann abandons his usual sharp eye for men filling widescreen spaces; it took more than an hour for "Pretty Boy" Floyd, Homer, and the other members of the Dillinger gang to register as faces, and not a jot more. A gangster pic without a colorful supporting cast is like a Western without a saloon brawl. The rest of the action is listless (a nightime shootout in a forest goes on a couple of minutes longer than necessary), and the transitions jarring. Does Mann mean to suggest that Dillinger spent almost ten years in jail for a petty crime, escaped, assembled a first-rate crew, robbed banks successfully, and became a public icon? It's a blur.
Within this posturing lies the seed of a good movie: the creation of a federal police force that grafted developments in forensics and criminology onto vigilantism. In borrowing much from the criminals they wanted dead or behind bars, J. Edgar Hoover's FBI took its cue from the weaselly, plump, neurotic man at its head who wanted his G-men to wear smart suits as they shot crooks in the back. A film about the chicanery of crime fighting -- the implicit cooperation between the Mafia and the FBI in taking care of small fry like John Dillinger -- sounds exactly like the kind of project to which Mann would be irresistibly attracted (the corporate maneuvering in The Insider has the verisimilitude of a Louis Auchincloss novel); and in Billy Crudup's weird, very entertaining performance as Hoover (and being J. Edgar Hoover was a performance, as the press-savvy chief recognized in a career spanning ten presidential administrations), I saw material rich with comic potential. Since Mann isn't known to giggle at meal times, I wouldn't doubt that this ironic approach was beyond his sensibilities. Better an interesting failure than an inert one, though.
Crudup, by the way, has fabulous cheekbones.
Friday, July 3, 2009
3 comments:
- pomme said...
euh! LOL!
- July 3, 2009 at 8:50 PM
- tray said...
I haven't seen such consensus on anything as I've seen on the crappiness of this movie since - well I guess Sarah Palin. (And before that, how wonderful the Clipse's awful second album was.)
- July 5, 2009 at 3:35 PM
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Aloha Friday
I'm starting a new weekly column I call Aloha Friday – so make sure to subscribe so you don't miss 'em.
Here's the deal. Between my day job and my blogging, I spend waaay too much time online each day. So naturally I see some really awesome stuff (and lots of really strange stuff, too).
Aloha Friday is all about saying aloha (goodbye) to the week, and aloha (hello) to the weekend. So let's kick it off with a glimpse at the best of the internet lately.
~ ~~
The photo above was taken at one of my fave places on earth: our wedding site, on the Big Island of Hawaii. But most of the country doesn't look quite that sunny right now. Are you a fan of winter weather? This little guy sure is.
This is not a dream.
A good reminder to strive for balance… something admittedly I struggle with (she says as she types a blog post at one in the morning).
Is this a real thing? I want it. Pints should not be shared. It's in the Constitution.
Flashback to childhood in the 80s. It wasn't that long ago, was it?
I'm anti-chatskis, but I do keep one of these on my desk at work. And it makes me happy.
Wanna join me for brunch this weekend? Awesome! Would you mind bringing a donut cake?
This time last year, I was going a little crazy over goat cheese.
Two years ago, I finally got around to sharing my wedding pictures.
And three years ago this week, I was sharing my engagement story. All together now: awwwwwww!
Have a great weekend!


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Do You DIY at Home?
We jump in and do it ourselves on a lot of home projects.
Let's see: we've installed a toilet; mounted a flat screen television; painted the trim, walls, and ceiling throughout the apartment, sewed pillows, tiled a backsplash, installed new power outlets/switches, hung a ceiling fan, and made some book shelves with the help of ISS Designs.
Some things we'd like to do include: tiling the bathroom, replacing the lavatory, refinishing the wood floors, and swapping out the door hardware. We don't know that we'll get to all of these projects in the future, but we revel in taking on home improvement projects. There's something very satisfying about learning how to do something new and bettering your home in the process. What sort of projects have you DIY'ed in your home?...