text:
The parathyroid calcium receptor determines parathyroid hormone secretion and the response of parathyroid hormone gene expression to serum Ca2+ in the parathyroid gland. Serum Ca2+ regulates parathyroid hormone gene expression in vivo post-transcriptionally affecting parathyroid hormone mRNA stability through the interaction of trans-acting proteins to a defined cis element in the parathyroid hormone mRNA 3'-untranslated region. These parathyroid hormone mRNA binding proteins include AUF1 which stabilizes and KSRP which destabilizes the parathyroid hormone mRNA. There is no parathyroid cell line; therefore, we developed a parathyroid engineered cell using expression vectors for the full-length human parathyroid hormone gene and the human calcium receptor.
Co-transfection of the human calcium receptor and the human parathyroid hormone plasmid into HEK293 cells decreased parathyroid hormone mRNA levels and secreted parathyroid hormone compared with cells that do not express the calcium receptor. The decreased parathyroid hormone mRNA correlated with decreased parathyroid hormone mRNA stability in vitro, which was dependent upon the 3'-UTR cis element. Moreover, parathyroid hormone gene expression was regulated by Ca2+ and the calcimimetic R568, in cells co-transfected with the calcium receptor but not in cells without the calcium receptor. RNA immunoprecipitation analysis in calcium receptor-transfected cells showed increased KSRP-parathyroid hormone mRNA binding and decreased binding to AUF1. The calcium receptor led to post-translational modifications in AUF1 as occurs in the parathyroid in vivo after activation of the calcium receptor.
The expression of the calcium receptor is sufficient to confer the regulation of parathyroid hormone gene expression to these heterologous cells. The calcium receptor decreases parathyroid hormone gene expression in these engineered cells through the parathyroid hormone mRNA 3'-UTR cis element and the balanced interactions of the trans-acting factors KSRP and AUF1 with parathyroid hormone mRNA, as in vivo in the parathyroid. This is the first demonstration that the calcium receptor can regulate parathyroid hormone gene expression in heterologous cells.
Parathyroid hormone (PTH) regulates calcium homeostasis and bone metabolism. Changes in extracellular Ca2+ ([Ca2+]o) are sensed by the parathyroid G-protein coupled calcium receptor (CaR) [1]. The CaR determines the response of the parathyroid to [Ca2+]o at the levels of PTH secretion, PTH gene expression and parathyroid cell proliferation [2,3]. Increased [Ca2+]o activates the CaR, resulting in a G-protein-dependent activation of PLC, PLA2 and PLD [4]. This results in decreased PTH secretion and parathyroid cell proliferation. Calcimimetics bind transmembrane (TM) 6 and TM7 of the CaR to allosterically alter the conformation of the CaR [5,6]. The calcimimetic R568 decreases PTH secretion, PTH mRNA levels and parathyroid cell proliferation [7,8]. Genetic deletion of Gq/11 specifically in the parathyroid leads to severe hyperparathyroidism (HPT) [9]. Similarly, CaR-/- mice are not viable due to the severe HPT [10] and can be rescued by mating with PTH-/- or GCM2-/- mice, where PTH is either absent or markedly reduced [11,12]. Therefore, the CaR and its signal transduction are central to parathyroid physiology and the maintenance of a normal serum PTH and intact Ca2+ homeostasis.
Low serum Ca2+ and chronic kidney disease lead to secondary hyperparathyroidism which is characterized by increased PTH mRNA levels in experimental models [13]. The increased PTH mRNA in vivo is post-transcriptional and is mediated by the interaction of trans-acting proteins to a defined cis-acting AU-rich element (ARE) in the PTH mRNA 3'-untranslated region (UTR) [14-16]. A 26-nucleotide sequence within the ARE is conserved among species and is both necessary and sufficient for protein binding and the regulation of PTH mRNA stability by dietary calcium or phosphorus depletion [16,17]. AU-rich binding factor 1 (AUF1) and Upstream of N-ras (Unr) are PTH mRNA trans-acting proteins that stabilize PTH mRNA [18,19]. The binding of these proteins to the PTH mRNA 3'-UTR is regulated in the parathyroid by chronic hypocalcemia, hypophosphatemia and experimental kidney failure as well as by the calcimimetic R568 [7,15,16]. We have recently identified the decay-promoting protein KSRP (KH domain splicing regulatory protein) as an additional PTH mRNA 3'-UTR binding protein that determines PTH mRNA stability in transfected cells [20]. KSRP-PTH mRNA interaction is increased in parathyroids from hypophosphatemic rats, where PTH mRNA is unstable, and decreased in parathyroids from hypocalcemic and experimental renal failure rats, where the PTH mRNA is more stable. The balanced interaction of PTH mRNA with AUF1 and KSRP determines PTH mRNA half-life and levels and hence serum PTH levels [20].
There is no parathyroid cell line and the signal transduction pathway whereby [Ca2+]o regulates PTH secretion has been characterized by using bovine parathyroid cells in suspension and rat parathyroid organ cultures [21,22]. Parathyroid hormone gene expression and its regulation in cells in vitro have been studied in primary cultures of bovine parathyroids [23,24]. HEK293 cells transfected with the CaR faithfully maintain an intact signal transduction after the stimulus of a high [Ca2+]o to activate the mitogen-activated protein kinase (MAPK) pathway [25,26]. We have now utilized this transfected heterologous cell system to study the mechanisms whereby the CaR regulates PTH gene expression. As the regulation of PTH gene expression by [Ca2+]o is predominantly post-transcriptional, we studied PTH mRNA stability in a system that was independent of any effect on PTH transcription through the PTH promoter. To do this we expressed PTH mRNA driven by a viral promoter to express large amounts of PTH mRNA in HEK293 cells. Differences in PTH mRNA levels would therefore represent only post-transcriptional regulation. Expression of the CaR markedly decreased PTH mRNA levels and stability and conferred responsiveness to [Ca2+]o and the calcimimetic R568 in these cells. This was mediated by the PTH mRNA 3'-UTR ARE. Moreover, expression of the CaR in the HEK293 cells led to a shift from the interaction of the PTH mRNA with the stabilizing protein, AUF1, to the destabilizing protein, KSRP. Furthermore, the expression of CaR modified AUF1 post-translationally as previously shown in vivo [7,27]. Therefore, the expression of the CaR in this heterologous cell system reproduces the signal transduction that determines PTH gene expression in the parathyroid.
The calcium receptor decreases parathyroid hormone mRNA levels post-transcriptionally in HEK293 cells
PTH gene expression is regulated post-transcriptionally by Ca2+ and calcimimetics affecting PTH mRNA stability [7,15]. To focus on the effect of the CaR on PTH mRNA stability in a cell system we constructed an expression vector containing the full-length human PTH gene including its three exons and two introns (hPTH) driven by a viral SV40 (not shown) or CMV promoter (Figure 1A). Expression of PTH from both viral promoters gave similar results and we used the CMV promoter for further studies.
Figure 1. Over-expression of the CaR decreases PTH mRNA levels in co-transfected HEK293 cells. HEK293 cells were transiently co-transfected in triplicate with expression plasmids for the hPTH gene and control GFP or GH and either the CaR (CaR+) or empty vector (CaR-). A. Schematic representation of the human PTH expression plasmid used for transient transfections. The boxes show the CMV promoter (grey) and the PTH exons with the untranslated regions (UTRs) (white) and coding regions (diagonal lines). The arrows show the pre, pro and mature PTH. B. Immunohistochemistry on whole cells using an intact cell enzyme-linked immunoassay for the cell surface expression of the CaR. Untransfected (background), CaR (+) or control plasmid (-) transfected cells were analyzed using a CaR antibody or IgG (-). C. Immunoblot analysis of extracts from HEK293 cells co-transfected with expression plasmids for the CaR and myc-AUF1 as control plasmid, using anti-CaR or myc antibodies. D-G. Effect of CaR on PTH expression. D. Northern blot for hPTH and co-transfected GFP with the CaR (+) or an empty vector (-). Ethidium bromide staining of the membrane is shown as a loading control. E. qPCR for PTH and co-transfected GH mRNA levels from cells with (red) and without (blue) the CaR. F. Secreted PTH from cells as above 1 h after an incubation in fresh medium, 1 mM Ca2+. G. qRT-PCR for PTH mRNA levels from cells without and with either the CaR or the PTH1R (checkered). Data in D-G are expressed as fold change (mean ± SE) (n = 3). *, P < 0.01, CaR: control.
The hPTH expression plasmid was transiently transfected into HEK293 cells. The transcribed PTH mRNA was correctly spliced resulting in PTH mRNA of the expected size (Figure 1D). The PTH mRNA was translated into mature human PTH that was measured in the medium by radioimmunoassay (Figure 1F). We then studied the effect of the CaR on PTH mRNA levels in HEK293 cells expressing the PTH gene by transient transfection. The expression of the CaR was confirmed by immunohistochemistry on whole cells using an intact cell enzyme-linked immunoassay to determine cell surface expression [28] (Figure 1B) and by western blot (Figure 1C). Transfection efficiency was >95%, as indicated by fluorescent microscopy of the cells co-transfected with a green fluorescent protein (GFP) expression plasmid (not shown). Co-transfection of the human (h) CaR together with the hPTH plasmid resulted in a marked decrease in PTH mRNA levels by Northern blot (Figure 1D) and real time RT PCR (qPCR) (Figure 1E) corrected for co-transfected control genes GH (Figure 1E) or endogenous HPRT (Figure 1G). The decrease in PTH mRNA by the CaR was reflected in a decrease in secreted PTH at 48 h (Figure 1F). Transfection with another G protein coupled receptor (GPCR), the PTH receptor (PTH1R) had no effect on PTH mRNA levels, confirming the specificity of the CaR effect on PTH mRNA (Figure 1G).
The calcium receptor decreases parathyroid hormone mRNA levels through the parathyroid hormone mRNA 3'-UTR AU-rich element
We have previously reported that the regulation of PTH mRNA levels is dependent upon a cis-acting instability ARE in the PTH mRNA 3'-UTR [15,16,20]. We then determined if the regulation of PTH mRNA levels by the CaR is exerted through the PTH mRNA ARE, using a growth hormone (GH) reporter gene containing either the rat PTH 63 nt ARE (GH63) or a truncated non-functional PTH mRNA 40 nt element [29]. Over-expression of the CaR decreased GH63 mRNA levels but had no effect on wild-type GH mRNA levels or on a GH mRNA with the truncated 40 nt PTH mRNA ARE (Figure 2A). Our results indicate that the CaR specifically decreases PTH mRNA levels through the PTH mRNA ARE.
Figure 2. The CaR decreases PTH mRNA levels and stability through the PTH mRNA 3'-UTR cis element. A. Effect of CaR over-expression on reporter GH mRNA containing the PTH mRNA ARE. qRT-PCR for GH and control HPRT mRNA levels in cells transfected with either control (CaR-) or CaR (CaR+) and expression plasmids for GH (left panel), GH containing the PTH mRNA 63 nt ARE (GH-PTH63) (middle panel) or GH containing a 40 nt truncated ARE (GH-tPTH40) (right panel). Results are fold changes compared with cells without the CaR expressed as mean ± SE of three experiments. *, P < 0.05. B. Effect of CaR on PTH mRNA decay. Representative IVDA of transcripts for the full-length hPTH mRNA and the PTH mRNA with an internal deletion of the 3'-UTR ARE, incubated with extracts from cells expressing either the CaR or empty vector. C. Quantification of the amount of intact transcripts remaining with time related to time 0 (mean ± SE, in three repeat experiments; *, P < 0.05). Blue square, full length PTH mRNA without CaR (CaR-) and red square, with CaR (CaR+); blue triangle, PTH w/o ARE without CaR (CaR-) and red triangle, with the CaR, (CaR+).
To study the effect of the CaR on PTH mRNA stability we used an in vitro degradation assay (IVDA) [16,20]. A radiolabeled polyadenylated in vitro-transcribed hPTH mRNA was incubated with extracts from cells transfected with either the CaR expression plasmid or a control plasmid. The amount of intact PTH mRNA remaining with time represents the decay of the transcript by the different extracts and is indicative of the decay rate in vivo [15,30]. The rate of PTH mRNA decay was increased by extracts from cells expressing the CaR compared with extracts from cells with control plasmid correlating with PTH mRNA levels in the transfected cells (Figure 2B, top gel and Figure 2C). We then performed IVDA using polyadenylated PTH mRNA with an internal deletion of the ARE (Figure 2B, bottom gel and Figure 2C). The PTH mRNA transcript lacking the ARE was stable throughout the experiment and in contrast to the full-length PTH mRNA, was not affected by expression of the CaR (Figure 2B and 2C). Our results indicate that the CaR specifically decreases steady-state PTH mRNA levels and PTH mRNA stability through the PTH mRNA ARE.
Protein-PTH mRNA interactions by RNA immunoprecipitation assays
In vivo in the rat parathyroid, PTH mRNA stability is determined by the regulated binding of AUF1 and KSRP to the PTH mRNA 3'-UTR [20]. To identify protein-mRNA interactions in the CaR transfected cells, we performed RNA immunoprecipitation (RIP) assays using extracts from HEK293 cells transfected with the CaR or control plasmids. Immunoprecipitation was performed with antibodies to AUF1, KSRP or control IgG followed by qRT PCR of the recovered RNA and input extracts. Parathyroid hormone mRNA was decreased in the CaR expressing extracts compared with control extracts (Figure 3A) as above (Figure 1D, 1E and 1G). The RIP assay showed that the amount of PTH mRNA bound to AUF1 was decreased in CaR-expressing cells compared with control cells (Figure 3B). In contrast, PTH mRNA bound to KSRP was increased in the CaR-expressing cells (Figure 3C). This binding pattern is consistent with the stabilizing function of AUF1 and the destabilizing function of KSRP on PTH mRNA [18,20]. The increased binding to KSRP and the decreased binding to AUF1 correlate with the lower levels of PTH mRNA induced by the CaR in the transfected cells.
Figure 3. Expression of the CaR in HEK293 cells decreases AUF1 and increases KSRP interaction with the PTH mRNA. RNA immunoprecipitation (RIP) analysis of extracts from cells transiently transfected with expression plasmids for PTH and GH as control and either the CaR plasmid or empty vector. Immunoprecipitation was performed using antibodies for AUF1, KSRP or control IgG. Input (A) and immunoprecipitiated (B, C) samples were analyzed by qPCR for PTH and GH mRNA. Results are presented as PTH mRNA corrected for GH mRNA. PTH mRNA in the immunoprecipitated samples was corrected for PTH mRNA in the input. The results are mean ± SE of three repeat experiments. *, P < 0.05 compared with cells transfected with empty vector (CaR-).
The calcium receptor leads to post-translational modification of the parathyroid hormone mRNA binding protein AUF1
In vivo dietary-induced hypocalcemia and hypophosphatemia as well as adenine-induced renal failure and the calcimimetic R568 lead to post-translational modifications of AUF1 as shown by 2D gels [7,27]. To determine whether the effect of the CaR on PTH mRNA stability in the engineered cells involves post-translational modifications of AUF1 as it does in the parathyroid, we performed 2D gels on extracts from HEK293 cells transfected with the CaR expression plasmid or an empty vector as control. AUF1 has four isoforms of p37, p40, p42 and p45. There was no difference in AUF1 protein levels in the 1D gels between cells with and without the CaR, apart from a small decrease in p45 in the CaR-expressing cells (Figure 4A). However, 2D gels showed that endogenous AUF1 isoforms p37, p40 and p42 had a different distribution of the spots between extracts of cells with and without the CaR (Figure 4B). These results suggest that the CaR induces post-translational modifications in AUF1 in the transfected cells.
Figure 4. AUF1 is post-translationally modified in HEK293 cells transfected with CaR. A. 1D gels for AUF1 and α-tubulin as a loading control. B. 2D gel analysis for endogenous AUF1 in extracts from cells transiently transfected with the CaR (CaR+) plasmid or empty vector (CaR-). Molecular weight markers are shown on the right and the four AUF1 isoforms are indicated. C. 2D gel analysis of extracts from cells transiently transfected with the CaR plasmid or empty vector and the myc-AUF1 isoforms p37, p40, p42 or p45 separately. D. 2D analysis of extracts from cells transiently transfected with the CaR plasmid or empty vector and myc-AUF1p40 without and after treatment with a non-specific phosphatase (CIP). The results all represent one of two repeat experiments with similar results.
To analyze the four isoforms of AUF1 separately, we utilized myc-tagged AUF1 expression plasmids. HEK293 cells were transiently transfected with expression plasmids for myc- p37, p40, p42 or p45 isoforms of AUF1 together with the CaR or control plasmids. Cell extracts were analyzed on 2D gels with an anti-myc antibody. CaR expression led to changes in mobility of myc-AUF1 p37, p40 and also p45 but not p42 (Figure 4C). Unlike the changes in myc-AUF1 p45, endogenous AUF1 p45 was only slightly modified by CaR signaling. The change in endogenous p42 was not reflected in the myc-tagged transfected p42. The reasons for these discrepancies are not clear. AUF1 p40 undergoes reversible phosphorylation which may regulate ARE-directed mRNA turnover [31,32]. We therefore added a non-specific phosphatase to extracts from cells expressing myc-p40 with or without the CaR. Calf intestinal phosphatase (CIP) treatment of control extracts modified AUF1 to the form present in CaR-expressing extracts (Figure 4D). Treatment with CIP had no further effect on the CaR-expressing extracts. These results indicate that the CaR modifies AUF1 post-translationally and suggests that at least part of this change involves phosphorylation of isoform p40.
Parathyroid hormone mRNA levels are regulated by [Ca2+]o and the calcimimetic R568 through the calcium receptor
We then studied the effect of a low-calcium medium on the regulation of PTH gene expression by the CaR in the transfected cells. HEK293 cells transiently co-transfected with expression plasmids for PTH and the CaR or a control plasmid were grown in a medium with either 0.2 or 1.2 mM Ca2+. Expression of the CaR decreased PTH mRNA in cells grown in physiological 1.2 mM Ca2+ concentration by both Northern blots and qRT PCR (Figure 5A and 5B) as in Figure 1. Importantly, the low-Ca2+ medium attenuated the decrease in PTH mRNA levels induced by the CaR (Figure 5A and 5B). There was no effect of a low-Ca2+ medium in cells that did not express the CaR.
Figure 5. Expression of the CaR decreases PTH mRNA levels and confers responsivity to a low [Ca2+]o and the calcimimetic R568. A. Representative Northern blot analysis for PTH mRNA in cells transiently transfected in triplicate with hPTH and either the CaR plasmid or empty vector. After transfection cells were grown in 1.2 or 0.2 mM Ca2+ medium for an additional 48 h. Ethidium bromide staining of the membrane is shown as loading control. B. qRT PCR for PTH corrected for co-transfected GH mRNAs for cells treated as in A. C. qPCR for PTH and HPRT mRNAs from cells expressing the hPTH gene and the CaR (CaR+) or empty vector (CaR-) in cells grown in 1.2 mM Ca2+ supplemented with either R568 or vehicle. The results in A-C represent one of three repeat experiments performed in triplicate with similar results. D. Representative Northern blot for PTH and GFP mRNA levels in cells transfected with PTH and GFP and either CaR (CaR+) or empty vector (CaR-). The cells were grown in 0.2 mM calcium supplemented with R568 or vehicle. Quantification of the results is shown below the gel. The results are presented as mean ± SE of two repeat experiments performed in triplicate. *, P < 0.05, CaR+:CaR-; **, P < 0.05, CaR+, treated:CaR+, untreated.
Calcimimetics are compounds that bind and activate the CaR [5]. We then performed the same experiments with and without the calcimimetic, R568 (Figure 4C). The CaR decreased PTH mRNA levels as before at 1.2 mM Ca2+ (Figure 5C). Activation of the CaR by R568 decreased PTH mRNA levels at 1.2 mM Ca2+ (Figure 5C) and also at 0.2 mM Ca2+ (Figure 5D). 0.2 mM Ca2+ prevented the decrease in PTH mRNA levels due to the CaR (Figure 5B and 5D). However, at 0.2 mM Ca2+ the addition of R568 still effectively decreased PTH mRNA levels (Figure 5D). There was no effect of CaR expression on co-transfected GFP mRNA levels (Figure 5D) used as a control for transfection efficiency and loading. There was also no effect of R568 on PTH mRNA levels in cells co-transfected with the control plasmid that did not express the CaR (Figure 5C and 5D). The effect of R568 on PTH mRNA levels in the CaR-transfected cells at 0.2 mM Ca2+ indicates that the calcimimetic is effective at activating the CaR even when the CaR is in a relaxed configuration. Therefore, in these engineered cells, the CaR is permissive for the effect of [Ca2+]o and R568 on PTH mRNA levels.
Primary bovine parathyroid cells in culture have been successfully used for the study of PTH secretion in the short term (hours) but not for longer term effects on PTH gene expression. This is because these primary monolayer cultures of parathyroid cells lose the expression of the CaR after 24 h in culture [33]. Parathyroid organoid cultures retain a prolonged calcium response and regulation of PTH mRNA stability by high [Ca2+]o and a calcimimetic [34,35]. However, there is no parathyroid cell line. Kifor et al [25] showed that the CaR activated the phosphotyrosine kinase in both CaR-transfected HEK293 cells and native bovine parathyroid cells. In CaR-expressing HEK293 cells, [Ca2+]o activates PI-PLC, PLA2 and PLD, similar to the effect in bovine primary parathyroid cell cultures [25,26,36]. There have been no studies on the effect of the CaR on the regulation of PTH gene expression using the transfected HEK293 cells. We therefore expressed the CaR together with the hPTH gene in HEK293 cells. The effect of [Ca2+]o and the calcimimetic R568 on PTH expression in the parathyroid in vivo is predominantly post-transcriptional, regulating PTH mRNA stability and not transcription through the PTH promoter [7,15]. This allowed us to use a viral promoter upstream of the hPTH gene to study the effect of the CaR signaling on PTH mRNA levels. The PTH gene was efficiently transcribed, processed and translated to mature immunoreactive PTH. Significantly, PTH mRNA and secreted PTH levels were decreased in cells expressing the CaR compared with control cells. The calcium concentration in the medium was 1.2 mM, which would activate the CaR to reduce PTH mRNA levels in the CaR-transfected cells. Therefore, the CaR suppresses PTH gene expression post-transcriptionally by acting on PTH mRNA driven by a viral and not the native PTH promoter. In vitro degradation assays using extracts from cells with or without the CaR showed that expression of the CaR led to a decrease in full-length PTH mRNA half-life but not the half-life of a PTH mRNA with an internal deletion of the PTH mRNA ARE. The decreased PTH mRNA stability correlated with decreased PTH mRNA steady state levels.
Dietary-induced changes in serum calcium and phosphate levels regulate PTH gene expression in vivo post-transcriptionally [14,15]. This regulation is mediated by the changes in binding of stabilizing trans-acting factors, AUF1 and Unr, and the destabilizing protein KSRP, to the defined cis-acting ARE in the PTH mRNA 3'-UTR [16,18-20]. The role of the PTH mRNA ARE in the regulation of PTH mRNA levels by the CaR was demonstrated by transfection experiments using a GH reporter mRNA containing the PTH mRNA 63 nt ARE. The CaR decreased GH-PTH ARE mRNA levels but had no effect on native GH mRNA or a GH mRNA containing a truncated PTH mRNA ARE. Calcium receptor-transfected HEK293 cells showed increased KSRP-PTH mRNA binding and decreased AUF1-PTH mRNA binding by RIP analysis, correlating with the decreased PTH mRNA levels and stability in the CaR-transfected cells. Therefore, the signaling of the CaR in the parathyroid is maintained in the transfected cells as reflected in changes in AUF1 and KSRP PTH mRNA interactions and the role of the PTH mRNA ARE in this regulation.
AUF1-PTH mRNA binding is increased by hypocalcemia or chronic kidney disease, or decreased by hypophosphatemia or administration of the calcimimetic R568 [7,15]. These changes in serum Ca2+ or phosphate and kidney disease are associated with post-translational modifications of AUF1. AUF1 isoforms p40 and p42 are modified by dietary-induced hypocalcemia, and the secondary hyperparathyroidism of chronic kidney disease and its treatment by R568 [7,27]. We now show that expression of the CaR in the engineered cells also induces post-translational modifications in both endogenous AUF1 and myc-tagged transfected AUF1 isoforms that are similar to the modifications in vivo in the parathyroid. The modification of at least AUF1 p40 involves phosphorylation where expression of the CaR is associated with dephosphorylation. Post-translational modifications of this isoform, AUF1 p40, are altered concomitant with changes in RNA binding activity and stabilization of ARE-containing mRNAs in phorbol ester-treated monocytic leukemia cells [32]. AUF1 p40 recovered from polysomes was phosphorylated on Ser83 and Ser87 in untreated cells but lost these modifications following phorbol ester treatment. It was suggested that selected signal transduction pathways may regulate ARE-directed mRNA turnover by reversible phosphorylation of AUF1 p40 [31,32]. Therefore, both in the CaR-transfected cells and in the phorbol ester-treated cells, dephosphorylation of AUF1 p40, is associated with decreased AUF1 activity. In the case of PTH mRNA, inactivity of AUF1 results in a less stable PTH mRNA after expression of the CaR, as AUF1 is a PTH mRNA stabilizing protein [18,27].
We also show that expression of the CaR conferred responsiveness to low [Ca2+]o and the calcimimetic R568, underlining the relevance of this cellular model to the parathyroid. A low- [Ca2+]o medium increased PTH expression only in the cells expressing the CaR. Similarly, R568 decreased PTH expression through the CaR at both 1.2 and 0.2 mM [Ca2+]o. Therefore, regulation of PTH mRNA levels by expression of the CaR itself and both its activation by the calcimimetic and its relaxation by a low [Ca2+]o are maintained in these transfected cells. This is the first demonstration that the CaR can regulate PTH gene expression in heterologous cells. This regulation is obtained through the post-transcriptional mechanisms that involve changes in PTH mRNA-protein interactions in the parathyroid (Table 1). It is likely that PTH secretion would exhibit a similar regulation by calcium as PTH mRNA levels, given its dependence on the latter. However, secretion was not measured as a function of the extracellular calcium concentration in the heterologous HEK293 cell system, in which there is a prominent component of constitutive secretion, unlike the parathyroid cell [37,38]. Secretion in HEK293 cells may be independently regulated by the CaR although we have not studied this question. The parathyroid cell is unique in that the stimulus for secretion, gene expression and cell proliferation is a low [Ca2+]o and not a high [Ca2+]o. Inverse control of secretion by a low [Ca2+]o of PTHrP secretion also occurs in the epithelial cells of the lactating breast and certain breast cell lines [39,40]. In addition high [Ca2+]o decreases hormone secretion in the secretion of renin by renal juxtaglomerular cells [41]. Calcium receptor activation increases cell proliferation in cultured cells [42], but inhibits parathyroid proliferation in vivo [43]. It is remarkable and intriguing that despite these differences, the transfected cell system retains so many of the native characteristics of the unique parathyroid cell, specifically the relationship between calcium and PTH mRNA stability. The CaR-mediated decrease in PTH expression in these cells mirrors the marked increase in serum PTH in vivo in mice and men with inactivating mutations of the CaR [6,10,36,43,44]. These results support the concept that the parathyroid cell is geared to constitutively synthesize and secrete PTH, and it is the presence of a functioning CaR that tonically inhibits PTH expression and secretion and allows responsivity to [Ca2+]o.
Table 1. Regulation of PTH gene expression in HEK293 cells transfected with the CaR and hPTH plasmids and in the rat parathyroid by calcium
The expression of the CaR in transfected HEK293 cells decreases PTH gene expression post-transcriptionally and is sufficient to confer the regulation of PTH gene expression by [Ca2+]o and a calcimimetic, R568. The CaR decreases PTH gene expression in these engineered cells through the balanced interactions of the trans-acting factors KSRP and AUF1 with PTH mRNA and the PTH mRNA ARE, as in vivo in the parathyroid. This is the first demonstration that the CaR can regulate PTH gene expression in heterologous transiently transfected cells.
Cells and transfection
HEK293 cells were transiently co-transfected in a 10 cm dish with 5 μg expression plasmids for the PTH gene and the CaR plasmid or a control plasmid using a Calcium Phosphate Transfection Kit (Sigma, St Louis, MO, USA). At 48 h the cells expressing the CaR or controls were detached and replated in 24 well plates and treated as indicated, in triplicates for 48 h. Cells were grown in DMEM supplemented with 10% Fetal Bovine Serum (Biological Industries, Beit Haemek, Israel), L-Glutamine (Biological Industries, Beit Haemek, Israel) and Pen-Strep solution (Biological Industries, Beit Haemek, Israel). Calcium concentration in the medium was 1.2 mM. In some experiments Ca2+-free DMEM medium was used (0.2 mM Ca2+) and CaCl2 was added as indicated. R568, a gift from Amgen (Thousand Oaks, CA, USA) was dissolved in DDW and added to the medium at a final concentration of 1 μM.
RNA extraction and analysis
RNA extraction was performed using Tri reagent (Molecular Research Center, Cincinnati, OH, USA). mRNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Wilmington, DE, USA). For real-time PCR 1 μg mRNA was used as template for RT PCR with Maxime RT PreMix (Random primer) from iNtRON (Gyeonggi-do, Korea). qPCR was performed using SYBR Green ROX Mix (ABgene, Surrey, UK) according to the manufacturer's instructions in 7900HT Fast Real-Time PCR Systems from Applied Biosystems.
The following primers were used: hPTH (5'-gggtctgcagtccaattcat-3' and 5'-cagatttcccatccgatttt-3'), hGH (5'-gggaggctggaagatggc-3' and 5'-cgttgtgtgagtttgtgtcgaac-3'), hHPRT (5' tttgaatcatgtttgtgtcattagtga and 5' ttccaaactcaacttgaactctcatc). For Northern analysis RNA was run on agarose formaldehyde gels and blotted using a Hybond-N membrane (Amersham Biosciences, Little Chalfont, UK). Membranes were hybridized to fragments for hPTH or GFP [20,29]. In some experiments ethidium bromide staining of membranes was used for quantification of 18S or 28S as loading controls.
For the hPTH expression plasmid, a HpaII fragment including the three exons and two introns of the hPTH gene from plasmid pPTHg108 [20] was inserted downstream of the CMV promoter in pcDNA3 expression plasmid (Invitrogen, San Diego, CA, USA). The hCaR plasmid was kindly provided by M Lohse (Wurzburg, Germany), the GFP pEGFP-C1 (Clontech, Palo Alto, CA, USA) and hGH (kindly provided by O Meyuhas, Jerusalem, Israel [45]) were used as controls. The GH-PTH63 or GH-tPTH40 expression plasmids contained 63 bp or a truncated 40 bp fragments of the PTH mRNA 3'-UTR ARE that were inserted between the coding region and the 3'-UTR of GH mRNA [29]. hPTH1R was provided by MA Levine, Cleveland, OH, USA). In some experiments cells co-transfected with the GFP plasmid were analyzed by fluorescent microscopy to estimate transfection efficiency, which was always >90%.
Immunoreactive parathyroid hormone
Medium was replaced 1 h prior to collection and analyzed using the Immulite 2000 Intact PTH assay (Los Angeles, CA, USA)
In Vitro Degradation Assays
Radiolabeled transcripts (200,000 cpm) were incubated with 40 μg protein extract from cells in a volume of 50 μl and in a reaction buffer containing 3 mM Tris HCl, pH 7.5, 2 mM MgCl2, 3 mM NaCl, 10 mM ATP and 30 units RNasin. At timed intervals samples were removed, RNA extracted, separated on agarose gels and analyzed by autoradiography as described [15].
The following plasmids were used as templates for RNA transcription: pBluescript II KS plasmid containing either the full-length rat PTH cDNA (772 bp) or a PTH cDNA with an internal deletion of the PTH mRNA ARE and a stretch of ~150 dT nucleotides that by in vitro transcription produced a poly(A) tail. The plasmids were linearized with SmaI [20].
2D gel electrophoresis
Analysis was performed as previously described [27].
RNA immunoprecipitation
Transiently transfected cells were grown in a 14 cm dish for 48 h, collected in ice-cold PBS, pelleted and re-suspended in RIPA buffer (containing 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitors) supplemented with RNase inhibitors (Promega, Madison, WI, USA) and homogenized by pipetting. Equal amounts of whole cell extracts were immunoprecipitated with Protein A agarose-bound anti-KSRP or AUF1 antibody beads (Calbiochem, Darmstadt, Germany) or IgG as control after incubation for 2 hr at 4°C. The beads were then washed with modified RIPA buffer (supplemented with 1 M NaCl, 1% Sodium Deoxycholate, 1 mM EDTA and 2 M urea). RNA was extracted and analyzed by qPCR for PTH and GH mRNA using SYBR Green ROX Mix as described above.
Intact cell enzyme-linked immunoassay
The assay was performed as previously described [27]. In brief: 48 h after transfection cells were detached in PBS supplemented with BSA and EDTA. The cells were then pelleted and incubated for 1 h at 4°C with the anti-CaR antibody followed by PBS wash (× 3) and incubation with the secondary HRP-conjugated antibody. Cells were then washed (× 3) prior to adding the HRP substrate and the fluorescent signal was visualized using an electrochemiluminescent reader.
Anti-KSRP was a gift from R Gherzi, Genoa, Italy. Anti-AUF was from Upstate (Charlottesville, VA, USA). Anti-myc was from Cell Signaling (Boston, MA, USA). Anti-CaR was from Abcam (Cambridge, UK).
Statistical analysis was performed using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). Values are reported as mean ± SEM unless stated otherwise. A 2-tailed Student's T-test was used to assess differences from the control group. A P value of less than 0.05 was considered significant.
ARE: AU-rich element; AUF1: AU rich binding factor; CaR: calcium receptor; CIP: calf intestinal phosphatase; GFP: green fluorescent protein; HEK: human embryonic kidney; HPT: hyperparathyroidism; IVDA: in vitro degradation assay; KSRP: KH domain splicing regulatory protein; PTH: parathyroid hormone; qRT PCR: quantitative reverse transcriptase polymerase chain reaction; RIP: RNA immunoprecipitation; TM: transmembrane; UTR: untranslated region.
Authors' contributions
Most experiments were performed by HG who also conceived and designed the experiments. VL-M performed the experiments in Fig 4C–D and MN and TM assisted in different experiments. JS and TN-M conceived, designed and wrote the manuscript. All authors read and approved the final manuscript.
This work was supported in part by the American Society of Bone and Mineral Research Bridging Fund and grants from the Israel Academy of Sciences and Amgen.
Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert J: Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid.
Nature 1993, 366:575-580. PubMed Abstract | Publisher Full Text
Silver J, Naveh-Many T, Kronenberg HM: Parathyroid hormone: molecular biology. In Principles of bone biology. Edited by Bilezikian JB, Raisz LG, Rodan GA. San Diego: Academic Press; 2002:407-422.
Rodriguez M, Nemeth E, Martin D: The calcium-sensing receptor: a key factor in the pathogenesis of secondary hyperparathyroidism.
Am J Physiol Renal Physiol 2005, 288:F253-F264. PubMed Abstract | Publisher Full Text
Hofer AM, Brown EM: Extracellular calcium sensing and signalling.
Nat Rev Mol Cell Biol 2003, 4:530-538. PubMed Abstract | Publisher Full Text
Nemeth EF, Steffey ME, Hammerland LG, Hung BC, Van Wagenen BC, DelMar EG, Balandrin MF: Calcimimetics with potent and selective activity on the parathyroid calcium receptor.
Proc Natl Acad Sci USA 1998, 95:4040-4045. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Hu J, Spiegel AM: Naturally occurring mutations of the extracellular Ca2+-sensing receptor: implications for its structure and function.
Trends Endocrinol Metab 2003, 14:282-288. PubMed Abstract | Publisher Full Text
Levi R, Ben Dov IZ, Lavi-Moshayoff V, Dinur M, Martin D, Naveh-Many T, Silver J: Increased parathyroid hormone gene expression in secondary hyperparathyroidism of experimental uremia is reversed by calcimimetics: correlation with posttranslational modification of the trans acting factor AUF1.
J Am Soc Nephrol 2006, 17:107-112. PubMed Abstract | Publisher Full Text
Colloton M, Shatzen E, Miller G, Stehman-Breen C, Wada M, Lacey D, Martin D: Cinacalcet HCl attenuates parathyroid hyperplasia in a rat model of secondary hyperparathyroidism.
Kidney Int 2005, 67:467-476. PubMed Abstract | Publisher Full Text
Wettschureck N, Lee E, Libutti SK, Offermanns S, Robey PG, Spiegel AM: Parathyroid-specific double knockout of Gq and G11 alpha-subunits leads to a phenotype resembling germline knockout of the extracellular Ca2+ -sensing receptor.
Mol Endocrinol 2007, 21:274-280. PubMed Abstract | Publisher Full Text
Ho C, Conner DA, Pollak MR, Ladd DJ, Kifor O, Warren HB, Brown EM, Seidman JG, Seidman CE: A mouse model of human familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.
Nat Genet 1995, 11:389-394. PubMed Abstract | Publisher Full Text
Kos CH, Karaplis AC, Peng JB, Hediger MA, Goltzman D, Mohammad KS, Guise TA, Pollak MR: The calcium-sensing receptor is required for normal calcium homeostasis independent of parathyroid hormone.
J Clin Invest 2003, 111:1021-1028. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Tu Q, Pi M, Karsenty G, Simpson L, Liu S, Quarles LD: Rescue of the skeletal phenotype in CasR-deficient mice by transfer onto the Gcm2 null background.
J Clin Invest 2003, 111:1029-1037. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Silver J, Kilav R, Naveh-Many T: Mechanisms of secondary hyperparathyroidism.
Am J Physiol Renal Physiol 2002, 283:F367-F376. PubMed Abstract | Publisher Full Text
Kilav R, Silver J, Naveh-Many T: Parathyroid hormone gene expression in hypophosphatemic rats.
J Clin Invest 1995, 96:327-333. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Moallem E, Silver J, Kilav R, Naveh-Many T: RNA protein binding and post-transcriptional regulation of PTH gene expression by calcium and phosphate.
J Biol Chem 1998, 273:5253-5259. PubMed Abstract | Publisher Full Text
Kilav R, Silver J, Naveh-Many T: A conserved cis-acting element in the parathyroid hormone 3'-untranslated region is sufficient for regulation of RNA stability by calcium and phosphate.
J Biol Chem 2001, 276:8727-8733. PubMed Abstract | Publisher Full Text
Bell O, Silver J, Naveh-Many T: Identification and characterization of cis -acting elements in the human and bovine parathyroid hormone mRNA 3'-untranslated region.
J Bone Miner Res 2005, 20:858-866. PubMed Abstract | Publisher Full Text
Sela-Brown A, Silver J, Brewer G, Naveh-Many T: Identification of AUF1 as a parathyroid hormone mRNA 3'-untranslated region binding protein that determines parathyroid hormone mRNA stability.
J Biol Chem 2000, 275:7424-7429. PubMed Abstract | Publisher Full Text
Dinur M, Kilav R, Sela-Brown A, Jacquemin-Sablon H, Naveh-Many T: In vitro evidence that upstream of N-ras participates in the regulation of parathyroid hormone messenger ribonucleic acid stability.
Mol Endocrinol 2006, 20:1652-1660. PubMed Abstract | Publisher Full Text
Nechama M, Ben Dov IZ, Briata P, Gherzi R, Naveh-Many T: The mRNA decay promoting factor K-homology splicing regulator protein post-transcriptionally determines parathyroid hormone mRNA levels.
FASEB J 2008, 22:3458-3468. PubMed Abstract | Publisher Full Text
Brown EM, Gardner DG, Aurbach GD: Effects of the calcium ionophore A23187 on dispersed bovine parathyroid cells.
Endocrinology 1980, 106:133-138. PubMed Abstract
Almaden Y, Canalejo A, Hernandez A, Ballesteros E, Garcia-Navarro S, Torres A, Rodriguez M: Direct effect of phosphorus on parathyroid hormone secretion from whole rat parathyroid glands in vitro.
J Bone Miner Res 1996, 11:970-976. PubMed Abstract
Silver J, Russell J, Sherwood LM: Regulation by vitamin D metabolites of messenger ribonucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells.
Proc Natl Acad Sci USA 1985, 82:4270-4273. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Moallem E, Silver J, Naveh-Many T: Regulation of parathyroid hormone messenger RNA levels by protein kinase A and C in bovine parathyroid cells.
J Bone Miner Res 1995, 10:447-452. PubMed Abstract
Kifor O, Diaz R, Butters R, Brown EM: The Ca2+-sensing receptor (CaR) activates phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells.
J Bone Miner Res 1997, 12:715-725. PubMed Abstract | Publisher Full Text
Kifor O, MacLeod RJ, Diaz R, Bai M, Yamaguchi T, Yao T, Kifor I, Brown EM: Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells.
Am J Physiol Renal Physiol 2001, 280:F291-F302. PubMed Abstract | Publisher Full Text
Bell O, Gaberman E, Kilav R, Levi R, Cox KB, Molkentin JD, Silver J, Naveh-Many T: The protein phosphatase calcineurin determines basal parathyroid hormone gene expression.
Mol Endocrinol 2005, 19:516-526. PubMed Abstract | Publisher Full Text
Ray K, Fan GF, Goldsmith PK, Spiegel AM: The carboxyl terminus of the human calcium receptor. Requirements for cell-surface expression and signal transduction.
J Biol Chem 1997, 272:31355-31361. PubMed Abstract | Publisher Full Text
Kilav R, Bell O, Le SY, Silver J, Naveh-Many T: The parathyroid hormone mRNA 3'-untranslated region AU-rich element is an unstructured functional element.
J Biol Chem 2004, 279:2109-2116. PubMed Abstract | Publisher Full Text
Fritz DT, Ford LP, Wilusz J: An in vitro assay to study regulated mRNA stability.
Sci STKE 2000, 2000:L1. Publisher Full Text
Wilson GM, Lu J, Sutphen K, Sun Y, Huynh Y, Brewer G: Regulation of A + U-rich element-directed mRNA turnover involving reversible phosphorylation of AUF1.
J Biol Chem 2003, 278:33029-33038. PubMed Abstract | Publisher Full Text
Wilson GM, Lu J, Sutphen K, Suarez Y, Sinha S, Brewer B, Villanueva-Feliciano EC, Ysla RM, Charles S, Brewer G: Phosphorylation of p40AUF1 regulates binding to A + U-rich mRNA-destabilizing elements and protein-induced changes in ribonucleoprotein structure.
J Biol Chem 2003, 278:33039-33048. PubMed Abstract | Publisher Full Text
Brown AJ, Zhong M, Ritter C, Brown EM, Slatopolsky E: Loss of calcium responsiveness in cultured bovine parathyroid cells is associated with decreased calcium receptor expression.
Biochem Biophys Res Commun 1995, 212:861-867. PubMed Abstract | Publisher Full Text
Ritter CS, Slatopolsky E, Santoro S, Brown AJ: Parathyroid cells cultured in collagen matrix retain calcium responsiveness: importance of three-dimensional tissue architecture.
J Bone Miner Res 2004, 19:491-498. PubMed Abstract | Publisher Full Text
Ritter CS, Pande S, Krits I, Slatopolsky E, Brown AJ: Destabilization of parathyroid hormone mRNA by extracellular Ca2+ and the calcimimetic R-568 in parathyroid cells: role of cytosolic Ca and requirement for gene transcription.
J Mol Endocrinol 2008, 40:13-21. PubMed Abstract | Publisher Full Text
Pearce SH, Bai M, Quinn SJ, Kifor O, Brown EM, Thakker RV: Functional characterization of calcium-sensing receptor mutations expressed in human embryonic kidney cells.
J Clin Invest 1996, 98:1860-1866. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Groskreutz DJ, Sliwkowski MX, Gorman CM: Genetically engineered proinsulin constitutively processed and secreted as mature, active insulin.
J Biol Chem 1994, 269:6241-6245. PubMed Abstract | Publisher Full Text
Beuret N, Stettler H, Renold A, Rutishauser J, Spiess M: Expression of regulated secretory proteins is sufficient to generate granule-like structures in constitutively secreting cells.
J Biol Chem 2004, 279:20242-20249. PubMed Abstract | Publisher Full Text
Mamillapalli R, VanHouten J, Zawalich W, Wysolmerski J: Switching of G-protein usage by the calcium-sensing receptor reverses its effect on parathyroid hormone-related protein secretion in normal versus malignant breast cells.
J Biol Chem 2008, 283:24435-24447. PubMed Abstract | Publisher Full Text
VanHouten J, Dann P, McGeoch G, Brown EM, Krapcho K, Neville M, Wysolmerski JJ: The calcium-sensing receptor regulates mammary gland parathyroid hormone-related protein production and calcium transport.
J Clin Invest 2004, 113:598-608. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Ortiz-Capisano MC, Ortiz PA, Garvin JL, Harding P, Beierwaltes WH: Expression and function of the calcium-sensing receptor in juxtaglomerular cells.
Hypertension 2007, 50:737-743. PubMed Abstract | Publisher Full Text
McNeil SE, Hobson SA, Nipper V, Rodland KD: Functional calcium-sensing receptors in rat fibroblasts are required for activation of SRC kinase and mitogen-activated protein kinase in response to extracellular calcium.
J Biol Chem 1998, 273:1114-1120. PubMed Abstract | Publisher Full Text
Pollack MR, Brown EM, Chou Y-HW, Hebert SC, Marx SJ, Steinman B, Levi T, Seidman CE, Seidman JG: Mutations in the human Ca2+ -sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.
Cell 1993, 75:1297-1303. PubMed Abstract | Publisher Full Text
Pearce SH, Williamson C, Kifor O, Bai M, Coulthard MG, Davies M, Lewis-Barned N, McCredie D, Powell H, Kendall-Taylor P, Brown EM, Thakker RV: A familial syndrome of hypocalcemia with hypercalciuria due to mutations in the calcium-sensing receptor [see comments].
N Engl J Med 1996, 335:1115-1122. PubMed Abstract | Publisher Full Text
Levy S, Avni D, Hariharan N, Perry RP, Meyuhas O: Oligopyrimidine tract at the 5' end of mammalian ribosomal protein mRNAs is required for their translational control.
Proc Natl Acad Sci USA 1991, 88:3319-3323. PubMed Abstract | Publisher Full Text | PubMed Central Full Text


text:
The American solution to stop the debt spiral is: • Act now.
Ryan accused Obama of trying to "dodge and demagogue" the debt problem he created.
Romney made a vast fortune in part by loading up businesses with debt, laying off employees, and canceling their health benefits.
debt (dět)
Something that is deficient or required to restore a normal state.
The Mosaic law encouraged the practice of lending (Deut. 15:7; Ps. 37:26; Matt. 5:42); but it forbade the exaction of interest except from foreigners. Usury was strongly condemned (Prov. 28:8; Ezek. 18:8, 13, 17; 22:12; Ps. 15:5). On the Sabbatical year all pecuniary obligations were cancelled (Deut. 15:1-11). These regulations prevented the accumulation of debt.

subset: commoncrawl


text:
1997 Chevrolet Camaro RS 5-speed w/ 859 miles ATTENTION FOURTH GENERATION CAMARO COLLECTORS AND ENTHUSIATS! ONLY 859 ORIGINAL MILES. YES, 859 ORIGINAL DOCUMENTED MILES! SHOW QUALITY CONDITION... More
Here's your chance to own a young survivor with very low mileage. This 1995 Chevrolet Camaro Z/28 has been pampered by its previous... More
For sale in our Chicago showroom is a 1999 Chevrolet Camaro SS. This Fourth-Generation Camaro was originally equipped with the 320 horsepower 5.7... More
For Sale in our Detroit Showroom is this clean custom 1993 Chevrolet Camaro. This Camaro has been built to be a street beast from... More
Up for sale in our Tampa showroom is a very nice 1994 Chevrolet Camaro convertible 69 Resto Mod. All the look and classic... More
30th Anniversary Edition Camaro SS Highly Detailed & Meticulously Maintained Charcoal Interior 350ci LT1 V8 Upgraded Automatic Transmission Chrome Torque Thrust Wheels Brand New Tires MotoeXotica Classic Cars is pleased to... More
This 1992 Chevrolet Camaro Heritage Edition Z/28 has a 5.7 Liter TPI Fuel Injected V8, 4 Speed Automatic Transmission w/ Overdrive, Only 44,275 Original... More
This 1992 Chevrolet Camaro z28 is for sale in our Louisville KY showroom. 1992 marked the final year of the 3rd gen Camaro. Originally... More
NOTHING SAYS SUMMER LIKE A RED CONVERTIBLE. THIS CAR HAS 30,800 ACTUAL MILES ON IT. IT LOOKS BRAND NEW & DRIVES AS GOOD AS... More
SAME OWNER SINCE 1995. YOU WOULD BE HARD PRESSED TO FIND ANOTHER CAMARO THIS CLEAN THAT HAS BEEN DRIVEN. THIS CAR HAS BEEN WELL... More
1997 Camaro SS Z28 Hardtop Excellent condition inside and out 16,400 miles Financing, extended warranty, shipping assistance Fees and Taxes: $399.00 Doc Fee on all Purchases. Out of state buyers... More
5.7 with only 70k miles, automatic, just driven 150 miles and it ran like brand new, originally a Black car.... More
For sale is a low mile 1993 Chevrolet Camaro B4C Special Service Package located in our Indianapolis Showroom. The Camaro optioned with the B4C... More
For sale is our St. Louis showroom is a one owner high ranking AutoCheck 1992 Chevrolet Camaro RS anniversary edition convertible. Talk about investment... More
For sale is a low mile 1997 Chevrolet Camaro Z28 30th Anniversary Edition located in our Indianapolis Showroom. To commemorate the 30th anniversary of... More


text:
Showing 1-25 of 145 results
Valeton La' Charger Modern Crunch$55.003.5 stars
Lone Wolf Audio Left Hand Wrath$250.004 stars
JHS Firefly$199.004 stars
Nemphasis The Muff4 stars
Suhr Riot Reloaded$200.004.5 stars
A stack-in-a-box with practical voicing options for classy amp-style distortion.
Suhr Rufus$200.004 stars
A fuzz for all seasons, with variety and versatility in spades.
Effectrode Mercury Fuzz$319.004.5 stars
Quality valve-driven pedals that could become an essential part of your tone.
EC Murinae$149.004.5 stars
DRW Si Fuzz Ace$199.004 stars
With a different feature set and voice, the Si is a solid alternative to the ...
DRW Ge Fuzz Ace4 stars
If you want a Fuzz Face-type pedal, this has the sound and is compact, robust ...
JAM Rattler$209.004.5 stars
JAM Fuzz Phrase$285.004 stars
Way Huge Havalina Germanium Fuzz$171.004 stars
A new flavour of Tone Bender-style fuzz - but a tasty one nonetheless!
Way Huge Swollen Pickle Jumbo Fuzz Dirty Donny Edition$149.004 stars
The Dirty Donny has a massive range of sounds, some in Big Muff territory, some ...
Empress MultiDrive4.5 stars
Empress Heavy$299.004 stars
Yerasov Brown Creature BC-1$89.004.5 stars
Fuzzy Rhodes FR753.5 stars
Rainger FX El Distorto$215.004.5 stars
Rainger FX Dr Freakenstein's Dwarf$170.004 stars
Rainger FX Dr Freakenstein Fuzz$334.994 stars
Dunlop Jimi Hendrix Band Of Gypsys FFM6 Fuzz Face Mini$171.004 stars
Xvive V3 Metal$88.003 stars
Xvive V1 Classic Rock$88.003 stars
If you're after a value-for-money distortion, it's a worthy contender.
Walrus Audio Jupiter Fuzz$179.004 stars
Dr Scientist Frazz Dazzler$200.004.5 stars
Lets you turn up the fuzz with an expression pedal - cool or what?
Skreddy Hybrid Fuzz Drive$249.004.5 stars
Earthquaker Devices Terminal Fuzz$165.004 stars
A versatile alternative for anyone yearning for a vintage Shin Ei FY-2.


text:
Eye eye! The Bluewaters Dubai Eye Ferris Wheel
Ferris wheel watchers will likely think this baby will be pipping the London Eye to the biggest Ferris wheel in the world post, but they'd be wrong writes Alex Mcnabb.February 14, 2013 10:46
Register for our free newsletter
Philippines confirms second MERS case
Abu Dhabi's Dana Gas hits 9-wk high after wins Kurdistan case
Saudi Telecom Co to pay Q2 dividend of 1 riyals/share
UAE non-oil business growth slows to 22-month low in June
MIDEAST STOCKS-Gulf likely to be subdued by oil price drop, Greece
Abu Dhabi's TAQA drops 5.4 pct after saying no merger talks
Qatar's Doha Bank raises 2 bln riyal Tier 1 capital-boosting bond
Dubai's Drake & Scull increases GCC ownership limit to 100 pct
Abu Dhabi's TAQA says not holding merger talks
Gold languishes below $1,170 ahead of U.S. jobs data
Ferris wheel watchers will likely think this baby will be pipping the London Eye to the biggest Ferris wheel in the world post, but they'd be wrong writes Alex Mcnabb.February 14, 2013 10:46
This is an investigative journalist's dream. The UK's press fell on the story, baying with glee at the chance to uncover more horsing around with our food - and they've come up trumps.February 13, 2013 8:44
BlackBerry has certainly managed to create some headlines, although not everyone's drinking the Kool-Aid, writes Alex Mcnabb.February 12, 2013 11:22
A smartphone will happily gorge its way through thirty kilobytes of data in about the time it takes a fly to hit a windscreen, writes Alex McNabb.February 10, 2013 5:05
To get all the facts straight, Kipp has contacted the head of communications for Middle East and North Africa to understand what was going on.February 7, 2013 11:28
It's been an interesting week in politics, both past and present. I started it off by sitting down to watch…February 7, 2013 11:22
Alex McNabb writes: Samsung's support operative came back in response to my email, confirming my worst fears. "You can't disable the touchpad on a Samsung Series 5 Ultra."February 6, 2013 12:15
I think the answer lies in my lack of self-control. Not only do I lack self-control, I know I lack self-control, writes Peter Ellen.February 4, 2013 5:30
Workplace stress presents a major challenge for managers because it is difficult to identify and because of this many are unaware how to spot early warning signs; the extent of its impact is also often underestimated or overlooked says Michelle Hunter, Business PsychologistFebruary 4, 2013 9:42
You can set out to create a piece of content that people will really, really want to share and watch it die the death of the neglected, while at the same time a puppy being scared by a hoover being switched on spreads across the Internet like nightshade falling across the earth in a one minute motion-capture.February 3, 2013 6:55


text:
Originally written on Fox Sports | Last updated 7/25/12
The Phoenix Suns have re-signed guard Shannon Brown. The contract is for two years with the second year partially guaranteed. Brown averaged 11 points and 2.7 rebounds in a career-high 23.7 minutes per game last season. He appeared in 59 games, 19 as a starter. He came to the Suns as a free agent in 2011, signing a one-year deal. Shooting guard is a position of need for the Suns, who had signed restricted free agent Eric Gordon to a contract but it was matched by New Orleans. The team has worked out a three-team trade that would send Minnesota guard Wesley Johnson and a first-round draft pick to the Suns.
Comments are closed.
What do you think?To leave a comment, you can:
NBA News
Delivered to your inbox
You'll also receive Yardbarker's daily Top 10, featuring the best sports stories from around the web. Customize your newsletter to get articles on your favorite sports and teams. And the best part? It's free!
Follow us on Twitter
|Best of Yardbarker
|NFL News
|MLB News
Today's Best Stuff
For Bloggers
Join the Yardbarker Network for more promotion, traffic, and money.
Company Info
What is Yardbarker?
Yardbarker is the largest network of sports blogs and pro athlete blogs on the web. This site is the hub of the Yardbarker Network, where our editors and algorithms curate the best sports content from our network and beyond.
|© 2015 Yardbarker, Inc. All rights reserved. Use of this website (including any and all parts and components) constitutes your acceptance of these Terms of Use and Privacy Policy. (What's New?)


text:
Global Warming: Arctic Storms Releasing Methane
Arctic storms may be speeding up global warming due to a faster release of methane gas into the environment. Large quantities of methane gas are being released off of the east Siberian shelf due to submarine permafrost that is slowly disappearing.
All of this was found at a great cost during August 2010. 11 sailors on board of a tug boat drowned, while trying to save a fishing boat in the arctic waters of Siberia. What they did not know, was that they were trying to save Russian researchers that were trying to figure out if arctic storms that unsettle the ocean waters will increase the methane gas released from the ocean floor, thus causing trouble with global warming.
The researchers were drilling into a methane gas hot spot in the Laptev Sea from their fishing boat and trying to measure how much gas was released with their sonar. The scientists discovered that the once solid permafrost was no longer frozen under the sea and was releasing 500 tonnes of methane gas into the atmosphere for every square kilometers of sea bottom every day.
Plumes of methane were found along the whole coastline of the research area, the East Siberian Arctic Shelf and off Norway's coast at Svalbard. The researchers also measured how two storms, one from 2009 and the other from 2010, would affect the levels of methane released into the sea. They found the sea storms made the methane gas release faster into the earth's atmosphere.
It was determined that global warming was linked to how the sub-sea permafrost was melting off the sea floor and that it is uncomfortably close to the thaw point of terrestrial permafrost. The findings make things all the more scarier in what is happening with global warming. It was found that if a massive "pulse" of 50 billion tonnes of methane gas were released at once, it could warm the earth by a degree or more.
Although, the data did not wholly show if a major pulse is going to happen. The amount of methane gas is still quite small, but it helps the science community to understand how these arctic storms speed up the release of the methane gas that can cause global warming to accelerate. Although, the release of these gases is small, it still is a time bomb ready to go off. There is about 17 teragrams of methane released from the sea floor each day from the Ease Siberian Arctic Shelf, making one teragram equal to about 1.1 million tons. And the Earth emits 500 million tons of natural and man-made methane into the Earth's atmosphere each year.
These researchers risked their lives to show us proof of global warming and that Arctic storms are releasing methane into such desolate and dangerous areas of the earth, and that the chances are high that this problem will affect the whole planet. While the researchers where observing the second storm, they had to radio in for help because it became so dangerously strong. So when the rescue tug boat was sent to the researchers, the boat then sunk before they were able to reach them, all of the researchers did survive unscathed. To share the bravery of one crew, the researchers dedicated their research paper "to the memory of the crew of Russian vessel RV Alexei Kulakovsky".
By Tina Elliott
5 Responses to Global Warming: Arctic Storms Releasing Methane
News worldwide should show respect for the brave Russian crew of the lost tug RV Alexei Kulakovsky and also to the brave scientists and others involved in rescue of all.
I didn't know the tug had sunk but I do know there are some pretty serious Russian crew of nuclear icebreakers.. All things considered it looks like the Arctic Sea is getting dangerous, too dangerous to study by drilling test bore into sediments.
It seems remote sensing satellite technology capable of watching progression of Methane into the upper Stratosphere where it accumulates in a swath around Earth. Merlin Lidar Satellite isn't set for launch by French till 2014. https://directory.eoportal.org/web/eoportal/satellite-missions/m/merlin -What I'd like to know is why the US and Russia along with China didn't get this technology flying long before this.
Remote sensing in real time of Methane concentrations is critical to understanding how to stop the rise of wildcat storms and the destruction of the Polar Jet that guides weather.
Double checking spectral reflection of methane specific infrared laser beam by near Earth orbiting telescope that doesn't at least have the jitters is something that can give insight..
Multiple source full coverage technology is needed to get information on what's working to regain margin for error so efforts to save HZ don't go in vain.
Thermodynamically Earth is destabilized by price on all things undercutting of ecosystem.
That needs a bit of a alteration involving the largest entropy reduction effort ever, bar the swamping of effort by Runaway CH4 and extinction hysteresis..
Knowledge is power and if the proper framework change is made to the value of money then the full potential of world enterprise can be aligned with the Open systems of Nature in a grand effort not constrained to failure.
I'm guessing Russian intelligence at least understands the seriousness of the situation but that the world banking cartel controlling futures so profits keep rolling in won't relent.
There's also a good chance that the resulting climatological adjustments (catastrophic or not) will lead to the ice age that's past due rather than an overly hot earth.
Actually, it's gradually speeding-up process: Methane heats Atmosphere & the rising temperature kicks off more upon more Methane. However, at critical point, the're great chance of sudden/explosive acceleration of its emission.


text: https://bbs.archlinux.org – Arch uses ex/vi for vi, not the vi build of vim: https://bugs.archlinux.org/task/20778 and http://ex-vi.sourceforge.net/.You can use vim as the default editor by setting the EDITOR environment variable in your startup configs. And, I guess, break the habit of typing 'vi' when you really mean vim. thisoldman https://bbs.archlinux.org/profile.php?id=25262 2012-10-26T23: (HowTos)


text:
Quest:Proving Grounds (Alliance)
this wiki
|Proving Grounds
|Lieutenant Haggerdin
|Lieutenant Haggerdin
Requires Level 51
|Alterac Valley
or 3960 at Level 90
|Stormpike Guard +250
Ironforge +250
Gnomeregan Exiles +62
|[Stormpike Insignia Rank 1] [The Frostwolf Artichoke]
Objectives Edit
Travel to the Icewing Caverns located southwest of Dun Baldar in Alterac Valley and recover the Stormpike Banner. Return the Stormpike Banner to Lieutenant Haggerdin in the Alterac Mountains.
Description Edit
You've arrived just in time, <class>. These Frostwolf savages just won't give up! We must show them the might of the Stormpike. They must bow to our superiority.
Before you make your way to the field of battle, you will want to join the rank of Stormpike.
Southwest of Dun Baldar, you will find the Icewing Caverns. Planted deep within the caverns is a Stormpike Banner. Venture forth and recover that banner. Return it to me, and you will have earned a Stormpike initiate's insignia.
Rewards Edit
Progress Edit
Beware the harpies of the region, <class>. They'll not think twice to rip out your throat!
Completion Edit
Take and wear this insignia with pride, <name>. As your rank among the Stormpike grows, so too will the insignia. Return to me as you gain honor with Stormpike, and I shall replace your insignia with other, more powerful insignias.
Perhaps someday you will grow to be a legend on this field of battle. Should that day ever come, you will be granted the Eye of Command.
Gains Edit
Upon completion of this quest you will gain:
- 6600 XP (or 39 at level 70)
- 250 reputation with Stormpike Guard
- 250 reputation with Ironforge
- 62 reputation with Gnomeregan Exiles
Patch changesEdit
- Patch 1.5.0 (2005-06-07): Added.


text:
MOVE IT is the UK's biggest dance event taking place annually in London.
The event only comes around once a year - providing dance enthusiasts, students, learners, lovers, teachers, advanced and professional dancers the unique opportunity to enjoy all things dance over one incredible weekend.
MOVE IT 2016 will take place at London ExCel from 18-20 March 2016.
The MOVE IT team is thrilled to announce that the event has been shortlisted as a finalist in the AEO exhibition excellence awards 2015.
MOVE IT PRO offers seminars and masterclasses for teachers, professionals and aspiring professional dancers looking to be inspired and guided through the professional dance industry.
MOVE IT offers an incredible lineup of over 240 dance classes - including a complete mix of genres and the current and future trends in the UK dance scene.
The MOVE IT Main Stage is the host of spectacular performances from some of the most exciting artists and dance companies from all over the UK.
The Genée International Ballet Competition, the Royal Academy of Dance's flagship event, returns to London from 10–19 September 2015.
MOVE IT is proud to continue it's support for official charity for danceaid.
danceaid harnesses the energy of dance to transform the lives of vulnerable children around the world.


text:
|Country Inns By Carlson Texas City hotels are provided below. Search for cheap and discount Country Inns By Carlson hotel rates in Texas City, TX for your upcoming individual or group travel. We list the best Country Inns By Carlson Texas City hotels so you can review the Texas City Country Inns By Carlson hotel list below to find the perfect place.
This technology will list below Texas City, TX hotels/motels that are comparable to the quality of Country Inns By Carlson.
Booking a group or meeting? Set up a group hotel block in Texas City TX
Next: Show All Available Comparable Hotels with Pricing >


text:
The Christmas Blessingby Donna VanLiere
View All Available Formats & Editions
In The Christmas Shoes, young Nathan Andrews was a child who lost his mother to cancer. Now his deepest wish is to become a doctor. When a stranger named Robert gave him the money to buy his mother a pair of shoes for her last Christmas, both Robert and Nathan learned the deepest lessons of love and giving. Now a medical student in his third year, Nathan/i>… See more details below
In The Christmas Shoes, young Nathan Andrews was a child who lost his mother to cancer. Now his deepest wish is to become a doctor. When a stranger named Robert gave him the money to buy his mother a pair of shoes for her last Christmas, both Robert and Nathan learned the deepest lessons of love and giving. Now a medical student in his third year, Nathan realizes there are still things to be learned about faith, blessings, and sacrifice. Lessons he will learn from Meghan Sullivan-a young woman born with a hole in her heart that has not kept her from becoming a champion runner. And lessons learned from a young boy named Charlie, who teaches how to live a life of true courage. Together, they will help guide Nathan through the darkest period in his life. The Christmas Blessing is an inspiring about hope existing in the darkest places, and love is always the greatest gift of all.
Editorial Reviews
Publishers Weekly
"The plot's resolution will lift the reader's spirits to a new level."
RT Book Reviews (Top Pick!)
"Enough sentimentality to bring me to tears and strong characters I could visualize.... a perfect holiday gift to give or get."
Book Reporter
Product Details
- 9781429936767
- Publisher:
- St. Martin's Press
- Publication date:
- 10/28/2003
- Sold by:
- Macmillan
- Format:
- NOOK Book
- Pages:
- 224
- Sales rank:
- 306,718
- File size:
Meet the Author
Donna VanLiere is a New York Times and USA Today best-selling author. Her much-loved Christmas Hope series includes The Christmas Shoes and The Christmas Blessing, both of which were adapted into movies for CBS Television; The Christmas Secret; The Christmas Journey; and The Christmas Hope, which was adapted into a film by Lifetime. She is also the author of The Angels of Morgan Hill and Finding Grace. VanLiere is the recipient of a Retailer's Choice Award for Fiction, a Dove Award, a Silver Angel Award, an Audie Award for best inspirational fiction, and a nominee for a Gold Medallion Book of the Year. She is a gifted speaker who speaks regularly at conferences. She lives in Franklin, Tennessee, with her husband and their children.
Donna VanLiere is a New York Times and USA Today best-selling author. Her much-loved Christmas Hope series includes The Christmas Shoes and The Christmas Blessing, both of which were adapted into movies for CBS Television, The Christmas Secret, The Christmas Journey and The Christmas Hope, which was adapted into a film by Lifetime. She is also the author of The Angels of Morgan Hill and Finding Grace. VanLiere is the recipient of a Retailer's Choice Award for Fiction, a Dove Award, a Silver Angel Award, an Audie Award for best inspirational fiction, and a nominee for a Gold Medallion Book of the Year. She is a gifted speaker speaks regularly at conferences. She lives in Franklin, Tennessee, with her husband and their children.
More from this Author
Read an Excerpt
Chapter One
All change is a miracle to contemplate; but it is a miracle which is taking place every instant.
I gunned the engine, pulled the truck out of my parking space, and flew over the speed bumps on my way out of the apartment complex. A young mother grabbed her toddler and gave me a dirty look. I thumped the face of my watch, and the second hand seemed to groan before deciding to move. Too late now, I'll never make it, I thought, glancing at the clock in my dashboard.
I couldn't believe it; I was never late. I'd noticed that my watch was having problems a couple days earlier and had been relying on an extra clock in my bathroom to make sure I was showered and out the door on time. As I was shaving I must have accidentally pulled out the cord just enough to stop the clock from running. The tires squealed as I pulled out onto the main road, and the gardener working at the entrance to the complex gave me my second nasty look of the morning, even shaking his head for effect.
If I made all the stoplights through town, I could get to the hospital in fifteen minutes. Turning into the hospital lot, I glanced at the clock-fourteen minutes-a new personal record. There was no time to circle for a spot, so I parked at the far end of the lot and ran for the main entrance. Maybe he hasn't started yet. Who was I kidding? Dr. Goetz never failed to start on time. I ran faster between the rows of cars.
As part of my third-year medical rotations, the university had placed me under the tutelage of Dr. Crawford Goetz-the best cardiologist in the hospital. Cardiology wasn't part of a normal rotation block, but the university felt that a rotation in cardiology would only enhance a student's studies. So, I was stuck for the next four weeks with Dr. Goetz. He was a Harvard and Vanderbilt man, the chief of cardiology, father of four, grandfather of two, and a thorn in my flesh. He specialized in pediatric cardiology, but since the hospital had only a small number of child patients a year, as department head, Dr. Goetz would also oversee the treatment of adult patients.
In each of our rotations, a medical student was part of a team that consisted of an attending physician, three to four students, and an upper-level resident. Peter Vashti was the upper-level resident on Dr. Goetz's team. My clipboard with the day's rounds was hanging at the nurses' station, the last to be picked up. The other students and Peter were already following Dr. Goetz from room to room. I checked the room number for the first patient to be seen and ran to catch up, sneaking in behind William Radcliff, an old friend and fellow student who, to my good fortune, stood six-five. Dr. Goetz was sitting on the patient's bed, a forty-seven-year-old man recovering from open-heart surgery.
"She's working like a thirty-year-old's heart," Dr. Goetz said.
"Does that translate to the rest of his body?" the man's wife asked, cracking a wad of gum. Dr. Goetz laughed. He had a carefree, easy way with his patients and their families; too bad that didn't translate to his students.
"So everything feels normal?" Dr. Goetz asked, resting his hand on the patient's shoulder.
"He's cranky again," the wife said, her gum exploding like a firecracker.
"Is that good or bad?"
"I don't know if it's good or bad, but for him it's normal," his wife continued.
The patient looked sheepish. Poor guy, no wonder he had heart surgery. She was relentless.
"All right, Jason," Dr. Goetz said, smiling. "You're ready to go home." The man shook Dr. Goetz's hand and I could see his eyes fill with tears; he started to speak, then stopped. He didn't want to get emotional in front of a handful of medical students. He pumped Dr. Goetz's hand again, nodded, and looked down at the sheet resting on his lap. Dr. Goetz squeezed his shoulder and turned to leave, nodding for us to follow.
Filing back into the hallway, we could hear Jason's wife get an early start on what could be heart attack number two. "What do you mean you're not going to wear the piece? Just because your heart's working again doesn't mean your hair's going to grow back. Put this on. Put this on, or I'm not walking out these doors with you, I mean it. I will not walk out these doors." For the sake of his heart, I hoped his head would shine like the new dawn as he left the hospital.
"Who's our next patient," Dr. Goetz asked, scribbling something onto Jason's chart. "Andrews?"
I looked down at the chart in my hands. "The patient in room 2201."
"Mr. Andrews," he said, as if giving a speech to a room of five hundred. "Just as you were not given a number at birth, but a name, you will find that your patients came into the world in the exact same manner. Learn who they are, not where they're located."
I could feel sweat break out on my upper lip. I never intended to seem demeaning toward the patient. "I didn't mean it that ..." I began, but it was too late. Dr. Goetz had already learned the name of the patient and was leading the students through the halls.
"And Mr. Andrews, as a reminder, your rotation begins at six A.M. Not six eighteen." I felt my chest tighten. I should have known that Dr. Goetz would pick up on my tardiness.
During a break in rounds, I retreated to the lounge and sank into the sofa. I leaned my head against the wall and rubbed my temples. If I'd known there was going to be someone like Dr. Goetz in my future, I never would have signed up for medical school in the first place. I glanced at my watch and noticed it had stopped running again. I tapped the face, but the second hand wouldn't budge. I took the watch off and flipped it over to thump the battery casing. I ran my finger over the inscription: With all the love in the world, Mom.
My mother died about a year after she stood with me on the hill overlooking the valley. Maybe she knew she'd never see me grow up; perhaps she was preparing me for the long valley I would go through without her, or maybe preparing her family and herself for death was the final step of faith she would take.
I remember my father coming into my room during the early morning hours of that Christmas. He said that my mother had stepped into Heaven. He let my sister Rachel sleep; she was much too young to understand what was happening anyway. I ran to the living room, where my mother lay still on the hospital bed; my grandmother was holding her hand, weeping. I watched my mother for the longest time, praying she'd move again, that she'd reach for me and say, You need to get back into bed, Little Man, but she couldn't reach for me, and I knew it. She was thirty-four years old.
Wilson's Department Store was about to close on that Christmas Eve as I ran from one department to the next looking for the perfect gift until the shoes caught my eye on a sales rack. I ran them to the front register and pulled a crumpled wad of bills and loose change out of my jeans pocket. When the clerk told me I didn't have enough money, I was heartbroken. I just had to buy those shoes for my mother. I turned to a man behind me, and, before I knew what was happening, he paid for the shoes, and I ran out the door for home. When I helped my mother unwrap the shoes, she held them to her chest and made me feel as if I'd just handed her Heaven itself. We buried her in them. I started leaving shoes on her tombstone again when I was sixteen. The owner of Wilson's somehow found a similar pair every year and ordered them for me.
During the last weeks of her life, my mother wrote a series of letters to my sister Rachel and me. In one addressed to me she wrote,
Dear Nathan, I have had many joys in my life but none that have compared to you and Rachel. I always want you to know that I fell more in love with you every day. Please don't ever dread Christmas, Nathan, but remember to look for the miracles instead. It may be hard to see them at times but they will always be there because Christmas is the season for miracles.
She finished the letter and signed it, With all the love in the world, Mom.
I was helping my mother string lights on the shrubs outside our home the winter before she got sick when she first told me about the miracles of Christmas. "Jesus was born at Christmas," she said, wrapping a long strand around a juniper yew. "He left Heaven to live here." She bent over the back of the yew and tugged at the lights, stuck on a low branch. I pulled along with her, and together we continued wrapping the bush. "That's kind of like us becoming a worm and living in the dirt," she said, wiping her nose. "Love came down on Christmas, Nathan. That's the greatest miracle of all. That's the true blessing of Christmas and why it will always be the season for miracles." She stood back and admired her work, frowning at the tangled mess. "It'll look better when the lights are on." She dug into the box and pulled out another jumbled string, talking as she worked. "If you get too busy, you won't see the miracles that are taking place right in front of you," she said, replacing a blown light.
Before she died, my mother bought special gifts for Rachel and me; she wanted my father to give them to us on our sixteenth birthdays. Rachel got a gold locket and I got this watch-a flat, gold-faced Timex with a simple black band. The inscription was a reminder of something I'd always asked her.
"Is your love for me as big as Texas?"
"Bigger," she'd say.
"As big as the United States?"
"As big as the world?"
"It's even bigger than the world! But if you combined all the love in the world, it might come close to how much I love you," my mother told me.
I'd worn the watch every day since my father gave it to me, as promised, on my sixteenth birthday,
Soon after my mother's death I told my father and grandmother that I wanted to be a doctor. When people asked what I wanted to be when I grew up I responded the same. I wanted to be a doctor so I could help people just like my mother.
Before I knew it, I was through college and into medical school. What a tribute to your mother's memory, an aunt would say or, What a tremendous way to honor your mother, an old family friend would comment. I felt the pressure mounting-people were counting on me to become a physician-my mother's memory depended on it. But after three months of rotations and watching people suffer and die, and now a week with Dr. Goetz, I questioned whether I'd made the right decision. In all honesty, when someone died it left me emotionally drained, and I was taken back to the morning my mother passed away. I felt as if I didn't measure up, that I wasn't cut out for it. I opened my eyes and realized I needed to get back to rounds.
Our team gathered outside the patient's room, and Micah, another third-year med student on our team, stepped forward and began to give the patient's blood pressure, pulse, heart rate, and the results of a heart test administered the previous afternoon. Micah was the "gunner" of our group-a med student's term to describe a fellow student who was always the first to answer, the first to volunteer for a procedure, the first to give stats on someone else's patient, and the first to get on other students' nerves. The term had been around long before we ever applied to medical school. William and I shot each other glances as Micah handed out Xeroxed copies of an article on angioplasty from one of our textbooks, one of at least twelve articles so far, all of them filed after our rounds in the nearest garbage can. William and I suffered in silence; it was all we could do, there was a gunner on every rotation.
Helen Weyman was the next patient on our rounds. She was a fifty-two-year-old woman complaining of chest pain who had a history of cervical disc disease. I had done Helen's workup when she was admitted to the hospital the previous afternoon. I went over her progress notes with the team before entering her room. It was customary that the attending physician took over once the group entered the patient's room; it was our time to stand back and learn, but I felt it was important to greet my patients first.
"Good morning, Helen," I said, standing at her side.
"I see your daughter was able to bring your knitting to you. Now you're not so bored, I hope." Dr. Goetz glanced at me. "What are you making?" I asked.
"A baby blanket for my next grandchild ... number three. I've made a blanket for all of them. She's due in the next week or two."
I picked up the blanket and turned it over in my hands. "You've even got her name in here!" I sensed Dr. Goetz waiting for me to finish. "Let's go ahead and take a listen to your heart again this morning." I listened to her heart and felt for her pulse. I was taking up too much time. "Dr. Goetz would like to listen to your heart today as well." I moved away from the bed. Dr. Goetz took my place and examined her. As he did, he asked her about all her grandchildren, where they lived, how long she'd been married, and if she'd make him a pair of slippers. She laughed, and I watched as Dr. Goetz won over yet another patient. Before leaving the room I squeezed Helen's shoulder and told her I'd be by later to check on her.
I walked with William toward the cafeteria for lunch when my pager went off. I walked to Helen's room. The baby blanket was still sitting on her lap. Her daughter Mary, looking very pregnant and uncomfortable, was sitting in the chair next to the bed. "Is everything all right, Helen?" I asked.
She leaned forward and rubbed her hand over her lower back. "My back has been hurting."
I helped Helen into a more comfortable position. "You've been immobile longer than usual, and that may be putting pressure on those discs in your back. Does that feel better?"
She paused for a moment. "Yes, thank you, I think it helped."
"So you don't think it's anything serious?" Mary asked.
"No, it may be just some inflammation around those discs. But we should rule out any other possibilities," I said, handing the knitting back to Helen. "How much longer before this is done?"
"Just a couple more days, I think," she said, taking the needles in her hand.
I left her room and went to the nurses' station to discuss follow-up with the nurse on duty and to page one of the residents when Mary came rushing from her mother's room.
"My mother needs help!"
A nurse ran past me and headed to Helen's room. I followed close behind. I had just stepped inside when the nurse called in a loud, firm voice.
"Page, Dr. Vashti."
I stood in the hallway, right outside Helen's door, feeling helpless as Peter wheeled Helen to the OR.
Copyright ©2003 by Donna VanLiere
Customer Reviews
Average Review:
and post it to your social network
Most Helpful Customer Reviews
See all customer reviews >


text:
Software Engineering, Project Management, and Effectiveness
"Anyone can hold the helm when the sea is calm." — Publilius Syrus
Change is tough. Especially leading it.
Whether you are leading yourself, others, or organizations through a change, it helps to have tools on your side.
Recently, I read Leadership Transformed, by Dr. Peter Fuda.
It uses 7 metaphors to guide you through leadership transformation:
It might seem simple, but that's the point. Metaphors are easy to remember and easy to use.
For example, you can use the Movie metaphor to increase your self-awareness and reflection that allow you to first "edit" your performance, and then direct a "movie" that exemplifies your leadership vision.
The other benefit of simple metaphors is they allow both for creative interpretation and creative expression.
I appreciated the book the further I went along. In fact, what really clicked for me was the fact that I could easily remember the different metaphors and the big idea behind them. It was a nice brain-break from memorizing and internalizing a bunch of leadership frameworks, principles, and patterns.
Instead, it's just a simple set of metaphors that remind us how to bring out our best during our leadership transformations.
The metaphors are actually well-chosen, and they really are helpful when you find yourself in scenarios where a different perspective or approach may help.
Even better, the author grounds his results in some very interesting data, and aligns it to proven practices for effective leadership.
Here is my book review: Book Review: Leadership Transformed: How Ordinary Managers Become Extraordinary Leaders
I included several highlights and "scenes" from the book, so you can get a good taste of the book, movie trailer style.
If you end up reading the book, I encourage you to really dive into the background and the anatomy of the Leadership Impact tool that Dr. Fuda refers to. It's incredibly insightful in terms of leadership principles, patterns, and practices that are fairly universal and broadly applicable.


text:
Italian 2.80 Carat London Blue Topaz and 2.40 ct. t.w. Tsavorite Feather Ring in Sterling Silver
Item#: 835719
A plume bejeweled. Glam green and blue peacock feather ring comes to life with 2.40 ct. t.w. tsavorites and a 2.80 carat London blue topaz. Made in Italy. Sterling silver ring with a gunmetal finish.
Product Details (for Italian 2.80 Carat London Blue Topaz and 2.40 ct. t.w. Tsavorite Feather Ring in Sterling Silver. Size 5)
1 1/8 inches
Sterling close STERLING SILVER: Also known as "Standard Silver," sterling silver is silver alloyed with other metals to increase the hardness and durability. Generally, sterling is 92.5% silver and 7.5% other metals. Some sterling silver jewelry is plated with 14kt-18kt gold and referred to as "vermeil," or "gold over sterling." Silver
Polished close POLISHED: A buffed-out finish used to produce a mirror-like shine.
Gemstone 1:
Topaz close TOPAZ: Available in a stunning selection of shades, the topaz's sky blue hue is one of the most popular colors for jewelry designs. Once thought to offer protection, today topaz is favored for its durability and affordability. Yellow topaz is the birthstone for November while blue topaz is for December. (Color: Blue, Quantity: 1, Shape & Cutting Style: Pear close PEAR: Similar to the outline of a pear fruit, this is a round shape that comes to a point on one side. , Weight: 2.80 carats, Size: 11X7 mm)
Gemstone 2:
Tsavorite close TSAVORITE: a deep green variety of garnet. It has similarities to emerald, but is rarer and more durable. First uncovered in the 1960's in Tanzania, it was given its name on account of the discovery of mines in Kenya's Tsavo National Park in the early 1970's. (Color: Green, Quantity: 67, Shape & Cutting Style: Round, Weight: 2.40 ct. t.w., Size: 1.5-2.25 mm)
All weights and measures are approximate.
Ross-Simons Presentation Box Your purchase includes an elegant Ross-Simons presentation box.
100% Moneyback Guarantee every item, every order, every day
Free Shipping on orders $250 or more
Only $7.95 Flat Rate for any order under $250
24/7 Live Help toll free at 1-800-835-0919
100% Secure Checkout verified by Norton Security
Recently Viewed Items:
Learn More About Sterling Silver Polished Finish Topaz Tsavorite Free Ring Sizer


text:
T.O.--Too Old!
Originally written on Helmet2Helmet | Last updated 5/19/12
Still out of a job and with time not on his side, free agent receiver Terrell Owens is pushing hard to find a new home. That home, if he had his druthers, would be somewhere in the Bay Area. During an interview with "The Drive" on 95.7 The Game in San Francisco, which airs Monday, Owens said he would like to play for either the 49ers or Raiders. Of course, T.O. started his career with the Niners, where he had much of his success, earning four Pro Bowl trips. It seems he might've been content calling it quits there, but it's simply a pipedream with Randy Moss now in town. "Would I have loved to have ended my career there, obviously signing a one- or two-year deal, absolutely. … It is what it is at this point. I don't make those decisions and they were well aware I was out there so they felt they had the best man on their roster." As for Oakland, it's a scenario that began gaining speculation when Darrius Heyward-Bey tweeted a photo of Owens working with Raiders wideouts a...
20 Comments:
I think T.O. ought to go for it. If he's too old, his body will let him know!
Very few teams with any sense would take him. It's always about him. His mouth has cost him. No one wants to play with a loud mouth. The game is better off without him!
Time to go into retirement and live off of the interest you made from the millions you earned while playing.
Wait you didnt invest and save for your future, did you. Hmm no thought went through that peon mind of yours about 20 years from now. I guess that was hard cause 99% of your thoughts was where you could put your dick next. Stupid
Well at least you dont have a whole bunch of ex's and children to support. You were smart and used protection. O wait how many kids do u have with different women. Stupid
Well at least you have the love of the children that you spent years raising, Wait none of them can stand the sight of you cause you abandoned them all. Wait that makes you,the xact thing your father was.
All of these have one thing in common. Black man, Black man, Black man. Cromartie your time is coming.
Only one thing comes to my mind, as Nelson Muntz from the Simpsons puts it. HAHAHAHAHAHAHA
John Edwards spring to mind. White man, White man, White Man. Only he's going to jail. Wait. Anothing thing springs to mind. HAHAHAHAHAHAHAHA!
You are a racist DOUCHE BAG!!!!!! Go **** off somewhere.
You can call that racist if you like. Guess what, whatever you call it, it doesnt change that its fact.
No wait lets have a round of blaming someone or something else first.
HAHAHAHAHA in a year or 2 your going to be 100% broke, everyone will hate you and you will be trying to sell autographs for a living. Have fun living off of $15,000 a year.
38 is not to old to play.
hell im 61 and i still lift carpets that weigh 300 to 400 pounds and move vfurniture by my self
you are as big of an idiot as owens is.
Just wants to play.....wants to be looked at and talked about.....so?
i bet he does. he probably wants enough money to pay off his child support, not that he would give it to his children because he is too selfish to do anything for anyone else, but himslef. he is a sad pathetic human being. i wish him luck in his old age when he will be alone and broke.
.......T.O. a 49er, had your chance, made a great catch against Green Bay in the playoffs, I was there and it was a great catch, but you never learned how to act like you appriciated what you had, wore out your welcome. I beleive in second chances but you abused your second, third, forth and so on. you don't deserve another chance cause you don't know how to handle it, good luck in retirement, hopefully you will find humility in your next indevore
do't you jsut love the way our wonderful sports media relentlessly shoves TO and D Rosenhous in your face and over the sports shows for what seemed like an eternity----showing a couple of eg0-maniacs blowing off their idiot mouths and then finally TO is scratching for a gig and he mentions that they "parted ways"---no media inquiry---no media hype---no media anything----we sure got some phonies in the media in this pathetic country, don't we----ames you proud to be an american
Look, the man didn't respect the game when he was playing. It was all about HIM. He made 48 MILLION and could keep his pants zipped. I am so sick of the NFL players who can't handle the limelight or their finances. TO, your ten minutes are over. Go away and find a real job to pay all the child support
who really gives a bleep what this idiot wants, why waste time on him.
the NFL doesn't want him. he is a cancer to every team he's ever been on.
You all thats hating on T.O. is a trip go to work, at least one thing we can say is he has not been arrested or on drugs just a guy who wants the ball. Yea he has a MOUTH look at what YOU ALL are saying...BIG MOUTHES TO
Comments are closed.
What do you think?To leave a comment, you can:
NFL News
Delivered to your inbox
You'll also receive Yardbarker's daily Top 10, featuring the best sports stories from around the web. Customize your newsletter to get articles on your favorite sports and teams. And the best part? It's free!
Follow us on Twitter
|Best of Yardbarker
|NFL News
|MLB News
Today's Best Stuff
For Bloggers
Join the Yardbarker Network for more promotion, traffic, and money.
Company Info
What is Yardbarker?
Yardbarker is the largest network of sports blogs and pro athlete blogs on the web. This site is the hub of the Yardbarker Network, where our editors and algorithms curate the best sports content from our network and beyond.
|© 2015 Yardbarker, Inc. All rights reserved. Use of this website (including any and all parts and components) constitutes your acceptance of these Terms of Use and Privacy Policy. (What's New?)


text:
Randomized trials are ones where similar patients are randomly assigned to a certain treatment group -- such as playing video games -- while others are assigned to a different kind of group. These trials look for a specific outcome over a certain period of time.
Among stroke patients in observational studies, those playing virtual reality games improved their upper arm strength by 14.7 percent and motor function -- or the ability to perform standard tasks -- by 20 percent.
Those in the randomized trials who played virtual reality games had a 4.89 times greater chance of improving arm strength, compared with those did not play these games but had standard rehabilitation, the researchers found.
Treatment varied in each study, however, most patients played 20 to 30 hours over four to six weeks of therapy, the authors noted. The systems used included three traditional video game systems (Glasstron, IREX, PlayStation Eye Toy) and nine virtual reality systems (including Virtual Teacher, CyberGlove, VR Motion, PneuGlove and Wii).
Saposnik also noted that some studies looked at video gaming used in addition to standard therapy, something he said may have skewed the results somewhat in favor of the new therapy.
Video gaming may help stroke patients because of the brain's unusual potential for remodeling, in which it creates new nerve cell connections, some studies suggest. What is needed for such remodeling, recent research indicates, is training that is challenging, repetitive, task-specific, novel and motivating -- all hallmarks of virtual reality games, which can also give immediate real-time feedback.
"Our study confirms the potential benefit of virtual reality in stroke rehabilitation identified in small studies," Saposnik said. "Further larger randomized trials are needed before changing practice. However, we are [going] in the right direction," he added.
Commenting on the study, Dr. Ralph L. Sacco, presi
|Copyright©2010 ScoutNews,LLC.
All rights reserved


text:
This is the book lover's community that has been waiting for you. Join us for fun and meet people who share the same love of books that you do. Java Junction is filled with publisher and author groups. Special Interests is filled with hobbies, fun things, cooking tips, recipes, and great things to do. Author's Coffee Corner - Stop by our Author group area and find hundreds of authors who have their Author area with us.
Coffee Time Romance Event Calendar | Author & Publisher Events Calendar
Hello. It says your a moderator. How long does it usually take for our initial forum get to know you post to be approved? I'm having trouble finding my way around the site. Is there a help section?


text:
Haddad, F; Patel, S; (2009) Triplane Fractures of the Ankle. British Journal of Hospital Medicine , 70 (1) 34 - 40.
Full text not available from this repository.
|Triplane Fractures of the Ankle
|UCL classification:
|UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Surgery and Interventional Science (Division of)
Archive Staff Only: edit this record