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1.
Effect of trifluoperazine and colchicine on LDL-receptor synthesis in smooth muscle cells exposed to hypercholesterolemic medium in vitro have been studied. While trifluoperazine at 25 microM concentration caused stimulation of LDL-receptor synthesis, colchicine acted as a dose-dependent inhibitor of LDL-receptor synthesis. Thus calmodulin down regulates LDL-receptor synthesis independent of microtubular involvement.  相似文献   

2.
We recently demonstrated that the preventive effect of trifluoperazine (a potent inhibitor of calmodulin, protein kinase C, and phospholipase A2) on cholesterol-induced atherogenic activity of smooth muscle cells was mediated through its ability to inhibit smooth muscle cellular DNA synthesis coupled with stimulation of LDL receptor synthesis. The present study addressed the effect of trifluoperazine on cholesterol metabolism of aortic SMCs enriched with cholesterol through the nonreceptor pathway and revealed that (a) TFP caused inhibition of cholesterol synthesis compared with control cells bathed with hypercholesterolemic medium alone. (b) The drug also caused inhibition of free cholesterol and cholesteryl ester accumulation within smooth muscle cells compared to control cells. These results demonstrate that the preventive effect of TFP on atherogenic activity of smooth muscle cells may also be due to its ability to affect the altered/modified cholesterol metabolism of smooth muscle cells exposed to hypercholesterolemic medium in vitro.  相似文献   

3.
The LDL receptor synthesis of human skin fibroblasts in the presence of the specific calmodulin antagonists trifluoperazine, condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde (compound 48/80) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) (W-7) was studied. Labelling of cells with [35S]methionine followed by immunoprecipitation of radioactive LDL receptor protein with monospecific antibodies revealed that calmodulin antagonists caused a 3-fold increase in the radioactivity of the LDL receptor protein as compared with values found in control cells. A corresponding increase of high-affinity binding and internalization of 125I-labelled LDL was observed. The drugs did not influence the overall protein synthesis or the half-life of the LDL receptor. A concomitant suppression of cholesterol synthesis from [14C]mevalonolactone was found to be an independent effect. The calmodulin antagonist-produced stimulation of LDL receptor synthesis could not be simulated by preincubation of cells with cyclic nucleotide analogues, cholera toxin or 3-isobutyl-1-methylxanthine, known as specific effectors of adenylate cyclase and cyclic nucleotide phosphodiesterase, respectively. Modulation of calcium concentration in the incubation medium had no reproducible effect on the rate of LDL receptor synthesis. The results implicate calmodulin as an intracellular suppressor of LDL receptor synthesis in human skin fibroblasts.  相似文献   

4.
K Kariya  Y Takai 《FEBS letters》1987,219(1):119-124
In quiescent cultures of rabbit aortic smooth muscle cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) induced DNA synthesis to some extent in the presence of rabbit plasma-derived serum but inhibited the rabbit whole blood serum (WBS)-induced DNA synthesis and increase in cytoplasmic free Ca2+ concentration Ca2+]i). Prolonged treatment of the cells with phorbol-12,13-dibutyrate (PDBu) caused the partial down-regulation of protein kinase C to a level of 25-35% of that in control cells. In these PDBu-pretreated cells, TPA neither induced DNA synthesis nor inhibited the WBS-induced DNA synthesis, but still inhibited the WBS-induced increase in [Ca2+]i. These results suggest that there are down-regulation-sensitive and -resistant types of protein kinase C in rabbit aortic smooth muscle cells; that the down-regulation-sensitive type has the proliferative and antiproliferative actions whereas the down-regulation-resistant type lacks them; and that the down-regulation-resistant type has the activity to inhibit the WBS-induced increase in [Ca2+]i.  相似文献   

5.
The calmodulin and C-kinase antagonists melittin, calmidazolium, N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W7), and trifluoperazine (TFP) also inhibit the activity of the human erythrocyte Ca2+-dependent protease, calpain I. W-5, the nonchlorinated derivative of W-7, was ineffective as an inhibitor of calpain I just as it is for calmodulin and protein kinase C. Dose response studies provided the following IC50 values: melittin, 2.6 microM; calmidazolium, 6.2 microM; trifluoperazine, 130 microM; W-7, 251 microM. These IC50 values indicate that the compounds have affinities 10 to 600 fold less for calpain I than for calmodulin; however, the affinities of the inhibitory compounds are comparable for calpain I and protein kinase C. Kinetic analysis indicates that the compounds are competitive inhibitors of calpain I with respect to substrate.  相似文献   

6.
Modulation of smooth muscle calponin by protein kinase C and calmodulin   总被引:2,自引:0,他引:2  
When smooth muscle calponin was incubated with protein kinase C, 1 mole of phosphate was incorporated per mole of calponin. The apparent Km value for calponin of the protein kinase was about 0.4 microM. The phosphorylation of calponin by protein kinase C was inhibited markedly by calmodulin in a calcium-dependent manner. Kinetic analysis of calmodulin-induced inhibition of calponin phosphorylation by protein kinase C revealed that calmodulin inhibited the phosphorylation in a noncompetitive fashion with calponin and the determined Ki value was 0.4 microM. These results suggest that interaction of calmodulin with calponin may play a regulatory role in the phosphorylation by protein kinase C and smooth muscle contraction.  相似文献   

7.
Porcine uterine smooth muscle phosphorylase kinase has been partially purified. The enzyme was activated about 1.5-2.0-fold by exogenous calmodulin. Half maximal stimulation was observed at about 100 nM calmodulin. The activation was dependent on calcium and was maximum at pH 7.5 in the range of pH from 6 to 9. This activation was completely abolished by 100 microM trifluoperazine. The result suggested that unlike slow and cardiac muscles, phosphorylase kinase of uterine smooth muscle showed similar response to calmodulin with that of fast muscle. The physiological role of the calcium and calmodulin-dependent activation of myometrium phosphorylase kinase is briefly discussed.  相似文献   

8.
Exposure of porcine vascular smooth muscle cells to platelet-derived growth factor (PDGF; 18-180 ng/ml) but not epidermal growth factor (EGF; 30 ng/ml), somatomedin C (SmC; 30 ng/ml), or insulin (10 microM), results in a rapid, reversible, time- and concentration-dependent disappearance of vinculin staining in adhesion plaques; actin-containing stress fibers also become disrupted following exposure of cells to PDGF. Disappearance of vinculin staining from adhesion plaques is also caused by 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 200-400 nM), though the time course of the disappearance of vinculin staining under these conditions takes longer than in cells exposed to PDGF. The PDGF-induced removal of vinculin from adhesion plaques was inhibited in a concentration-dependent fashion by 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8; 0.25-4 microM) and leupepetin (2-300 microM), and by n-alpha-tosyl-L-lysine chloromethylketone (TLCK; 100 microM) and trifluoperazine (TFP; 2.5 microM). Addition of PDGF to vascular smooth muscle cells caused a rapid, transient increase in cytosolic free calcium, from a basal resting level of 146 +/- 6.9 nM (SEM, n = 62) to 414 +/- 34 nM (SEM, n = 22) as determined using the calcium-sensitive indicator Fura-2 and Digitized Video Microscopy. This increase in cellular calcium preceded the disappearance of vinculin from adhesion plaques and was partially blocked by pretreatment of cells with TMB-8 but not leupeptin. This rise in cytosolic free calcium was found to occur in approximately 80% of the sample population and displayed both spatial and temporal subcellular heterogeneity. Exposure of cells to TPA (100 nM) did not result in a change in cytosolic free calcium. Both PDGF (20 ng/ml) and TPA (100 nM) caused cytosolic alkalinization which occurred after PDGF-induced disruption of vinculin from adhesion plaques, as determined using the pH-sensitive indicator BCECF and Digitized Video Microscopy. PDGF stimulated DNA synthesis and vinculin disruption in a similar dose-dependent fashion. Both could be inhibited by leupeptin or TMB-8. These results suggest that 1) exposure of vascular smooth muscle cells to PDGF is associated with the disruption of vinculin from adhesion plaques, 2) PDGF-induced vinculin disruption is regulated by an increase in cytosolic calcium (but not cytosolic alkalinization), and involves proteolysis; 3) activation of protein kinase C also causes vinculin removal from adhesion plaques but by a calcium-independent mechanism, and 4) the cellular response to PDGF-stimulated increases in cytosolic free calcium is heterogeneous.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

9.
Tamoxifen inhibits bone resorption by disrupting calmodulin-dependent processes. Since tamoxifen inhibits protein kinase C in other cells, we compared the effects of tamoxifen and the phorbol ester, phorbol myristate acetate, on osteoclast activity. Phorbol esters stimulate bone resorption and calmodulin levels four-fold (k0.5 = 0.1-0.3 microM). In contrast, tamoxifen inhibited osteoclast activity approximately 60% with an IC50 of 1.5 microM, had no apparent effect on protein kinase C activity in whole-cell lysates, and reduced protein kinase C alpha recovered by immunoprecipitation 75%. Phorbol esters stimulated resorption in a time-dependent manner that was closely correlated with a similar-fold increase in calmodulin. Protein kinase C alpha, beta, delta, epsilon, and zeta were all down-regulated in response to phorbol ester treatment. Tamoxifen and trifluoperazine inhibited PMA-dependent increases in bone resorption and calmodulin by 85 +/- 10%. Down-regulation of protein kinase C isoforms by phorbol esters suggests that the observed increases in bone resorption and calmodulin levels are most likely due to a mechanism independent of protein kinase C and dependent on calmodulin. In conclusion, the data suggest that protein kinase C negatively regulates calmodulin expression and support the hypothesis that the effects of both phorbol esters and tamoxifen on osteoclast activity is mediated by calmodulin.  相似文献   

10.
The present study examined the involvement of protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), protein tyrosine kinase (PTK) and Ca2+/calmodulin mediated pathways on the uptake of L-DOPA through the L-type amino acid transporter in Neuro 2A cells, an in vitro model of neuronal cells. Non-linear analysis of the saturation curve for L-DOPA revealed a Km value (in microM) of 54+/-2 and a Vmax value (in nmol mg protein/6 min) of 34+/-1. L-DOPA uptake was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 mM), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC50=82 microM). The Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 microM) uptake with IC50's of 33 and 105 microM, respectively. The inhibitory effect of BHC on the accumulation of L-DOPA was of the competitive type, whereas that of calmidazolium and trifluoperazine was of the non-competitive type. Modulators of PKA (cyclic AMP, forskolin, isobutylmethylxanthine and cholera toxin), PKG (cyclic GMP, zaprinast, LY 83583 and sodium nitroprusside), PKC (phorbol 12,13-dibutirate, phorbol 12-myristate 13-acetate and chelerythrine) and PTK (genistein and tyrphostin 25) failed to affect the accumulation of a non-saturating (2.5 microM) concentration of L-DOPA. It is concluded that L-DOPA uptake in Neuro 2A cells is promoted through the L-type amino acid transporter and appears to be under the control of Ca2+/calmodulin mediated pathways.  相似文献   

11.
The present study examined the involvement of protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), protein tyrosine kinase (PTK) and Ca(2+)/calmodulin mediated pathways on the luminal uptake of L-DOPA through the L-type amino acid transporter in immortalized rat capillary cerebral endothelial (REB-4) cells. Non-linear analysis of the saturation curve for L-DOPA revealed a K(m)value (in microM) of 71+/-9 and a V(max)value of 17+/-1 (in nmol mg protein/6 min). L-DOPA uptake at the luminal cell border was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 m m), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC(50)=140 microM). The Ca(2+)/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 microM) uptake with IC(50)s of 23 and 33 microM, respectively. The inhibitory effect of BHC on the accumulation of L-DOPA was of the competitive type, whereas that of calmidazolium and trifluoperazine was of the non-competitive type. Modulators of PKA (cyclic AMP, forskolin, isobutylmethylxanthine and cholera toxin), PKG (cyclic GMP, zaprinast, LY 83583 and sodium nitroprusside), PKC (phorbol 12,13-dibutyrate, staurosporine and chelerythrine) and PTK (genistein and tyrphostin 25) failed to affect the accumulation of a non-saturating (2.5 microM) concentration of L-DOPA. It is concluded that L-DOPA uptake in RBE-4 cells is promoted through the L-type amino acid transporter and appears to be under the control of calmodulin mediated pathways.  相似文献   

12.
13.
Calmodulin-activated protein kinase activity in the endoplasmic reticulum fraction of rat adipocytes was identified and characterized. The major endogenous protein substrate of the calmodulin-activated kinase activity has an apparent molecular weight of 54,000 as determined by sodium dodecyl sulfate gel electrophoresis. The calmodulin-activated component of the activity was saturated at 10 microM ATP. Calcium or calmodulin alone did not increase the activity, but the simultaneous presence of calcium and calmodulin increased activity three to four-fold. Half-maximal activation of this activity occurred at 8 microM Ca2+. The addition of increasing amounts of calmodulin caused a concentration-dependent activation in the presence of calcium, which was saturable at high calmodulin concentrations. Magnesium was required for activity, with half-maximal activity occurring at 230 microM. The antipsychotic drug trifluoperazine inhibited the activation of the protein kinase activity by calmodulin, but had a negligible effect on the basal activity. Half-maximal inhibition occurred at 63 microM. Phosphorylation of the 54,000 mol. wt band was independent of cAMP, cGMP and the combination of cAMP and cAMP-dependent protein kinase. Calmodulin-activated protein kinase phosphorylated both phosphoserine and phosphothreonine residues in the 54,000 mol. wt substrate. These experiments have partially characterized a calmodulin-activated protein kinase activity from adipocytes, which appears to be a unique activity of unknown function.  相似文献   

14.
Theca cells were collected from the second largest preovulatory follicle. Chelation of extracellular calcium with EGTA attenuated LH (10 ng)-induced androstenedione production by theca cells, and this effect was more pronounced in calcium-deficient than in calcium-replete incubation medium. Incubation of theca cells with steroidogenic agonists in the presence of the calcium channel blocker verapamil (100 microM) suppressed androstenedione production stimulated by LH (a 57% decrease), the adenylate cyclase activator forskolin (a 59% decrease) and the cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP (a 61% decrease). Furthermore, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a putative inhibitor of intracellular calcium mobilization, suppressed LH-induced androstenedione production in a dose-dependent fashion. The calmodulin inhibitors trifluoperazine (100 microM) and R24571 (50 microM) inhibited androstenedione production stimulated by hormonal (LH) and non-hormonal (forskolin, 8-bromo-cAMP) agonists (decreases ranging from 76 to 98%). While increasing the intracellular calcium ion concentrations with the calcium ionophore A23187 did not affect basal concentrations of androstenedione, treatment of LH-stimulated cells with the ionophore caused dose-dependent inhibition of androstenedione production; these effects were enhanced by coincubation with phorbol 12-myristate 13-acetate (a known activator of protein kinase C). We conclude that the mobilization of calcium is critical for agonist-stimulated steroidogenesis in hen theca cells, apparently requiring the interaction of calcium with its binding protein, calmodulin. Furthermore, increased cytosolic calcium concentrations may be involved in the suppression of androstenedione production, possibly as a result of an interaction with protein kinase C.  相似文献   

15.
Biphasic effects of 1,25-dihydroxyvitamin D-3 on DNA synthesis were shown in primary cultured (24 h) chick embryo myoblasts exposed to physiological concentrations of the hormone. The sterol stimulated [3H]thymidine incorporation into DNA in proliferating myoblasts, e.g., at early stages of culture prior to cell fusion or in high serum-treated cells. The opposite effects were observed during the subsequent stage of myoblast differentiation in low-serum media. The mitogenic effect of 1,25-dihydroxyvitamin D-3 was correlated with an increase in c-myc mRNA and a decrease in c-fos mRNA levels, whereas its inhibitory action on DNA synthesis was accompanied by increased myofibrillar and microsomal protein synthesis and an elevation of creatine kinase activity, the latter suggesting a stimulation of muscle cell differentiation by the sterol. These data are in agreement with the results of previous morphological studies. Treatment of myoblasts with the calcium ionophore X-537 A or the phorbol ester TPA caused only a transient stimulation of [3H]thymidine incorporation into DNA, which occurred earlier than the response elicited by 1,25-dihydroxyvitamin D-3, suggesting that changes in intracellular Ca2+ and kinase C activity are not major mediators of the hormone effects. A similar temporal profile of changes in calmodulin mRNA levels as that of [3H]thymidine incorporation into DNA was observed after treatment of myoblasts with the sterol, in accordance with the role of calmodulin in the regulation of cell proliferation. 1,25-dihydroxyvitamin D-3 may play a function in embryonic muscle growth and differentiation.  相似文献   

16.
In certain cell systems, including neonatal vascular smooth muscle (VSM) cells, phorbol esters are growth inhibitory. Here we show that 1,2-dioctanoyl-sn-glycerol (DiC8), when added 2 h after alpha-thrombin, reverses by greater than 95% the induction of DNA synthesis in VSM cells by alpha-thrombin. Sphingosine, a naturally occurring lysosphingolipid inhibitor of protein kinase C, and its synthetic analogues N-acetylsphingosine and C11-sphingosine were used to investigate this phenomenon further. Neither phorbol 12-myristate 13-acetate (PMA;200 ng/ml) nor sphingosine (up to 10 microM) alone had any effect upon basal DNA synthesis in VSM cells. Like DiC8, PMA totally blocked the induction of DNA synthesis by alpha-thrombin. This inhibitory effect of PMA was reversed by sphingosine in a dose-dependent manner with complete reversal at 10 microM. Neither N-acetylsphingosine nor C11-sphingosine exhibited any effect on DNA synthesis in VSM cells. The effect of sphingosine and its analogues on the activity of protein kinase C extracted from VSM cells was measured by histone III-S phosphorylation. Protein kinase C activity was inhibited 50% by 300 microM sphingosine, but less than 15% by similar concentrations of N-acetylsphingosine and C11-sphingosine. To assess the effects of sphingosine and analogues on protein kinase C in intact cells, we examined the effect of the lipids on [3H]phorbol dibutyrate binding. Sphingosine (at greater than 5 microM), but not N-acetylsphingosine or C11-sphingosine, blocked [3H]phorbol dibutyrate binding in a dose- and time-dependent fashion. Thus the mechanism of growth inhibition by DiC8 and PMA in neonatal VSM cells appears to be through activation of protein kinase C by these compounds. Sphingosine reverses this growth inhibition through interference with the binding to protein kinase C of phorbol esters or other activators of this enzyme.  相似文献   

17.
Several recent publications indicate that Ca2+ is required for protein synthesis in mammalian cells, including the Ehrlich ascites tumor cell. The present communication examines whether the effects of Ca2+ might be mediated through calmodulin or a related protein. Four calmodulin antagonists belonging to different chemical categories were used to provide evidence of calmodulin involvement. Three of the antagonists inhibited protein synthesis in intact cells; 50% inhibitory concentrations were 10 microM calmidazolium, 12 microM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and 17.5 microM trifluoperazine (TFP). Initiation was preferentially inhibited as indicated by an increase in the 80S monomers accompanied by a significant disaggregation of polyribosomes. All the antagonists also inhibited protein synthesis initiation in the cell-free protein-synthesizing system; 50% inhibitory concentrations for compound 48/80, calmidazolium, TFP, and W7 were 10 microM, 125 microM, 300 microM and 500 microM, respectively. A weak analogue of W7 inhibited only 20% at 1000 microM. Inhibition in the cell-free system was reversed by the addition of exogenous calmodulin in all four cases. The levels of 43S complexes were significantly elevated with all four antagonists, indicating a block in the utilization of 43S complexes. The similarity of the effects of four distinct classes of antagonists and their ready reversal by exogenous calmodulin leads us to suggest that there may be a role for calmodulin or a very similar calcium-binding protein in protein synthesis.  相似文献   

18.
The mechanism by which human alpha-thrombin activates the Na+/H+ exchanger was studied in cultured neonatal rat aortic smooth muscle cells. Thrombin (0.4 unit/ml) caused a rapid cell acidification followed by a slow, amiloride-inhibitable alkalinization (0.10-0.14 delta pHi above base line). In protein kinase C down-regulated cells (exposed to phorbol 12-myristate 13-acetate for 24 or 72 h), the delta pHi induced by thrombin was only partially attenuated. This protein kinase C-independent activation of the Na+/H+ exchanger was blocked by pertussis toxin (islet activating protein (IAP)), reducing delta pHi by 50%. IAP did not directly inhibit Na+/H+ exchange activity as assessed by the response to intracellular acid loading. Thrombin also stimulated arachidonic acid release by 2.5 fold and inositol trisphosphate release by 6.2 fold. IAP inhibited both of these activities by 50-60%. Intracellular Ca2+ chelation with 120 microM quin2 prevented the thrombin-induced Ca2+ spike, inhibited thrombin-induced arachidonic acid release by 75%, and inhibited thrombin-induced activation of the Na+/H+ exchanger in protein kinase C-deficient cells by 65%. Increased intracellular [Ca2+] alone was not sufficient to activate the Na+/H+ exchanger, since ionomycin (0.3-1.5 microM) failed to elevate cell pH significantly. 10 microM indomethacin inhibited thrombin-induced delta pHi in both control and protein kinase C down-regulated cells by 30-50%. Thus, thrombin can activate the Na+/H+ exchanger in vascular smooth muscle cells by a Ca2+-dependent, pertussis toxin-sensitive pathway which does not involve protein kinase C.  相似文献   

19.
The role of Ca2+ was studied in the release of the organic osmolyte sorbitol from rat IMCD cells in response to hypoosmotic stress. When cells were exposed to hypoosmotic media, sorbitol release was greatly reduced in Ca-free media which, on readmission of Ca2+, returned to control values. Under isoosmotic conditions, the ionophore A23187 stimulated sorbitol release without any effect on cell volume. Addition of trifluoperazine, a calmodulin inhibitor, but not the protein kinase C inhibitor H-7, inhibited the osmotically-activated sorbitol release. These results suggest that sorbitol release is a calmodulin-dependent event, possibly activated by a rise in intracellular calcium as a result of cell swelling.  相似文献   

20.
Glucagon caused a marked decrease in the total L-pyruvate kinase activity of control hepatocytes maintained in monolayer culture (t1/2 = 54 h), while the addition of insulin to hepatocytes isolated from a fasted rat caused a four- to fivefold increase in the total enzyme activity. Maintenance of L-pyruvate kinase in control cultures of hepatocytes was shown to require insulin. However, when 1 microM glucagon was present in the medium, the total L-pyruvate kinase activity was not maintained even in the presence of 1 microM insulin, but rather the total L-pyruvate kinase activity of the cells steadily declined from 12.1 to 5.7 units/mg DNA by the 6th day in culture. The increase in the total L-pyruvate kinase activity of fasted hepatocytes cultured in the presence of insulin was shown to result from an increase in protein synthesis, since actinomycin D and cycloheximide blocked the insulin-induced increase in the enzyme activity. The addition of 1 microM glucagon to cultures of fasted hepatocytes also blocked the insulin-induced increase in total L-pyruvate kinase activity. Since glucagon decreased the total L-pyruvate kinase activity in control hepatocytes and blocked the increase in L-pyruvate kinase activity in fasted hepatocytes, it is suggested that, in addition to the phosphorylation of L-pyruvate kinase by a cAMP-dependent protein kinase, glucagon also acts to decrease the synthesis of L-pyruvate kinase in vitro.  相似文献   

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