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Paying homage to the greatest cinematic triumph of the 21st century—that immortal masterpiece, Tron: Legacy—the sequel to Lawz Spoken's 2010 collaboration with OnlyOne, Chron, is aptly titled Chron Legacy. Only this one is billed to "Illmaculate & Lawz Spoken, co-starring OnlyOne" (OnlyOne in this case presumably being Hologram Jeff Bridges). Tonight's the release show for Chron Legacy, and it will see Illmaculate and OnlyOne performing conventional sets, although one can hope they'll also take some time for some of the battle raps that made them names in the Portland hiphop community. Lawz Spoken—AKA Mstr Cntrl, which I assume is meant to be said aloud as "Mister Cantrell"—is not on the bill tonight, which means we shall have to impatiently wait until his return in Chron 3: Live Free or Chron Harder. NED LANNAMANN
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Sensory neurons can detect ischemia and transmit pain from various organs. Whereas the primary stimulus in ischemia is assumed to be acidosis, little is known about how the inevitable metabolic challenge influences neuron function. In this study we have investigated the effects of anoxia, aglycemia, and acidosis upon intracellular Mg2+ concentration [Mg2+]i and intracellular pH (pHi) in isolated sensory neurons. Anoxia, anoxic aglycemia, and acidosis all caused a rise in [Mg2+]i and a fall in pHi. The rise in [Mg2+]i in response to acidosis appears to be due to H+ competing for intracellular Mg2+ binding sites. The effects of anoxia and aglycemia were mimicked by metabolic inhibition and, in a dorsal root ganglia (DRG)-derived cell line, the rise in [Mg2+]i during metabolic blockade was closely correlated with fall in intracellular ATP concentration ([ATP]i). Increase in [Mg2+]i during anoxia and aglycemia were therefore assumed to be due to MgATP hydrolysis. Even brief periods of anoxia (<3 min) resulted in rapid internal acidosis and a rise in [Mg2+]i equivalent to a decline in MgATP levels of 15–20%. With more prolonged anoxia (20 min) MgATP depletion is estimated to be around 40%. With anoxic aglycemia, the [Mg2+]i rise occurs in two phases: the first beginning almost immediately and the second after an 8- to 10-min delay. Within 20 min of anoxic aglycemia [Mg2+]i was comparable to that observed following complete metabolic inhibition (dinitrophenol + 2-deoxyglucose, DNP + 2-DOG) indicating a near total loss of MgATP. The consequences of these events therefore need to be considered in the context of sensory neuron function in ischemia.
- magnesium
- ischemia
sensory neurons from the cervico-thoracic dorsal root ganglia (DRG) and vagal afferent nerve fibers originating from the nodose ganglion (NG) play a major role in the transmission of ischemic pain from the heart and other organs (3, 5, 7, 13, 30, 72, 75). Sensory neurons can be excited by a variety of pathophysiological stimuli (3, 4, 17, 30, 34), but under ischemic conditions it is thought that the primary stimulus is the intense acidosis that results from anaerobic respiration (5, 12, 31, 38, 40, 41, 46, 53). Little is known about how the metabolic challenge presented by anoxia and aglycemia influences sensory neuron function in ischemia. In the central nervous system (CNS), ischemia results in a rapid depletion of cellular energy reserves (36, 45) that has profound effects on ion homeostasis and electrical signaling (15, 28). To investigate the effects of anoxia and aglycemia on metabolism and ion homeostasis in sensory neurons, we have used an in vitro model; i.e., isolated sensory neurons. In this study we have focused on the effects of ischemia-like conditions on cytosolic Mg2+, pH, and ATP levels.
Magnesium is an abundant and essential intracellular ion that can regulate the activity of numerous enzymes, G proteins, ion transporters, and ion channels (62). The majority of magnesium within cells is, however, usually either bound or is located in intracellular organelles (22, 25) such that the concentration of free magnesium ions is relatively low; i.e., 0.3–1.25 mM (29, 44, 65). Moreover, although intracellular Mg2+ concentration ([Mg2+]i) is regulated, transmembrane fluxes of Mg2+ are invariably slow (24, 60, 63). As a consequence, under normal conditions, [Mg2+]i tends to remain fairly constant. One notable exception to this is where there is net ATP hydrolysis (65). ATP has a relatively high affinity for Mg2+ such that almost all ATP within the cell is in the form MgATP−. In contrast, AMP has a low affinity for Mg2+. Thus whenever there is net hydrolysis of MgATP (to ADP and then AMP), magnesium ions are released into the cytosol (39, 50, 65). Thus an increase in cytosolic magnesium is not only invariably a consequence of metabolic compromise but also provides for a simple method with which to monitor changes in cytosolic MgATP [via measurement of [Mg2+]i (32, 43)]. Although indirect, this technique has the advantage that one can observe dynamic changes in ATP levels in real time in a single neuron.
Interference with metabolism can also result in disturbances of cellular pH regulation (11, 14, 58, 77, 78). Changes in intracellular pH (pHi) can have far-reaching consequences for cell function because a great many processes involving enzymes, transporters, ion channels, and signaling pathways are sensitive to pH to varying degrees. In this study we have therefore investigated the effects of ischemia-like conditions, including anoxia, anoxic-aglycemia, and acidosis on [Mg2+]i and pHi in sensory neurons from rat dorsal root ganglia (DRG) and nodose ganglia. Our data indicate that a significant rise in [Mg2+]i, fall in pHi, and ATP depletion can occur even during very brief periods of anoxia (up to 3 min), and over longer periods (15–20 min) elevation of [Mg2+]i and decline in ATP can be substantive especially if accompanied by aglycemia.
Neuron dissociation.
Adult Wistar rats aged between 6 and 8 wk (130–150 g) were euthanized by an overdose of halothane (4%) followed by exsanguination in accordance with schedule 1 of the United Kingdom Animals (Scientific Procedures) act 1986 and United Kingdom Home Office Code of Practice for the Humane Killing of Animals under Schedule 1 of the Animals (Scientific Procedures) Act 1986. Cervical-thoracic DRG (C4-Th6) and no dose ganglia (NG) were removed under sterile conditions and immediately transferred into ice-cold Ca2+- and Mg2+-free phosphate-buffered saline (PBS), pH 7.4. After being cleaned, the ganglia were incubated at 37°C for 30–35 min in a dissociation media comprising 10 mg collagenase type I (Worthington), 1 mg/trypsin (Sigma), 60 μM CaCl2, and 33 μM MgCl2 in PBS. After the enzyme treatment, ganglia were washed once in PBS (Ca2+- and Mg2+-free) and once in Dulbecco's modified Eagle's medium (DMEM, containing 10% fetal bovine serum and 1.2 mM l-glutamine), before mechanical dispersion by trituration in 1.5 ml of DMEM. The dissociated cells were then washed twice by centrifugation (at 1,000 g for 5 min) followed by resuspension in fresh DMEM. After the final wash, the cell pellet was resuspended in 500 μl basal TNB-100 containing Protein-Lipid-Complex (Biochrom, Berlin, Germany), penicillin (100 IU/ml), streptomycin (100 μg/ml), and 10 μM/ml nerve growth factor (NGF). The cell suspension was then seeded onto coverslips coated with poly-l-lysine and laminin and incubated in sterile culture dishes in a humidified chamber at 37°C and 5% CO2-95% air for 2 h. After this incubation period, an additional 3 ml of TNB-100 containing 10 μM/ml NGF was added to each culture dish. The neurons were then kept in the incubator before being used in experiments. Only small-sized neurons with diameters ranging between 15 and 27 μm (from both cervical-thoracic DRG and NG) were used in this study. In parallel studies, >70% of neurons meeting these size criteria responded to brief application of capsaicin (10–100 nM for 10 s) with an increase in intracellular Ca2+ concentration (measured using Fura-2; data not shown).
Culture of F11 cells.
To provide a large number of essentially identical cells for the chemical estimation of cellular ATP levels, we used a DRG-derived cell line. The F11 cell line is a mouse neuroblastoma × rat dorsal root ganglia hybridoma (55). The cell line used in these experiments was also stably transfected with TRPV1 (33, 55). F11 cells were the kind gift of Mathias Dreger (Dept. of Physiology Anatomy and Genetics, University of Oxford). F11 cells were cultured in Ham's F12 media supplemented with Glutamax-I (Life Technologies), 20% FBS (GIBCO-BRL), 2% hypoxanthine-thymidine-aminopterin supplement (Biochrom KG), 100 μg/ml streptomycin, and 100 μg/ml penicillin at 37°C and 5% CO2 in plastic tissue culture flasks (Nunc, DK).
Fluorescence recording.
Fluorescence measurements were performed using an inverted microscope (Nikon) equipped for multiwavelength epifluorescence microscopy. The excitation light was provided by a 75-Watt xenon lamp plus an appropriate band-pass filter. Mag-indo-1 was illuminated continuously at 340 ± 5 nm (plus >64 × neutral density filtration), and emitted fluorescence was filtered and measured at 405 ± 10 nm and 495 ± 12 nm. Carboxyseminaphthorhodafluor-1 (SNARF-1) was illuminated continuously at 540 ± 12 nm (plus 512 × neutral density filtration), and emitted fluorescence was filtered and measured at 590 ± 5 nm and 640 ± 5 nm. Emitted fluorescence was detected by tri-alkali photomultiplier tubes (PMT Thorn, EMI, UK) that were air-cooled to −20°C. The output from each PMT was fed through a current-to-voltage converter, filtered at 10 Hz, digitized at 250 Hz, and then averaged over 500-ms intervals. Data acquisition and analysis was performed using a CED 1401, or micro 1401, and a PC running Spike 2 software (Cambridge Electronic Design).
Calibration of Mag-indo-1 in saline.
Calibration of Mag-indo-1 (Sigma, Dorset, UK) in simple saline calibration solutions was performed at a final concentration of 5 μM (prepared from a stock solution of 1 mg/ml in dimethyl sulfoxide, DMSO). The calibration solutions contained (in mM) 0–150 KCl, 0–100 MgCl2, 5 NaCl, 20 HEPES, and 1 EGTA, pH 7.2, at room temperature (21–23°C or 37°C). MgCl2 and KCl concentrations in these solutions were varied as follows (MgCl2:KCl, in mM), 0:150, 0.3:149.5, 1:148.5, 5:142.5, 10:135, 30:105, and 100:0. The pH sensitivity of Mag-indo-1 was determined by titrating the above solutions to alternative pH values, between 8.0 and 5.5, using KOH or HCl. Fluorescence measurements were obtained from 20- to 200-μl aliquots of the above solutions placed on a glass bottomed culture dish situated on the stage of an inverted microscope. For measurements at 37°C the culture dish was heated, and the temperature of the Mag-indo-1 solution was continuously monitored. Correction for background fluorescence was performed by taking measurements from an aliquot of HEPES solution (pH 7.2) lacking Mag-indo-1.
The calibration equation [Mg2+] = (R − Rmin)/(Rmax − R)·Kd. Ffλ2/Fbλ2, where R is emission fluorescence ratio F405/F495, was fitted to calibration data using a Marquardt-Levenberg algorithm (Sigma-plot) to determine all three parameters (i.e., Rmin, Rmax, and Kd·Ffλ2/Fbλ2). These parameters correspond to the fluorescence ratio in the absence of Mg2+ (Rmin), the fluorescence ratio at saturating Mg2+ (Rmax), and the product of the dissociation constant for Mg2+ and the ratio of fluorescence at 495 nm for the Mg2+-free form of the dye divided by that for the Mg2+-bound form of the dye (Kd·Ffλ2/Fbλ2).
Dye loading.
For in vivo measurements of [Mg2+]i or pHi, cells were incubated with the acetoxymethyl ester forms of Mag-indo-1 or carboxy-SNARF-1 (all from Molecular Probes, Leiden, NL) at a concentration of 5 μM in either HEPES-buffered Tyrode or bicarbonate-buffered Tyrode solution. Dye loading was carried out at room temperature in a dark chamber for 20 min (Mag-indo-1-AM) or 15 min (SNARF-1-AM).
In situ calibration of Mag-indo-1.
In situ calibrations, i.e., in the intracellular environment, were attempted using Mg2+ calibration solutions containing 0–150 mM KCl, 0–100 mM MgCl2, 5 mM NaCl, 1 mM EGTA, 20 mM HEPES, and 1 μM ionomycin (plus other constituents where indicated), pH 7.2. To quench Mag-indo-1 fluorescence, cells were incubated with a solution containing 150 mM KCl, 5 mM NaCl, 1 mM MnCl2 20 mM HEPES, and 1 μM ionomycin, pH 7.2.
In situ calibration of carboxy-SNARF.
The in situ calibration of carboxy-SNARF-1 was carried out using the nigericin method as described previously (8). pH calibration solutions contained 140 mM KCl and 10 μM nigericin plus one of the following buffers: 20 mM 2-(N-morpholino)ethane sulfonic acid or 20 mM HEPES or 20 mM 3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid. The pH of these three solutions was adjusted to 5.0, 7.0, and 9.0, respectively, using HCl or KOH. Neurons were exposed to each of these solutions in order of ascending pH value to determine the calibration constants Rmax, pK, and Rmin (see Ref. 8).
ATP measurement.
For measurement of cellular ATP levels F11 cells were resuspended in a Tyrode solution preequilibrated with 5% CO2, containing either 11 mM glucose (control) or omitting glucose and containing 2–4 dinitrophenol (DNP, 250 μM) and 2-deoxyglucose (2-DOG, 10 mM). Aliquots of 100 μl of this cell suspension were placed in a 96-well plate and then incubated at 37°C in a humidified chamber containing 5% CO2 in the gas phase for 0 (control), 0.5, 1, 2, 3, 5, 8, 11, 15, 18, and 25 min (+DNP and 2-DOG). ATP was measured using a modified luciferase assay (ATPlite, PerkinElmer, Wellesley, MA) and a Wallac 1450 MicroBeta TriLux luminometer according to the manufacturer's instructions.
Physiological saline solutions.
Standard bicarbonate buffered Tyrode contained (in mM) 117 NaCl, 4.5 KCl, 2.5 CaCl2, 1 MgCl2, 23 HCO3−, and 11 glucose and was equilibrated with 5% CO2, pH 7.4. Ca2+-free bicarbonate-buffered Tyrode solutions were devoid of CaCl2 and contained 1 mM EGTA. Glucose was replaced in glucose-free solutions by an equimolar amount of sucrose.
Normoxic and normocapnic solutions were prepared by bubbling bicarbonate-buffered Tyrode solution with 5% CO2-95% air. Anoxic solutions were prepared by bubbling Tyrode with 5% CO2-95% N2 for at least 30 min followed by the addition of 0.5 mM Na2S2O4 (64) ∼3 min before use. Measurement of bath Po2 using optical oxygen meter (Microx TX2, PreSens, Germany) did not reveal any detectable difference between anoxic solutions in the bath and the zero-point calibration. All normoxic and anoxic bicarbonate-buffered Tyrode solutions were maintained at 37°C (in a water bath) and a constant pH of 7.4.
Hypercapnic acidosis was achieved by bubbling standard bicarbonate-buffered Tyrode solutions with 20% CO2-80% air (pHo 6.8). Metabolic acidosis (pHo 7.0) was achieved by reducing NaHCO3 to 10 mM (NaCl was increased to 130 mM to maintain osmolarity) and bubbling with 5% CO2-95% air. To achieve a combined respiratory and metabolic acidosis Tyrode solution with reduced NaHCO3 (10 mM) was bubbled with 20% CO2-80% air (pHo 6.2).
Physiological salines were continuously bubbled with gasses in glass bottles in a water bath (at 37°C) and were delivered to the experimental chamber via medical grade stainless steel tubing articulated with short sections of Pharmed tubing (Norton Performance plastics) to ensure the stability of CO2 and O2 levels in these solutions. A zero dead space two-way tap was placed just upstream of the recording chamber to allow change of solutions entering the chamber, and a heater was placed between the tap and the recording chamber to compensate for any temperature loss between solution reservoirs in the water bath and solutions entering the chamber (thus maintaining the temperature at 37° C in the recording chamber). The recording chamber itself had a low volume (∼100 μl) and solution flow rate was ∼2 ml/min.
Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, 1 μM) was added to the experimental buffer from a 10 mM stock solution in DMSO. DNP (250 μM) was added from a stock solution of 250 mM in water. 2-DOG (10 mM) and propionate (10, 15, and 20 mM) were directly added to the experimental buffer solutions (all reagents from Sigma, Dorset, UK).
Values were expressed as means ± SE, Statistical significance was tested using paired Student's t-test for cell experiments. Statistical tests on calibration data were performed using one-way analysis of variance, and post hoc analyses were carried out using Bonferroni's multiple comparison, calculated by Statistical Package for the Social Sciences (SPSS) 12.0 software for windows.
Mag-indo-1 calibration in saline: effects of temperature and pH.
Calibration of Mag-indo-1 in saline revealed a marked effect of temperature. At 37°C and pH 7.2, there was a small increase in the 405/495 fluorescence ratio at 0 mM Mg (R0 = 0.07 ± 0.001 at 37°C vs. 0.05 ± 0.003 at 22°C P < 0.05) but little effect at 300 mM Mg (R300 = 1.20 ± 0.002 at 37°C vs. 1.19 ± 0.005 at 22°C, Fig. 1A). The main effect of increasing temperature was a decrease in the calibration constant Kd·Ffλ2/Fbλ2 from 9.46 at 22°C to 4.8 at 37°C (values for Rmin and Rmax obtained from curve fitting were 0.052 and 1.22 at 22°C and 0.063 and 1.24 at 37°C). These data are consistent with increasing temperature reducing the Kd for magnesium (42) and highlight the importance of conducting calibrations at an appropriate temperature.
Changes in pH also affected the fluorescence ratio (Fig. 1B). This was most evident at [Mg2+] below 10 mM and at pH ≤ 6.5. Lowering pH to ≤6.5 also increased Rmin and decreased Rmax (Fig. 1D). The calibration constant Kd·Ffλ2/Fbλ2 was relatively insensitive to pH in the range 7.2–6.5 but increased at more alkaline and more acid pH (Fig. 1E). One possible explanation for these observations is that at low pH, Mag-indo-1 may become protonated, and this form could have different fluorescent properties to those of deprotonated and Mg2+-bound forms. Despite these effects, the standard calibration equation still produced a good fit to the data even at extreme pH (Fig. 1B). Close inspection of these data and calibration curves over a range of [Mg2+] likely to be encountered in cells (0.5–5 mM) revealed that minor variation in pH (i.e., from 7.2 to 7.0) has no significant effect on [Mg2+] measurement (Fig. 1, B, C, and F). More severe acidosis (pH ≤ 6.0) will, however, increase the fluorescence ratio and, unless calibration constants appropriate to the pH are used, cause overestimation of [Mg2+]i.
Mag-indo-1 calibration in situ.
Some calcium indicators exhibit different properties in the intracellular environment to those in simple saline (69). We therefore attempted an in situ calibration in neurons loaded with Mag-indo-1-AM. Exposure to a Ca2+-free depolarizing solution (50 mM K+) containing 1 mM Mn2+ (to promote Mn2+ influx via voltage-gated Ca2+ channels) caused a rapid quenching of the Mag-indo-1 fluorescence signal at 495 nm to 15.5 ± 1.2% of control (n = 4). Addition of ionomycin (1 μM) caused a slight further quenching of fluorescence at 495 nm to 12.1 ± 2.5% of control (Fig. 2A). Permeabilization of Mag-indo-1-loaded neurons with digitonin also dramatically reduced fluorescence at 405 and 495 nm to 8 ± 2.3% and 1 ± 0.3% (n = 4) of control. These data indicate that there is little interference from cellular autofluorescence or the presence of any form of Mag-indo-1 that is not readily able to bind divalent cations (by virtue of being only partially hydrolyzed, degraded, bound to another molecule, or sequestered in some inaccessible space).
Attempts to estimate Rmax in situ were partially successful. Incubation of Mag-indo-1-loaded neurons in a calibration solution containing 1 μM ionomycin and 100 mM Mg2+ resulted in a progressive increase in fluorescence ratio that failed to reach a steady state within 15 min (Fig. 2D). This situation could not be improved by the use of higher concentrations of ionomycin (65) or by using another ionophore 4-Br-A23187 (59) either alone or in combination with monesin and nigericin. The most likely explanation for this observation is that neither ionophore allows a sufficiently rapid equilibration of large amounts of Mg2+ across the cell membrane. Similar problems have been encountered by others; e.g., Tashiro and Konishi (66) used incubation times of between 5 and 14 h to try to calibrate Mag-fura using 4-Br-A23187, monesin, and nigericin. For our purposes incubation times of this duration were considered too long as both dye leakage and cell swelling would perturb measurements. Switching from a magnesium-containing solution to one containing 10 mM Ca2+, however, resulted in a very rapid increase in fluorescence ratio (Fig. 2D), which stabilized after a few seconds. This reflects the fact that Mag-indo-1 has a much higher affinity for Ca2+ than for Mg2+ (42), and consequently less Ca2+ needs to enter the cell to saturate the Mag-Indo-1. We were therefore able to obtain an in situ value for Rmax with Ca2+ (Rmax·Ca = 1.08 ± 0.14, n = 4) from which we estimated the in situ Rmax for Mg2+ (Rmax·Mg) by multiplying by a small conversion factor (∼1.2) derived from the ratio of the values for Rmax·Ca and Rmax·Mg obtained in saline. Using this approach gives a value for Rmax·Mg in situ of ∼1.3, which is essentially the same as that obtained in saline (1.3 at pH 7.2). Intracellular calibration of Mag-indo-1 or the related probe furaptra (Mag-fura-2) in cardiac and skeletal muscle has also been shown to yield similar values to those obtained in saline (23, 65, 74).
Attempts to obtain a value for Rmin by incubation in Mg2+- (and Ca2+)-free solutions containing 1 μM ionomycin failed. Although the fluorescence ratio declined significantly in this calibration solution, a steady-state value was not attained even after 40 min incubation (Fig. 2C), suggesting a failure to completely deplete the cell of Mg2+. Consequently, as there was good agreement between Rmax·Ca values obtained in situ and in saline, we used calibration data obtained in saline, pH adjusted where necessary, for the estimation of [Mg2+]i in this study.
Mag-indo-1 calibration: possible interference from intracellular calcium.
Magnesium-sensitive indicators invariably have a higher affinity for calcium than that for magnesium. For Mag-indo-1 the Kd for Ca2+ is ∼34 μM, whereas that for Mg2+ is 2.03 mM (42). However, [Mg2+]i is approximately four orders of magnitude greater than intracellular Ca2+ concentration ([Ca2+]i) (resting [Ca2+]i ≅160 nM in both NG and DRG neurons, Henrich and Buckler, unpublished observations). Thus, under resting conditions, the intracellular concentration of Ca2+-bound Mag-indo-1 is approximately 1/100th of that of the Mg2+-bound form. Under conditions of metabolic inhibition or anoxia, [Ca2+]i is increased in these neurons but by no more than about 150 nM in Ca2+o-free conditions (Henrich and Buckler unpublished observations), which would be equivalent to a rise in [Mg2+]i of ∼10 μM. Thus under the conditions employed in this study, changes in [Ca2+]i will have no significant effect upon the estimation of [Mg2+]i.
Correlation between [Mg2+]i and ATP depletion.
To confirm the correlation between depletion of cellular MgATP and rise in [Mg2+]i, we compared a luciferase-based assay of MgATP with measurement of [Mg2+]i in F11 cells. A neuronal (DRG) cell line was chosen to ensure, as far as possible, that a uniform population of cells would be used for both measurements. F11 cells were exposed to a combination of the uncoupler DNP and the glycolytic inhibitor 2-DOG, and then either [Mg2+]i was measured continuously by fluorescence microscopy in F11 cells growing on a glass coverslip or ATP content was analyzed at specific time points in aliquots of an F11 cell suspension. As can be seen from the results (Fig. 3), there was a strong correlation between ATP depletion and elevation of [Mg2+]i. Moreover these data show that exposure to a combination of both uncoupler and 2-DOG results in near-complete (>90%) ATP depletion within a relatively short period of time (i.e., ≧11 min).
Resting levels of free magnesium and pHi in sensory neurons.
Under control conditions (bicarbonate-buffered media + 5% CO2, pHo = 7.4 at 37°C), pHi for DRG and NG neurons was 7.13 ± 0.04 and 7.24 ± 0.05, respectively. Resting [Mg2+]i, estimated using pH 7.2 calibration data obtained in saline, was about 1.5 mM in both types of neuron (Table 1). The removal of extracellular Ca2+ had no effect upon [Mg2+]i. The removal of both extracellular Ca2+ and Mg2+ caused a slight reduction in [Mg2+]i in NG neurons but had no effect in DRG neurons (Table 1), suggesting that at rest transmembrane Mg2+ fluxes were sufficiently slow as to be unable to rapidly modulate [Mg2+]i. All of the following experiments are therefore conducted in Mg2+- and Ca2+-free media to exclude Mg2+ or Ca2+ influx from interfering with our measurement of [Mg2+]i.
Effects of brief anoxia on [Mg2+]i and pHi.
Exposure to brief anoxia led to a rapid increase in Mag-indo-1 fluorescence ratio to an apparent plateau that was sustained throughout the exposure period (3 min, Fig. 4). The mean rise in [Mg2+]i at the end of this 3-min anoxic period was 0.21 ± 0.04 mM in NG neurons and 0.28 ± 0.02 mM in DRG neurons. On return to normoxic conditions there was a slow recovery of [Mg2+]i toward preanoxic levels, although in 30% of neurons this recovery was incomplete. Brief anoxia also caused a fall in pHi of <0.1 (Fig. 5, Table 2). In about half of the neurons studied, there was a partial recovery of pHi following this initial acidosis, whereas in others it was sustained throughout the anoxic period (see Fig. 5, A–D). To completely exclude the possibility that the observed rise in Mg2+ during anoxia was an artifact caused by an effect of pH upon Mag-indo-1, we recalculated the change in Mg2+ using calibration data obtained in saline at pH 7.0 for anoxic conditions and pH 7.2 for control conditions. This did not substantially change our estimates of the rise in [Mg2+]i during anoxia (Δ[Mg2+]i = 0.23 mM in NG neurons and 0.3 mM in DRG neurons see Table 3). Thus even overcompensation for any possible direct pH effects on Mag-indo-1 did not affect our observation that brief anoxia causes an ∼20% increase in intracellular free magnesium.
Effects of prolonged anoxia and anoxic aglycemia on [Mg2+]i and pHi.
When neurons were exposed to longer periods of anoxia (20 min), we observed a further, slower increase in [Mg2+]i (see, e.g., Fig. 4B). Prolonged anoxia caused little further acidification, compared with brief anoxia, in either NG or DRG neurons (Fig. 5 and Table 2). After 20 min of anoxia, the rise in [Mg2+]i (calculated using pH 7.0 calibration data) was 0.43 mM in NG neurons and 0.69 mM in DRG neurons (Table 3). Exposure to prolonged anoxia in the absence of glucose (anoxic aglycemia) resulted in an increase in [Mg2+]i (by 1.35 mM in DRG and 0.67 mM in NG, Table 3) that was larger than that seen in anoxia alone (P < 0.05 for both DRG and NG neurons). Under these conditions, the rise in [Mg2+]i was often biphasic (Fig. 4, C and F). Anoxic aglycemia did not, however, have a greater effect on pHi than anoxia alone (Fig. 5, C and F, and Table 2).
Effects of metabolic inhibition on [Mg2+]i and pHi.
Since the above experiments were conducted in Mg2+-free media, the rise in [Mg2+]i must derive from internal sources. The most likely source is release of Mg2+ formerly bound to ATP into the cytosol as a consequence of metabolic inhibition and net ATP hydrolysis. To confirm that inhibition of energy metabolism would indeed cause a rise in [Mg2+]i in these cells, we tested the effects of two uncouplers. FCCP induced a rapid increase in Mag-indo-1 fluorescence ratio (Fig. 6D) and a substantial acidification of ∼0.5 pH units (Fig. 6A and Table 2). With the use of calibration data obtained at pH 6.5 in saline for Mag-indo-1 (to compensate for the fall in pHi), the FCCP-induced rise in [Mg2+]i was ∼0.4 mM (Table 3). This rapid rise in [Mg2+]i is consistent with the expectation that FCCP would not only halt oxidative phosphorylation but probably also accelerate ATP hydrolysis via the mitochondrial ATP synthase. In view of the acidification caused by FCCP, however, we sought to confirm this result using another mitochondrial uncoupler 2,4-DNP. DNP had a smaller effect on pHi than FCCP (Fig. 6B and Table 2) but also caused a rapid rise in [Mg2+]i of 0.65 and 0.37 mM in DRG and NG, respectively (Fig. 6E and Table 3). At the concentrations used both uncouplers appeared equally effective in depolarizing sensory neuron mitochondria (as assessed using Rhodamine-123, Buckler and Henrich, unpublished observations), but FCCP appeared to be a more effective protonophore at the cell membrane causing a greater intracellular acidification.
To further assess the effects of inhibition of energy metabolism on [Mg2+]i, without unnecessary complications from changes in pHi, we therefore chose to use DNP rather than FCCP as an inhibitor of oxidative phosphorylation. Combining DNP with glucose removal and 2-DOG to inhibit both oxidative phosphorylation and glycolysis caused a similar rise in [Mg2+]i during brief exposure (3 min) to that observed in response to DNP alone (Fig. 6, E and F, and Table 3). The fall in pHi within a 3-min exposure to DNP + 2-DOG was slightly smaller than that seen with DNP alone (Table 2). This might reflect a reduction in the production of acid by anaerobic metabolism. Longer exposure (20 min) to this solution caused a much larger increase in [Mg2+]i (1.2 and 0.65 mM in DRG and NG neurons, respectively, see Tables 2 and 3).
Effects of anoxia and metabolic inhibition on [Mg2+]i in Na+-free conditions.
Intracellular Mg2+ concentration in most cells is normally regulated via an plasma membrane Na+/Mg2+ exchanger (63, 68) albeit slowly. To determine the maximum rise in [Mg2+]i attainable with complete ATP depletion in the absence of any Mg2+ extrusion, we exposed neurons to a combination of DNP, aglycemia and 2-DOG in Na+-free solution until the rise in [Mg2+]i reached a maximum (∼20 min, see Fig. 7C). Under these conditions free [Mg2+]i rose by 2.1 and 1.1 mM in DRG and NG neurons, respectively (see Table 3). This rise in [Mg2+]i was significantly greater than that seen in Na+-containing saline (P < 0.05 for both DRG and NG neurons). In contrast, the rise in [Mg2+]i following 20 min of anoxia in Na+-free conditions was smaller (0.5 mM in DRG and 0.3 mM in NG, see Table 3 and Fig. 7D). Na+-free conditions alone had no significant affect upon resting [Mg2+]i.
Effects of Na+-removal on intracellular pHi.
The removal of sodium from the extracellular solution under normoxic conditions caused only a very small and relatively slow cellular acidification; in DRG neurons pHi fell from 7.17 (±0.02) to 7.15 (±0.02, n = 7, P < 0.05) and in NG neurons pHi fell from 7.15 (±0.02) to 7.12 (±0.02, ns) over a 3-min interval. The degree of acidosis caused by anoxia was little affected by removal of extracellular sodium (Fig. 7 B), but the acidification caused by DNP + 2-DOG was significantly greater in Na+-free Tyrode compared with normal Tyrode (P < 0.001 in DRG, P < 0.05 in NG, see Table 2).
Effects of intracellular acidosis on [Mg2+]i.
The above data clearly suggest that a major source of rise in Mg2+ in response to anoxia is likely to be due to release from hydrolyzed ATP. In all of our experiments involving anoxia or metabolic inhibitors we also observed a fall in pHi albeit, in most cases, by only a small amount (0.1–0.3 pH units). Although such a change in pHi is unlikely to have a significant impact upon the accuracy of our measurement of [Mg2+]i acidosis could directly alter [Mg2+]i in these cells through actions on Mg2+ buffering or transport. We therefore investigated the effects of intracellular acidification alone upon [Mg2+]i in magnesium-free media by the addition of the weak acid propionate at 10, 15, and 20 mM. In addition, we also subjected neurons to physiological acid challenges, such as might be encountered during ischemia, using hypercapnic acidosis (high CO2, pH 6.8), isocapnic acidosis (low HCO3−, pH 7.0), mixed acidosis (high CO2 and low HCO3−, pH 6.2). All of these maneuvers induced a fall in pHi (Fig. 8, A–D). Intracellular acidosis was most marked with the hypercapnic and mixed acidosis, which caused a rapid fall in pHi of ∼0.4 (see Table 4). This effect is probably due to the rapid permeation and hydration of CO2. Any subsequent pHi recovery during these experiments was notably rather slow (see Fig. 8, C and D). In each case, we then used the measured pHi (Table 4) to determine the most appropriate pH-corrected Mag-indo-1 calibration data to use to estimate corresponding changes in [Mg2+]i (Table 5). With all methods used to induce intracellular acidification, we observed a small rise in [Mg2+]i (Fig. 8, E–H, and Table 5), the magnitude of which was closely correlated with the size of the intracellular acidosis (Fig. 9). Thus internal acidification alone appears to also have a direct effect upon [Mg2+]i (see discussion).
In the present study we investigated the effects of anoxia and aglycemia on [Mg2+]i, as an index of ATP depletion in sensory neurons, and upon pHi. Evaluation of the suitability of Mag-indo-1 revealed potential limitations to its use under very acid conditions but not within the range of pHi encountered in this study. We observed rapid changes in both [Mg2+]i and pHi in response to anoxia and anoxic aglycemia. In addition we found that intracellular acidosis alone increases cytosolic magnesium. The significance of these observations is discussed below.
Resting intracellular magnesium concentration and effects of removal of extracellular magnesium.
Resting [Mg2+]i for both types of neuron was ∼1.5 mM, which is higher than previously reported in DRG neurons from newborn rats, [between 0.26 and 0.48 mM; (21, 35)]. In these latter studies neurons were cultured for 3–7 days, and [Mg2+]i was determined at room temperature using Mag-fura-2. The disparity between our and other studies could therefore reflect differences in age, culture conditions, temperature, or the indicator used. Similar differences between [Mg2+]i measurements made using Mag-indo-1 and Mag-fura-2 have also been observed in cardiac myocytes (65, 67).
Removal of extracellular magnesium has little effect on [Mg2+]i (Table 1) suggesting that resting transmembrane Mg2+ fluxes in these neurons are relatively slow. Similar observations have been reported in smooth muscle (56) and cardiac myocytes (65).
Interaction between neuronal pHi and [Mg2+]i.
Exposure of sensory neurons to hypercapnic acidosis, isocapnic acidosis, or the weak acid propionate caused an intracellular acidification with concomitant elevation of [Mg2+]i (Fig. 8, A–H, and Tables 4 and 5). Compensation (or even over compensation) for any minor effects of pH upon Mag-indo-1 did not significantly alter our estimates of this rise in [Mg2+]i. We therefore believe this rise in [Mg2+]i to be a genuine effect of intracellular acidosis. It was nevertheless noted that the time course of change in [Mg2+]i was exceptionally closely correlated with the change of pHi during both the application of an acid challenge and upon its removal. This is particularly apparent when comparing the effects of isocapnic acidosis, where changes in pHi and [Mg2+]i were relatively slow (Fig. 8, B and F), with hypercapnic acidosis where change in pHi and [Mg2+]i occur very rapidly (Fig. 8, C, D, G, and H). Indeed during hypercapnic acidosis the temporal correlation between the anticipated rapid change in pHi and the change in [Mg2+]i is striking (see Fig. 9A). We also noted a simple linear relationship between change in pHi and [Mg2+]i in both neuron groups (Fig. 9, D and E). Because these experiments were conducted in the absence of external magnesium (and calcium), the rise in [Mg2+]i cannot be attributed to magnesium (or calcium) influx and must therefore originate from some internal source. From the extreme rapidity of the changes in [Mg2+]i that occur upon addition and removal of weak acids (CO2 and propionate see Fig. 8, E, G, H, and Fig. 9A), the most likely explanation is that protons compete for, and displace Mg2+ ions from, cytosolic binding sites/buffers. A similar close association between pHi and [Mg2+]i has also been reported in leech neurons and was attributed largely to H+ displacing Mg2+ from MgATP (26). In our study, however, even if [MgATP]i were 10 mM, a fall in pH from 7.2 to 6.8 would release <0.2 mM Mg2+ (see Ref. 47), this is half the observed rise in [Mg2+]i in response to a hypercapnic acidosis. Rise in intracellular H+ must therefore cause displacement of Mg2+ from other binding sites not just MgATP (see below).
Effect of metabolic inhibition and anoxia upon pHi.
When sensory neurons were exposed to anoxia or DNP (or cyanide, data not shown), there was a small reversible fall in pHi similar in magnitude to that reported in carotid body type I cells (9), brainstem neurons of the nucleus tractus solitari, ventrolateral medulla, inferior olive and hypoglossal neurons (11), and hippocampal neurons (14, 78) but smaller than in dorsal vagal neurons (58) and cardiac myocytes (48). There are a number of possible causes for such a fall in pHi including: 1) the generation of H+ from net hydrolysis of ATP, 2) anaerobic metabolism and lactate production, 3) enhanced acid influx, and 4) the inhibition of acid extruding pathways, e.g., as described for hippocampal neurons (77, 78). In addition, from the observation that acidosis can displace Mg2+ from internal buffering sites, the converse must also occur; i.e., a rise in magnesium should displace protons from these same buffer sites.
Although we have not explicitly investigated these mechanisms, we have made a number of observations that may shed some light on this issue. We noted that upon exposure to Na+-free media, the fall in pHi was very slow (see results). As Na+-removal leads to the inevitable inhibition of Na+-dependent acid efflux, this maneuver gives an approximate measure of background acid loading due to acid-equivalent influx and metabolic acid generation under normal control conditions. The observation that the application of anoxia (Fig. 7B) causes a much more rapid cellular acidification than does Na+ removal (see e.g., Fig. 5) clearly indicates that anoxia must increase acid influx or acid generation or H+ displacement from internal buffers (or a combination thereof). This rapid initial acidification soon slows, however, and a near steady-state acidosis is then attained (the exact profile of pH change was variable between neurons some showing a partial recovery following the initial acidosis; see Fig. 5). Although this could reflect activation of acid efflux pathways by the fall in pHi (70), thus counteracting the enhanced acid load, the observation that the (20 min) anoxia-induced acidosis was not significantly enhanced in Na+-free medium suggests that this is unlikely. Our preferred explanation at present is therefore that the initial acidification in response to anoxia represents a predominantly transient acid load. After this event any sustained acidosis may be due to a much lesser continuing acid loading process and/or inhibition of acid extrusion. A rapid transient acid load in response to anoxia would also help explain why a partial pH recovery was observed in some neurons (e.g., Fig. 9B).
All of the above pathways may therefore be involved in mediating the observed changes in pHi in response to prolonged anoxia. Clearly, however, further studies are required to evaluate the precise causes and duration of the acid load and to evaluate the effects of anoxia upon Na+-dependent acid extrusion before a full explanation can be given.
Effects of metabolic inhibition on [Mg2+]i.
Exposure to conditions that are likely to cause energy depletion lead to an increase in [Mg2+]i. As these experiments were conducted in Mg2+-free media, this must reflect Mg2+ release from internal sources and is believed to be due to the hydrolysis of MgATP to Mg2+ + AMP.
The association between decline in ATP levels and elevation in [Mg2+]i during metabolic inhibition was confirmed in the DRG-derived F11 cell line (Fig. 3). Because we also observed a fall in pHi under these conditions, it is, however, important to consider the extent to which the rise in [Mg2+]i might be due to H+ displacing Mg2+ from internal buffers. Figure 9, C and D, compares the effects of anoxia, anoxic aglycemia, and DNP + 2-DOG with those of direct cytosolic acidification using weak acids upon both [Mg2+]i and pHi. This comparison shows that the rise in [Mg2+]i in response to anoxia, etc. is greater than that predicted from the fall in pHi alone. Thus changes in [Mg2+]i under these conditions primarily reflect MgATP depletion rather than cell acidification. One exception to this is FCCP. Even very brief exposure to FCCP caused a much greater acidification than that seen with anoxia or anoxic aglycemia or DNP + DOG. This probably reflects the protonophoretic properties of FCCP. This acidification and the consequent rise in [Mg2+]i will almost certainly have masked the effects of MgATP depletion upon [Mg2+]i.
The extent to which a small part of the rise in [Mg2+]i in response to anoxia could be due to H+ displacing Mg2+ from buffers, or conversely whether it is the release of Mg2+ from MgATP that displaces H+ from buffers, remains a moot point. It is probable, however, that both a fall in pHi and rise in Mg2+ reflect the same event; i.e., the onset of ATP depletion.
The maximum rise in [Mg2+]i observed with total metabolic blockade (DNP + 2-DOG) was increased in Na+-free media, whereas the small rise caused by anoxia was unaffected by sodium removal. These data suggest that a large increase in [Mg2+]i leads to the activation of a Na+-dependent Mg2+ extrusion. This is consistent with the view that Mg2+ extrusion in mammalian cells is 1) mediated via a Na+/Mg2+ exchanger (63, 68) and 2) highly [Mg2+]i dependent (71).
[Mg2+]i and magnesium buffering.
As previously discussed, the effects of acidosis upon [Mg2+]i can only be accounted for if a significant proportion of intracellular Mg2+ is bound to intracellular buffers. Magnesium buffering also appears to occur when Mg2+ is released from MgATP. Even when Na+-dependent Mg2+ extrusion was blocked the maximum rise in [Mg2+]i seen with complete metabolic blockade (DNP + 2-DOG) was only 1 and 2 mM for NG and DRG neurons, respectively. This increase in [Mg2+]i is less than would be expected given normal neuronal [MgATP] of around 3 mM (16, 19). Whereas it is conceivable that ATP levels may be lower in sensory neurons than in CNS neurons, it is more likely that the rise in [Mg2+]i is simply constrained by Mg2+ buffering. Similarly, in cardiac myocytes the maximum increase in [Mg2+]i following metabolic inhibition is limited to around 1.5–2 mM (65). In chick ventricular myocytes, the apparent magnesium-buffering power during metabolic inhibition is 2.5; i.e., for every 2.5 Mg2+ ions released by MgATP hydrolysis, only 1 is free in solution and the other 1.5 ions bind to other buffers (39). If we assume the Mg2+-buffering capacity in sensory neurons to be the same, we can estimate [MgATP]i to be between 2.5 and 5 mM for nodose and DRG neurons respectively.
Magnesium buffering has only been extensively characterized in erythrocytes (18) where it is mediated primarily by ATP, 2,3 DPG, and hemoglobin (57). Although the nature of magnesium buffers in other cells has yet to be established, a number of binding sites for magnesium are known. In myocytes, creatine phosphate binds Mg2+ (20, 27), studies in brain suggest that phospholipids are also major binding sites for Mg2+ (49) and some cytosolic proteins e.g., troponin C, calmodulin, S100 protein, and actin [all of which are expressed in sensory neurons (54, 61, 76)], can also bind Mg2+. Many of these also bind calcium so their contribution to Mg2+ buffering may well depend on [Ca2+]i (10, 51, 52, 73). In this respect it is noteworthy that Mg2+ buffering in chick myocytes was lower in the presence of external calcium than in its absence (39).
Time course of ATP depletion in sensory neurons during anoxia and anoxic aglycemia as assessed by change in [Mg2+]i.
It is notable that in response to anoxia the rise in [Mg2+]i begins almost immediately. This is in marked contrast to resting cardiac myocytes where there is a delay of several min between exposure to hypoxia or metabolic inhibitors and rise in [Mg2+]i (6, 37, 65). This suggests that energy reserves, probably in the form of phosphocreatine, are much greater in muscle than in sensory neurons.
In response to prolonged anoxia or metabolic blockade, the rise in [Mg2+]i was mostly monophasic but in aglycemic anoxia there were often two distinct phases the first immediate the second beginning after a delay (Fig. 4, C and F). The maximum rise in [Mg2+]i was also greater in aglycemic anoxia than in anoxia alone (Table 3) but equivalent to that seen with metabolic blockade (compare Table 3 anoxia 0 glucose with DNP/2-DOG + Na+). These observations suggest that under anoxic conditions the fall in ATP is probably limited by anaerobic glycolysis. In the absence of exogenous glucose, however, decline in ATP levels can only be constrained for a brief period, presumably while utilizing endogenous carbohydrate reserves, before there is a further dramatic fall in ATP. The mean delay between applying anoxic aglycemia and the secondary rise in [Mg2+]i was 8.8 (±0.3; n = 6) min and 10.6 (±1, n = 6) min for DRG and NG neurons, respectively. These values are comparable to measurements in the hippocampus, wherein endogenous glucose, glycogen, and other glycolytic intermediates can support basal metabolic requirements for around 5.5 min after metabolic inhibition and removal of glucose (1).
If we assume 1) that ATP depletion is near complete following prolonged exposure to DNP + 2-DOG (as in the F11 cell line, see Fig. 3), 2) that Mg2+ buffering is relatively independent of [Mg2+]I, and 3) that Mg2+ extrusion is minimal, we can derive a relative scale for ATP depletion by expressing rise in [Mg2+]i as a percentage of that seen with DNP + 2-DOG (measured under Na+-free conditions). Using this approach, we obtained the following estimates: after 3 min of anoxia ATP levels fall by 14% and 21% in DRG and NG, respectively, and after 20 min, anoxia ATP levels fall by about 33% and 40%. We performed these calculations using data obtained for anoxia in normal Na+-containing media because Na+/K+-pump activity is a major consumer of ATP (2). Arguably these measurements could underestimate ATP depletion if there is loss of Mg2+ due to Na+/Mg2+ exchange. The rise in [Mg2+]i caused by anoxia in Na+-free conditions was not, however, any greater than that in Na+-containing solutions; indeed it was slightly less (which is consistent with reduced ATP consumption in Na+-free conditions).
In summary, we have utilized the magnesium indicator Mag-indo-1 and the pH indicator SNARF-1 to investigate the effects of ischemia-like conditions upon [Mg2+]i, pHi, and ATP levels in isolated sensory neurons. Our data indicate that in response to anoxia there is a rapid initial rise in [Mg2+]i that most probably results from a fall in MgATP to 75–80% of control within minutes. This is followed by a slower rise in [Mg2+]i suggesting that after 20 min, MgATP levels are reduced to around 60–70% of control. The consequences of short periods of aglycemic anoxia are initially comparable to those of anoxia alone, but after 8–10 min there is another abrupt and dramatic rise in [Mg2+]i similar to that seen with complete metabolic blockade suggesting near-total MgATP depletion. Thus with concomitant loss of both oxygen and glucose endogenous energy reserves can partially defend neuronal ATP levels but only for a relatively short period of time. These changes in [Mg2+]i suggest that there may be two distinct phases to changes in neuronal energy metabolism in response to ischemia: the first occurring almost immediately upon oxygen depletion in which there is a fall in [MgATP]i, which is then maintained until anaerobic substrates are used up at which point the remaining MgATP is rapidly exhausted. In addition to changes in [Mg2+]i, there was a small, but very rapid, cellular acidification that suggests a transient generation of acid from within the cell upon loss of oxygen. We also observed that acidosis alone caused a small increase in [Mg2+]i. Because the effects of anoxia and extracellular acidosis on both cytosolic pH and [Mg2+]i are likely to be additive, true ischemic conditions might produce even more profound changes in [Mg2+]i and pHi. Although the time course of these events may well be different in vivo, even short periods of ischemia could produce the anoxic conditions necessary to initiate these events. Increased [Mg2+]i, acidosis, and ATP depletion are likely to affect many aspects of cell function, including ion channel activity, cell signaling, and ionic homeostasis. The functional consequences of these metabolic events for neuronal responses to ischemia therefore need to be investigated.
This work was supported by a grant from the Wellcome Trust (to K. J. Buckler) and a DFG research fellowship (HE3678/1-1 to M. Henrich).
We thank Dr. Robert Wilkins for his kind advice regarding ATP measurement and Dr. Mathias Dreger for the gift of the F11 cell line.
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.
- Copyright © 2008 the American Physiological Society

subset: commoncrawl


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Stephen G. Kliegerman
President of Development Marketing
Frances Lucy
West Side Office
As the last finishing touches are being put in place, 208 West 96th Street, the Upper West Side's newest contemporary boutique condominium, officially launches sales this week. This milestone marks a new and exciting sales phase for Halstead Property Development Marketing, LLC, the exclusive selling agent, who is poised to introduce the building, featuring modern architecture and meticulously thought out floor plans with a distinctive metal façade and is comprised of nine move-in ready three-bedroom, three-bath full floor homes. [For more sales information, please contact Frances Lucy at (212) 381-2374 or visit www.208w96.com.]
A mere stone's throw away from the newly-constructed 1,2,3 Subway stop and surrounded by vibrant shops, restaurants and grocery stores, 208 West 96th Street combines the livability and excitement of the Upper West Side with downtown style. Homes at 208 West 96th Street are being offered from $2,275,000 and up with a ten-year 421A Tax Abatement. Each home features solid teak flooring throughout, key locked elevator access opening into a private foyer, Juliet balcony in the living room and breathtaking double-paned floor-to-ceiling glass windows. The large, light filled Master Suite features an enormous dressing area and private balcony, while the second and third bedrooms offer generous proportions. Special units include a penthouse that features additional amenities including a gas fireplace in the living room as well as a private rooftop terrace and the 2nd floor home that boasts a large outdoor private garden.
The heart of the luxurious open layout is the ultra chic, state-of-the-art kitchen featuring a SubZero refrigerator, Viking hood and range, Bosch dishwasher, Sharp microwave drawer, wine refrigerator as well as a unique and clever central vacuum system with a floor-level crumb vent, which is the only one of its kind offered in a Manhattan new development. All of the superior appliances are showcased in custom cabinetry with a walk-in pantry and freestanding island with breakfast bar and sleek white Corian countertops.
Spa-like bathrooms feature penny tile walls with Ceasarstone quartz countertops that are offset by custom honey-toned cabinetry, creating a soothing environment. Toto sinks and commode as well as Axor and Kohler fixtures complete the suite. An additional highlight is the radiant heated porcelain floor, extra long Duravit soaking tub and enclosed glass shower in the windowed Master bath.
The lobby of 208 West 96th Street echoes the elegant contemporary architecture with an undulating wall that plays with light and creates a sense of space. Residents will enjoy the convenience of a Virtual Doorman and valet closets for packages as well as refrigeration for specialty deliveries. In addition, each home has a private storage room as well as a separate common bike storage. The common rooftop terrace is highlighted by dramatic views of Upper Manhattan, Central Park and the Hudson River. Perfect for entertaining, private rooftop cabanas equipped with water, electricity and gas are available for purchase.
"We are quite proud of 208 West 96th Street because it embraces our company's focus and attention to detail found in all of the properties we develop," explains Barry Gottehrer, President of Manor Properties Group. "Through keeping the highest standards combined with a firm understanding of our clientele, we will continue to create luxury homes on the Upper West Side and beyond."
Responsibly built, 208 West 96th Street incorporates green, forward thinking features into every aspect of living. All of the windows in each of the homes have Low-E coating for added efficiency. High-end appliances are Energy Star rated including washer, HVAC, refrigerator and dishwasher along with water-saving dual flush technology on commodes. Other Eco-friendly practices include low VOC paint and floor sealer for home interiors, rubber pavers made from recycled materials on the rooftop terrace and an Otis Gen2 motorless elevator.
[The complete terms are in an offering plan available from the Sponsor. File No. CD09-0349].
[The complete terms are in an offering plan available from the Sponsor. File No. CD09-0349].
About Manor Properties Group, LLC
Manor Properties Group, LLC is a New York City-based developer of quality housing. The company also develops, owns and manages a number of residential and commercial properties in the New York tri-state area. The core business philosophy is based on four basic principles: hire the experts, focus on service, attend to details, and work with the community. For over 30 years, this philosophy has become the hallmark of Manor Properties Group's success. Manor Properties Group's experience ranges from large scale commercial and industrial park to condominium construction and conversion. Current residential projects include The Dover, boutique condominiums comprised of two totally renovated brownstones located at 252 and 256 West 123rd Street, and 208 West 96th Street, boutique luxury condominiums featuring 9 floor-through homes in the heart of the Upper West Side. For more information on Manor Properties Group, please visit www.manorny.com.
About Halstead Property Development Marketing, LLC
Halstead Property Development Marketing, LLC is the exclusive sales and marketing firm for numerous new developments in the tri-state area. They consult with developers on every aspect of the development process, including: pre-development planning such as comprehensive market research, detailed unit mix and layout analysis and amenities planning, all of which enables their clients to maximize sales prices, increase absorption rates and provide an overall superior product benefiting the consumer and developer. In addition, the division directs and coordinates the selection of all on-site personnel, conducts extensive sales training and monitoring, designs exceptional sales environments, and creates integrated communications planning including marketing, advertising, public relations and branding. For more information on the Halstead Property Development Marketing Division, please visit:http://www.HalsteadPDM.com/
Tuesday, February 15, 2011
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Almodóvar's new film opens amid Spanish tumult
Pedro Almodóvar's film Bad Education made its debut in an opening that had been postponed after last week's terrorist bombings in Madrid. Almodóvar canceled the gala screening after Thursday's train bombings, which killed 201 people and injured more than 1,600. "We have been through a hard time of great pain, and so we decided to drop everything festive," he said at Tuesday's opening. Bad Education follows two boys who contend with sexual and psychological torment at a Roman Catholic school in the 1960s. "I'm getting more and more serious and possibly more and more sad," Almodóvar told a news conference, Spanish news agency Efe reported. "I'm aware of the change, but I don't know where it will take me. Both my school years and the 1980s left their mark on me, and that influence and the tone of the periods can be seen in the film--the '60s as dark years and the '80s full of color and freedom,'' he said. Bad Education will kick off the Cannes Film Festival in May.


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Three Iranian-Americans have left Tehran under a prisoner swap following the lifting of most international sanctions on Iran.
In a sign of sustained readiness to track Iranian compliance with remaining United Nations curbs, the United States imposed fresh sanctions on 11 companies and individuals for supplying Iran's ballistic missile programme.
The Obama administration imposed the new sanctions after delaying the action for more than two weeks during tense negotiations to free five US prisoners, according to sources.
Iran conducted a precision-guided ballistic missile test last October, violating a UN ban.
"This is a good day because once again we are seeing what's possible through strong American diplomacy," Barack Obama said at the White House.
"These things are a reminder of what we can achieve when we lead with strength and with wisdom."
"This is a good day. Once again, we're seeing what's possible with strong American diplomacy." —@POTUS #IranDeal https://t.co/yNV26XZmDQ
— The White House (@WhiteHouse) January 17, 2016
Iranian President Hassan Rouhani hailed the nuclear deal as a "golden page" in Iran's history and said the agreement could be used as a model to resolve other regional issues.
The lifting of sanctions and the prisoner deal considerably reduce the hostility between Tehran and Washington that has shaped the Middle East since Iran's Islamic Revolution of 1979.
A US official said a Swiss plane had left carrying Jason Rezaian, the Washington Post's Tehran bureau chief, Saeed Abedini, a pastor from Idaho and Amir Hekmati, a former Marine from Michigan, as well as some family members.
One more Iranian-American released under the same swap, Nosratollah Khosravi-Roodsari, was not aboard the aircraft. A fifth prisoner, American student Matthew Trevithick, was released separately yesterday, a US official said.
"We can confirm that our detained US citizens have been released and that those who wished to depart Iran have left," a senior US administration official said.
Several Iranian-Americans held in US prisons after being charged or convicted for sanctions violations have also been released, their lawyers said.
Speaking to parliament, Mr Rouhani, a pragmatist elected in 2013 on promises to end Iran's years of sanctions and isolation, said he looked forward to an economic future less dependent on oil exports.
These are nevertheless likely to jump now that the European Union, United States and UN have scrapped the sanctions in return for Tehran complying with the deal to curb its nuclear ambitions - ambitions that Tehran said were peaceful.
But Mr Rouhani noted bitter opposition to the lifting of economic curbs from Israel, some members of the US Congress and what he called "warmongers" in the region - an apparent reference to some of Iran's Gulf Arab adversaries, not least Saudi Arabia.
Presenting the draft budget for the next Iranian fiscal year, which begins in March, Mr Rouhani told parliament the deal was a "turning point" for the economy of Iran, a major oil producer which has been virtually shut out of international markets for the past five years.
He later said he expected 5% economic growth in the next Iranian fiscal year beginning in March and assured foreign investors of political and economic stability.
"The nuclear negotiations which succeeded by the guidance of the Supreme Leader and support of our nation, were truly a golden page in Iran's history," he said.
Tens of billions of dollars' worth of Iranian assets will now be unfrozen and global companies that have been barred from doing business there will be able to exploit a market hungry for everything from automobiles to airplane parts.
In The Hague, the US and Iran settled a longstanding claim, releasing to Tehran $400m in funds frozen since 1981 plus $1.3bn in interest, the US State Department said.
The funds were part of a trust fund once used by Iran to purchase military equipment from the US but which was tied up for decades in litigation at the Iran-US Claims Tribunal.
The settlement announcement was made after Tehran released the US detainees.
The nuclear deal has been widely welcomed across Europe.
Minister for Foreign Affairs Charlie Flanagan said: "I welcome the news that the International Atomic Energy Agency has verified the implementation of Iran's nuclear commitments under the Joint Comprehensive Plan of Action, in line with what was agreed last July.
"This is a critical landmark in the implementation of that agreement and EU sanctions will now be lifted.
"This agreement represents an important cooperative effort to deal with one source of tension in a region that already suffers from instability and conflict," added Mr Flanagan.


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BAKU, Azerbaijan, June 20 (UPI) -- The government in Turkmenistan said an Azeri border patrol broke the law by interrupting work in a disputed offshore field.
The foreign ministry in Turkmenistan accused the government of Azerbaijan, in a letter, of "unlawful actions" at a disputed offshore field in the Caspian Sea, the Russian news service RIA Novosti reports.
Turkmenistan and Azerbaijan haven't agreed on maritime borders since gaining independence from the Soviet Union in 1991.
Workers from Azerbaijan carried out an exploration program in the area in the 1980s.
Azeri authorities had reminded Turkmenistan that a 2008 agreement suspends exploration or development in the region until both sides determine maritime borders.
The field, called Kapaz on the Azeri side and Serdar in Turkmenistan, holds an estimated 580 million barrels of oil and 1.1 trillion cubic feet of natural gas.
Iran, meanwhile, said it discovered oil in the Caspian Sea in waters that might be claimed by Azerbaijan, the semiofficial Fars News Agency reports.


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[WOMEN'S WORLD CUP COUNTDOWN] Australia recalled its controversial WPS star Lisa de Vanna only a month after she was
kicked out of training for the Women's World Cup. The Matildas also selected Ellyse Perry, who has already won a World Cup -- in women's cricket -- and seven
De Vanna, who plays in WPS for magicJack, was kicked out of training camp when she refused to attend a photo shoot. Last year, she landed in hot water when a mother complained her daughter viewed photos of women using animal statues as props to simulate sexual acts on De Vanna's Facebook page
Perry, 20, was a member of Australia's 2010 Twenty20 championship team. "Having had that experience with cricket and also winning a World Cup I know how great that is and how enjoyable that is," she said.
Sermanni's youngest player is 16-year-old Caitlin Foord. The Australia squad also includes three 17-year-olds: Emily van Egmond, Sam Kerr and Teigen Allen.
Australia Roster:
Goalkeepers: Melissa Barbieri (Melbourne Victory); Casey Dumont (Brisbane Roar); Lydia Williams (Canberra United)
Defenders: Teigen Allen (Sydney FC); Laura Alleway (Brisbane Roar); Kim Carroll (Brisbane Roar); Elise Kellond-Knight (Brisbane Roar); Ellyse Perry (Canberra United); Clare Polkinghorne (Brisbane Roar); Servet Uzunlar (Sydney FC)
Midfielders: Tameka Butt (Brisbane Roar); Lauren Colthorpe (Brisbane Roar); Heather Garriock (LdB Malmo); Collette McCallum (Perth Glory); Sally Shipard (Canberra United); Emily van Egmond (Canberra United)
Forwards: Lisa De Vanna (Brisbane Roar); Caitlin Foord (Sydney FC); Samantha Kerr (Perth Glory); Leena Khamis (Sydney FC); Kyah Simon (Sydney FC)


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GSTAAD, Switzerland, July 8 (UPI) -- Fifth-seeded Andreas Seppi, the runner-up last year at the event, was defeated Tuesday in the first round of the Swiss Open tennis tournament.
Seppi was eliminated by qualifier Dominik, Meffert 6-2, 5-7, 6-4 on the second day of the clay-court tournament. Seppi was beaten by Paul-Henri Mathieu in last year's Swiss Open title match.
No. 6-seeded Nicolas Kiefer, the only other seeded player on the court Tuesday, advanced to the second round with a 6-3, 6-2 romp over Gianluca Naso.
Also Tuesday Stephane Bohli beat Robin Roshardt, 6-2, 4-6, 6-4; Victor Hanescu topped Olivier Patience, 7-6 (7-4), 6-2; Jeremy Chardy defeated Luis Horna, 7-6 (7-5), 6-7 (6-8), 7-5; and Flavio Cipolla rallied for a 5-7, 6-4, 6-4 win over Jiri Vanek.
Top-seeded Stanislas Wawrinka, playing in his home country, takes on Bohli Wednesday in the second round.


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Compiled by Arthur Paul Moser
The first settlement in the region that is now Dallas County was made on what is known as Buffalo Head Prairie. Just who was the first settler is a little obscure, though the claim that Mark Reynolds and family, natives of Tennessee, settled in 1832 on Buffalo Head Prairie, northwest a short distance from the Blue Mounds, is tolerably well authenticated.
Soon after Reynolds settled in the county, he placed a large buffalo head which he found on the prairie on a pole, where it remained for years as a way to mark for hunters and emigrants, and thus the prairie became known as Buffalo Head.
Soon after the Vanderford, Haines, Cox, Wright, Wilkerson and Gregg families from Ohio settled on land, and within the next few years there was a healthy increase of home seekers from Pennsylvania, New York and other states of both East and South.
The greater part of the territory now comprising Dallas County, in 1843, was organized into a county called Niangua, the name a corruption of the Indian word Nehemgar.
December 16, 1844, the boundaries of the county were slightly changed and an act passed providing that all that portion of the county, heretofore known as Niangua shall hereafter compose and be known as the County of Dallas in honor of Honorable George M. Dallas of Pennsylvania, then Vice President-elect of the United States. (--Encyclopedia of the History of Missouri, Conard, Vol. 2, p. 218.)
Dallas County was first settled in 1833 by Mark Reynolds and family who moved on from Polk County, where he located in 1831. In 1837 the Wilkinson family arrived and the same year a large number of immigrants built their cabins--under authority of the Acts of January 29, 1841, and February 15, 1841, the county was organized, and from that date the development of the territory may be said to date. (--A Reminiscent History of the Ozark Region, Goodspeed Bros., Chicago, 1894, p. 23.)
It was organized Dec. 10, 1844, as "Niangua County", and Dec. 16, 1844, the name was changed to Dallas in honor of George M. Dallas, of Pennsylvania, who had recently been elected Vice President. (--How Missouri Counties, Towns and Streams Were Named, second article, p. 83.) (David W. Eaton, in a series of articles in The Missouri Historical Review.)
Dallas County (Parent County, Polk). Organized 1842 as Niangua County; in 1844, boundaries slightly changed and name changed to Dallas; court-house burned Oct. 18, 1863; second court-house burned July 30, 1864, and records destroyed; the replaced records were burned Sept. 3, 1867. (--Fourth Edition of the Handy Book for Genealogists, Geo. Everton, Sr., p. 98.)
NOTE: Despite the belief held by some people, the report that the court-house was burned Aug. 11, 1861, cannot be verified.
The Evans, Randelman, Reynolds and Williams families from Kentucky settled in this county in 1837-38. They were soon followed by members of the Vandeford (other sources spell the name Vanderford), Haines, Cox, Gregg, and Wilkerson (other sources spell the name Wilkinson), families from Ohio, and later there were many settlers from New York, Pennsylvania, and the New England States.
A party of hunters, among them Samuel Grigsby, found on the prairie a skeleton head of a large buffalo, and placed it on a stake. It became a noted way-mark for hunters, travelers and immigrants, and gave to the surrounding region its name of Buffalo Head Prairie. (--Gazetteer of Missouri, Campbell, 1874, p. 183.)
The county was first settled in 1831, and was formed from Polk County. (--Campbell's New Atlas of Missouri, p. 57.)
A part of Dallas County came from Greene Co. (--Past and Present of Greene Co., Fairbanks and Tuck, Vol. 1, p. 123. Also, History of Greene County, Missouri, Holcombe, 1883, p. 157.)
Niangua County, 1841; changed in 1844 to Dallas County. (--Our Store-House of Missouri Place Names, Robert L. Ramsay, p. 6.)
Nine Missouri counties have Indian names. Dallas at one time was known as Niangua. (--Our Store-House of Missouri Place Names, Robert L. Ramsay, p. 39.)
Springfield-Greene County Library


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Tag Archives: Done
Rashard Mendenhall likely done in Pittsburgh
We mentioned yesterday that the Pittsburgh Steelers will likely redo their backfield for next season.
Even the Steelers' official website agrees with the idea that at least Rashard Mendenhall won't be back in the Steel City next season.
Mendenhall will be a free agent this offseason and after a disappointing season in which he came back fro…
Source: Yardbarker: Pittsburgh Steelers
Doug Legursky Says He Is Done Dealing With Ignorant People On Twitter
Pittsburgh Steelers offensive lineman Doug Legursky announced Saturday evening on Twitter that he is done with the social media platform due to his inability to deal with ignorant people.
Source: Yardbarker: Pittsburgh Steelers
Max Starks thinks he's done in Pittsburgh
On Sunday, tackle Max Starks will play his 123rd game for the Steelers since they made him their third-round pick in the 2004 draft. Starks believes that it will be his last as a member of the team. Starks will be a free agent after the season and told Mike Bires of the Beaver County…ProFootballTalk » Pittsburgh Steelers
Mike Wallace won't play Sunday, likely done in Pittsburgh
Pittsburgh Steelers wide receiver Mike Wallace won't play in Sunday's season finale against the Browns because of a hip/hamstring injury.
There's a good chance that Wallace has played his snap for the Steelers now that he's scheduled to become a free agent once the new league year begins in March.
I can't imagine Wallace returning to Pitt…
Source: Yardbarker: Pittsburgh Steelers
AFC North Roundup: Pull Out the Fork, They're Not Done Yet Edition
Source: Behind the Steel Curtain – All Posts
Steelers on track to overtake AFC North, but still much work to be done
One of the great things about the NFL is how things change from week to week. It wasn't that long ago that we were wondering if the Steelers could win back to back games at all this season and now they've accomplished that. The AFC North division is clearly up for grabs, as no team has asserted itself any longer. Injuries have played a part in …
Source: Yardbarker: Pittsburgh Steelers
Steelers get it done on the ground with a bunch of backups
It makes perfect sense. The league's second-worst rushing offense, missing four key parts, turned in their best rushing performances of the season. The Steelers outgained the Bengals 167-80 on the ground, with their top two running backs (Rashard Mendenhall and Isaac Redman) on the inactive list along with a pair of starting offensive linemen (Ma…
Source: ProFootballTalk » Pittsburgh Steelers
Steelers & Mike Wallace Can Now Work On Getting A New Long-term Contract Done By Opener
Pittsburgh Steelers wide receiver Mike Wallace is now in house and has signed his one-year, $ 2.74 million restricted tender. With that course of action now over, negotiations can now reopen and the two sides can continue to try to get a long term-deal hammered out prior to the season opener a week from this coming Sunday in Denver.
As I have point…
Source: Yardbarker: Pittsburgh Steelers
Need Steelers Tickets? Shop Ticket Liquidator or try our Steelers eBay Ticket Page.
Antonio Brown Happy to Get a Deal Done to Stay in Pittsburgh
It's a happy day in Steeler Nation, as Antonio Brown is going to be around for a long time to come after inking a new deal. Brown spoke to the media after Saturday practice, and seemed happy about the signing. "It's special, I'm with a unique organization as the Steelers. Knowing that I'm going to […]…
Source: Steelers Gab
Steelers get their deal done with David DeCastro
And then there were eight (unsigned first-round picks). After Detroit signed 23rd overall pick Riley Reiff this morning, the Steelers have followed suit, getting 24th pick David DeCastro signed, according to Ed Bouchette of the Pittsburgh Post-Gazette. The guard from Stanford will get a four-year deal worth $ 7.814 million, per the slotted system th…
Source: ProFootballTalk » Pittsburgh Steelers


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Morning class was fun, we did mock interviews for a live broadcast directing exercise. I played a dragon hunter who was being interviewed by Sarah Millican, who was played by one of my male classmates, fun fun fun.
In the afternoon I finished the main edit of the drama students show, all I have to do now is add titles, add credits, add one sound effect and export it. But I'll have to wait for the head of drama to provide me with the titles and credits before I can do it. Hopefully she'll get back to me tomorrow.


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Written on: 06/12/2008
Nice empolyees, really polite. I like the stuff but sometimes it seems that there is nothing there that i'm looking for.


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'Nanolab' runs real-time cell tests at molecular level
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San Jose, Calif. Researchers are tapping nanotechnology to deliver a device that could some day conduct as many as 2,000 tests on individual biological cells in real-time.
The so-called nanolab chip could leapfrog today's gene and protein microarrays to create a much faster, more granular device for diagnosis and drug screening applications.
The chip uses microfluidics to transport individual cells across the device, nanowire sensors less than 10 nanometers in diameter to identify genes and proteins in the cell and nanomechanical sensors to detect protein and gene interactions. The chip may be able to accomplish with a few cells and minutes of tests what today's microarrays need relatively large biological samples and hours or a day to achieve. Like other advanced projects, the researchers are applying microelectronics technology to medical diagnostics (see stories, page 26).
Researchers at the California Institute of Technology, UCLA and the Institute for Systems Biology (Seattle) are each building a part of the device. "We are at the stage of trying to integrate all these technologies together," said James R. Heath, a professor of chemistry at CalTech in Pasadena. "That turns out to be a significant challenge."
By year's end, the seven-member group hopes the device can identify a dozen protein or gene signatures in 20 minutes by integrating two or more of the device subsystems now in development. A year later, the group aims to detect as many as 50 signatures in less than a minute based on integrating three or more subsystems.
"Once we get there, we will be able to demonstrate it's possible to go to 1,000 to 2,000 signatures," said Heath.
"This brings molecular biology to reality," Heath said. "If you can do these tests in real-time using just one to five cells, it changes your ability to diagnose diseases and screen for new drugs."


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** Super Coolbie **
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Posts: 86582
TV actor Anas Rashid talks at length about television,fitness and his Gujarat connect,in an interview
Shruti Jambhekar
He weilded the sword with flourish in the historical show,Prithviraj Chauhan,played the romantic lead in the afternoon soap,Aise Karo Na Vida and is now back in another starring role in the prime time daily Diya Aur Baati Hum.Anas Rashid was in town to promote his show and got into the festive mode as he hopped over to light some diyas,burst a few crackers.Once the photoshoot wrapped up,Anas got talking shop.He admitted with a visible sigh of relief,It is a great feeling to be back on prime time after almost three years.I knew that Id be back with a bang and here Im !
In real life,Anas confesses that he relates upto 90 per cent with this screen character.The only difference is Im arrogant,while Sooraj is not like that. Anas,like most of his contemporaries had no plans to becoming an actor.I tried various things like a job in marketing,I owned a restaurant,then bought a gymnasium,an apparel boutique and lots more.Later on when friends insisted I try out modeling,I did a Punjabi music video.I shot lots of music videos and thought Id give TV a shot.Thats when I moved to Mumbai. He made his debut in Ekta Kapoors Kahiin Toh Hoga,where he essayed the role of Kartik Ahluwalia.Since then,theres no looking back for this hard-working actor.
As someone whos been part of the television industry for five years,says theres been tremendous change in television industry over the last few years.The economic slowdown dealt a big blow and top actors were sidelined for almost three years.Only now the good times are rolling as the soaps are made on huge budgets. He goes on to add,I too had to compromise on my salary when I was doing Aise Karo Na Vida,as the producers and channels talked of the soap being telecast on afternoon slot and I had to abide by it.I had no choice because as an actor its important to be visible,or else you lose out to competition.
What does he think of the small screens tilt towards small towns/ rural areas in terms of content.Well,majority of population in our country resides in small towns and villages.I come from a small town myself.Frankly speaking,when I did Kahiin Toh Hoga,it was tough to relate to a family having 300-400 crore business et al.At least now,common people can relate to the content on TV shows.
A fitness freak,Anas confesses,I am very conscious about my diet and exercise.Infact,I have reduced a lot for the current soap because post Aise Karo Na Vida,I had put on some on weight.
Anas whos been to Ahmedabad several times says with a smile,I have stopped counting the number of times I have visited Ahmedabad! I love this city for the immense love I get from the people here. It doesnt end here,Anas has a deep connection with Vadodara too,I stayed in Vadodara for two years when I was shooting Prithviraj Chauhan.I loved food,culture and a smalltown feeling it gave me,and of course,Barodians have showered a lot of love on me.I miss Vadodara so many times, he signs off.
Anas Rashid with co-actor Deepika Singh get in the festive mood
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The 'Yeh Un Dino Ki Baat Hai' actor talks about the show that ...
He certainly cannot contain his excitement..
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7 Things for ASC Industry Leaders to Know for Thursday
Here are seven things for ASC industry leaders to know for Thursday, March 28, 2013.
Institute of Medicine report suggests Medicare spending gaps are due to waste.
The IOM report "Geographic Variation in Health Care Spending and Promotion of High-Value Care – Interim Report" analyzed three decades of research and found disparities in Medicare spending that couldn't be accredited to patients' health status and age. Unnecessary spending and services could be the cause.
PPACA generates 111M hours of paperwork.
A study done by the American Action Forum has found that the Patient Protection and Affordable Care Act has created 111.4 million hours of paperwork. The AAF estimated that it would take 55,742 employees working 2,000 hours per year to complete the paperwork.
OIG issues fraud alert on physician-owned distributorships.
The HHS Office of Inspector General has issued a special fraud alert centered on specific elements of physician-owned distributorships. The OIG has named eight "suspect characteristics" of physician-owned entities.
Kaleida Health plans Buffalo ASC.
Kaleida Health plans to lease 20 percent of the Buffalo (N.Y.) Niagara Medical Campus and open an ambulatory surgery center in that space.
Raleigh Orthopaedic Surgery Center opens.
Rex Healthcare and Raleigh (N.C-based) Orthopaedic Clinic have announced the opening of the joint venture Raleigh Orthopaedic Surgery Center. The 27,707-square-foot center will offer outpatient, orthopedic services.
Helena SurgiCenter appoints Heather Lambott clinical director.
Helena (Mont.) SurgiCenter, a multispecialty ASC, has named Heather Lambott as its new clinical director.
AAOS approves first appropriate use criteria.
The American Academy of Orthopaedic Surgeons has approved its first appropriate use criteria, which concentrates on the treatment of distal radius fractures. This AUC covers 10 treatments and 216 common patient scenarios.
More Articles on ASC Issues:
8 Steps for Smooth ASC Patient Flow
An Evolving Industry: 6 Trends in Ambulatory Surgery Centers
Get Claims Out of the Door Faster: 6 Tips for ASC Revenue Cycle Improvement
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Welcome to Las Descargues
Our traditional Haute Quercy farmhouse gîte is situated in the hamlet of Las Descargues at the end of a warm, south-west facing ridge overlooking the Bave Valley between St Céré and Figeac in the Lot.
Built in 1748, some forty years before the French Revolution it's a house full of character and fascinating structures all retained and preserved during a sympathetic restoration of the property. There are large oak beams, weathered limestone and granite walls almost one metre thick, inglenook fireplaces you can sit in and a bread oven you can't.
The area is a paradise for the outdoor enthusiast and for those with an interest in natural history, with butterflies and moths, beetles, birds, flowers, in fact wildlife in profusion in a mosaic of unspoilt natural habitats.
We provide comfortable gîte accommodation for families seeking the freedom to do their own thing or we can structure a holiday to meet the needs of those with an interest in the wildlife of this beautiful part of France.


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I don't have any particular flavor or texture descriptors in mind to throw around. I found the tea fresh, bright, incredibly pure (one of my favorite aspects of EoT's pressings) and light. I thought the qi from this 2011 Nannuo was less immediate and capturing than any of the 2010 tea's I sampled.
It was good, but I agree with Hobbes, I don't believe it's US$72 good. I preferred the Mansai and that tea is ten bucks cheaper. Taking both 2011 teas into mind, I do think it's fair to say that the quality of these productions has increased. I preferred them both to the three 2010 examples I sampled. At this point, my interest in trying the single cake of the sold-out 2011 Mannuo I managed to acquire could not be much higher.


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Beautiful. I've done this one before but this time around I beat my last time. Thanks for sharing all your photos.
Beautiful horse, Laura!!
When I stopped and got out of the car, he/she got all excited and wanted to check me out. It was fun. Thanks, Lyndee, Pumpkin, Pat and Warbler.
It needs a carrot Laura... Another good photo!
Love the picture ... Beautiful horse
Horse. Horse posing for Laura.
I don't know a lot about horses, but she looks like a pretty nice looking one to me.


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Published: April 8 2006 03:00 Copyright The Financial Times
In 1926, Aldous Huxley reported that Shanghai was "life itself – dense, rank, richly clotted". Today, the adjectives – also once used for Cairo, Damascus, Bombay, Calcutta and Benares – evoke not so much a place as the prejudices and fears of the straw-hatted European traveller in the pre-war years – someone fastidiously upholding the aesthetic norms of his bourgeois civilisation that, unbeknown to him, was soon to go up in flames.
To such a traveller, defying the heat and dust in his white suit, the first sight of Shanghai's waterfront, the Bund, dominated by the customs house clock tower and the HSBC dome, or of Bombay heralded by the Gateway of India, was usually reassuring. Here, in the heart of the bewildering Orient, was something he could hold on to.
To be an Indian in Shanghai is to know a similar sensation of familiarity, if tinged with unease. It is also to be inevitably reminded of Bombay, the city most complicit with Shanghai in 19th-century inequity. Both port cities began to flourish after the British bullied China into opening up its markets to India-grown opium. The political and economic networks of British imperialism created a native class of comprador traders in the two cities, attracted to them a cosmopolitan cast of businessmen and adventurers and set them apart from their vast, steadily impoverished hinterlands.
During a recent stay I often gazed upon the Bund from a stylish new hotel in the Pudong. The architecture before me was more eclectic than that in colonial Bombay. It was also more pompous. But then the British abroad were always prone to self-aggrandisement in stone and their European rivals, trying hard to keep up, conjured even greater fantasies of grandeur.
The imposing solidity was once meant to awe the natives into obsequiousness. But things had changed dramatically in the last half century. The natives now not only "swarmed", as the European traveller might have said, in the buildings on the Bund, they had also erected their own grand monuments on the once-desolate mud flats of the Pudong. Still, as in Bombay, it was hard to appreciate the architecture, colonial as well as post-colonial, for its own sake. I couldn't rid myself of the feeling that what I saw was a faÃade and that behind it lay another country and a history that still shaped, in significant ways, the present.
In 1921, Gandhi claimed that Bombay's big buildings hid "squalid poverty and dirt". He was referring to the dubious sources of the city's wealth. But it wasn't just the trade in contraband goods but a kind of institutionalized brutality and callousness that underpinned daily life in both cities. As Shanghai's great chronicler Lynn Pann describes it, in 1935 alone, the municipal corporation in Shanghai collected more than 5,000 corpses of poor people from the pavements of the International Settlement.
The British claim to represent civilised western values in India somewhat limited the potential for exploitation in Bombay and the deaths by starvation. But no such commitment to civilisation was deemed necessary in Shanghai, where modern capitalism assumed its most rapacious forms, and where an axis of gangsters, politicians and foreign businessmen effectively ruled the city until the communist takeover in 1949. Bombay had its sadistic police officers but there were more of them in Shanghai, where Sikh policemen imported from India were always ready to fire upon unruly Chinese.
Chinese and Indian thinkers and activists in the early 20th century could see that their richest and most glamorous cities were incompatible with nationalist pride. In 1921, while exhorting Indians to economic self-sufficiency and a ban on foreign imports, Gandhi asked Bombay to be "ready to lose what she has". The same year the Communist party of China was born in Shanghai and began to feed upon the rage and frustration of the labouring poor and the anti-imperialism of the enlightened middle class. Not surprisingly, development on the socialist model, and the eradication of poverty in the countryside, preoccupied the post-colonial elites of India and China.
Consequently, both Shanghai and Bombay lost much of their power and prestige to inland, bureaucratic cities such as Beijing and Delhi, and had to wait many decades for the ideological moment when the creation of wealth in the cities was deemed more important than the removal of poverty. In recent years, a new form of globalisation, in which India and China are no longer subject countries but players on an apparently levelled field, has revived both Bombay and Shanghai. Originally created by global capitalism, neither metropolis has needed to undergo the brutal and traumatic modernisation suffered recently by such cities as Beijing, Xian, Bangalore and Delhi. However, the national experience of the past half century has given a markedly different character to the two cities once bound by empire.
In previous decades, historical amnesia and real-estate sharks helped destroy many of Bombay's fine old buildings. Shanghai, on the other hand, was a beneficiary of the odd communist feeling for the past that also preserved Prague and helped rebuild Warsaw. Many of its European-era buildings have survived. Wearing the distinguished mantle of old age and decay, they now face their greatest danger from developers wishing to fill their grand spaces with the sterile sparkle of shopping malls.
In post-Mao China, Shanghai quickly regained its role as the engine of Chinese modernity. It took an early lead in this regard over Bombay, India's window on the west. Visiting the city in 1983, soon after Deng Xiaoping launched his economic reforms, Jan Morris complained of inefficiency, drabness and a general aesthetic collapse. Nothing was further from my own experience this autumn with hotels, restaurants, taxis, public parks, and museums in Shanghai, which make the power and wealth of the new Chinese civilisation appear an undeniable reality.
Jinmao Tower looms gracefully on the Pudong and already seems to possess the solidity and iconic status of the Rockefeller Center in New York. The shiny cathedrals of consumerism on Nanjing and Huaihai Road, and the boutiques and cafÈs of Xintiandi persuade more quickly than the shopping malls of suburban Bombay that they are here to stay. Shanghai's film studios produce cinema of arguably greater quality than those of Bollywood. New art galleries and nightclubs open almost every week. Modern Shanghai has its own trendy writers, if not of the stature of Lu Xun, Mao Dun and Eileen Chang, and they prefer to explore the fresh possibilities of individual freedom – sex, drugs and rock music – available in the metropolis rather than recount a painful recent history of arbitrariness and destitution in the countryside.
The two cities also deal differently with their hinterlands. Bombay has many empowered ethnic and religious xenophobes, often supported by its affluent classes, but they cannot keep at bay the destitute and hopeful immigrants from the rest of India. Together they contribute to the squalor of the city, which is as obvious now as it was during Gandhi's time, although it also speaks of a messily democratic country where slum-dwellers form a sizeable electoral base no politician dares lose. In a throwback to Shanghai in the 1930s, a kind of mafia capitalism still flourishes in Bombay. Gangsters, politicians and businessmen together rule the city. In the recent past, their in-fighting has plunged large neighbourhoods into violence.
Shanghai, on the other hand, seems more orderly and remarkably free of poor people. One day on the Bund I found a beggar – the only one I saw in several walks around the city – and he was so melodramatically seedy that I half-wondered if he had been put there by the tourist board as a reminder of the city's sordid imperial past.
The neon lights of Pudong skyscrapers throb luridly at night, making the "peaceful rise" of China appear, apart from everything else, an occasion for lovers of kitsch. But things are not so peaceful behind the glittering surfaces. The soil is subsiding in newly built-up Pudong; chemical poisons contaminate river waters elsewhere in China; and aggrieved peasants hold hundreds of demonstrations every week.
None of this seems to worry the hundreds of thousands of Chinese cheerfully moving through the shopping malls and the waterfront park in Shanghai. One feels in these great crowds, overwhelming even to an Indian, not so much life itself, dense, rank or clotting, as the poignancy of the desires of the Chinese people for a better life. It is always a shock to remember the immense suffering China has known in the previous century and it seems petty to begrudge the Chinese shoppers a bit of consumerist self-indulgence.
But, watching the old waterfront, or the lights of Pudong from the terrace at M on the Bund, you still feel the presence of an even greater and much more restless Chinese mass in the inscrutable countryside, cruelly shut out from the new urban prosperity their labour and taxes have contributed so much to. It is hard not to wonder if it is always true, in unfree and free nations alike, that, as Walter Benjamin put it, every step towards civilisation is also a step towards barbarism.
Pankaj Mishra is the author of the acclaimed 'An End to Suffering: The Buddha in the World'. He was a guest of the Pudong Shangri-La in Shanghai
Pudong Shangri-La, 33 Fu Cheng Road, Shanghai, tel: +86 21-6882 8888


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Picked up a set of Lidl ergo grips last year and they came with some really comfortable plastic bar ends that look just like these:
and many other similar ones on ebay. I want another set for another bike but not seen them in Lidl since and wondered if anyone had seen any? They are solid thermoplastic and I modify them slightly so they can replace one of my ODI lock on collars but I won't/can't do the mod to aluminium or carbon clamp ones.


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Friends of the Library monthly meeting
- When: Tuesday, October 9, 2012, noon
- Where: Hood River Library, Hood River
- Cost: Not available
- Age limit: Not available
- Categories: Gatherings, Community meeting
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