From the Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire.
Xiujun Zhang, Qing Feng, Rick H. Cote; Efficacy and Selectivity of Phosphodiesterase-Targeted Drugs in Inhibiting Photoreceptor Phosphodiesterase (PDE6) in Retinal Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2005;46(9):3060-3066. doi: 10.1167/iovs.05-0257.
purpose. Phosphodiesterase (PDE) inhibitors are important therapeutic agents, but their effects on photoreceptor PDE (PDE6) and photoreceptor cells are poorly understood. The potency and selectivity of various classes of PDE inhibitors on purified rod and cone PDE6 and on intact rod outer segments (ROS) were characterized.
methods. The inhibition constant (K i) of isozyme-selective PDE inhibitors was determined for purified rod and cone PDE6. Perturbations of cGMP levels in isolated ROS suspensions by PDE inhibitors were quantitated by a cGMP enzyme-linked immunoassay.
results. Most PDE5-selective inhibitors were excellent PDE6 inhibitors. Vardenafil, a potent PDE5 inhibitor (K i = 0.2 nM), was the most potent PDE6 inhibitor tested (K i = 0.7 nM). Zaprinast was the only drug that inhibited PDE6 more potently than did PDE5. PDE1-selective inhibitors were equally effective in inhibiting PDE6. In intact ROS, PDE inhibitors elevated cGMP levels, but none fully inhibited PDE6. Their potency for elevating cGMP levels in ROS was much lower than their ability to inhibit the purified enzyme. Competition between PDE5/6-selective drugs and the inhibitory γ-subunit for the active site of PDE6 is proposed to reduce the effectiveness of drugs at the enzyme-active site.
conclusions. Several classes of PDE inhibitors inhibit PDE6 equally as well as the PDE family to which they are targeted. In intact ROS, high PDE6 concentrations, binding of the γ-subunit to the active site, and calcium feedback mechanisms attenuate the effectiveness of PDE inhibitors to inhibit PDE6 and disrupt the cGMP signaling pathway during visual transduction.
Visual transduction is mediated by complex biochemical pathways that precisely regulate cGMP levels in the signal-transducing outer segment portion of vertebrate retinal photoreceptors (for reviews, see Refs. 1 2 3 ). Disruptions of cGMP metabolism in retinal photoreceptors have serious consequences for visual functioning. Most genetic mutations that are correlated with retinitis pigmentosa and related diseases are found in genes coding for proteins of the phototransduction cascade, including photoreceptor phosphodiesterase (PDE6), guanylate cyclase (GC), and their associated regulatory subunits. 4 5 In many such cases, persistent elevation of cGMP concentration in retinal photoreceptors results in disruption of retinal development and/or photoreceptor apoptosis.
Remarkably little is known of the effects of PDE5-selective and other family-specific drugs on PDE6 and on cGMP metabolism in photoreceptors. Preclinical and clinical data on the effects of sildenafil have revealed significant but transitory effects on visual function, presumably through inhibition of photoreceptor PDE6. 12 Tadalafil and vardenafil, two other approved drugs, show lesser effects on visual function. 13 No systematic research of purified rod and cone PDE6 inhibition by various classes of PDE-selective inhibitors has been published in the literature, and only isolated data exist on potential differences between inhibition of the rod and cone PDE6 isozymes. 14 15 Electrophysiological measurements of individual rod photoreceptors exposed to IBMX (3-isobutyl-1-methylxanthine) 16 17 or zaprinast 18 demonstrate direct effects of PDE6 inhibition on the light responsiveness of rod photoreceptors, consistent with a drug-induced elevation in cGMP content. 19 Effects of PDE inhibitors on the electroretinogram or on human psychophysical measurements of visual function (reviewed in Ref. 12 ) are also consistent with direct inhibition of PDE6 in rods and cones.
In this study, we surveyed the potency and selectivity of PDE inhibitors targeted to PDE1 through -5, to inhibit purified rod and cone PDE6. We also examined the effects of nonselective and PDE5/6-selective inhibitors on cGMP concentration in the signal-transducing outer segment of rod photoreceptors. We found a general lack of discrimination of rod and cone photoreceptor PDE6 with respect to most drugs that have been designed to target a specific PDE family. However, in terms of the effectiveness of PDE inhibitors to elevate cGMP levels in intact ROS, we identified several mechanisms that oppose and minimize the ability of PDE inhibitors to disrupt cGMP metabolism in photoreceptor cells.
Frogs (Rana catesbeiana) were obtained from Charles Sullivan, Inc., and kept in controlled lighting conditions (12 hours dark–light) for 2 weeks before use. Animals were treated in accordance with ARVO guidelines, and protocols were approved by the institutional animal care and use committee. Bovine retinas were purchased from W. L. Lawson, Inc. (Lincoln, NE); E4021 was a gift from Eisai Co., Ltd. (Tokyo, Japan); vardenafil was provided by Bayer Pharmaceuticals; and sildenafil and tadalafil were synthesized. All other reagents were from Sigma-Aldrich (St. Louis, MO). All PDE inhibitors were prepared as stock solutions in DMSO and diluted in buffer before use, so that the final concentration of DMSO was always <1%. Ringer's solution consisted of (in mM): 105 NaCl, 2 KCl, 2 MgCl2, 1 CaCl2, 5 glucose, and 10 HEPES (pH 7.5). The ROS homogenization buffer contained (in mM): 100 Tris (pH 7.5), 10 MgCl2, 0.5 EDTA, 1 dithiothreitol, 0.5 mg/mL BSA, and mammalian protease inhibitor cocktail (Sigma-Aldrich). PDE assay buffer contained 20 mM Tris (pH 7.5), 10 mM MgCl2, and 0.5 mg/mL BSA.
Membrane-associated bovine rod and soluble cone PDE6 was purified from frozen bovine retinas exactly as described recently. 20 Activation of rod and cone PDE6 was performed by limited trypsin proteolysis of the inhibitory γ-subunit. 20 Rod or cone PDE6 was incubated with each drug for 15 minutes at room temperature before addition of the substrate. PDE activity was measured by either a phosphate release microplate assay (2 mM cGMP, 0.2 nM PDE6) or by a radiotracer assay (1.0 μM cGMP, 2.0 pM PDE6); 21 similar K i values were obtained with both assays.
The concentration of rhodopsin in ROS suspensions was determined spectrophotometrically. 25 To buffer the intracellular free calcium concentration of ROS at their dark-adapted level (∼500 nM for amphibian ROS 26 ), intact ROS were incubated in Ringer's supplemented with 1.09 mM EGTA for 10 minutes before addition of a PDE inhibitor.
cGMP was extracted by quenching with 50% HCl/ethanol, followed by centrifugation. The acidic supernatant containing cGMP was dried in a vacuum concentrator. The cGMP concentration was determined by cGMP enzyme-linked immunoassay (Amersham Pharmacia Biotech) with reference to standards treated identically to the experimental samples.
K i was calculated from the sigmoidal concentration dependence curve, with the following equation 29 : K i = IC50/(1+[S]/K m), where IC50 is the concentration of inhibitor that reduces catalytic activity in vitro by 50%, S is the substrate concentration, and K m is the Michaelis constant. The following K ms were used: 14 μM for purified bovine rod PDE6, 30 7 μM for purified bovine cone PDE6 (Valeriani BA, Cote RH, unpublished data, 2004), and 20 and 60 μM for activated or nonactivated frog PDE6, respectively. 31 All experiments were repeated at least three times, and average results are reported as the mean ± SD. Curve fitting was performed with the computer program SigmaPlot (SPSS, Inc., Chicago, IL).
We first tested a set of nonspecific and family-specific PDE inhibitors for their ability to inhibit purified PDE6. Both rod and cone PDE6 was tested in the activated state, in which the inhibitory γ-subunit is absent. Dose–response curves were generated for each inhibitor, and the drug inhibition constant (K i) was calculated based on the IC50 and knowledge of the K m for each enzyme.
Table 1summarizes the results of testing 15 PDE inhibitors that represent both nonspecific inhibitors (e.g., IBMX) and class-specific inhibitors of PDE1 through PDE5. (Family-specific inhibitors of PDE7 through PDE11 are not currently available.) We found that inhibitors of PDE3 and PDE4 were much less potent in inhibiting rod or cone PDE6 than their own PDE families. The selectivity (defined as the ratio of inhibition constants for PDE6 versus PDE-X) ranged from 40 to 50 (for the PDE4 inhibitor rolipram) to ∼300 (for the PDE3 inhibitor cilostamide) and up to 5700 for the PDE4 inhibitor YM976. The PDE2 inhibitor EHNA showed low potency and a modest ability to discriminate PDE2 from PDE6. In contrast, both PDE1 inhibitors tested (8-methoxymethyl-IBMX and vinpocetine) did not discriminate PDE1 from rod PDE6. 8-Methoxymethyl-IBMX showed a 10-fold selectivity for cone PDE6 compared with PDE1, although in neither case was the drug very potent (cone PDE6 K i = 0.4 μM). We conclude that vinpocetine and 8-methoxymethyl-IBMX are more accurately defined as PDE1/6-specific inhibitors.
Most of the so-called PDE5-selective inhibitors tested in this study should be considered PDE5/6 inhibitors (Table 1) . Whereas vardenafil (K i < 1 nM) is the most potent inhibitor of PDE6, it showed only threefold selectivity or less for PDE5 over PDE6. Both E4021 and sildenafil are potent PDE6 inhibitors (K i ≤ 10 nM), but also lack discrimination of PDE5 versus PDE6. Only tadalafil (selectivity ratio of 210 [cone PDE6] or 640 [rod PDE6]) and T-1032 (selectivity ratio, 20–60) represent authentic PDE5-selective inhibitors. Zaprinast, a first generation PDE5-targeted inhibitor (which also weakly inhibits PDE1 with a 10-fold higher K i), has 10-fold higher potency for PDE6 than for PDE5 (Table 1)and should be considered a "PDE6-selective" inhibitor.
When examining pharmacologic differences between rod and cone PDE6, we found that 8-methoxymethyl-IBMX and its parent compound, IBMX, showed a three to fourfold preference for inhibiting cone PDE6 compared to the rod isozyme. None of the inhibitors tested in Table 1preferred binding to rod PDE6 compared with cone PDE6.
Based on a similar report on the effects of IBMX on the cGMP content of rabbit retina where almost equal reductions in the rates of cGMP hydrolysis and synthesis were observed, 32 it seemed likely that inhibition of guanylate cyclase compensates for the inhibitory effects of IBMX on PDE6 in frog ROS in Figure 1 . Because IBMX is known to elevate internal calcium concentrations in ROS 33 which would then inhibit the calcium-sensitive guanylate cyclase, we decided to "clamp" the free calcium concentration of ROS at its dark-adapted level of ∼500 nM. By itself, buffering the calcium concentration to 500 nM in the Ringer's solution elevated cGMP levels by ∼50% (Fig. 1) , consistent with an earlier study. 34 However, when preincubation with a calcium-buffered Ringer's was combined with treatment with 400 μM IBMX, we observed a fivefold elevation of cGMP concentration in frog ROS (Fig. 1) . Thus, IBMX inhibition of PDE6 in intact, dark-adapted ROS resulted in the elevation of cellular cGMP concentration when guanylate cyclase activity was held constant by buffering free calcium. To isolate the effect of PDE5/6 inhibitors on PDE6 in intact ROS, the remaining experiments were performed in buffered calcium conditions.
To see whether high-potency PDE5/6 inhibitors behave differently from IBMX, we compared the time course of cGMP elevation when sildenafil, vardenafil, or IBMX were incubated with ROS maintained in a 500 nM calcium-Ringer's solution. Figure 2shows that cGMP levels in ROS reached an elevated, stable plateau within 2 minutes after exposure to the PDE inhibitor. The relatively slow action of PDE inhibitors on cGMP metabolism in intact ROS contrasts with the instantaneous inhibition of purified, activated PDE6 by these drugs in vitro (data not shown), and the rapid (<1 second) inactivation of PDE6 inferred in electrophysiological recordings. 35 The new steady state level of cGMP after drug exposure suggests that PDE6 is not completely inhibited at the inhibitor concentrations tested in Figure 2 .
We next determined the dose–response relationship of IBMX, sildenafil, and vardenafil on dark-adapted ROS maintained in a calcium-buffered Ringer's solution. For all three inhibitors, the aqueous solubility limit of each drug was reached before a plateau in the cGMP stimulation could be observed (Fig. 3) . For 2.0 mM IBMX or 125 μM vardenafil, a fivefold elevation of cGMP levels was observed (Fig. 3A) , whereas 110 μM sildenafil induced a less than twofold increase in cGMP content in ROS (Fig. 3B) . Because no evidence of saturation was observed for these drugs, these concentrations represent a minimum estimate of the effective dose needed to produce a half-maximum change (EC50) in ROS cGMP levels.
The relative potency with which PDE inhibitors affected purified PDE6 catalysis in vitro (i.e., K i) compared with the perturbing cGMP levels in intact ROS (i.e., EC50) is difficult to quantify. However, for a first approximation, we can calculate the predicted IC50 for an inhibitor to block PDE6 activity in the presence of the cGMP concentration in dark-adapted ROS (50 μM), assuming no diffusional barriers. As shown in Table 2 , the predicted IC50 for IBMX (73 μM) is at least 14-fold lower than the minimum EC50 estimated in Figure 3 . In contrast, the predicted IC50 for vardenafil (140 nM) is at least 350-fold lower than the minimum EC50 for intact ROS.
In dark-adapted ROS, rod PDE6 exists in its nonactivated state in which the catalytic dimer (αβ) is inhibited by binding of two inhibitory γ-subunits (for review, see Ref. 36 ). Figure 4demonstrates that vardenafil's inhibitory potency for the PDE6 αβγ2 holoenzyme is reduced 12-fold compared with the activated PDE6 αβ catalytic dimer. In contrast, IBMX inhibition of PDE6 holoenzyme is reduced only threefold relative to activated PDE6. This discrepancy may be accounted for by competition between the γ-subunit and PDE inhibitor for a common binding site in the catalytic pocket (see the Discussion section).
To test this idea further, we examined the ability of IBMX, sildenafil, and vardenafil to compete at the active site, not only with cGMP but also with the γ-subunit. Figure 5shows that at low drug concentrations both vardenafil and sildenafil—but not IBMX—stimulated the PDE6 holoenzyme two- to threefold when 2 mM cGMP was used as the substrate. (This paradoxical stimulation of PDE6 αβγ2 activity at low drug concentrations was first observed with zaprinast 14 and E4021. 37 ) At higher drug concentrations, the expected inhibitory action of all three inhibitors was seen. Under identical conditions, activated PDE6 catalytic dimers (αβ) did not show stimulation of catalysis at low drug concentrations (data not shown). These results are consistent with high-affinity, bulkier PDE5/6 inhibitors competing with γ-subunit binding in the catalytic pocket, whereas the smaller IBMX molecule inhibits catalysis without greatly affecting γ-subunit binding (see the Discussion section).
A central issue in the clinical development of PDE inhibitor therapy is the specificity of the drug for the targeted PDE. Table 1is the first systematic analysis of the inhibition of rod and cone PDE6 by inhibitors that were designed to target PDE families 1 through 5. With the exception of PDE3- and PDE4-selective inhibitors and a few PDE5-selective inhibitors, most of the tested compounds in Table 1lacked selectivity (K i ratio < 10) for the PDE family they are intended to target. The 10-fold selectivity of zaprinast for PDE6 compared to PDE5 makes this compound the only "PDE6-selective" inhibitor that we tested. The high selectivity of the PDE5 inhibitor tadalafil for PDE5 over PDE6 bodes well for future drug development in which vision-impairing side effects resulting from PDE6 inhibition may be minimized further.
None of the compounds we tested showed major differences in their inhibition of rod and cone PDE6 isozymes, although two xanthine analogues (IBMX and 8-methoxymethyl-IBMX) showed three- to fourfold selectivity for cone PDE6. Differences in amino acid residues contacting these inhibitors and/or differences in conformation of the active site 38 may explain this preference of cone PDE6 for xanthine-based inhibitors.
The ability of PDE6 to bind to several different classes of PDE inhibitor may reflect the unique catalytic properties of the photoreceptor enzyme. Unlike the other 10 PDE families, PDE6 operates with very high catalytic efficiency for cGMP (k cat/K m = 4 × 108 M−1 · s−1; see Ref. 36 ). Although the low affinity of substrate (K m = 20 μM for cGMP) and the high catalytic constant (up to 8000 cGMP hydrolyzed per second) of PDE6 are ideally suited for the millisecond time-scale activation of PDE6 required for visual transduction, 2 these same properties may allow a wide variety of inhibitor compounds to enter the catalytic pocket and inhibit catalysis.
The observation that PDE inhibitors only modestly elevated cGMP levels in intact ROS unless the free calcium concentration was buffered (Fig. 1)can be explained as follows: Inhibition of PDE6 by drug entry into the outer segment transiently elevates cGMP levels. This causes cGMP-gated ion channels to open, allowing entry of sodium and calcium into the ROS. Elevation of intracellular calcium inhibits guanylate cyclase and offsets the reduced hydrolysis of cGMP by the inhibited PDE6, resulting in little or no increase in cGMP concentration. 32 On buffering free calcium in the medium, this calcium feedback mechanism is blocked, allowing continued synthesis of cGMP in conjunction with reduced cGMP hydrolysis, and a net increase in intracellular cGMP concentration.
The plateau in steady state cGMP levels after incubation of IBMX or PDE5/6-selective inhibitors with ROS suspensions (Fig. 2)was unexpected, because PDE6 inhibition in conjunction with continued cGMP synthesis by guanylate cyclase in the calcium-buffered Ringer's should lead to continual increases in intracellular cGMP concentration. Also unexpected was the failure of all tested PDE inhibitors to show saturation behavior when cGMP levels in ROS were assayed as a function of inhibitor concentration (Fig. 3) . In contrast, both nonactivated and activated PDE6 in vitro displayed stereotypical dose–response behavior with IBMX or vardenafil (Fig. 4) . Assaying PDE6 inhibition in situ in intact ROS (Fig. 3)must take into consideration the very high PDE6 concentration in the outer segment of rod photoreceptors (40 μM catalytic subunit concentration in frog rods 39 ). Although the high PDE6 concentration associated with the outer segment membranes facilitates the single-photon sensitivity of photoreceptors, 40 it also requires very high drug concentrations to stoichiometrically inhibit PDE6 in ROS. (For example, in Figure 3the highest tested concentrations of sildenafil and vardenafil are only approximately threefold greater than the PDE6 subunit concentration in ROS.) In summary, the unexpected behavior illustrated in Figures 2 and 3can be accounted for if some of the PDE6 in intact ROS cannot be inhibited under our experimental conditions.
In the case of the closely related PDE5 family of phosphodiesterases, allosteric activation of PDE5 by cGMP binding to the GAF domain enhances the binding affinity of sildenafil, tadalafil, and vardenafil. 41 42 Figure 4demonstrates a parallel phenomenon for PDE6, wherein activated PDE6 catalytic dimer (αβ) was more potently inhibited by PDE inhibitors than the nonactivated holoenzyme (αβγ2). This effect was more pronounced for vardenafil (Fig. 4A)than for IBMX (Fig. 4B) . Furthermore, low concentrations of sildenafil or vardenafil—but not IBMX—can cause an apparent stimulation of nonactivated PDE6 when the cGMP substrate concentrations are very high (Fig. 5) . This latter effect is probably due to mutually exclusive competition between the γ-subunit, drug, and substrate for a common binding site in the catalytic pocket, in conjunction with an ∼1000-fold greater affinity of γ-subunit (K d ∼ pM 43 44 ) than of drug (K i ∼ nM; Tables 1 2 ) for the active site.
These results have important implications for the architecture of the catalytic site of PDE6. IBMX, a xanthine derivative, has been shown to occupy a subpocket within the active site of PDE5 normally occupied by the guanine ring of cGMP 38 and exhibits many of the same interactions observed with the authentic substrate. Although IBMX, sildenafil, and vardenafil all share contact points in this region, the two PDE5/6-selective inhibitors also contact the catalytic pocket of PDE5 at additional sites that account for the 1000-fold higher affinity of these drugs. 45 Many of these PDE5 contacts interacting with the ethoxyphenyl and methoxypiperazine groups of sildenafil are close to amino acids that in PDE6 have been implicated in γ-subunit binding to the catalytic domain. 46 47 If the bulkier PDE5/6-selective inhibitors overlap in their binding sites with the γ-subunit interaction sites in the catalytic pocket of PDE6, it would explain the mutually exclusive competition of γ-subunit and drug that weakened the affinity of these drugs for nonactivated PDE6 (Fig. 4)and led to the paradoxical activation of PDE6 holoenzyme at low drug concentrations (Fig. 5) .
In summary, when assessing the clinical efficacy of PDE inhibitor therapy for treatment of human diseases, the following effects on photoreceptor PDE6 must be considered: the ability of systemically administered PDE inhibitors to cross the blood–retinal barrier to reach the photoreceptors (not addressed in this study); the intrinsic pharmacologic selectivity of the drug for PDE6 inhibition; the unique cellular context of the rod outer segment in which PDE6 resides; the state of activation of PDE6 (and hence its competition with the γ-subunit); and the interrelatedness of cGMP and calcium metabolism in visual signaling. Regarding calcium, the fact that elevation of cGMP levels by PDE inhibitors increases intracellular calcium concentration in photoreceptors may have serious consequences, considering that photoreceptor apoptosis and retinal degeneration are believed to result from sustained cGMP elevation and/or calcium overload. 48 49 For these reasons, administration of PDE inhibitors for novel therapeutic uses must be evaluated for potential adverse effects on photoreceptor viability as well as visual function.
Supported by National Eye Institute Grant EY05798, Bayer Healthcare AG, and the New Hampshire Agricultural Experiment Station, Scientific Contribution 2259.
Submitted for publication February 24, 2005; revised March 31, 2005; accepted April 6, 2005.
Corresponding author: Rick H. Cote, Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824; rick.cote@unh.edu.
* Average of K i values for each family of PDE obtained from the literature. IBMX is nonselective (NS).
where the IC50 was obtained from the dose–response curve for each inhibitor for rod and cone PDE6 and the K m for bovine rod and cone PDE6 are 14 μM or 7.0 μM, respectively.
‡ Selectivity is defined as the ratio of the K i for the two indicated PDEs: 6R, rod PDE6; 6C, cone PDE6, and; X, PDE families 1 through 5.
§ Reported value is the IC50, because K i could not be calculated.
Synergistic effect of inhibiting PDE6 and buffering intracellular Ca2+ on cGMP levels in intact ROS. Purified ROS suspensions (rhodopsin, 4.8 μM) were prepared in complete darkness. Two portions were incubated with either 1.09 mM EGTA ([Ca2+]free, 500 nM; squares) or 400 μM IBMX (triangles). A third sample was first preincubated for 10 minutes with EGTA followed by addition of IBMX (circles). Portions were quenched at the indicated times with 50% HCl/ethanol. The cGMP concentration was determined by an enzyme-linked immunoadsorbent assay and reported relative to the rhodopsin content of the sample. Data points at the mean ± SD of results in three experiments.
* The K i values were determined as described in Table 1 , using nucleotide-depleted frog ROS homogenates to measure nonactivated (10 μM cGMP, 4.0 nM PDE6) or activated (1.0 μM cGMP, 20 pM PDE6) PDE6.
Time course of drug-induced increase in cGMP concentration in intact ROS. Purified ROS (0.008 mole cGMP per mole rhodopsin) were preincubated with 1.09 mM EGTA for 10 minutes, to buffer the free calcium concentration. At time 0, 400 μM IBMX (○), 50 μM sildenafil (▵), or 100 μM vardenafil (□) was added and the samples quenched in 50% HCl/ethanol for cGMP determinations. The results were normalized to the average maximum stimulation for each drug (units: moles cGMP per mole rhodopsin): IBMX, 0.040; vardenafil, 0.035; and sildenafil, 0.014. Data points are the mean of results in three experiments in which the SD was ≤13% of the mean.
PDE inhibitors induced dose-dependent elevations of ROS cGMP levels that do not saturate. Ca2+-buffered ROS suspensions (10-minute preincubation with 1.09 mM EGTA) were treated with the indicated concentration of IBMX (○), sildenafil (▵), or vardenafil (□) for 10 minutes and then acid quenched for cGMP extraction and quantitation. The data points are the mean ± SD of results in three experiments.
PDE6 holoenzyme was less susceptible to inhibition by PDE inhibitors. ROS suspensions were homogenized, and soluble proteins and metabolites were separated from PDE6-containing ROS membranes by centrifugation. The ROS membranes were further depleted of endogenous nucleoside triphosphates by incubation at 22°C for 30 minutes. Nonactivated PDE6 holoenzyme (4 nM; open symbols) was incubated with vardenafil (A) or IBMX (B) for 15 minutes before addition of 10 μM [3H]cGMP, to assay catalytic activity. Activated PDE6 (20 pM; filled symbols) was incubated with vardenafil (A) or IBMX (B) for 15 minutes before adding 1 μM [3H]cGMP. The results shown are typical of those in at least three other experiments.
PDE5/6-selective inhibitors, but not IBMX, stimulated catalysis at high cGMP concentrations. ROS membranes (2.0 nM PDE6 concentration) depleted of soluble proteins and nucleotides were incubated with IBMX (○), sildenafil (▵), or vardenafil (□) for 15 minutes. Catalytic activity was determined by a colorimetric assay with 2 mM cGMP. The data were normalized to the basal PDE6 activity for plotting and represent the mean ± SD (n = 3). *Statistically significant, P < 0.05.
The authors thank Bev Valeriani (University of New Hampshire) for providing the purified cone PDE6 used in some of the experiments.
PughEN, LambTD. Phototransduction in vertebrate rods and cones: molecular mechanisms of amplification, recovery and light adaptation.StavengaDG DeGripWJ PughEN eds. Molecular Mechanisms in Visual Transduction. 2000;183–255.Elsevier Science BV New York.
FrancisSH, TurkoIV, CorbinJD. Cyclic nucleotide phosphodiesterases: relating structure and function. Prog Nucleic Acids Res Mol Biol. 2001;65:1–52.
SchudtC, DentG, RabeKF. Phosphodiesterase Inhibitors. 1996;Academic Press New York.
RispoliG, GillespiePG, DetwilerPB. Comparative effects of phosphodiesterase inhibitors on detached rod outer segment function.BorsellinoA CervettoL TorreV eds. Sensory Transduction. 1990;157–167.Plenum Press New York.
PentiaDC, HosierS, CollupyRA, ValerianiBA, CoteRH. Purification of PDE6 isozymes from mammalian retina. Methods Mol Biol. .In press.
CoteRH. Structure, function, and regulation of photoreceptor phosphodiesterase (PDE6).BradshawRA DennisEA eds. Handbook of Cell Signaling. 2003;453–457.Academic Press San Diego.
Cosegregation of codon 807 mutation of the canine rod cGMP phosphodiesterase beta gene and rcd1.


Alan Sachs is the head of global research and development at Life Technologies. Previously, he was vice president of RNA Therapeutics for Merck Research Laboratories. He assumed leadership of Sirna Therapeutics in San Francisco and established the global RNA Therapeutics Department for Merck Research Laboratories effective January 4, 2007. Prior to this, Dr. Sachs assumed responsibility for the scientific leadership at Rosetta Inpharmatics LLC effective July 2002, responsibility for the Rosetta site effective March, 2004, and overall responsibility for the Department of Molecular Profiling in 2006. Dr. Sachs joined Merck & Co., Inc. as Director of Clinical Genomics for MRL in July 2001.
Prior to joining Merck, Dr. Sachs was Associate Professor of Molecular and Cell Biology while at the University of California at Berkeley, and a Whitehead Institute Fellow at the Whitehead Institute in Cambridge. Dr. Sachs is one of the leading figures in the field of mRNA translation and regulation, and has made landmark contributions, particularly to the understanding of the role of the poly(A) tail in translation and mRNA stability.
Dr. Sachs graduated from Cornell University with a B.A. in Biochemistry; received his Ph.D. in Cell Biology at Stanford University; his M.D. from Stanford Medical School; and completed a post-doctoral fellowship in the Department of Biochemistry, Stanford University.


text:
Students inspired to play like champs
HAMILTON, ONT. - At Hamilton's St. Thomas More Catholic Secondary School, members of the senior soccer teams are learning more than just tactics, strategy and skills specific to their sport; they're learning how faith contributes to their game.
Published in Catholic Education
Students of other faiths thrive at Catholic school
PHOTO GALLERY: Students from other faiths are included at Brebeuf College, an all boys' Catholic high school in Toronto. See photos from the students' Holy Week pilgrimage to Rome.
This photo gallery is an example of what Digital Edition subscribers have access to. The Digital Edition features the same articles as the print edition PLUS colour photos, videos, audio clips and links. If you are not a subscriber, you can try a free subscription by going here:http://www.catholicregister.org/digitaledition
Toronto's Brebeuf College School has a vibrant faith life, and not only for Catholic students.
While attending a school of a different faith can be daunting, the experience has proven worthwhile for the school's Catholic and non-Catholic students alike.
Published in Youth Speak News
Nun has found her happy place
In Sr. Anna Chan's earlier career in retail management, she sought happiness through a love of fine clothes, shoes and bags.
"I was looking for happiness," said Chan, "but I was looking in the wrong place."
Published in Call to Service
Retreat and Renewal Centres
The Catholic Register is proud to present its annual feature on retreat & renewal centres which help the mind, body and soul.
You can browse the articles below and see all the ads on the Digital Edition. The original pull-out section appeared in the April 19, 2015 issue.
Retreats & Renewal Centres: Catholic Register Special Feature 2015
- Retreat will be a workout for body and soul, by Jean Ko Din, The Catholic Register
- Where strangers leave as friends, by Michael Chen, The Catholic Register
- Taking retreats online, by Jean Ko Din, The Catholic Register
- TD Ameritrade founder pours energy, resources into new retreat centre, by Joe Ruff
- Carmelite invokes Elijah at retreat for Pope and Curia, by Laura Ieraci
Published in About the Catholic Register
Retreat will be a workout for body and soul
It's not just our bodies that need a workout, our souls need training too.
That's what St. Paschal Baylon Church's young adult ministry will be doing this year for its annual retreat April 25-26.
Published in Features
Where strangers leave as friends
Earlier this year, I had a "dilemma." There were five retreats happening in the same month — some landing on the same weekends. Which retreat should I go on? There was a Chinese Catholic students' retreat, a quad university chaplaincies including my alma mater, one with the University of Toronto, another one on discernment and the silent retreat with the York University chaplaincy. In the end, I chose the days of discernment retreat. Funny enough, I ended up making a decision to go on a retreat to learn how to make better life decisions.
Taking retreats online
For the busy person who can't get away for a spiritual retreat weekend, all they may need is to turn on their computer.
Virtual retreats are a fairly new concept — a retreat conducted through online media, often based on a time and place of the individual's choosing.
TD Ameritrade founder pours energy, resources into new retreat centre
OMAHA, Neb. - Working to establish and build a business that would become one of the largest discount brokerages in the United States — TD Ameritrade — Joe Ricketts found some 20 years ago he was drifting away from the Catholic faith and values that formed him growing up in Nebraska City.
Carmelite invokes Elijah at retreat for Pope and Curia
VATICAN CITY - Just as the people they preach to must be reawakened to God, so to must the Church's pastors — right up to those holding the highest positions in the Church — a Carmelite priest told Pope Francis and members of the Curia at their recent retreat.
To be a Catholic man of the Church
I recently attended a men's retreat and got three surprises. First surprise, a men's and women's retreat were happening at the same venue. Second, no one would be allowed to talk because it was a silent retreat. (Okay, participants were allowed to talk but only during the talks and during Mass.) The third surprise, and most important, is what I learned about masculinity and the role that men play in the Church. We also learned about what it means to be a "husband to the Church."
Published in YSN: Speaking Out
Regis' Lenten retreat takes aim at busy youth
TORONTO - Everyone needs spiritual direction at some point in their life, said Sr. Mechtilde O'Mara.
Published in Canada: Toronto-GTA
Pope begins retreat, encourages prayer, fasting, charity
VATICAN CITY - Pope Benedict XVI asked Catholics for their prayers as he began his weeklong Lenten retreat Feb. 26.
Before reciting the Angelus prayer at midday with visitors in St. Peter's Square, he also prayed that all Catholics would "embrace the spirit of this holy season, through prayer, fasting and almsgiving."
Finding solitude amidst the silence on retreat
STAMFORD, CONN. - They say silence is golden. Well, silence is also tough. I learned just how challenging it is to be completely silent during a young women's retreat with the Sisters of Life in Stamford over the Feb. 10-12 weekend.
Going into the retreat, I knew there were going to be periods of silence. But I didn't quite know the full extent of it. Following our first talk of the weekend on the theme "Love Never Fails," the sisters informed us — to my dismay — we would start our silence after compline, or night prayer. We'd break the talking fast the next day with dinner at 6 p.m. About 20 hours, with talking only allowed within the context of confession and Mass.
Wake up to the Risen Son
I am not a morning person. I automatically hit the snooze button a couple of times before I finally drag myself out of bed. But during Lent I got a wake up call that I couldn't ignore.

redpajama_set_name: RedPajamaCommonCrawl


A unique event location located on the magnificently beautiful national heritage site at the Amsterdam North Sea Canal. With its many unique buildings on this historic terrain, this location is suited for every kind of event and the location is equipped with every comfort. These ingredients make Taets Art and Event park the ultimate location for a unique and inspirational conference.
Are you looking for accommodation close to the conference venue?
We have arranged a special lower price for accommodation at Inntel Hotels Amsterdam Zaandam.
Offer: Craft Deluxe Twin Bedroom - €140 per night for 1 person. Breakfast and the use of wellness facilities are included. Limited availability until January 12, 2018.


But did you know that this landmark is part of the Birmingham Museum of Art's permanent collection? In 1990, when the casting was complete and the figures permanently installed, the Museum agreed to take over the fountain's care, custody, and control, thus ensuring it would be available for future generations to enjoy.
As with all our other outdoor sculptures, The Storyteller needs to be examined and assessed twice yearly in order to determine if our maintenance protocol is keeping it stable and safe. Protecting it from the elements, like acid rain, and the occasional unintended harmful actions of fountain visitors, requires a strict program of examination, washing, and waxing.
Logistics, however, can be daunting when you're caring for a sculpture that sits squarely at the juncture of five major traffic arteries and serves as a gathering place and photographic backdrop for throngs of residents and visitors. In order to accomplish our treatment, we must first enlist the aid of the City of Birmingham Department of Public Works to drain and pressure wash the fountain's basin interior. As soon as the fountain is emptied, our art handlers quickly move into place to examine the sculptures up close to determine the efficacy of our treatment program and document current condition. After the exam is complete, they begin the process of carefully washing the figures with a specialized detergent and, if needed, removing any mineral deposits that may be deposited onto the surface. After the washing is completed, and the figures are dry (which doesn't take long in that unrelenting noon day sun!), they apply several protective coats of a clear or pigmented wax to all the surfaces. The wax saturates the surface and gives the patina a luminous, rich quality but, most importantly, it serves as a protective barrier against harmful substances such as dirt, grime, and acid rain. The fountain basin is then quickly refilled, providing once again a beautiful backdrop for those visitor photographs. In a few months the conservation process begins again because one chapter of The Storyteller's "never ending story" is the Museum's deep commitment to protecting this important work of art.


Built in the 1980s as a single-occupier corporate office, Tanfield's deep-plan floorplates and monolithic form made it unsuitable for letting to multiple tenants. BDP's design solution retained the best features of the existing structure, whilst driving a new day-lit atrium through the heart of the building to provide access to office suites formed by sub-division of the large floorplates. Together with new rooftop office accommodation, the expanded floor area is now suitable for a range of tenants in high quality office suites, entered from an impressive atrium, and benefiting from efficient modern services, lighting and infrastructure.
The site is dominated by the Administrative Offices for Standard Life Investments – Tanfield House. The building is located in Inverleith / Canonmills to the north of Edinburgh New Town. Bounding the site to the south is the Water of Leith. Bounding the site to the east are tenements, along Tanfield / Inverleith Row, with retail at ground floor.
Originally the site was home to Tanfield House, a fairly small property to the north of the site. In the early 19th century two rival gas companies, The Oil Gas Company and The Edinburgh Gas Company, developed the land. The Oil Gas Company failed in 1839 and their vacated premises became Tanfield Hall, where the first Assembly of the Free Church of Scotland was held in 1843. The Edinburgh Gas Company ceased trading in the 1930's having expanded across the river to the Glenogle Road site. These buildings combined with later additions were used for storage and industry until the early 1980's. Key elements included a wool store, bonded warehousing, and a printing & bookbinding works.
In 1991 Tanfield House for Standard Life Assurance Company (Standard Life Investments) by Michael Laird Partnership (now Michael Laird Architects) [link] opened. The offices with basement car park were built over the remains of underground gasometer tanks. As part of the Tanfield office development a major underground gas main that dissected the site was diverted towards the western perimeter. The unlisted façade of the Wool Store onto Inverleith Row was retained and the new Standard Life building stepped back from the street. In the early 21st century Standard Life decided to dispose of the site and its innovative building.
Tanfield House is located by the Water of Leith in the Inverleith Conservation area.
The Standard Life Data Centre (also known as the Podium Building) on the south bank of the Water of Leith was also developed by Michael Laird Partnership for Standard Life.
A competition was held for Tanfield in Spring 2005, organised by Standard Life through Jones lang La Salle.
The aspirations of City of Edinburgh Council are set out in the Planning Brief for Tanfield. New residential accommodation will be encouraged, in conjunction with other land uses if necessary to maintain the mixed use character of the locality. There may be a requirement for some replacement/retention of the existing Standard Life employment. New development within the Inverleith Conservation Area – designated 'outstanding' – shall be required to preserve or enhance its character and appearance. Any redevelopment should refer to the future of the Standard Life Data Centre. A pavilion building on Brandon Terrace may be acceptable.
Any redevelopment of the site other than for Class 4 use will need to be justified to CEC. The Tanfield site could be described as brownfield and its central location combined with the slow Edinburgh office market make residential logical, assisting the Edinburgh & Lothian Strategic Plan's aims to increase Edinburgh's housing stock.
Tanfield House is not listed (incl. retained Wool Store façade) nor is it in the Edinburgh World Heritage Site.


Saoirse Úna Ronan (/ˈsɜːrʃə ˈuːnə ˈroʊnən/ SUR-shə; born 12 Aprile 1994) is an Erse actress.
↑ Symkus, Ed (7 April 2011). "Saoirse Ronan gets physical as action heroine 'Hanna'". GateHouse News Service. The MetroWest Daily News. Archived frae the oreeginal on 22 June 2013. Retrieved 12 April 2011.
↑ Debruge, Peter (4 October 2007). "Saoirse Ronan Atonement actress creating Lovely buzz". Variety. Archived frae the oreeginal on 22 June 2013. Retrieved 13 December 2008.
↑ Jocelyn (28 March 2011). "[Exclusive Interview] Saoirse Ronan Talks Hanna (part 1)". Archived frae the oreeginal on 22 June 2013. Retrieved 28 April 2011.
This page wis last eeditit on 17 Mairch 2019, at 09:12.


SNOWBOARDER Magazine's Crewzin', back for it's second season, gives you a behind the scenes look at how some of the top film crews in the game get work done. This week is another update from the Technine crew during their 2011 Chicago trip. This episode stars Jonah Owen, Dylan Thompson, and Andrew Brewer.


text:
var test = require('tape');

var events = require('events');

function listener() {}
function listener2() {}

test('listeners with only one item', function (t) {
  var e1 = new events.EventEmitter();

  e1.on('foo', listener);
  var fooListeners = e1.listeners('foo');
  t.deepEqual(e1.listeners('foo'), [listener]);

  e1.removeAllListeners('foo');
  t.deepEqual(e1.listeners('foo'), []);
  t.deepEqual(fooListeners, [listener]);

  t.end();
});

test('listeners is a copy', function (t) {
  var e2 = new events.EventEmitter();

  e2.on('foo', listener);
  var e2ListenersCopy = e2.listeners('foo');
  t.deepEqual(e2ListenersCopy, [listener]);
  t.deepEqual(e2.listeners('foo'), [listener]);

  e2ListenersCopy.push(listener2);
  t.deepEqual(e2.listeners('foo'), [listener]);
  t.deepEqual(e2ListenersCopy, [listener, listener2]);

  t.end();
});

test('listeners with two items', function (t) {
  var e3 = new events.EventEmitter();

  e3.on('foo', listener);
  var e3ListenersCopy = e3.listeners('foo');
  e3.on('foo', listener2);
  t.deepEqual(e3.listeners('foo'), [listener, listener2]);
  t.deepEqual(e3ListenersCopy, [listener]);

  t.end();
});

redpajama_set_name: RedPajamaGithub


text:
Storck UK was founded in 1988, almost 30 years ago.
The company has grown continuously over the years, building up successful brands including Werther's Original, Bendicks and Toffifee.
The sales area covers 2 countries and approx. 69 million inhabitants.
Basingstoke, UK
Storck Worldwide
Learn more about Storck and our brands around the world.
Contact us for further information on a career at Storck.


Hi I have an homework which is I'm having hardtime to solve.
This would then be homogeneous in $a$ and $b$ (meaning the sum of the powers of $a$ and $b$ is constant in the numerator and denominator (separately)) so you can reduce it to an expression in just one variable: Factor out the highest power of $b$ in the numerator and the highest power of $b$ in the denominator, and let $x=\dfrac ab$.
And now you can apply long-division techniques, substituing $x$ back to $a$ and $b$ in the end.
This is assuming there is a typo and the way I fixed it above is correct. If there is no typo, then...I don't know.


text:
This privacy policy sets out how we, Graham Crowley, uses and protects any information that you provide to us.
We are committed to ensuring that your privacy is protected. Should we ask you to provide certain information by which you can be identified you can be assured that it will only be used in accordance with this privacy statement.
What type of information do we collect?
We receive, collect and store any information you enter on our website or provide to us in any other way. In addition, we collect the Internet protocol (IP) address used to connect your computer to the Internet; Where there are login accounts we collect login; e-mail address; password; computer and connection information and purchase history. We may use software tools to measure and collect session information, including page response times, length of visits to certain pages, page interaction information, and methods used to browse away from the page. Where e-commerce elements exist we also collect personally identifiable information (including name, email, password, communications); payment details (including credit card information), comments, feedback, product reviews, recommendations, and personal profile.
When you conduct a transaction on our website, or complete sections to request information or to contact us, as part of the process, we collect personal information you give us such as your name, address and email address. Your personal information will be used for the specific reasons stated within this policy.
To provide you with ongoing customer assistance
To be able to contact our Visitors and Users with general or personalised service-related notices and promotional messages;
To create aggregated statistical data and other aggregated and/or inferred Non-personal Information, which we or our business partners, including Attain Solutions Ltd, may use to provide and improve our respective services;
How do we store, use, share and disclose your site visitors' personal information?
Our company is hosted on the Wix.com platform. Wix.com provides us with the online platform that allows us to sell and promote our products and services to you. Your data may be stored through Wix.com's data storage, databases and the general Wix.com applications. They store your data on secure servers behind a firewall.
Where applied all direct payment gateways offered by Wix.com and used by our company adhere to the standards set by PCI-DSS as managed by the PCI Security Standards Council, which is a joint effort of brands like Visa, MasterCard, American Express and Discover. PCI-DSS requirements help ensure the secure handling of credit card information by our store and its service providers.
How long do we keep the information for?
We will retain data from Customers that have purchased items from us for period which covers the term of any warranty.
Other data will be held for a period until either data is cleansed or is requested to be removed
How do we communicate with you?
How do we use cookies and other tracking tools?
Our website uses tracking cookies (see below) – we use this information to provide data on how visitors use our website. This information is accessed primarily via Google Analytics and Google Search Console. We track this information to make improvements to the website, user experience and to analyse the activity on each area of our site.
Where digital marketing activity is used we will use cookies to retarget the site visitors using social media platforms, Adwords and similar platforms. The purpose is to target messages to visitors that have shown an interest in the product or services.
Our website may contain links to other websites of interest. However, once you have used these links to leave our site, you should note that we do not have any control over that other website. Therefore, we cannot be responsible for the protection and privacy of any information which you provide whilst visiting such sites and such sites are not governed by this privacy policy statement. You should exercise caution and look at the privacy statement applicable to the website in question.
How can you withdraw your consent?
If you don't want us to process your data anymore, please contact us at Graham Crowley, Greystone House, High Street, Wickham Market, Suffolk
Questions and your contact information
If you would like to: access, correct, amend or delete any personal information we have about you, you are invited to contact us at Graham Crowley, Greystone House, High Street, Wickham Market, Suffolk
Purpose for the cookie
ForceFlashSite
Placed for the session
When viewing a mobile site (old mobile under m.domain.com) it will force the server to display the non-mobile version and avoid redirecting to the mobile site
This cookie aids with security of the website
smSession
Persistent cookie is on for two days or two weeks
Identifies logged in site members
This is an essential cookie written to help with site security in preventing Cross-Site Request Forgery attacks.
svSession
Persistent cookie that lasts fort wo years
Identifies unique visitors and tracks a visitor's sessions on a site
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Indicates how a site was rendered.
Persistent cookie which is used for two weeks
bSession
This cookie improves functionality in the browser session
TS*
TS01*******
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This is used to the end of the browser session
This is an essential cookie and used throughout the site. It contains the user's session ID and does not contain any personal data about the user.
TSC_cookie
This is used for 2 years from set/update
This cookie is typically written to the browser upon the first visit to our site from that web browser. If the cookie has been deleted by the browser operator, and the browser subsequently visits our site, a new utma cookie is written with a different unique ID. This cookie is used to determine unique visitors to our site and it is updated with each page view. Additionally, this cookie is provided with a unique ID that Google Analytics uses to ensure both the validity and accessibility of the cookie as an extra security measure.
This cookie operates in conjunction with the utmb cookie to determine whether or not to establish a new session for the user. In particular, this cookie is not provided with an expiration date, so it expires when the user exits the browser. Should a user visit our site, exit the browser and then return to your website within 30 minutes, the absence of the utmc cookie indicates that a new session needs to be established, despite the fact that the utmb cookie has not yet expired.
This is used for 30 minutes from set/update
This cookie is used to establish and continue a user session with our site. When a user views a page on our site, the Google Analytics code attempts to update this cookie. If it does not find the cookie, a new one is written and a new session is established. Each time a user visits a different page on our site, this cookie is updated to expire in 30 minutes, thus continuing a single session for as long as user activity continues within 30-minute intervals. This cookie expires when a user pauses on a page on our site for longer than 30 minutes.
This is used for 6 months from set/update
This cookie stores the type of referral used by the visitor to reach our site, whether via a direct method, a referring link, a website search, or a campaign such as an ad or an email link. It is used to calculate search engine traffic, ad campaigns and page navigation within our own site. The cookie is updated with each page view to our site.
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This cookie collects information about how visitors use our website. We use the information to compile reports and to help us improve the site. The cookies collect information in an anonymous form, including the number of visitors to our website, where our visitors to our website have come from and the pages they have visited. The information obtained does not identify anyone.
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gid is used for 24 hours and ga for 2 years
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These cookies provide information about how the user uses the website and any advertising that the user may have seen before visiting the said website.
MS CONV and MSANALYTICS, AID, MUIDB, DSID, IDE, MUIDB , MSCC (Bing) and Adwords Conversion (Google)
These cookies are active for 2 years from set/update
These third party cookies allow us to identify which of our advertisements you have clicked on. Each individual advertiser uses its own tracking cookies and the data taken is not confidential or interchangeable.
19DSID, IDE
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Where pages have Google Maps, Google will use a number of cookies to determine location information.


text:
'Cosby Show' Creator Revives Fight With Business Managers
Posted by Johnson & Johnson, LLP | May 13, 2015 | 0 Comments
May 07, 2015 4:27pm PT by Austin Siegemund-Broka
Ed Weinberger claims his former accountants led him to lose years of revenue.
The co-creator of The Cosby Show, Taxi and other sitcoms will get to pursue his claims that his former business managers failed to monitor years of revenue he should have received for one of his creations.
Ed Weinberger's case concerns revenue he says he never received from 1999 to 2012 for one of his lesser-known series, the NBC sitcom Amen. In a ruling Wednesday, a California appeals court reversed the 2013 dismissals of his claims against the firms of Freedman Broder & Company and Myers and Associates on the grounds he filed too late.
Weinberger sued in 2012, claiming his payment structure for Amen comprised $8 million upfront and other compensation contingent on the production company Carson Productions recouping the full $8 million investment.
He says he tasked William Broder of Freedman Broder, his business manager in 1991, with monitoring the 1989 deal with Carson. "In 1995, both Carson and Broder told Weinberger that he should not expect to receive contingent compensation from Amen because his advance payment had been so large, the show's production costs had been high, and the show enjoyed only mediocre success," reads the appellate ruling.
But by 1998 Carson was $1.45 million from recouping Weinberger's $8 million payment. The following year Broder stopped requesting accounting statements from the company without telling Weinberger.
In 2004 Broder stopped representing Weinberger, who then hired Ronald Myers of Myers & Associates, who received information on the contingent compensation agreement for Amen. "However, Weinberger also allege[s] that Broder intentionally failed to advise Myers that Broder had not checked on the status of Weinberger's contingent compensation rights for the past five years. Myers never monitored Weinberger's rights," reads the ruling.
Myers quit representing him in 2008. In 2011 Weinberger finally requested an Amen accounting from Carson. He learned his $8 million was recouped in 1999 and the company owed him significant contingent compensation. Because the statute of limitations for breaches of contract is four years, the company paid him what it owed since 2007, but he couldn't recover the contingent compensation he was owed before then. He sued Broder and Myers and their companies for malpractice, fraud and breach of fiduciary duty.
In separate motions, the business firms held Weinberger should have requested an accounting of Amen from Carson when Broder stopped receiving statements in 1999, or when Myers concluded its work with him in 2008. Therefore, his lawsuit would be outside the two-year statute of limitations to bring accounting malpractice claims. The Los Angeles Superior Court held he didn't qualify for the delayed discovery rule, which extends the time for plaintiffs to file if they had reason not to know of the wrongdoing.
The appellate court decided his delay was reasonable.
Citing case law that gives plaintiffs time in filing complaints of professional malpractice "on the basis that the expertise expected of professionals is beyond the ability of laypersons to evaluate," the court found Weinberger arguably was entitled to expect his business managers not to conceal earnings from him, especially after Broder told him of the unlikelihood of Amen earning more money. "We therefore view this as a case where the fact of injury was hidden due to the alleged failings of Weinberger's accountants during the consecutive periods during which they represented him."
"We also acknowledge the logic of respondents' contention that once Weinberger began handling his own affairs in 2008 he was in a position to determine the facts at that time simply by asking Carson for an accounting statement," continues the ruling. But the court then presents an unusual conclusion: "What triggered Weinberger's decision to ask Carson for an accounting statement in 2011…go[es] to Weinberger's state of mind at that time. This ambiguity in the pleadings does not clearly and affirmatively show that the discovery rule does not apply here," presenting a factual question for the litigation process.
Read the full ruling.
Weinberger is represented by James Ryan of Johnson & Johnson. Freedman Broder is represented by Randall Dean of Chapman, Glucksman, Dean, Roeb & Barger, and Myers is represented by Stephen Tully of Garrett & Tully.
The Hollywood Reporter has reached out to the business managers for comment.
May 11, 5:36 p.m. A previous version of this story incorrectly named one defendant "Robert Myers," not "Ronald Myers." THR regrets the error.


Macy's offers the Calvin Klein Men's Solid Liquid Touch Quarter-Zip Sweater in several colors (Gray Heather pictured) for $34.99. Coupon code "PRIV" cuts that to $26.24. Choose in-store pickup to dodge the $9.95 shipping fee. That's $53 off list and the lowest price we could find. It comes in sizes XS to XXL. Deal ends April 14.


text:
Distribute Your Content
© 2021, Future Today, Inc.
FUTURE TODAY EXPERIENCES MASSIVE GROWTH, STREAMING OVER 43 MILLION HOURS OF CONTENT MONTHLY
Expands Channels-as-a-service Platform with Major Updates to Streaming and Advertising Technology Stack
April 20, 2020 - Menlo Park, CA
Future Today, the only full-stack streaming solution that packages branded channels and delivers the audiences to watch them, today announced significant growth in distribution, advertising, viewership and content, while adding major updates to their Channels-as-a-Service technology platform in the last year, including:
Advertising & Viewership
Grew viewership by 120% year-over-year, delivering content to more than 12M active households each month
Increased viewing time by 85% year over year, streaming more than 43 million hours of content in the month of March, alone
The company grew advertising partners from single to double digits, representing a 500% year-over-year growth in brands Future Today works directly with
Launched more than 65+ channels with leading MVPDs, including Comcast Xfinity and Cox Contour TV
Launched eight linear FAST channels on Vizio, Sling TV, Samsung TV+ and The Roku Channel
The company now serves more than 100MM households across the US on every major streaming platform
"With over 93% of our viewership coming from the U.S., we have a unique ability to deliver highly desirable and engaged audiences for our advertisers, while driving significant new revenue for our content partners," said Vikrant Mathur, CEO at Future Today. "Our latest technology, advanced tools and expertise make it easier than ever to build and sustain a growing AVOD or FAST channel OTT business."
In last 12 months, Future Today has experienced extraordinary growth and has aggressively expanded its content library to offer more than 225,000 movies, TV shows and episodes with the addition of 15,000 hours of premium content. Additionally, Future Today:
Launched 50 new channels including Pinkfong (Baby Shark), CoComelon, and Like Nastya to deliver and manage their AVOD and FAST channels to achieve ubiquitous consumer access.
Has rapidly expanded its children's content offerings, adding new content from several partners including, Lego, Spin Master, 9 Story, and Corus
As such, Future Today has become the largest publisher of family-friendly content on streaming platforms, with a portfolio of over 150 apps/channels dedicated to the genre and over 55,000 movies, tv shows and educational videos. HappyKids, the company's flagship kids channel, is the #1 AVOD Kids channel on CTV platforms in the US.
Channels-as-a-Service Technology
"As the only turnkey, end-to-end streaming stack available, we can launch dozens or even hundreds of channels in a matter of days, on nearly every top streaming platform and device. This is why content owners from around the world turn to Future Today as their streaming TV partner," said Vikrant Mathur, CEO at Future Today.
Future Today's updates to its technology stack are designed specifically to help content owners, video distributors and advertisers deliver a superior OTT experience for viewers, while driving new viewership, more engaged audiences and increased ad revenue. This includes the introduction of:
Channels–as-a-Service technology stack – features significant advances to its video streaming and advertising capabilities
Safe Advertising for Kids and Family – expands on Future Today's COPPA compliant, brand-safe environment to not only ensure personal data and privacy are protected, but also that every ad served is manually reviewed and vetted as appropriate for the kids
Contextual Data & Advertising – Future Today has expanded its contextual data capabilities across all channels, to maximize engagement and ROI for advertisers, while increasing fill rate and CPM for content owners and distributors
Enhanced Content Recommendations – updates to Future Today's AI-powered Content Recommendation Engine to serve the most relevant and desirable content viewers, helping grow new audiences, increase viewing time and boost ad revenue
Support for linear FAST channels – ability for content partners to include linear channels as part of their current VOD offering thereby allowing viewers to either find a specific show in the VOD library or couch-surf by browsing through the linear EPG.
The company offers a holistic solution for content owners seeking to launch new streaming channels, grow audiences and monetize their content across a multitude of OTT platforms and services. To accomplish this, Future Today has developed a proprietary and comprehensive portfolio of streaming technologies, services and solutions that include video management, content management and publishing, app development and maintenance, cross-channel promotion, advertising, monetization and more.
About Future Today
Future Today is a leader in the ad-supported streaming media universe with its flagship channels – FilmRise, Fawesome and HappyKids – ranking in the top free channels across nearly every OTT consumer platform. The company's proprietary, cloud-based technology platform manages OTT services for more than 350 content owners, producers, distributors and major media companies helping them launch and monetize complex Connected TV channels across devices in a matter of days. Future Today's comprehensive portfolio of technology and services includes video management, content management and publishing, app development and maintenance, cross-channel promotion, advertising, monetization and more. Learn more about Future Today here.
Andrew Laszacs
Bob Gold & Associates
310–320–2010 – office
310–940–4458 – cell
FutureToday@bobgoldpr.com
Please be in touch – we look forward to hearing from you.
Future Today Inc.
3723 Haven Ave, #133,


A new gluten free pasta for kids that isn't just guaranteed 'free from' but is certified organic and comes in fun dinosaur shapes for younger children.
Finding tasty gluten free food suitable for children can be a real challenge. That is why the Ulula team is so pleased to be able to stock this latest addition to our kids pasta range. Now you can buy top quality gluten free pasta for kids without having to worry, as this new pasta from Seitz is produced in a factory that doesn't handle any food stuffs containing gluten. Seitz is a long established family owned business from Germany that has many years experience of producing gluten free products, pasta in particular.
Our new gluten free pasta for kids comes in the great dino shapes that have proven so popular in our non allergy-aware children's pasta range. Now having a special dietary need doesn't mean your child has to feel different to the rest of the family or friends when they come round for tea. And, of course, being certified organic you know your child isn't going to be eating lots of chemical nasties with these fun pasta shapes – an important consideration when feeding kids with special dietary needs.
The Seitz pasta shapes are made from organic corn flour and organic rice flour and have a slightly longer cooking time than you'd perhaps expect, around 11-13 minutes – just check and adjust cooking time to your preferred texture. Most importantly, and unlike many gluten free pastas for kids on the market, the Sietz dinos retain their distinctive shapes when fully cooked. So, no more sloppy mess that doesn't appeal to anyone, let alone a young child!
The Seitz Gluten Free Pasta for Kids in Dino Shapes come in child friendly pack sizes of 250g and, depending on your child's age and appetite, our testers suggest that would give your little one about five portions. Single packs cost only £2.29 and you can buy a multi-pack of three for £6.57.
Let us know how you find the Seitz gluten free pasta for kids. Why not send the receipe for your child's favourite gluten free sauce or accompaniment and we'll share it here with other parents.
This entry was posted in Allergy Aware Foods and tagged Free From, Gluten Free, Pasta on March 30, 2015 by ululablog.