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The role of the Na+ pump α2-subunit in Ca2+ signaling was examined in primary cultured astrocytes from wild-type (α2 +/+ = WT) mouse fetuses and those with a null mutation in one [α2 +/− = heterozygote (Het)] or both [α2 −/− = knockout (KO)] α2 genes. Na+ pump catalytic (α) subunit expression was measured by immunoblot; cytosol [Na+] ([Na+]cyt) and [Ca2+] ([Ca2+]cyt) were measured with sodium-binding benzofuran isophthalate and fura 2 by using digital imaging. Astrocytes express Na+ pumps with both α1- (≈80% of total α) and α2- (≈20% of total α) subunits. Het astrocytes express ≈50% of normal α2; those from KO express none. Expression of α1 is normal in both Het and KO cells. Resting [Na+]cyt = 6.5 mM in WT, 6.8 mM in Het (P > 0.05 vs. WT), and 8.0 mM in KO cells (P < 0.001); 500 nM ouabain (inhibits only α2) equalized [Na+]cyt at 8 mM in all three cell types. Resting [Ca2+]cyt = 132 nM in WT, 162 nM in Het, and 196 nM in KO cells (both P < 0.001 vs. WT). Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER) Ca2+ pumps and unloads the ER, induces transient (in Ca2+-free media) or sustained (in Ca2+-replete media) elevation of [Ca2+]cyt. These Ca2+ responses to 10 μM CPA were augmented in Het as well as KO cells. When CPA was applied in Ca2+-free media, the reintroduction of Ca2+ induced significantly larger transient rises in [Ca2+]cyt (due to Ca2+ entry through store-operated channels) in Het and KO cells than in WT cells. These results correlate with published evidence that α2 Na+ pumps and Na+/Ca2+ exchangers are confined to plasma membrane microdomains that overlie the ER. The data suggest that selective reduction of α2 Na+ pump activity can elevate local [Na+] and, via Na+/Ca2+ exchange, [Ca2+] in the tiny volume of cytosol between the plasma membrane and ER. This, in turn, augments adjacent ER Ca2+ stores and thereby amplifies Ca2+ signaling without elevating bulk [Na+]cyt.
- astrocytes
- catalytic subunit
- fura 2
- sodium-binding benzofuran isophthalate
- sodium-potassium-adenosine 5′-triphosphatase isoforms
- transgenic mice
numerous physiological processes are regulated by cytosolic Ca2+ signals in all cells. It is, therefore, important to understand how these signals are controlled. To this end, we studied the influence of Na+pump (Na+-K+-ATPase) expression on the regulation of the cytosolic free Ca2+ concentration ([Ca2+]cyt) and the control of Ca2+ signaling in primary cultured mouse cortical astrocytes.
The rationale for these studies is the evidence that both a plasma membrane (PM) ATP-driven Ca2+ pump (PMCA) (19) and a PM Na+/Ca2+ exchanger (NCX) (10) help to control resting [Ca2+]cyt in astrocytes (7) as in many other types of cells. The NCX is regulated by the Na+ pump via its influence on the Na+electrochemical gradient across the PM. Most of the intracellular Ca2+ in quiescent cells is stored in the endoplasmic reticulum (ER). By controlling [Ca2+]cyt, the PMCA and NCX indirectly influence the ER Ca2+ store content. During cell activity, much of the "signal Ca2+" comes from the ER stores, although some also enters the cells through PM Ca2+-permeable channels.
The Na+ pump consists of α- and β-subunits in a 1:1 ratio (5, 24). The β-subunit is a highly glycosylated 40- to 60-kDa protein that may be involved in chaperoning and membrane trafficking of the larger (≈112 kDa) α-subunit (12). The α- (catalytic) subunit contains the Na+, K+, and ATP binding (and hydrolytic) sites, as well as a binding site for cardiotonic steroids such as ouabain, which inhibits the pump (5, 31). Four Na+ pump α-subunit isoforms have been identified: α1, α2, α3 (41, 44, 46), and α4(47). The latter is found only in the testis and will not be discussed here. Most cells express α1 and one of the other isoforms, all of which have different kinetic properties. The α1 has a higher affinity for Na+ than α2 and α3 and respectively] (Ref. 48; see also Refs. 40and 45). In rodents, the α1-isoform has an especially low affinity for ouabain; in contrast, the α2- and α3-isoforms have high affinity for ouabain (5, 35). Moreover, these isoforms are differently distributed in the PM (25, 26). In at least several types of cells (astrocytes, neurons, and arterial myocytes), α2 and α3 are confined to PM microdomains that overlie sarcoplasmic reticulum or ER (S/ER). In contrast, α1 is more uniformly distributed in the PM of these cells (25, 26). It is noteworthy that the NCX also is confined to PM microdomains that overlie the S/ER, whereas the PMCA is more uniformly distributed (25, 28).
These observations led to the suggestion (6, 8) that low-dose ouabain might regulate cell Ca2+ signaling by inhibiting only α2 or α3 and controlling the [Na+] primarily in the tiny ("junctional") space between the aforementioned PM microdomains and the subjacent junctional S/ER. Thus Na+ pumps with α2- or α3-subunits would be expected to regulate, via NCX, not only the local [Ca2+] in this junctional space (JS), but also the [Ca2+] in the junctional S/ER that plays a key role in Ca2+ signaling. To test this hypothesis, we measured the bulk cytosolic concentrations of Na+([Na+]cyt) and [Ca2+]cyt in resting astrocytes and the rise in [Ca2+]cyt induced by blocking the S/ER Ca2+ pump (SERCA). Astrocytes express only α1 and α2 Na+ pump isoforms (26, 46). Therefore, these parameters were studied in astrocytes from normal [wild-type (WT)] mice and from mice missing one or both of the high alleles (23).
Knockout Mice
Mice with null mutations in one or both α2 genes [i.e., heterozygotes (Het) = α2 +/−, and homozygote knockouts (KO) = α2 −/−, respectively] were generated as described (23). WT (α2 +/+) mice from the same litters were also studied. The mice were genotyped by Southern blot analysis of genomic DNA prepared from embryo tails (23). The Het mice appeared normal and developed normally into adults. The KO mice die very shortly after birth (23), but the fetuses appeared to be normal. All astrocytes were cultured from near-term fetuses.
Astrocyte Cell Cultures
Astrocyte primary cultures were initiated from embryonic day 18–19 mouse cerebral cortex by using a modification of the method of Booher and Sensenbrenner (11) as described (17). The cortex was separated from the meninges and the hippocampus and was placed in culture medium [DMEM-F12 (1:1) with 10% FBS, penicillin G (50 U/ml), and streptomycin (50 μg/ml)]. The cells from each mouse cortex were mechanically dissociated by sequential passage of the cortex through 80- and 10-μm nylon mesh. The resulting cell suspension was plated onto poly-l-lysine-coated 25-mm glass coverslips (≈50,000 cells/coverslip). The medium was changed ondays 4 and 7. The cells were characterized as protoplasmic (type 1) astroglial cells (4). Experiments were performed on subconfluent cultures on days 7–9 in vitro.
Immunoblot Analysis of Expressed Na+ Pump α-Subunit Isoforms
Membrane preparation.
Mouse astrocytes were cultured in 10-cm dishes for 2 wk. Cells were then harvested with buffer containing 140 mM NaCl and 25 mM imidazole-HCl (pH 7.4) and were pelleted (3,000 g, 4°C, 20 min). The cell pellet was resuspended in lysis buffer containing (in mM) 140 NaCl, 2 EDTA, 10 sodium azide, 20 Tris base, and 250 sucrose; the buffer also included four tablets per 100 ml of a complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The resuspended cells were homogenized with a Polytron (Brinkmann, Westbury, NY), and the homogenate was centrifuged at 480 g(4°C, 30 min). The supernate was centrifuged at 17,200 g(4°C, 30 min) to pellet membrane fragments and vesicles. The membrane pellet was then resuspended in lysis buffer with 1% deoxycholate and 1% Triton X-100 and incubated on ice for 30 min. After centrifugation (17,200 g, 4°C, 20 min), the supernatant fluid containing membrane proteins was collected and stored at −80°C. The protein concentration was determined with the bicinchoninic acid assay (Bio-Rad Laboratories, Richmond, CA) by using bovine serum albumin as a standard.
Skeletal muscle membrane preparation.
Male mice, 12 wk old, were used for quantitation of Na+pump α-isoform expression. Extensor digitorum longus (EDL) muscles from both legs were dissected and frozen (−80°C) for later use. EDL muscles (4–6 from each genotype: WT and α2 +/−) were homogenized with a Polytron in 1-ml ice-cold homogenization buffer [in mM: 250 sucrose, 30 imidazole (pH 7.5), and 1 EDTA]. The homogenates were centrifuged at 3,000g, 4°C, for 20 min to remove cellular debris. The supernatants were centrifuged at 17,200 g, 4°C, for 30 min. The membrane pellet was resuspended in lysis buffer containing protease inhibitors, 1% deoxycholate, and 1% Triton X-100 and treated as in the preceding section.
Immunoblot analysis.
Membrane (PM) proteins were solubilized in sodium dodecyl sulfate (SDS) buffer containing 5% 2-mercaptoethanol and were separated by 7.5% polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to a nitrocellulose membrane (Amersham, Piscataway, NJ); transfer was checked by Ponceau S staining. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 137 mM NaCl, 20 mM Tris, pH 7.6, and 0.1% Tween 20 for at least 2 h at room temperature. Nitrocellulose membranes were incubated overnight at room temperature with polyclonal antibodies raised against Na+pump α1- or α2-subunit isoforms, or with an α-subunit isoform nonspecific antibody (gifts of Dr. Thomas Pressley). Some membranes were probed with monoclonal or polyclonal antibodies raised against the cardiac/neuronal NCX, NCX1 (R3F1, a gift from Dr. Kenneth Philipson; π11–13 from Swant, Bellinzona, Switzerland). In some cases, gel loading was controlled with polyclonal or monoclonal anti-actin antibodies (Sigma Chemical, St. Louis, MO).
The nitrocellulose membranes were washed with Tris-buffered saline with Tween 20 and then incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG for 1 h. The immune complexes on the membranes were detected by enhanced chemiluminescence-plus (Amersham) and exposure to X-ray film (Eastman Kodak, Rochester, NY) for 30–60 s.
Quantitation of Na-K-ATPase isoform levels.
The band intensities of the immune complexes on the film were scanned (Epson Expressions, Epson America, Long Beach, CA) and quantified by densitometry with the use of Kodak ID image analysis software (Kodak Digital Science, Eastman Kodak). Samples containing various amounts of membrane protein were analyzed in a single blot, and each blot was exposed for two to three different times to ensure linearity of signal intensity. Changes in band densities (relative to WT band densities) for α1 and α2 were measured with isoform-specific polyclonal antibodies and were correlated with changes in α1 + α2 measured with a nonselective polyclonal antibody ("LEAVE"; see Ref.37). We assume, as did He et al. (20), who used different antibodies, that the nonselective antibody cross-reacts equally well with the two α-subunit isoforms after they are unfolded in SDS buffer.
Primary cultured mouse cortical astrocytes were fixed and cross-reacted with polyclonal or monoclonal antibodies raised against Na+pump α1- or α2-subunit isoforms (gifts of Drs. Thomas Pressley and Kathleen Sweadner; Refs. 13,37, 46). The primary antibodies were then cross-reacted with fluorescent-labeled secondary antibodies: Alexa-Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR) for monoclonal antibodies and Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). This enabled us to visualize the distribution of the primary label with a fluorescence microscope (Nikon Diaphot TMD; Nikon, Melville, NY). Details are published (26).
In some experiments, the cells were identified as astrocytes by labeling with polyclonal antibodies raised against glial fibrillary acidic protein (Boehringer Mannheim). In these experiments, nuclei also were identified by labeling for 5 min with a 50 μM solution of the nucleic acid stain, 4′,6′-diamidino-2-phenylindole (DAPI).
Fluorescent dye loading.
Astrocytes on coverslips were loaded with the Ca2+-sensitive fluorochrome fura 2 by incubation for 30 min in medium containing 3.3 μM fura 2-AM (22–24°C, 5% CO2–95% air). Alternatively, cells were loaded with the Na+-sensitive fluorescent dye sodium-binding benzofuran isophthalate (SBFI) by incubation for 1 h at 22–24°C in medium containing 10 μM SBFI-AM. After loading with either dye, the coverslips were transferred to a tissue chamber mounted on a microscope stage, where they were superfused for 15–20 min (35–36°C) with standard physiological salt solution to wash away extracellular dye.
Digital imaging methods.
Fura 2 fluorescence (510 nm emission; 380 and 360 nm excitation) and SBFI fluorescence (510 nm emission; 340 and 380 nm excitation) were imaged with a Zeiss Axiovert 100 microscope (Carl Zeiss, Thornwood, NY). The dye-loaded cells were illuminated with a diffraction grating-based system (Polychrome II, Applied Scientific Instruments, Eugene, OR) (18). Fluorescent images were recorded by using a Gen III ultrablue intensified charge-coupled device camera (Stanford Photonics, Palo Alto, CA). Image acquisition and analysis were performed with a MetaFluor/MetaMorph Imaging System (Universal Imaging, Chester, PA). Images were captured at rates of one per minute (under resting conditions) to one per second (when Ca2+ was changing rapidly); eight frames were averaged to improve the signal-to-noise ratio in each image. [Ca2+]cyt was calculated by determining the ratio of fura 2 fluorescence excited at 380 and 360 nm as described (17). [Na+]cyt was calculated by determining the ratio of SBFI fluorescence excited at 340 and 380 nm. SBFI calibration was carried out in the cells, in situ, at the end of each experiment, as described (17).
The standard physiological salt solution contained (in mM) 140 NaCl, 5.0 KCl, 1.2 NaH2PO4, 1.4 MgCl2, 1.8 CaCl2, 11.5 glucose, and 10 HEPES (titrated to pH 7.4 with NaOH). In Ca2+-free solutions, CaCl2 was omitted, and 50 μM EGTA was added. Stock solutions of fura 2-AM (1 mM) and SBFI-AM (10 mM) were prepared in DMSO.
FBS was obtained from Paragon Bioservices (Baltimore, MD); all other tissue culture reagents were obtained from GIBCO-BRL (Grand Island, NY). Fura 2-AM and SBFI were obtained from TefLabs (Austin, TX). Ouabain, cyclopiazonic acid (CPA), DMSO, poly-l-lysine, DAPI, penicillin G, and streptomycin were purchased from Sigma. All other reagents were analytic grade or the highest purity available.
Data analysis.
The numerical data presented in results are the means fromn single cells (one value per cell). The numbers of different animals and different litters are also presented, where appropriate. Data from two to three litters were obtained for most protocols and were consistent from litter to litter. Student'st-tests for paired or unpaired data were used to calculate the significance of the differences between means.
Expression of Na+ Pump α-Subunit Isoforms in Cultured Astrocytes
The expression of Na+ pump α1- and α2-isoforms in astrocytes cultured from the brains of WT, Het, and KO mouse embryos is compared in Fig.1. Figure 1 A shows the α2 genotype data from Southern blot analysis of the genomic DNA from the 12 embryos in a single litter. Figure1 B shows the Western blot analysis of the expressed α2 protein from these same embryos. Astrocytes from Het mice express about one-half as much α2 as do cells from WT mice, and cells from KO mice do not express any α2. Figure 1 C, which shows α1 expression data from the same litter, indicates that astrocytes with all three genotypes express approximately equal amounts of the Na+ pump α1-isoform. These Western blot data from primary cultured astrocytes are comparable to the results obtained in fresh cardiac tissue from α2 WT, Het, and KO mice (23).
The Na+ pump α2-isoform in astrocytes is confined to PM microdomains that overlie ER, where it colocalizes with the NCX (24), with which it is functionally coupled (see below). Several groups have reported that the Na+ pump α2-isoform and the NCX are reciprocally regulated under some conditions (e.g., Ref. 32; reviewed in Ref.10). Therefore, we also compared NCX expression in the astrocytes from WT and α2 Het and KO mice. Surprisingly, NCX expression was not significantly upregulated in Het and KO astrocytes (Fig. 2).
Quantitation of Na+ Pump α2-Isoform Expression
To interpret the functional effects of reduced α2-isoform expression, it is important to know what fraction of the total Na+ pump α-subunit (α1 + α2) is α2. This information cannot be obtained directly from Western blots cross-reacted with the isoform-specific antibodies. It should be possible, however, to obtain some quantitative information by also using an antibody that is not isoform specific and that recognizes both α1 and α2 (LEAVE) (37). In control experiments on mouse skeletal muscle (which also expresses only α1 and α2), we found that, in α2 Hets, total α-subunit expression was decreased by ∼30% (Fig. 3). Expression of α2 was decreased by ∼50% in the EDL of α2 Het mice, compared with WT (Fig. 3), whereas an ∼30% increase in α1 expression was observed. These results are comparable to those reported by He and colleagues (20), despite the use of different antibodies. The data suggest that ∼80% of total expressed α in EDL is α2(Fig. 3 B); this is close to the published value of 87% (20).
When this isoform-nonselective antibody was tested on astrocytes, however, there was no detectable decline in the Western blot total α-subunit band density in Het astrocytes and a 20% decline in KO astrocytes, compared with that in controls (Fig. 3). Because there is little or no upregulation of the α1-isoform in α2 Het and KO astrocytes (Figs. 1 C and 3), the implication is that the α2-isoform accounts for no more than ∼20% of the total α-subunit in these cells.
Localization of α1 and α2
It is important to ask whether astrocytes, which normally express both the α1- and α2-isoforms, but not α3 (26, 34), are able to grow normally when α2 is knocked out. Information on this point is provided by the immunocytochemical data from WT and α2 KO cells in Fig. 4 and the fura 2 images in Fig.5 A. As illustrated, WT and KO cells have a similar appearance. Cells of both genotypes express Na+ pumps with α1-subunits distributed uniformly over the cell surface, as detected with either polyclonal or monoclonal antibodies (Fig. 4) (25, 26). In contrast, the α2-isoform is expressed in WT but not KO cells (Fig. 4). Despite the absence of α2, the cells could be identified as astrocytes by cross-reaction with anti-glial fibrillary acidic protein antibodies (Fig. 4 B,middle). Also, cell nuclei could be detected by staining with DAPI (Fig. 4 B, right). In WT cells, α2 is distributed in a lacy, reticular pattern.
The black-and-white images in Fig. 5 A (top) show fura 2-stained astrocytes from WT, Het, and KO mice (left toright). The morphology of the cells from the three genotypes are all very similar.
Immunocytochemical data (26) reveal that the α2 epitope "colocalizes" with SERCA in astrocytes. As shown below (Fig. 6), functional α2 is located in the PM in WT astrocytes because the α2 can be blocked with low-dose (500 nM) ouabain, a hydrophilic, membrane-impermeant cardiotonic steroid. Thus these Na+ pumps must be located in PM microdomains that overlie sub-PM ER. Even with image deconvolution and reconstruction, the z-axis resolution is only ∼0.7 μm, vs. 0.25 μm in the x- and y-axes (25). Therefore, epitopes in the PM and in the underlying ER, <0.1 μm away, will appear to colocalize (26), even though they are in different membranes.
[Na+]cytin Astrocytes From Het and KO Mice
The normal bulk [Na+]cyt was 6.5 ± 0.1 mM in quiescent WT astrocytes. The level was not significantly higher (6.8 ± 0.4 mM; P > 0.05) in Het cells. [Na+]cyt was, however, modestly, but significantly, elevated (8.0 ± 0.3 mM; P< 0.001) in quiescent cells from KO mice (Fig. 6 B,left).
To test further the roles of the α1- and α2-isoforms in maintaining [Na+]cyt, the effects of 500 nM and 1 mM ouabain were compared. The lower dose should block only the α2-isoform (IC50 = 20–100 nM), whereas 1 mM ouabain should block both isoforms in rodents (α1 IC50 = 50–100 μM) (5,35). As revealed by the representative data in Fig.6 A (solid lines and inset), 500 nM ouabain increased [Na+]cyt only to 8.0 mM in WT astrocytes after a 10-min incubation (blue line). It had no effect on [Na+]cyt in the KO cells (red line) because there was no α2, and the [Na+]cyt was already at this level. Data from a number of such cells, and from Het cells, in which the findings were similar to those in WT cells, are summarized in Fig. 6 B,left (controls) and middle (+500 nM ouabain).
In contrast to the low-dose ouabain, 1 mM ouabain increased [Na+]cyt at comparable initial rates in all three cell types. Representative data for a WT cell and a KO cell are indicated by the dashed blue and red lines, respectively, in Fig.6 A. Data for a 10-min exposure to 1 mM ouabain are summarized in Fig. 6 B, right. Thus the α1-isoform is the primary determinant of bulk [Na+]cyt, and α2 apparently has only a minor influence on bulk [Na+]cyt.
Resting [Ca2+]cyt in Cells From Het and KO Mice
The distribution of [Ca2+] in quiescent cells from WT, Het, and KO cells is illustrated by the representative Ca2+ images in Fig. 5 A. These images indicate that, on the average, resting [Ca2+]cyt is slightly elevated in cells from Het mice and even more so in cells from KO mice. A frequency histogram of [Ca2+]cytvalues in WT and KO cells is presented in Fig. 5 B. There is considerable overlap between the [Ca2+]cytvalues in the two types of cells; nevertheless, it is clear that [Ca2+]cyt is skewed toward higher levels in the KO cells. The cell images in Fig. 5 A reveal that the lowest [Ca2+]cyt values are observed in some WT cells, but not in any of the KO cells. Conversely, some KO cells have higher resting [Ca2+]cyt levels than do any WT cells. Although not illustrated in the histogram (Fig.5 B), the data from Het cells are intermediate between those of WT and KO cells.
Resting [Ca2+]cyt data are summarized in Fig.7 B, left. The average resting [Ca2+]cyt is 132 ± 2 nM in the WT astrocytes (n = 670) and is elevated by ∼23% in Het cells and by ∼49% in KO cells. Particularly noteworthy is the fact that resting [Ca2+]cytis significantly elevated in the Het cells (P < 0.001; Fig. 7 B), despite a normal [Na+]cyt (Fig. 6).
Releasable Ca2+ and Ca2+ Signaling in Cells From Het and KO Mice
Storage of Ca2+ in the ER is governed by the ambient [Ca2+]cyt and by SERCA. The [Ca2+] gradient across the ER membrane is maintained by SERCA. Thus, if resting [Ca2+]cyt is elevated in cells from Het and KO mice, we would expect to observe increased storage of (releasable) Ca2+ in the ER. Inhibition of SERCA by agents such as thapsigargin and CPA promotes the unloading of ER Ca2+ in astrocytes and thereby induces large, transient elevation of [Ca2+]cyt (17, 18; and see Ref.38).
Figure 7 illustrates the effects of a maximal effective concentration of CPA (10 μM) on the elevation of [Ca2+]cyt in cells from WT, Het, and KO mice incubated in the presence of normal (1.8 mM) extracellular Ca2+. The representative time course data from individual cells in Fig. 7 A show that the peak of the [Ca2+]cyt transient is augmented, as is the amplitude of the plateau (until CPA is washed out) in cells from Het as well as KO mice. The initial peak Ca2+ transient corresponds to Ca2+ release from the ER. The plateau represents the balance between Ca2+ entry through store-operated Ca2+ channels (SOCs) and removal of free Ca2+ from the cytosol by PMCA and NCX and by mitochondria and perhaps a CPA-insensitive SERCA (but see Ref. 18). The SOCs are opened by ER Ca2+ store depletion (18,38). The plateau depends on extracellular Ca2+(Ref. 17, and see Fig.8 A) and on continued block of SERCA by CPA (Fig. 7 A). The summary data in Fig.7 B (right) reveal that the mean peak [Ca2+]cyt transient elevation is significantly greater in Het cells and KO cells than in WT cells.
Comparable data for cells incubated in Ca2+-free medium are illustrated by the initial responses (leftCa2+ transients) in Fig. 8 A. There is no plateau in this case, because the plateau (Fig. 7 A) is maintained by Ca2+ entry through SOCs [i.e., capacitative Ca2+ entry (CCE)] (38). Neverthleless, the peak CPA-induced [Ca2+]cyt transient is augmented in both Het and KO cells, although the mean increase in the peak did not reach statistical significance in the Het cells (Fig.8 B, middle). When external Ca2+is replaced, however, the Ca2+ transients that result from Ca2+ entry through SOCs are significantly greater in Het cells and KO cells than in WT cells (Fig. 8 A,second Ca2+ transients; Fig. 8 B,right). This augmentation can be explained if Ca2+ efflux via NCX is impaired and/or Ca2+entry via NCX is enhanced because of local, sub-PM Na+accumulation, as discussed below. In addition, there may be augmented Ca2+ entry through SOCs due to increased saturation of the ER Ca2+ stores in the Het and KO astrocytes (21).
Astrocytes cultured from the brains of rodents normally express both the α1- and α2-isoforms of the Na+ pump catalytic subunit (26, 46). The fact that α1 and α2 have very different kinetic properties, especially their different affinities for Na+and for ouabain (see Introduction), suggests that they have different functions. Here, using data from gene-targeted mice, we provide direct evidence for this view.
Reduced α2 Expression Does Not Affect Cell Morphology
Numerous reports indicate that Na+ pump inhibition by both low-dose and high-dose ouabain modulates early response genes and transcription factors and promotes mitogenesis (e.g., Refs.1, 15, 30, 36). Although issues of mitogenesis were not directly addressed in the present study, some relevant data were obtained. First, the 18- to 19-day-old Het and KO fetuses all appeared normal [not shown, but see James et al. (23), who also reported that Het mice develop normally into adults]. Second, the morphology of primary cultured cortical astrocytes from Het and KO fetuses was indistinguishable from that of WT astrocytes. Third, the rates of proliferation of Het and KO cells were not significantly different from those of WT cells (not shown). This is not surprising because the KO fetuses appeared to develop normally.
Reduction of α2 Expression Has Little Effect on "Bulk" [Na+]cyt
The SBFI data demonstrate that reduction of α2expression by ≈50% in the α2 Het astrocytes has a negligible effect on measured ("bulk") [Na+]cyt. In fact, complete KO of α2 expression increased [Na+]cyt by only ∼1.5 mM (from 6.5 to 8 mM). These small effects on bulk [Na+]cytmight be attributed simply to a small reduction in total α-subunit expression, because α2 accounts for only ∼20% of the total α-subunit and an even smaller fraction of the total Na+ pump flux in astrocytes (see below). Other evidence discussed below, however, indicates that the consequences of reduced α2 expression are isoform specific. Moreover, the changes in [Na+]cyt are clearly attributable to reduced Na+ extrusion by the Na+ pumps with α2-subunits, because the effects are mimicked by 500 nM ouabain, which does not block α1 in rodents. Indeed, it is noteworthy that the Na+ content in EDL muscles from both α1 Hets (mice with one α1 null mutation) and α2 Hets is indistinguishable from that of WT muscles (20).
Reduced α2 Expression Elevates Resting [Ca2+]cyt and Augments Ca2+ Transients and CCE in Astrocytes
Reduction of α2 expression by ≈50% in α2 Hets significantly increased resting [Ca2+]cyt and augmented Ca2+transients and CCE. While similar, but more pronounced, effects were observed in KO astrocytes, the data in Het cells are particularly noteworthy because bulk [Na+]cyt was not elevated significantly in the Het cells. These effects on [Ca2+]cyt are clearly attributable to inhibition of Na+ pumps with α2-subunits because low-dose (100–500 nM) ouabain has a comparable effect in astrocytes (9). Low-dose ouabain also augments Ca2+ transients without elevating bulk [Na+]cyt in arterial myocytes (2), which express the high ouabain affinity α3-isoform.
The hearts and skeletal muscle of α2 Het mice exhibit increased contractility (20, 23). In contrast, in mice with a null mutation in one α1 gene and one-half the normal α1 expression, cardiac and skeletal muscle contractility are reduced. Inhibition of α2 activity with low-dose ouabain in the α1 Het then enhances the contractility in both types of muscles, despite the further reduction of total α-subunit activity (20, 23).
In summary, the aforementioned observations all demonstrate that selective reduction of the activity of Na+ pumps with α2- or α3-subunits augments Ca2+ signaling in a variety of cell types. The implication is that a major role of these high-ouabain-affinity α-subunit isoforms is the modulation of Ca2+ homeostasis and Ca2+ signaling.
It is widely accepted that inhibition of Na+ pumps and reduction of the [Na+] gradient across the PM ([Na+]o > [Na+]cyt, where [Na+]o is extracellular [Na+]), e.g., by ouabain, promotes Ca2+ entry via NCX in most types of cells (10). Ouabain does not, however, promote Ca2+ entry or augment Ca2+ signaling when NCX expression is blocked by antisense oligonucleotides (42) or by a null mutation (39). Furthermore, the colocalization of NCX and Na+ pumps with α2- (or α3-) subunits in astrocytes and other cell types (25) is consistent with other evidence that the NCX and α2/α3 Na+ pumps are tightly coupled (reviewed in Refs. 2,10).
Reduction of α2 Activity Augments Ca2+ Signaling by Regulating [Na+] in a Sub-PM Cytosolic Compartment
How does reduced activity of the high-ouabain affinity Na+ pump α-subunit isoforms have such large effects on Ca2+ homeostasis and Ca2+ signaling, despite little or no effect on bulk [Na+]cyt? Consider that, at [Na+]cyt = 6.5 mM, α1 = 12 mM] operates at 14% of its maximal rate, whereas α2 = 22 mM] operates at only 2.5% of its maximal rate (48). Moreover, the turnover number (i.e., molecular cycling rate) for α2 is only 0.6 times that of α1(40). Thus the expected relative α2-to-α1 flux ratio is only ∼0.02:1, because only ≤20% of the expressed α in astrocytes is α2 (Fig. 3). That is, only ∼2% [or 5%, using the Na+ affinity (K Na) values of Segall et al. (40)] of the total Na+ pump flux in resting astrocytes would be mediated by α2! This seems unlikely.
To explain how reduction of Na+ pump α2/α3-subunit activity can modify Ca2+ homeostasis via NCX without detectable elevation of bulk [Na+]cyt (in α2 Hets), we must assume that these Na+ pumps regulate the local [Na+] and, via NCX, the [Ca2+] in a distinct subcompartment of cytosol. Diffusion of Na+ and Ca2+ between this compartment and bulk cytosol must be markedly restricted (2). Several investigators have provided evidence that cardiotonic steroids exert their cardiotonic effect by increasing [Na+] in a sub-PM cytosolic compartment that functionally couples the NCX to the Na+pump (14, 33, 43a). The presence of such a compartment also explains how the lowK Na(α2 ) andK Na(α3 ) can function while the "housekeeping" highK Na(α1 ) normally maintains bulk [Na+]cyt well below 10 mM. This compartment appears to be located between the PM and adjacent, junctional ER (jER) (2, 7, 8). The unit, consisting of the jER, the overlying PM microdomain, and the intervening tiny volume of cytosol (in the JS; see Fig. 9), has been named the "PLasmERosome" (8).
Immunocytochemical data (16, 25, 26), as well as preliminary coimmunoprecipitation data (29), indicate that these PM microdomains contain Na+ pumps with α2- or α3- (but not α1-) subunits, NCX, and transient receptor potential channel proteins, which may be components of SOCs (22) (Fig. 9 A). These proteins and those in the jER and perhaps other, as yet unidentified membrane proteins are all involved in Ca2+ signaling. In contrast, other regions of the PM are rich in PMCA and Na+pumps with α1-subunits (Fig. 9). Evidence that some SERCA and inositol trisphosphate receptor isoforms coimmunoprecipitate with NCX or transient receptor potential channel proteins (29) implies that the jER is structurally coupled to overlying PM microdomains.
This structural and functional coupling is illustrated in Fig. 9. Figure 9 B depicts the elevation of [Na+] and [Ca2+] in the JS and the consequent rise in ER [Ca2+] as a result of α2 KO (whether by a null mutation or by low-dose ouabain).
This model can be used to explain the augmented external Ca2+-dependent transient and sustained elevations of [Ca2+]cyt evoked by ER Ca2+ store depletion in Het and KO astrocytes (Figs. 7 A and 8). These external Ca2+-dependent signals are presumably mediated by Ca2+ entry through SOCs (18, 38). SOCs are permeable to Na+ as well as Ca2+(3). Thus, when the SOCs are opened in Het and KO cells, Na+ will tend to accumulate in the tiny JS between the PM and jER if Na+ extrusion through nearby α2Na+ pumps is reduced or abolished. As a consequence, Ca2+ extrusion should be reduced, and Ca2+entry increased, through adjacent NCX. The resultant local accumulation of Ca2+ could account for enhanced filling of jER Ca2+ stores, as well as spillover to bulk cytosol, and thus the rise in resting [Ca2+]cyt and the augmented Ca2+ signals.
PLasmERosomes obviously play a key role in regulating Ca2+signaling. Therefore, it seems appropriate to refer to them as "Ca2+ signaling complexes." Indeed, preliminary Ca2+ imaging studies indicate that SOC-mediated Ca2+ signals are apparently initiated in these Ca2+ signaling complexes (16). Clearly, a major task for future studies is to test this hypothesis directly.
Summary and Conclusion
One of the key results of this study is that knockout of α2 has a very small effect on bulk [Na+]cyt (a rise of <2 mM). Thus Na+ pumps with α2-subunits play only a minor role in maintaining the low resting [Na+]cytin astrocytes. In contrast, even a 50% reduction in α2expression, which does not affect bulk [Na+]cyt, is associated with significant elevation of resting [Ca2+]cyt and augmentation of Ca2+ signaling. The implication is that a major role of Na+ pumps with α2 (and α3; Ref. 2) subunits is the modulation of Ca2+ signals.
We thank Drs. Thomas Pressley, Kathleen Sweadner, and Kenneth Philipson for generous supplies of antibodies, and Hugo Gonzales-Serratos for help with the dissection of EDL muscles.
This study was supported by National Institutes of Health grants NS-16106 and HL-45215 (to M. P. Blaustein), HL-41496 (to J. B. Lingrel), a Grant-in-Aid from the American Heart Association Mid-Atlantic Affiliate (to V. A. Golovina), and a Grant to Promote Research from Miami University (to P. F. James).
Address for reprint requests and other correspondence: V. A. Golovina, Dept. of Physiology, Univ. of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201 (E-mail:vgolovin{at}umaryland.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 3, 2002;10.1152/ajpcell.00383.2002
- Copyright © 2003 the American Physiological Society


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Vision and Mission of launching Challenger in Space
The Vision of launching Challenger in space was to make an ordinary Citizen of America to go to Space for the first time in World. Christa McAuliffe was selected for taking her up to the Space. She was a Beloved and Favourite Teacher of her Students in a school. It was planned that she will show her students some Experiments in Zero Gravity live on TV. There were 5 Astronauts and 2 Payload Specialists chosen to float in the Space.
Pre-Launch Conditions
Challenger was originally set to launch from KSC in Florida at 14:42 Eastern Standard Time (EST) on January 22. Delays in the previous mission, STS-61-C, caused the launch date to be moved to January 23 and then to January 24. The launch was then rescheduled to January 25 due to bad weather at the Transoceanic Abort Landing (TAL) site in Dakar, Senegal. NASA decided to use Casablanca as the TAL site, but because it was not equipped for night landings, the launch had to be moved to the morning (Florida time). Predictions of unacceptable weather at KSC on January 26, caused the launch to be rescheduled for 09:37 EST on January 27.
The launch was delayed the next day, due to problems with the exterior access hatch. First, one of the micro-switch indicators, used to verify that the hatch was safely locked, malfunctioned. Then, a stripped bolt prevented the closeout crew from removing a closing fixture from the orbiter's hatch. By the time repair personnel had sawed the fixture off, crosswinds at the Shuttle Landing Facility exceeded the limits for a Return to Launch Site (RTLS) abort. While the crew waited for winds to die down, the launch window expired, forcing yet another scrub.
A Conference call took place between Thiokol and NASA
Forecasts for January 28 predicted an unusually cold morning, with temperatures close to −1 °C (30 °F), the minimum temperature permitted for launch. The Shuttle was never certified to operate in temperatures that low. The O-rings, as well as many other critical components, had no test data to support any expectation of a successful launch in such conditions.
At the Fixed Date of Launch…
The Challenger Space Shuttle launched on 28th January 1986. All the spectators were eagerly keeping an eye on the Shuttle. The students of Christa McAuliffe were jumping with joy at the time of launch and its going up movement in sky. Approximately 17 percent of Americans witnessed the launch live because of the presence of Payload Specialist Christa McAuliffe, who would have been the first teacher in space.
And the Disaster Started…
The Space Shuttle Challenger disaster occurred when the NASA Space Shuttle orbiter Challenger broke apart 73 Seconds into its flight, leading to the deaths of its Seven Crew members, which included Five NASA Astronauts and Two Payload Specialists. The spacecraft disintegrated over the Atlantic Ocean, off the coast of Cape Canaveral, Florida, at 11:39 EST (16:39 UTC). Disintegration of the vehicle began after an O-ring seal in its right solid rocket booster (SRB) failed at liftoff.
After the Burning Explosion…
There was No Escape System in the Challenger for the Crews. This Led to the death of 7 Crew Members. The whole America came under a huge hole of Grieve. All the Smiles at the time of launching turned into Sadness. All the spectators just kept watching at the the blast attentionally.
This was an Overview of Challenger Disaster just for the Remembrance of 7 Great Crews who lost their lives for entire nation.
Do not forget to comment below.

subset: commoncrawl


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There are some major problems with the group "Anonymous" doing this.
1) they are Anonymous and thus have no accountability.
2) What if they are wrong?
3) whats to keep them from placing the IPs of political enemies in the list?
If they can do it, so can law enforcement. Whats the great revelation here?
Conclusion: Can anyone trust this group, "Anonymous." With no accountability, I'm afraid to treat this list seriously.
(Well, I kinda doubt that Cyclops08 is your real name in life...I guest we can't "trust" what you say, here...or anywhere else, eh?)
What, indeed?
A few years go, Sky TV in the UK got a name right, but the wrong,
actual person, for some pedophile that was released from prison,
in a broadcast.
A mob burned the wrong guy, and his family, out of a house and home.
They probably are wrong on so many levels. No evidence that they got end IP addresses for people using the Tor network and not just the last relay. Even if that hurdle were jumped, IP addresses are not unique digital identifiers for individuals and change frequently so you almost certainly have a number of misidentified IP addresses here. There's a reason why we have trained law enforcement officials investigate crimes and not script-kiddies.


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Mangaluru: Additional Director General of Police (Administration) Alok Mohan held a press meet at the Police Commissioner's office here, on November 3.
Addressing the mediapersons, Alok Mohan said that 5 people have been involved in the murder of Madoor Isubu in the district sub-jail premises on November 2.
When asked how the inmates gained access to lethal weapons inside the jail, Alok Mohan said that the investigations in this regard are going on. "The weapons came from outside and we are investigating the case. So far, we have not recovered any weapons. There is a huge tree adjacent to the boundary of the jail. There is a possibility of dropping weapons at that spot through the compound wall," he added.
When asked on the number of injuries on Isubu's body, Alok Mohan said that there were 40 stab wounds on Isubu's body while no stab marks were found on Ganesh Shetty'sbody. Ganesh may have died due to the use of a blunt weapon or due to head injuries. Both Ganesh Shetty and Madoor Isubu were in different cells.
When enquired about the direct allegation by an undertrail about a jail officer bringing weapons inside the jail, Alok replied that the an investigation is going on. "Those who were involved in the crime, directly or indirectly, will be punished. If the jail officers are involved in the murder case, they will be severely punished. The truth will come out. Anyone can put allegations, but until and unless the truth is out, we cannot blame anyone. So far, we have not found any weapons. We are trying to find out the weapons using metal detectors."
"So far, we have booked 5 people who have executed the whole crime and we are looking for the involvement of others, both inside and outside the jail. When Madoor Isubu was walking in the morning, he was attacked by the undertrails. The jail is a safe place and such incidents should not have happened. This attack was pre-planned; the investigation is underway to find out the involvement of outsiders. In the old cell, there are no CCTV camera's but yes in the new block, we have installed CCTV cameras."
Alok Mohan also said that the AGDP of Prison Kamal Panth is investigating the case. "We have formed a high level committee to investigate the case and the police commissioner himself is monitoring the case."
When asked about the number of attackers, Alok said that the prison department and police department are two different entities. "Are you safe in Mangaluru or not?" he questioned to the reporters. The reporters replied and said, "We are not safe". He assured to have tight security in Mangaluru. When asked of the fate of the tree over the prison wall, Alok said, "You see it next week."
IGP Amrit Paul, Police commissioner S Murugan, SP Dr Sharanappa, DCP of Crime Dr Sanjeev Patil and DCP of Law & Order Shantharaj were also present.


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MOVIE SYNOPSIS [ General Audiences ] With a good performance of Rease Etzler (see cast), this film directed by Jeffrey Moscone was premiered in cinema in 2012. The movie "Final Tears" comes from USA. You can watch Final Tears online on TV cable or theaters with original audio in English at different dates. This film premiered in theaters in 2012.
Rating: 5 by 5 users./
Runtime: 54 minutes
Rease Etzler, Libertad Green, Jessie Holland, Gregory Logan, Jeffrey Moscone and Esmeralda Paz.
54-minute film (full movie) directed by Jeffrey Moscone.
The original audio of Final Tears has dialogues in Dolby Digital, it was produced in USA.
It premiered in cinemas or television in 2012.
Screenplay written by Jeffrey Moscone.
Jeffrey Moscone.
This film was produced and / or financed by Fetching Cat Productions.
Without commercial breaks, the full movie Final Tears has a duration of 54 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in EnglishDolby Digital in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, Final Tears in FULLTV Movies.


text:
Nick Markakis remains in the second spot in the lineup, with Adam Jones batting third. He's looking for his first extra-base hit since May 15, a span of 84 at-bats. His average is down to .236 with four homers, 19 RBIs, a .298 on-base percentage and a .304 slugging percentage. He's totaled at least 40 doubles in each of the last four seasons, but he's stuck on four this year.
I'm watching Markakis take batting practice now and he's launching a few balls into the right fields seats and onto the flag court. Another ball slammed off the Esskay sign on the out-of-town scoreboard.
Manager Buck Showalter addressed the speculation that Markakis is dealing with an undisclosed health issue, which the right fielder has denied.
"I'm real confident when the smoke clears, where Nick is going to be, the body of work," Showalter said.
"It's easy to say 'yes, he's right,' but if there was something wrong with Nicky, he wouldn't tell anybody. We had one night here where he got two hits and had a big game, and found out afterward he had a 100 degree temperature.
"I think he's fine. I know he does a lot of extra work. He's doing all the things he can do, in some cases maybe too much, but you're never going to take the want-to out of that. He's got too much of it going on.
"He's a guy you trust, you trust with a lot of things, and he'll figure it out, along with some help. I know we've had a lot of time and effort spent addressing it."


text:
Job Details
Group Chief Electrical Engineer
An international consultancy and construction company UK-International
12 - 15 years 45 -60 Lacs Per Annum 29th December 2016
Location - London, UK.................................. Candidate will be responsible for Monitor, review the Electrical Assessors appraisal of Risk Assessments and Method Statement (RAMS), recording observations in yellow jacket accordingly.
- Report directly to the Director of Technical Services
- Appointing Business Division and/or Business Unit Electrical Engineers and Electrical Assessors.
- Responsible for Business Division and/or unit Electrical Engineers and Electrical Assessors.
- Investigating any Electrical incidents or near misses.
- Ensure all mandatory training for Electrical Core Standards is in place.
- Manage Electrical Training budget and seek approval from Director of Technical services.
- Approval Selection and competence review of the Electrical Engineers Assessors (LV & HV) before formally appointment
- Identify and advise on any change in policy & procedure, through learning, legislative requirement or best practice
- The authority to suspend works should deficiencies or lack of control be established
- Support and provide adequate resource for electrically related near miss / accident / incident investigations
- Technically competent and qualified, with 10 years post qualification practical experience
- Have previously held an appointment as a HV Appointed Persons
- Builds collaborative working relationships
- Communicates effectively with all stakeholders
- Looks for ways to improve current procedures
- Effective planning and organisation skills
- Strong willed – has the ability to make tough decisions for the good of the group.
- A High Voltage Authorised Persons course – to be refreshed at periods no greater than 5 years
- Current IEE Edition Wiring Regulations qualifications
- City & Guilds 2391 Electrical Test and Inspection
- City & Guilds 2360 Electrical Installations part C, or ONC, or City & Guilds 283 Electrical Technicians courses
- Functional Area: Electrical
- Industry: Construction
- Job type: Full-Time
- highest Qualification: BE
- Designation: Group Chief Electrical Engineer
- Skills:
- Company Name:Touching Heights Business Solutions LLP
- Company Description:
Providing Man Power Resources for PAN India Location, Giving the HR Practical Training courses, Giving the Man Power Resources for IT as well as non IT, giving the candidate negotiation offers training formality


text:
USGS: Outbreak of coral disease is an epidemic
USGS: Outbreak of coral disease is an epidemic
HONOLULU (AP) — A federal agency says in a report that an outbreak of coral disease along Kauai's North Shore is an epidemic.
Dr. Thierry Work, head of Infectious Disease for the U.S. Geological Survey, told The Garden Island (http://is.gd/FueFgN) that this is the first time white coral disease on this scale has been documented in Hawaiian corals.
Work says in late-September he and other scientists got a look at the unusual outbreak along Kauai's North Shore. They photographed diseased coral and took samples, while also applying a marine epoxy to try and stop the disease's progression.
Work says the disease is rapidly spreading. A report released Wednesday says microscopic changes in tissue suggest the corals are under some type of stress.
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text:
Chelsea have restored their 10 point lead at the top of the Premier League table with a win over London rivals West Ham on Monday night.
A much-changed Blues XI which saw Cesc Fabregas start ahead of Nemanja Matic were knocked out of the EFL Cup at the same ground in October.
Chelsea grabbed the opener when N'Golo Kante broke up an attacking move on the left, fed Hazard who played a one-two during a swift counter-attack and dribbled past Randolph in goal.
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Chelsea's second came when Diego Costa kneed in a flick from a Cesc Fabergas corner kick.
The goal was Costa's 18th goal of the season and his 17th in the Premier League.
Manuel Lanzini grabbed a late consolation for the Hammers, who remain just outside the top-10, as he rifled home his sixth league goal of the season.
The win see's Chelsea led the log by 10 points with 27 games of the season already played. the Blues face Manchester United next week monday for an FA Cup tie.
Source: Sports.naija.ng


text:
Head or Heart
- Artist: Christina Perri
- Format: CD
- Release Date: 4/1/2014
|your cart is empty
2014 album from the singer/songwriter. In matters of love, should you rely on your heart or your head? This is the question Christina Perri posed as she began writing her second album, a collection of cathartic songs that seek to reconcile how we make decisions in love and life. HEAD OR HEART - was recorded over the past year, with further production from Jake Gosling, John Hill and Butch Walker. The album was mixed by Grammy Award winning engineer Manny Marroquin (Kanye West, John Legend, Alicia Keys).
To play the media you will need to either update your browser to a recent version or update your version of Flash Player.


text:
NCG TESTS: Garmin Approach S1 watchFebruary 28, 2012 Golf Equipment
NCG's team of experts review all the latest kit independently. Find out what they thought of the Garmin Approach S1 watch...
A fantastic, innovative device that is genuinely hard to fault. It is essentially a watch that doubles up as a GPS device, giving you numbers to the front, middle and back of the green. It is small and light so doesn't hinder the swing, and both affordable and stylish. If numbers are all you need (it's ideal for playing on courses you know) there are few devices better. If you want more information, perhaps Garmin's new Approach G6 is a better option.
TESTED: January 2012
TESTERS: Joe Whitley (7hcp), Dan Murphy (3hcp), Mark Townsend (7hcp)
GPS wristwatch designed for golf; gives yardages to front, middle and back; measures shot distances; wateproof; doubles up as standard watch
COURSES PRE-LOADED: 7,100 in Europe
SUBSCRIPTION FEES: None
w: garmin.co.uk
t: 02380 524 000


text:
The Voice winner 'made scapegoat'
Leanne Mitchell, the winner of last year's The Voice whose debut album was a huge flop, has complained that she has been made a "scapegoat".
The singer spoke out just days after the climax of this year's series and following criticism she has endured since managing to get to only 134 in the album chart with her first release.
She complained that no one was "on my side from the start" and countered suggestions she did not want to work to make the album a success. Leanne said she had no hand in the timing of the record - which came out almost a year after her win but had been completed many months earlier - and said she had trusted people around her to make the right decisions.
Will.i.am - one of the coaches from The Voice - has already claimed that the record label bore some of the blame. He said in an interview recently: "The record company should be freakin' embarrassed. Somebody should get slapped because you waited too long. It's hopeless to release a record so long after the series finished.
Leanne hit back this week in a message which she posted online after keeping silent in the days following her chart flop last month. She said: "Ever get the feeling you've been used as the scapegoat?
"For the record, I'd written and finished the majority of my album by the end of last summer, around September time. I don't know why it was decided to wait so long to release it, but unfortunately, being very new to all this and probably too naive, I had to trust the people around me in making the right decisions.
"I have absolutely worked my backside off to WRITE this album, not copping out on all covers. And as some people have speculated, I absolutely wanted and still want to work for it."
Leanne said she had worked hard since she was 13 "doing what I love, and felt that maybe it would have all been worth it for this past year". She went on: "I wonder if everyone that has said/written negative things about me have even listened to the album? Again, unfortunately, I don't think anyone was particularly on my side from the start."
The singer said she did not want to appear as though she was "bitter", and she actually feels "very lucky" to have had the experiences of the past year. "If people actually gave me a chance, and didn't judge me on what has been said or written, maybe they'd see some people are just in it for the music," she added.
This year's winner Andrea Begley looks on course to make the top 20 of the singles chart this weekend. Midweek positions released by the Official Charts Company show she is at number 19 with her cover of Evanescence's My Immortal. Show runner-up Leah McFall could see three songs in the top 40 at the weekend. Her version of I Will Always Love You is at number 22 in the midweeks, while I Will Survive is at 24 and Killing Me Softly is 34.


text:
2012 Honda Accord - Consumer Review
Build Quality
Reliability Value
Vehicle2012 Honda Accord LX 4dr Sedan (2.4L 4cyl 5A)
ReviewI own a 2012 Honda Accord LX. Drivers side seat just slightly above the lumbar support is hurting my back. Adjusting the seat or adding some padding does not help. Extreme discomfort after 25 minutes of driving time. Very poor seat design. Very disappointed in Honda.
Do a "Google" search to read about many others having back pain due to the seat design.
I like the 2012 Honda Accord's but what good is this car if I cannot drive it without being in severe pain.
Are you the author of this review?Update Your Review
on 04/01/14 20:43 PM (PDT)
Totally agree with you. I've paid particular attention the 8th generation 2008 to 2012 Accord sedan driver's seats, and there is just too much lumbar curve built in, even with a power adjust flattened out. It punches into my back and is REALLY annoying just on a test sit in the car. Thankfully, I never bought one. To compare, have a sit in a 2013 or later Accord sedan. Luckily, they did a great job this time around. The whole seat seems really good, the lumbar curve is mild and the 10 way power seats have adjustable lumbar if you need more. Sorry about your car. You're not imagining it. I hope you can gety it sorted out (get into a more comfortable car!)Report it
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Your shopping cart is empty!
Delicious mouth-watering 1 Kg Pine Apple Cake from 5 Star Bakery deliver through online via floristchennai.com. This product requires a minimum of 24 hours for delivery. Mouthwatering cakes are available at floristchennai.com. We deliver on time, also we have same day delivery and midnight delivery. Yummy, Pine Apple cake will more fresh with a quality of ingredients used in it. So it will taste good. Buy Cake in Online cake shop which experts will bring you good taste and flavor.


text:
Azzaro - Visit
Thread: SoTD, Wednesday July 20
Caron Pour Homme ****/? (New decant)
Continuing my L'Artisan sampling with Tea For Two.
Veiver & Rum by Helan.
A la Nuit
"Wovon man nicht lesen kann, darüber muss man schreiben."
Culti Acqua Profumo
D'Orsay Le Dandy with top notes of tobacco and Whisky: the middle is spicy with ginger and the bottom note is woody and balsamic.
Overcome by Fumes
CDG Series 1: TEA
:::::: Visit My Blog: La gardenia nell'occhiello ::::::
Continuing the French 'classic' theme, after M. Balmain and Chanel PM today its Moustache de Rochas edt Concentree
Royal Deligh - Creed. Have a good day!
2/3rds Eau Parfumée au Thé Verte Extreme + 1/3 Yang
Patou 1000.
Have a great day!
'I am not difficult, I am definite!' - Hedy Lamarr
.....Essenza di Zegna
My addiction: http://www.basenotes.net/wardrobe/5355
L'Instant De Guerlain Pour Homme.
With the stick deodorant and my new shower gel.
Taylor's Shaving Shop
Le Roy Soleil
Aromatic, woody, floral
http://blogsorbeta.blox.pl: CdG Series 8 Energy
(Oct. 3rd).
Givenchy Gentleman! Superb classic fragrance! [smiley=cool.gif]
Trying a blend of Creed - Silver Mountain Water and Spring Flower.........smells pretty good so far
L'Eau Kenzo pour Homme
GIT - One of the most versatile class fragrances on the market
Monsieur Galimard
In rotation: Greenbriar (new), Silver Mountain Water, Dunhill for Men (1934), Acqua di Parma Colonia, Habit Rouge EDC, Ho Hang, B*Men, Agua Brava
Hermes(Hermessence) - Vetiver Tonka
"I hold it that a little rebellion now and then is a good thing, and as necessary in the political world as storms in the physical...It is a medicine necessary for the sound health of government." - Thomas Jefferson
Sung Homme.
Just because it happened to you doesn't make it interesting.
In theory, there is no difference between theory and practice. In practice, there is.
My sales thread: http://www.basenotes.net/threads/304...85#post2614885
Wanted: YSL Nu EdP
Body Kouros
Ho Hang Club by Balencegia
Lanvin Pour Homme
Paco Rabanne I had to take a break from sweet.


text:
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text: Hi there I have had a regular client of mine drop their whole branding in my lap! Its not something I normally do. I have been reading up about font licensing.. Can you tell me what the general practice is for using a font for a client's brand? I realise that in a logo, text is outlined so the font is not passed on, but this company produces publications. They are based in the humanitarian sector. From what I understand I should buy the font on their behalf in their name so that they own the license, or get them to buy the license. They could then pass it to designers as needed when they work on publications in the short term, which I think is deemed 'sort of ok'. My client is clueless as to whether they own licenses for fonts they use and its tricky to ask without opening a can of worms, so I can't tell whats been done before! The desktop fonts are Frutiger and Times. I am desperate to change the Times, and the client regards it as 'old fashioned' but it will mean losing a few days of paid time. I would change it to another font that isn't so common, so designers would have to ask for it. But they pass native files around commonly anyway. Is what I have outlined above the 'right' thing please? ie buying the license in their name. Is this what brand designers commonly do? Or am I overdoing the issue? Any other suggestions around this issue is welcome. Rather in it over my head! Many thanks for any help or advice.


text:
The staff is nice, and well you can get a good hair cut, waxing, nails done, for a considerable price and time.
All reviews seem positive
From family friendly trips with kid friendly activities to local plumbers lawyers, spas, and contractors. Judy's Book has millions of listings and reviews for the best and not so great. Whether you're looking for a specific business or just trying to discover great places we make it easy. We're a family oriented site focusing on bringing trusted reviews and recommendations to moms and others. Green and Sustainable places are becoming more relevant to Judy's Book users, have a favorite place? Write a review and tell users why it's Green!


text:
Spring Valley Motors
8 reviews
735 Mills St, Kalamazoo, MI 49001, us
(269) 381-7077
Website URL:
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About this shop
- Hours
- Specialties
- Loaners
- No
- Notes
Score card
|They are always honest
|They are competent
|Their repair price is reasonable
|They complete the work in a timely manner
|They respond well when they screw something up
|They take the time to explain the problems and necessary repairs
|They treat male and female customers in the same manner
|They are always honest
|I would use this mechanic again
|I would recommend this mechanic to others
|They fix the problem the first time
|The shop is located in a safe neighborhood
|The hours of operation are convenient for customers
|They are near public transportation (or provide loaners, shuttle bus, rides as needed)
Nov 07, 2011
Apr 22, 2009
Need to invest in a diagnostics reader. Would save customers from needing to return.
Nov 12, 2004
OK, the lot the shop is on is kinda scary, a ramshackle gate and a gravel driveway past what looks like a junkyard of all things VW. But make it in to the shop with your VW, and you'll never go anywhere else. TOTALLY cool and trustworthy. I just moved out of the area and am pining for such a great place to trust my car to. I miss you, Kirk!
Oct 03, 2006
Don't be put off by the yard filled will Volkswagens in various stages of decay. All the cars strewn about are a gold mine for anyone who owns an old VW. Family owned/operated, and very helpful. If you own a VW, go no further. This is what I imagine a mechanic's shop being like in the 1940s (a good thing).
Nov 17, 2009
They have a shop rooster for cryin' out loud.
Dec 27, 2009
The boys at Spring Valley Motors were my mechanics for 10 years or so before I moved from Michigan to Oregon five years ago. I was in Michigan last summer helping my mom move into assisted living-a really difficult time. Her GMC Jimmy needed a lot of work; after spending way too much money on badly done brake job by someone else, I took it to Spring Valley, and it was overhauled within my spending/time range in a week. Not only are they always extremely competent, they are willing to explain, advise, and give customers rides. After several years of being an inner city biker/pedestrian/bus rider, I recently got another vehicle. Can only hope to find a shop of near the quality of Spring Valley here in Portland.
Dec 01, 2010
Everyone at Spring Valley has been just great. I've gone to them for the last 10 years, first with my VW and then with my Ford. They do great work, they are very fair with their prices and they don't try to fix things that don't need to be fixed. I will continue to go there each and every time I need work done, because I know my car will get fixed the right way. Kirk is a great guy, and he hires good people to work with, too. I would recommend Spring Valley Motors to anyone who needed work done on whatever car they have.
Jan 03, 2014
- Shellia
These guys were honest and explained the issue before they fix the car. We own older cars and it is great to find someone who is honest and doesn't try to rip me off because I am a woman!!!!
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text:
Garage door replacement hinge number one, used on overhead sectional doors. This hinge is installed down the center of the garge door to connect all panels together, also on the outer edge of the bottom panel and the second panel. Packaging includes carriage bolts, nuts, and complete instructions.
- Garage door hinge #1 2.75 in. W x 7.5 in. L
- Galvanized # 14-Gauge steel
- Accepts standard 7/16 in roller shaft
- 1 hinge per package, includes nuts and bolts
- For overhead sectional doors
- Installed down center of door and on lower panel
- Quick and easy to replace; original equipment quality


text:
1. Leung YPY, Kaplan GG, Coward S, Tanyingoh D, Kaplan BJ, Johnston DW, Barkema HW, Ghosh S, Panaccione R, and Seow CH on behalf of the Alberta IBD Consortium and the APrON Study TeamIntrapartum corticosteroid use significantly increases the risk of gestational diabetes in women with inflammatory bowel disease. Journal of Crohn's and Colitis, epub ahead of print. Jan (2015)Download abstract
2. Jia X, Pakseresht M, Wildgrube J, Sontag S, Andrews M, Wattar N, Begum Subhan F, McCargar L, Field CJ and the APrON study team. Women who take omega-3 long chain polyunsaturated fatty acid supplements during pregnancy and lactation meet the recommended intake. Applied Physiology Nutrition and Metabolism (accepted Jan 2015)+ View summary
3. Rash, J., Campbell, T., Letourneau, N., Giesbrecht, G.F.Maternal cortisol during pregnancy is related to infant cardiac vagal control. Psychoneuroendocrinology,54 ( April, 2015), 78–89.Download abstract
1. Kaplan BJ, Geisbrecht GF, Leung BMY, Field CJ, Dewey D, Bell RC, Manca DP, O'Beirne M, Johnston DW, Pop VJ, Singhal N, Gagnon L, Bernier FP, Eliasziw M, McCargar LJ, Kooistra L, Farmer A, Cantell M, Goonewardene L, Casey LM, Letourneau N, & Martin JW.The Alberta Pregnancy Outcomes and Nutrition (APrON) cohort study: Rationale and Methods. Maternal and Child Nutrition. Maternal and Child Nutrition. 2014+ View summary
2. Bridgman SL, Azad MB, Field CJ, Letourneau N, Johnston DW, Kaplan BJ, Kozyrskyj AL (in press)Maternal perspectives on the use of probiotics in infants: a cross sectional survey. BMC Complementary and Alternative Medicine.+ View summary
3. Faiqa Fayyaz, Flora Wang, Rene L. Jacobs, Deborah L. O'Connor, Rhonda C. Bell, Catherine J. Field and the APrON Study TeamFolate, vitamin B12, and vitamin B6 status of a group of high socioeconomic status women in the Alberta Pregnancy Outcomes and Nutrition (APrON) cohort+ View summary
4. Erin D. Lewis, Fatheema B. Subhan, Rhonda C. Bell, Linda J. McCargar, Jonathan M. Curtis, Rene´ L. Jacobs, Catherine J. Field and the APrON teamEstimation of choline intake from 24 h dietary intake recalls and contribution of egg and milk consumption to intake among pregnant and lactating women in Alberta+ View summary
5. Giesbrecht, G.F., & Dewey D.The effects of 'does not apply' on measurement of temperament with the Infant Behavior Questionnaire-Revised: A cautionary tale for very young infants. Early Human Development, 90(10), 627-634. 2014+ View summary
1. Giesbrecht, G.F., Poole, J., Letourneau, N., Campbell, T., Kaplan, B.J. and the APrON Study Team. Psychosom Med, November 2013.The buffering effect of social support on hypothalamic-pituitary-adrenal axis function during pregnancy. Psychosomatic Medicine+ View summary
2. Brenda MY Leung, Sheila W McDonald, Bonnie J Kaplan, Gerald F Giesbrecht, Suzanne C ToughComparison of sample characteristics in two pregnancy cohorts: community-based versus population-based recruitment methods. BMC Medical Research Methodology. December 2013, 13:149+ View summary
3. Gómez MF, Field CJ, Olstad DL, Loehr S, Ramage S, McCargar LJ & the APrON Study TeamUse of micronutrient supplements among pregnant women in Alberta: results from the Alberta Pregnancy Outcomes and Nutrition (APrON) cohort. Maternal and Child Nutrition 2013, doi: 10.1111/mcn.12038+ View summary
4. Brenda MY Leung, Bonnie J Kaplan, Catherine J Field, Suzanne Tough, Misha Eliasziw, Mariel Fajer Gomez, Linda J McCargar, Lisa Gagnon & the APrON Study TeamPrenatal Micronutrient Supplementation and Postpartum Depressive Symptoms in a Pregnancy Cohort. BMC Pregnancy and Childbirth 2013, 13:2 doi:10.1186/1471-2393-13-2+ View summary
5. Donna P Manca, Maeve O'Beirne, Teresa Lightbody, David W Johnston, Dayna-Lynn Dymianiw, Katarzyna Nastalska, Lubna Anis, Sarah Loehr, Anne Gilbert, Bonnie J Kaplan and the APrON study team The most effective strategy for recruiting a pregnancy cohort: a tale of two cities. BMC Pregnancy and Childbirth 2013, 13:75+ View summary
6. Gerald F. Giesbrecht, Tavis Campbell, Nicole Letourneau, Bonnie J. Kaplan, the APrON Study TeamAdvancing gestation does not attenuate biobehavioural coherence between psychological distress and cortisol. Biological Psychology. Volume 93, Issue 1, April 2013, Pages 45–51+ View summary
7. Mahsa Jessri, Anna P Farmer, Katerina Maximova, Noreen D Willows, Rhonda C Bell and APrON Study Team. BMC Pediatrics 2013, 13:77Predictors of exclusive breastfeeding: observations from the Alberta pregnancy outcomes and nutrition (APrON) study+ View summary
8. Kaplan BJ, Leung BMY, Giesbrecht GF, Field CJ, Bernier FP, Tough S, Cui X, Dewey D, & the APrON Study Team. Appl Physiol Nutr Metab, January 2013, 38(1):85-89.Increasing the quality of life from womb to grave: The importance of pregnancy and birth cohorts. Applied Physiology, Nutrition and Metabolism.+ View summary
1. Giesbrecht GF, Campbell T, Letourneau N, Kooistra L, Kaplan BJ, & the APrON Study Team.Psychological distress and salivary cortisol covary within persons during pregnancy. Psychoneuroendocrinology, 37:270-279.+ View summary
2. Gerald F. Giesbrecht & Nicole Letourneau & Tavis Campbell & Bonnie J. Kaplan & The APrON Study TeamAffective experience in ecologically relevant contexts is dynamic and not progressively attenuated during pregnancy. Archives of Women's Mental Health. December 2012, Volume 15, Issue 6, pp 481-485 + View summary
3. Begum F, Colman I, McCargar L, & Bell R.Gestational weight gain and early postpartum weight retention in a prospective cohort of Albertan women. Journal of Obstetrics and Gynaecology Canada.J Obstet Gynaecol Can. 2012 Jul;34(7):637-47.+ View summary
4. Gerald F. Giesbrecht, Douglas A. Granger, Tavis Campbell, Bonnie Kaplan, the APrON Study TeamSalivary Alpha-Amylase During Pregnancy: Diurnal Course and Associations With Obstetric History, Maternal Demographics, and Mood. Developmental Psychobiology. Volume 55, Issue 2, pages 156–167.+ View summary
1. Leung B, Kaplan BJ, Dewey D, Field CJ, Farmer A, O'Beirne M, & Johnston DW.Integrating KT within a research study: The APrON Experience. Interdisciplinary Teams – Making Research Make a Difference, KT Casebook, Alberta Innovates – Health Solutions, Vol 2, pp 5-12.+ View summary
2. Leung BM, Wiens KP, & Kaplan BJ.Does prenatal micronutrient supplementation improve children's mental development? A systematic review. BMC Pregnancy and Childbirth, 11:12.+ View summary
1. Leung BMY, Kaplan BJPerinatal depression: prevalence, risks, and the nutrition link-a review of the literature. Journal of the American Dietetic Association. 2009;109:1566-75.+ View summary


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San Francisco, CA (Hayward) Fire Protection Services
Established in 2006, our Hayward and Sacramento branch locations were opened to service our customers in San Francisco and Northern California. We specialize in fire protection service, inspections of all sprinkler and suppression systems and installs of fire sprinkler and suppression systems.
We strive to provide great customer service and offer a 24 hour response team.
Western States Fire Protection Co.
2489 Technology Dr.
Hayward, CA 94545
Phone: 510-783-2400
Fax: 510-783-2408
Click for a map of all office locations


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Mitt Romney wins popular vote in all five Republican contests
Andrew Moran
Apr 24, 2012 in
New York
- Will former Massachusetts Governor Mitt Romney inch closer to the nomination after Tuesday's nights five GOP contests? Can Texas Congressman Ron Paul amass delegates and make an impact? What about Newt Gingrich? Here is the complete live coverage.
Romney has been declared the winner in all five states: Connecticut, Delaware, New York, Pennsylvania and Rhode Island.
"After 43 primaries and caucuses, many long days and more than a few long nights, I can say with confidence – and gratitude – that you have given me a great honor and solemn responsibility. And, together, we will win on November 6th!" said Romney during a campaign speech in Manchester, New Hampshire, who focused his attention to the general election against President Barack Obama.
"Four years ago Barack Obama dazzled us in front of Greek columns with sweeping promises of hope and change. But after we came down to earth, after the celebration and parades, what do we have to show for three and a half years of President Obama? Is it easier to make ends meet? Is it easier to sell your home or buy a new one? Have you saved what you needed for retirement? Are you making more in your job? Do you have a better chance to get a better job? Do you pay less at the pump?"
A total of 231 delegates are up for grabs Tuesday. Connecticut will have 25 out of its 28 delegates at stake, while the remaining three are labelled as superdelegates with two being two Republican Committee representatives and one being the state party chairman.
Meanwhile, Delaware, a winner-take-all state, has 17 delegates on the line. New York will have 95 delegates being fought for and is a winner-take-all state, unless there is no clear winner, in which the delegates are then allocated proportionally.
Pennsylvania is a loophole primary and has 72 delegates at stake. 16 of Rhode Island's 19 delegates will be allocated proportionally and three delegates have already pledged to Romney.
This is a breaking news story so please check back throughout the night for continuous updates.
Below are the results for the remaining candidates the state of Connecticut (60 percent reporting):
Newt Gingrich: 10 percent | 5,253 votes
Ron Paul: 14 percent | 6,952 votes
Mitt Romney: 67 percent | 33,996 votes (declared winner)
Below are the results for the remaining candidates the state of Delaware (98 percent reporting):
Newt Gingrich: 27 percent | 7,659 votes
Ron Paul: 10 percent | 2,984 votes
Mitt Romney: 57 percent | 16,057 votes (declared winner)
Below are the results for the remaining candidates the state of New York (five percent reporting):
Newt Gingrich: 16 percent | 3,171 votes
Ron Paul: 16 percent | 3,168 votes
Mitt Romney: 56 percent | 11,325 votes (declared winner)
Below are the results for the remaining candidates the state of Pennsylvania (50 percent reporting):
Newt Gingrich: 11 percent | 21,198 votes
Ron Paul: 13 percent | 26,970 votes votes
Mitt Romney: 56 percent | 111,761 votes (declared winner)
Below are the results for the remaining candidates the state of Rhode Island (99 percent reporting):
Newt Gingrich: six percent | 789 votes
Ron Paul: 24 percent | 3,080 votes
Mitt Romney: 63 percent | 8,228 votes (declared winner)
The next GOP contests will be held on May 8 when the states of Indiana, North Carolina and West Virginia hold their respective primaries. A total of 132 delegates will be at stake.
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Association and Sequence Mining in Web Usage
Web servers worldwide generate a vast amount of information on web users' browsing activities. Several researchers have studied these so-called clickstream or web access log data to better understand and characterize web users. Clickstream data can be enriched with information about the content of visited pages and the origin (e.g., geographic, organizational) of the requests. The goal of this project is to analyse user behaviour by mining enriched web access log data. With the continued growth and proliferation of e-commerce, Web services, and Web-based information systems, the volumes of click stream and user data collected by Web-based organizations in their daily operations has reached astronomical proportions. This information can be exploited in various ways, such as enhancing the effectiveness of websites or developing directed web marketing campaigns. The discovered patterns are usually represented as collections of pages, objects, or re-sources that are frequently accessed by groups of users with common needs or interests. The focus of this paper is to provide an overview how to use frequent pattern techniques for discovering different types of patterns in a Web log database. In this paper we will focus on finding association as a data mining technique to extract potentially useful knowledge from web usage data. I implemented in Java, using NetBeans IDE, a program for identification of pages' association from sessions. For exemplification, we used the log files from a commercial web site.
If you experience problems downloading a file, check if you have the proper application to view it first. In case of further problems read the IDEAS help page. Note that these files are not on the IDEAS site. Please be patient as the files may be large.
Volume (Year): (2011)
Issue (Month): 2 ()
Pages: 31-36
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| Postal: No. 59-61, Nicolae Balcescu Street, Postal Code 800008, Galati
Phone: (0040) 336.130.242
Fax: (0040) 336.130.242
Web page: http://www.feaa.ugal.ro
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References listed on IDEAS
Please report citation or reference errors to , or , if you are the registered author of the cited work, log in to your RePEc Author Service profile, click on "citations" and make appropriate adjustments.:
- Lillian Clark & I-Hsien Ting & Chris Kimble & P. C. Wright & Daniel Kudenko, 2006. "Combining Ethnographic and Clickstream Data to Identify User Web Browsing Strategies," Post-Print halshs-00489627, HAL.
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Category Archives: Uncategorized
Sex Toys Bring Women New Feelings – They Don't Complain Any More!
The world's largest trade fair has come to an end. It has brought us a comprehensive overview of global adult toys industry as well as the premium brands, newcomers and latest trends. According to news reports, we found out that … Continue reading
No Longer Taboo – How to Introduce Sex Toys To Your Sex Life
When it comes to sex, there are no hard and fast rules on how to achieve the best mind blowing orgasms possible. You can actually be as kinky and creative as you possibly want in bed. Don't be afraid to … Continue reading
Why Adult Toys Are Not Bad Either
There have been leftovers of the free love generation who have managed to survive and the great thing is that in recent times there has been a sexual revival of sorts in America. A good example of this is that … Continue reading


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I'm new to working with SimTalk and am having trouble correctly using the waituntil statement. I need the workers to pick up the part off the pallet on their respective unload station and carry it over to the pallet on their reload station. The problem I'm having is getting the workers to wait until a pallet arrives at Reload before dropping off their part. I've tried placing waituntil statements in many different Entrance and Exit Controls to see if any work, but have had no success. Am I incorrectly using the waituntil statement or is it just not in the right place? I'm practically learning this software on my own so it's probably something simple.
I've attached the file saved with the error message shown for clarity.
Solved! Go to Solution.
Hello kg21em,
it is a good question. We need to synchronize the arrival of the container and the worker.
At first you have to prevent that the worker unloads the part when no container is on the station Reload:
Therefore we perform
var Worker: object := ?.cont
if Worker.occupied
reload.entranceLocked := true
in the entrance control of the Workplace.
Now no container can enter the station Reload.
In order to catch the event of arrival of the container we use a sensor at the end of the line Line3 before Reload.
Here we must stop the container, wait for the worker with a part, load the part onto the container, sent the worker back and open the station Reload:
Reload.exitLocked := true
waituntil workplace1.occupied and workplace1.cont.occupied prio 1
var worker: object := workPlace1.cont
Reload.entranceLocked := false
Reload.exitLocked := false
The attached model is for Plant Simulation 12.2
Thanks for the quick response!
I've added your solution in to affect the second worker as well, but this has created a second problem. It seems that some containers will get stuck at Reload2 and end up containing more than just one part.
I've attached the updated model.
09-05-2016 04:00 AM - edited 09-05-2016 04:03 AM
Hello kg21em,
the problem at ReadLoad2 is completely different problem. The worker should unload.
We must catch the event of a ready pallet a station Unload2. Therefore we use the exit control of Unload2.
I will not use the exit behavior Carry Part Away.
In the init method we initialize a variable Worker4Unload by the responsible worker:
Worker4Unload := WorkerPool1.cont
In the mentioned exit control we must stop the container.
Then we must wail until the worker is free.
Then the process can continue:
var part: object := @.cont
part.destination := Reload2
Thank you for all of the help! It works great now.
I've attached the latest update with the process fully working for anyone interested. It's still a bit messy as the proccessing time of certain stations needs to be changed.


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Walmart workers threw a 104-Year-Old woman a surprise birthday party.
Employees at a Walmart in Arkansas found out one of their customers was turning 104 years old last week. So when she came in to shop, they threw her a surprise party in the break room. And she was so emotional, she cried when she walked in.
Click Here to see more.


text:
Mon., Nov. 4, 2013, 3:54 p.m.
Kunka Rejects Requests To Drop Out
Joe Kunka, the dark horse in the three-way race for Coeur d'Alene mayor, told Huckleberries a few minutes ago that he's received dozens of phone calls asking him to drop out of the race. A conservative Republican he knows, named Bob, left a message on Friday, asking him to drop out and throw his support to Mary Souza. Kunka said the call hurt his feelings. But he didn't give it a second thought because he intends to see his campaign through to the end tomorrow night. Kunka said some of the calls said his campaign was hurting both of his opposing candidates, Souza and Steve Widmyer. Souza is endorsed by the Reagan Republicans and Reclaim Coeur d'Alene. Widmyer by Balance North Idaho, local Realtors, police and fire associations and the Spokesman-Review. Kunka is unaffiliated. Some of the calls said specifically that he was hurting Souza's chances to become mayor. None of the calls mentioned that he would help Widmyer by dropping out.
Question: Will Kunka pull more votes from Souza or Widmyer?
You must be logged in to post comments. Please log in here or click the comment box below for options.comments powered by Disqus


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Oil selector
Which recommended lubricant for my vehicle?
Our Lube advisors help you to find the best oil for your model and brand of vehicle.
You can have a look to our Lube advisors below for the following countries:
Otherwise, if you are looking for an engine oil guide, you can search for it in our online lubricants guide and catalog on http://catalog.lubricants.total.com/automotive
You'll be able to find the recommended motor oil, gear oil, axle oil, brake fluid… for many automotive applications: cars, scooters, motorcycles, trucks and buses, public works vehicles, leisure boats and agriculture equipment. You'll be able to check all our oil specs and official homologations


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|Printer-Friendly version
Review Date: January 06, 2006
Director: Stephen Gaghan
Writer: Stephen Gaghan
Producers: Jennifer Fox, Michael Nozik, Michael Nozik
George Clooney as Bob
Matt Damon as Bryan
Jeffrey Wright as Bennett
This film seems to want to say a lot of things, but never really comes out and says much of it, so it�s up to the learned viewer to sit there, with plenty of workbooks, an encyclopedia and a trusty political science student by their side, to attempt to de-puzzle all of what writer/director Stephen Gaghan is attempting NOT to tell us. For me, I just got tired of it. I don�t mind waiting half an hour or even a full hour (like this film) before anything starts to make any sense in a movie, but you know what�it should start to make sense after that, and other than a few connections, a couple of conclusions during the film�s final 10 minutes, some basic points (there�s corruption in the oil industry, whoopdee-shit), I didn�t appreciate much of what was played out in this film and for a movie watcher, a movie-fan�well, that just plain sucks. Famed film critic Roger Ebert adored this movie, even though even he claimed not to have understood it all, and although I certainly respect anyone�s opinion to that, I need a little more out of a movie myself, especially one that features a very interesting topic, plenty of capable actors (kudos to George Clooney for his performance here) and seemingly interesting dialogue and directing. It all looked and felt good and authentic, but it ultimately left me feeling empty and angry inside. Not angry at the film�s message, but angry at its makers for not creating something more digestible to the layman � and by that, I�m not asking them to �dumb it down�, but rather, �crank up��its coherence!
PS: Oh, and if anyone can tell me what the shit SYRIANA means�drop me a line, will ya?


text:
'Sports' version makes a perfect first R/C car, or even a second!
Released in November 2011, the MINI-Z buggy features a 4WD drive train supported by a 4-wheel independent double-wishbone suspension with oil shocks and a slipper clutch to protect the gears in a technologically advanced design that far exceeds any expectations of its palm top size. With a formidable reputation for performance established through the MINI-Z Cup Buggy class, this 'Sports' version of the proven MINI-Z buggy is equipped with the basic components of MINI-Z buggy's success, but at a great low price. Precision components are protected within the shell structure of the chassis that keeps out dirt and provides optimal rigidity. The 7-stage geared steering servo provides torque and precise response to deliver the MINI-Z's renowned linear steering feel and control. Also, the Sports version is equipped with a specially designed split circuit board that allows easy manual adjustment of the optional gyro unit. The included body is color printed in a uniquely styled design like the MINI-Z's most popular high-performance MP9 model and also includes fluorescent wheels for high-visibility. Just add batteries to start enjoying the serious R/C racing buggy performance of this factory assembled Readyset that makes an ideal first or even your second R/C car!
●Readyset package allows model to be enjoyed straight after purchasing batteries.
●Fully pre-assembled chassis with printed polycarbonate body.
●Real racing performance will please buggy owners. Great for indoor practice.
●Includes Perfex KT-19 transmitter. Allows various setting adjustments such as steering angle.
●FHS 2.4GHz R/C system removes the need to manage frequency bands.
●Frequency crystals are not required and up to 40 cars can be run at the same time.
●Optional gyro unit set (MZW431) can be installed. Gyro sensitivity can be manually adjusted on the chassis.
●Most optional parts already released for MINI-Z Buggy can be installed.
●This model is specially designed for the KT-19 transmitter. It is NOT compatible with the KT-18 and EX-5UR ASF transmitters (No.82011).
This item is over £100, so qualifies for free standard UK shipping!
To see shipping costs simply add the item to your basket and choose your country from the drop down list on the view cart page.
Use this form to ask us a quick one sentence question about this specific product. For more in depth queries, please feel free to email or call us.
Our system will drop you an automated e-mail as soon as this product comes back into stock.


text: For the sake of where both these cats are taking the game we never really entertained the beginning of this beef a week ago when apparently AKA sneaked dissed Cassper Nyovest. Well rather a Cassper Nyovest fan tagged AKA's tweet. Cassper Nyovest responded by saying his lost respect for AKA and AKA posted a picture of himself and Cassper Nyovest insinuating that he is a groupie. The tweets above were a continuation of what happened this weekend. Is it ever going to stop though?


text: BoE has published the results from its systemic risk survey for the second half of 2015 up to 25 September. The report finds that the perceived probability of a high-impact event in the UK financial system over the short term has fallen slightly, however the perception of such an event in the medium term has increased by a similar amount. It identifies the risk of economic downturn and cyber attacks as being prevalent concerns within the industry. (Source: BoE Publishes Systemic Risk Results H2 2015)


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Original Hewlett Packard brand CE320A / HP 128A black laser toner cartridges are high quality original laser cartridges designed exclusively for your Hewlett Packard printer. The HP 128A laser cartridge delivers crisp, high definition output perfect for text or images. You can order your original Hewlett Packard CE320A toner cartridge right here, no hassle, no delays. 100% Satisfaction Guarantee.
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text:
Australian Clinical Study of the Apnex Medical HGNS System to Treat Obstructive Sleep Apnea
This study has been completed.
Apnex Medical, Inc.
ClinicalTrials.gov Identifier:
First Posted: August 23, 2010
Last Update Posted: April 15, 2015
Information provided by (Responsible Party):
Apnex Medical, Inc.
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- No Study Results Posted
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No Study Results Posted on ClinicalTrials.gov for this Study
|Study Status:
|This study has been completed.
|Study Completion Date:
|May 2013
|Primary Completion Date:
|December 2010 (Final data collection date for primary outcome measure)
Eastwood PR, Barnes M, Walsh JH, Maddison KJ, Hee G, Schwartz AR, Smith PL, Malhotra A, McEvoy RD, Wheatley JR, O'Donoghue FJ, Rochford PD, Churchward T, Campbell MC, Palme CE, Robinson S, Goding GS, Eckert DJ, Jordan AS, Catcheside PG, Tyler L, Antic NA, Worsnop CJ, Kezirian EJ, Hillman DR. Treating obstructive sleep apnea with hypoglossal nerve stimulation. Sleep. 2011 Nov 1;34(11):1479-86. doi: 10.5665/sleep.1380.


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Teacher Education in Rhode Island
The information below is designed to help people explore potential careers, and find information about colleges offering related educational programs. If interested,each individual may contact multiple schools.
Also try online schools for teacher, for more choices and flexibility.
Featured Teacher Education in Rhode Island
Grand Canyon UniversityCampus
- Locations:
- Phoenix, AZ
Saint Joseph's UniversityOnline
- Locations:
- Online
George Washington UniversityOnline
Northcentral UniversityCampus
Earn your graduate degree online with Northcentral University.
- Locations:
- San Diego, CA
Colorado Technical University OnlineCampus
A degree from CTU can connect you to what matters most: powerful professional network, faculty who are real-world professionals and innovative technology. So once you earn your degree you hit the ground sprinting. Are you in?
- Locations:
- Colorado Springs, CO
Saint Marys University of MinnesotaCampus
At Saint Mary's University of Minnesota, we take pride in offering online graduate degrees designed to complement the schedules of working adults like you.
- Locations:
- Winona, MN
Concordia University - PortlandCampus
Online Learning at Concordia University-Portland
- Locations:
- Portland, OR


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Monthly Archives: November 2007
Beth and Greg got married last weekend at the Arrowhead Golf Course in Littleton (Roxborough). Arrowhead is one of those fabulous Colorado locations that is breathtaking any time of year, with the amazing rock formations and expansive views.
Here's Dad walking Beth down the aisle..
I loved working with the two of you!! Best wishes as you start anew on the East Coast!
~ Autumn


text: Maybe its just me, but has anyone else seen more Apple advertising lately? It seems like for the past 2 years, nothing. None of those dumb silouhette commercials, no billboards, no posters besides at Best Buy where they sell iPods. But now, its like they are running frantic to get their name out there again. I think they really are taking a hit from Android! I haven't seen a internet advertisement for apple or itunes or anything until today, I saw one for iTunes. Maybe I'm looking in all the wrong places like Android sites for advertising, but has anyone else noticed this?