Journal of Neurochemistry, 2001, 76, 442±449

Reduction of glyceraldehyde-3-phosphate dehydrogenase activity in Alzheimer's disease and in Huntington's disease ®broblasts
Jennifer L. Mazzola and Michael A. Sirover
Department of Pharmacology, Temple University, School of Medicine, Philadelphia, USA

Abstract New functions have been identi®ed for glyceraldehyde-3phosphate dehydrogenase (GAPDH) including its role in neurodegenerative disease and in apoptosis. GAPDH binds speci®cally to proteins implicated in the pathogenesis of a variety of neurodegenerative disorders including the b-amyloid precursor protein and the huntingtin protein. However, the pathophysiological signi®cance of such interactions is unknown. In accordance with published data, our initial results indicated there was no measurable difference in GAPDH glycolytic activity in crude whole-cell sonicates of Alzheimer's and Huntington's disease ®broblasts. However, subcellular-speci®c GAPDH±protein interactions resulting in diminution of GAPDH glycolytic activity may be disrupted or masked in whole-cell preparations. For that reason, we examined GAPDH glycolytic activity as well as GAPDH± protein distribution as a function of its subcellular localization

in 12 separate cell strains. We now report evidence of an impairment of GAPDH glycolytic function in Alzheimer's and Huntington's disease subcellular fractions despite unchanged gene expression. In the postnuclear fraction, GAPDH was 27% less glycolytically active in Alzheimer's cells as compared with age-matched controls. In the nuclear fraction, de®cits of 27% and 33% in GAPDH function were observed in Alzheimer's and Huntington's disease, respectively. This evidence supports a functional role for GAPDH in neurodegenerative diseases. The possibility is considered that GAPDH : neuronal protein interaction may affect its functional diversity including energy production and as well as its role in apoptosis. Keywords: Alzheimer's disease, ®broblasts, GAPDH, Huntington's disease. J. Neurochem. (2001) 76, 442±449.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC is commonly identi®ed as a key enzyme in glycolysis. However, recent studies from independent investigators demonstrate that GAPDH may be rede®ned as a complex multifunctional protein (reviewed in Sirover 1997, 1999). Diverse properties of GAPDH include membrane transport, membrane fusion, microtubule bundling, phosphotransferase activity, nuclear RNA export, DNA replication and DNA repair. The subcellular localization of GAPDH appears to be fundamental to its multiple functions, oligomeric structure and, possibly, its isomeric form. Thus, as a tetramer in the cytoplasm, GAPDH functions in glycolysis or as an RNA binding protein. In contrast, as a monomer in the nucleus, GAPDH functions in DNA repair. The subcellular localization of GAPDH appears to be tightly regulated both during proliferation (Cool and Sirover 1989) and during apoptosis (Ishitani and Chuang 1996; Ishitani et al. 1996, 1998; Saunders et al. 1997, 1999; Sawa et al. 1997).

The potential involvement of GAPDH in neurodegeneration is based on the demonstration that proteins important to the pathogenesis of several neuronal disorders strongly bind GAPDH with high af®nity in vitro. GAPDH strongly interacted with the b-amyloid precursor protein (b-APP) (Schulze et al. 1993). The latter is a protein directly implicated in Alzheimer's disease (AD). Furthermore,

Received July 24, 2000; revised manuscript received September 1, 2000; accepted September 4, 2000. Address correspondence and reprint requests to Jennifer Mazzola, Department of Pharmacology, Temple University School of Medicine, 3420 North Broad Street, Philadelphia, PA 19140, USA. E-mail: Abbreviations used: AD, Alzheimer's disease; AMC, age-matched control; b-APP, beta-amyloid precursor protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase [EC1.2.1.12]; HD, Huntington's disease; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; TBS-T, Tris-buffered saline with 0.1% Tween.


q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 442±449

ataxin 1. 1996). and atrophin. con¯uency was determined as described above (2 T75 ¯asks/determination). 1996). altered in spinocerebellar ataxia type 1. To radiolabel cellular DNA for these studies (3H)thymidine (50 mL. 1988. AG06241B (male. GM00305A (female. An LDH kit was purchased from Sigma (St Louis. NJ. 59 years of age) and GM04192 (male. in the present study. The homogenate was centrifuged at 375 g for 10 min. HD patients and age-matched control (AMC) subjects. GM04222 (male. Further. 5 mm KCl. SCA 1 (Servadio et al. 4 mm l-glutamine. Cells were disrupted by 15 strokes in a glass dounce homogenizer (Kontes. 1% aprotinin. Experiments were completed on 12 separate cell strains to give an n ˆ 4 for each group. Subcellular fractionation Subcellular fractionation was performed as described previously (Cool and Sirover 1989). Veri®cation of cell separation was determined by quanti®cation of lactate dehydrogenase (LDH) activity and by the distribution of (3H)thymidine radiolabeled DNA. 38 Ci/mmol) for 30 min prior to collection. (female. LDH activity (25 mg protein) was measured in a reaction mixture containing 0. In contrast. The pellet was resuspended in Buffer I then recentrifuged at 500 g for 10 min. Alzheimer's and Huntington's disease 443 several of the proteins mutated in the CAG trinucleotide repeat neurodegenerative disorders have been shown to bind selectively to GAPDH. 62 years of age). AG08044A (female. The prepared crude cell sonicates or subcellular fractions (25 mg) were added to a cuvette Experimental procedure Cell culture Human skin ®broblast cell strains were obtained from the Human Genetic Mutant Cell Repository at the Coriell Institute for Medical Research (Camden. The supernatant. The PBS was removed and the cells resuspended in Buffer I. 1 mg/mL). Therefore. forms a protein with an N-terminal polyglutamine domain. Thus. AG07375A (male. is the ®nding that b-APP (Shivers et al. which contained the cytosol. Koshy et al. Cells were pelleted by centrifugation at 500 g for 10 min. Haass et al. The precipitate was collected on glass ®bre ®lters. no detectable (3H)thymidine incorporation into DNA was observed (results not shown). Fibroblasts were grown in Dulbecco's modi®ed Eagle's medium supplemented with 10% fetal calf serum. 58 years of age). cells from 10 visibly con¯uent T75 ¯asks were collected using a rubber policeman in 1  phosphate-buffered saline (PBS). 1995) and GAPDH (Cool and Sirover 1989) are each located in the nucleus and the cytoplasm. We now report speci®c intracellular reduction in GAPDH glycolytic activity in both AD and in HD cells as compared to that observed in AMC cells. it was suggested that binding to GAPDH may serve as the new function (Roses 1996. Roses et al. 1991) and GAPDH (Cool and Sirover 1989) are both localized within the cytoplasm and the plasma membrane. Determination of GAPDH glycolytic activity GAPDH dehydrogenase activity was determined as described previously (Zhang and Snyder 1992). 56 years of age). 63 years of age). AG06848B (female. MO. Cells were cultured in T75 ¯asks at 378C in a humidi®ed atmosphere of 5% CO2 in air. NJ. These results provide evidence that inhibition of GAPDH activity in AD and in HD cells is due to posttranslational alterations of the GAPDH protein. 51 Ci/mmol) was added to each T75 ¯ask 10±14 days prior to cell collection. Protein concentration was determined using the Bradford method. 59 years of age). Huntington's disease (clinically affected) cell cultures used were GM01061A (male. huntingtin. Polyglutamine stretches are postulated to attribute a gain of function. 71 years of age). pH 7. Preparation of crude cell sonicates Cells were collected by trypsinization when visibly con¯uent and centrifuged at 1500 g for 15 min at 48C. it was our intention to further elucidate the role of GAPDH in neurodegenerative disease by determining GAPDH glycolytic activity and gene expression in subcellular fractions of ®broblasts from AD patients.1. 1996. The mutant CAG expansion is located in the coding region and. 56 years of age) and AG07376B (male. 60 years of age).GAPDH. Medium was replaced every three days. In parallel. cells were pulsed with (3H)thymidine (50 mL. as witnessed by our results. The ®lters were dried overnight at 258C. Each fraction was sonicated 1  20 s at 60 W using a needle probe. USA).19 mmol/L NADH in 1  PBS for 3 min at 258C. To determine incorporation of (3H)thymidine into DNA. Huntingtin (Hackam et al. The extent of disruption was monitored using light microscopy. The supernatant was recovered and termed the perinuclear fraction. subcellular fractionation analysis may be vital to an understanding of GAPDH function in neurodegenerative disease. mutated in dentatorubalpallidolusian atrophy (Burke et al. To obtain 3  107 cells for each experiment. the androgen receptor. upon translation. Cell pellets were resuspended in Buffer I (20 mm Tris±HCl. 61 years of age) and AG06010C q 2001 International Society for Neurochemistry. Vineland. 0. 1 mm MgCl).62 mmol/L pyruvate. Consistent with the notion of a GAPDH : neuronal protein complex in vivo. Alzheimer's disease (clinically affected) cell cultures used were AG05809B (female. cytoplasmic organelles and the plasma membrane. All subsequent separation protocols were performed at 48C. 71 years of age). 1998). Journal of Neurochemistry. The resulting pellet was dissolved in Buffer I and termed the nuclear fraction. was carefully removed and termed the postnuclear fraction. To con®rm con¯uency. USA) with a tight-®tting pestle. Radioactivity was determined using liquid scintillation spectroscopy the following day. 76. Age-matched control cell strains used were AG04560B (male. which causes the disease. each fraction (50 mL) was precipitated with 10% trichloroacetic acid (1 mL) and heat-denatured calf thymus DNA (100 mL. Cells were trypsinized for passaging when visibly con¯uent (approximately 3 weeks after previous passaging). USA). As expected. defective in spinobulbar muscular atrophy. The suspension was sonicated 3  20 s at 60 Watts at 48C using a needle probe. 51 years of age). GAPDH gene expression and subcellular distribution of the GAPDH protein was unchanged. 442±449 . 100 mg/mL streptomycin and 100 units/mL penicillin. These mutated proteins include the Huntington's disease (HD) protein. LDH activity was measured by the loss of absorbance at 340 nm.

GAPDH (1. 1998).5). only six strains are shown in Fig.10. L. As shown in Fig. The blots were washed with TBS-T between antibody incubations and before development on Hyper®lm ECL ®lm using the ECL detection reagents (Amersham. All samples were run in duplicate and puri®ed human GAPDH was used as a positive control for the ®rst and last sample in each set of GAPDH assays. USA). lane 2 is AG4560B. antihuman placental GAPDH monoclonal antibody 40. Non-speci®c binding sites on the blots were blocked by incubation with TBS-T (20 mm Tris±HCl. IL. Journal of Neurochemistry. Arlington Heights. No difference in GAPDH glycolytic activity was observed (Fig. lane 5 is GM01061A. Kish et al. GAPDH glycolytic activity was quanti®ed by (a) the production of NADH (mmole NADH formed/mg of cell protein) and (b) determination of speci®c activity for age-matched control. This is consistent with the known molecular mass of GAPDH. There were no statistical differences among AD (104 ^ 11 mU/mg protein).26 mm for the NADH extinction coef®cient. Arlington Heights. IL. q 2001 International Society for Neurochemistry. Immunoblot analysis Samples (3 mg) were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (Seal et al. Speci®c activity was expressed as milliunit (mU)/mg protein. pH 7. lane 3 is AG07375A. The reaction (total volume ˆ 1 mL) was initiated by the addition of the substrate. 20 mm sodium phosphate. 1b). Catalysis was determined by the formation of NADH as measured by gain in absorbance at 340 nm for 5 min. Statistical signi®cance was calculated using Kruskal± Wallis ANOVA of ranks. The concentration of NADH was calculated using 6. Results Initial experiments compared GAPDH glycolytic activity in crude cell sonicates from AD. 1999. Proteins were transferred electrophoretically to Hybond nitrocellulose membranes optimized for enhanced chemiluminescence (ECL. Amersham. lane 4 is AG05809B. USA) for 1 h. and 0. 1998. 1998) and postmortem brain tissue (Browne et al. IL. A. Arlington Heights. 2 to reduce redundancy. 1a). 2. Determination of GAPDH protein in crude cell sonicates was ascertained by immunoblot analysis of age-matched control (lanes 1 and 2). USA). Fig. 1999). 0. and AMC ®broblasts (122 ^ 18 mU/mg protein). diluted 1/50 in TBS-T with 5% milk for 2 h.25 mm NAD1. Each blot was incubated with the primary antibody. 1987). 76. HD and AMC ®broblasts. a single 37 kDa protein was detected in cell free extracts. Glycolytic activity curves are reported as mmole NADH/mg protein produced over time. 137 mm NaCl. 442±449 . Lubec et al. Results are the mean ^ SEM of separate determinations made from each of the 12 cell strains. These results in cell free extracts are consistent with previous studies using HD ®broblasts (Cooper et al.1% Tween 20) with 5% non-fat dried milk at 258 for 1 h.444 J. Although each of the 12 cell strains was examined. HD (130 ^ 17 mU/mg protein). 2 Expression of the GAPDH gene in AMC. This study was Fig. Sirover containing a reaction mixture (800 mL) of 100 mm sodium pyrophosphate (pH 8. and lane 6 is GM00305A. Immunoblot analysis was used to investigate the relationship between GAPDH activity and gene expression in AD. Tabrizi et al. AD and HD cells. and 3 mm dithiothreitol (258C).09 (Arenaz and Sirover 1983). This lack of alteration in dehydrogenase catalysis was veri®ed by analysis of GAPDH-speci®c activity (Fig. 1997. 1 Determination of GAPDH glycolytic activity in crude cell extracts. HD and AMC cells. Alzheimer's disease and Huntington's disease cells. Mazzola and M. Blots were washed 3  TBS-T (50 mL total) then incubated with secondary antibody (horseradish peroxide linked antimouse IgG diluted 1/1000 in TBS-T with 5% milk. A milliunit was de®ned as the amount of enzyme required to catalyse the production of 1 nmol NADH per minute (Cooper et al. Lane 1 is AG08044A.6 mm). Alzheimer's disease (lanes 3 and 4) and Huntington's disease (lanes 5 and 6) cells. Amersham.6.

In AMC. Although some inter-individual variation may be observed. Statistical analysis of postnuclear (c) and nuclear (d) GAPDH-speci®c activity was determined using Kruskal±Wallis ANOVA of ranks followed by the Student's t-test with **p ˆ 0. Additionally.045. In contrast. Similar results were obtained in all 12 cell strains (data not shown). 1. Postnuclear (a) and nuclear (b) GAPDH activity is expressed as the mean ^ SEM as in Fig. Preparation of crude cell extracts may dissociate GAPDH : neuronal protein complexes.002 and *p ˆ 0. Journal of Neurochemistry.GAPDH. the lack of LDH activity in the nuclear fraction Fig. 4 Comparison of Alzheimer's GAPDH activity to age-matched controls. As such. q 2001 International Society for Neurochemistry. AMC and AD). Alzheimer's and Huntington's disease 445 Fig. Subcellular fractionation was performed using 10 con¯uent T75 ¯asks on eight separate cell strains (n ˆ 4. the nuclear fraction displayed a complete absence of activity. (a) Age-matched control. the perinuclear fraction possessed low LDH activity demonstrated by reduced NADH loss over time (data not shown). 3 Quanti®cation of subcellular fractionation. these results demonstrate that the level of GAPDH gene expression is comparable in AMC. This pattern of LDH distribution is consistent with its localization as a cytoplasmic enzyme. performed with equal protein from each cell strain. Lactate dehydrogenase activity of postnuclear and nuclear fractions was determined as described in Experimental Procedures. examination of GAPDH activity in those samples may not re¯ect the effects of such intracellular interactions on GAPDH catalysis. AD and HD cells. 76. (b) Alzheimer's disease and (c) Huntington's disease cells. subcellular GAPDH activity and expression was investigated using nuclear. To validate the subcellular protein distribution. Control experiments were initially performed to document the validity of the cell fractionation protocols. In addition. For that reason. 442±449 . the intracellular localization of LDH activity was quanti®ed in each cell fraction. Incorporation of (3H)thymidine into DNA (d) was expressed as cpm/mg protein for the average of the postnuclear and nuclear fractions. AD and HD cells. perinuclear and postnuclear isolates. the postnuclear fraction exhibited high LDH activity as demonstrated by signi®cant loss of absorbance over the 3-min incubation period in all three cell types (Figs 3a±c).

The AD postnuclear fraction displayed a substantial diminution in glycolytic activity as compared to the AMC (Fig. However. Analysis of the GAPDH-speci®c activity revealed no statistically signi®cant difference between the HD postnuclear fraction (233 ^ 27 mU/mg protein) and the AMC postnuclear fraction (p ˆ 0. The data presented in Fig. 4a). This is in contrast to the results obtained from the cell free extracts as shown in Fig. Mazzola and M. the subcellular distribution of (3H)thymidine labelled DNA was examined. 4b). the reduction in GAPDH-speci®c activity (46 ^ 8 mU/mg protein) was also statistically signi®cant (p ˆ 0. statistical analyses were performed to quantify differences in subcellular-speci®c activity. 1.045) when compared to AMC nuclei (63 ^ 8 mU/mg protein) (Fig. HD. However. However. Speci®c activity was determined from the initial rates exclusively from the linear portion of the GAPDH activity curves as de®ned by linear regression analysis (with r2 . demonstrates the absence of unbroken cells contaminating that fraction.025. In contrast.025) as compared to AMC nuclei (Fig. the results demonstrate that the amount of immunoreactive GAPDH was unchanged in the different subcellular environments. GAPDH catalysis was also decreased in the AD nuclear fraction as compared with catalysis in AMC nuclei (Fig. Thus. To verify the integrity of the nuclear membrane. A. Subcellular fractionation was performed using 10 con¯uent T75 ¯asks on eight separate cell strains (n ˆ 4.002) in the AD (202 ^ 15 mU/mg protein) postnuclear fraction as compared to the postnuclear fraction from AMC cells (276 ^ 25 mU/mg protein). 5d). En toto. perinuclear GAPDH-speci®c activity was unchanged (p ˆ 0. 3d) demonstrate that the nuclear fraction of AMC.08) (Fig. To begin to determine the mechanism that underlies the observed decrease in glycolytic activity. than 0. 5b). 442±449 . No difference was observed between the HD perinuclear fraction (67 ^ 16 mU/mg protein) and the AMC perinuclear fraction (61 ^ 9 mU/mg protein) (p ˆ 0. Cumulatively. intracellular differences in AD and HD glycolytic activity q 2001 International Society for Neurochemistry. subcellular distribution of the GAPDH protein was determined in AMC.2) in AD cells (50 ^ 12 mU/mg protein) as compared to AMC cells (61 ^ 9 mU/mg) (data not shown). In contrast to the AD results. 1. In the AD nuclear fraction. nuclear and perinuclear fractions each revealed a signi®cant amount of GAPDH protein present in each cell strain (Figs 6a±c). and AMC subcellular fractions (only six cell strains are shown to eliminate repetitiveness). 4c demonstrate a statistically signi®cant reduction of 27% (p ˆ 0. AD or HD cells. nuclear HD catalysis (42 ^ 6 mU/mg protein) was signi®cantly reduced by 33% (p ˆ 0. To verify the reduction observed in intracellular GAPDH glycolytic activity in AD. 5a) and a greater reduction in the nuclear fraction (Fig. AD and HD cells contained high levels of radiolabeled DNA. 76. 5 Comparison of Huntington's GAPDH activity to age-matched controls.35) (data not shown). Results from this experiment (Fig. L. GAPDH glycolytic activity was then determined in each subcellular fraction. The reduction in AD cells was more pronounced and with less variation in the postnuclear fraction than in the nuclear fraction. Postnuclear (a) and nuclear (b) GAPDH activity is expressed as the mean ^ SEM as in Fig. Immunoblot analysis of the postnuclear. 5c). Journal of Neurochemistry.995). HD cells showed a modest reduction in the GAPDH curve of the postnuclear fraction (Fig. AMC and HD). Statistical analysis of postnuclear (c) and nuclear (d) GAPDH-speci®c activity was determined using Kruskal±Wallis ANOVA of ranks followed by the Student's t-test with *p ˆ 0. virtually no radioactivity was detected in the postnuclear fraction. no difference in GAPDH protein distribution was detected in any of the 12 cells strains between AD. A similar lack of radiolabel was observed in the perinuclear fraction (results not shown). data from both the LDH assay and the DNA precipitation assay document the purity of each fraction obtained during the cell separation. Sirover Fig.446 J. 4d).

Thus. a known GAPDH inhibitor. Such binding could result in the formation of non-glycolytically active oligomers from enzymatically active GAPDH tetramers. normal GAPDH dehydrogenase activity was reported in AD or in HD postmortem brain tissue (Browne et al. 1997) and in HD (PorteraCailliau et al. AD and HD cells. 1996). neuronal protein binding could facilitate tetrameric to monomeric GAPDH dissociation. rendering it less catalytically active. Cell strains are in same order as described in legend for Fig. 76. 1999). 1998). Tabrizi et al. the above ®ndings suggest that preparation of whole cell or brain tissue samples from AD or from HD patients destroy subcellular GAPDH interactions which inhibit its glycolytic activity. may re¯ect structural alterations separate from changes in the primary amino acid structure of the GAPDH protein. 1997. Our results also indicate that nuclear GAPDH may be altered. Smale et al. These latter ®ndings are consistent with previous studies which demonstrated normal GAPDH glycolytic activity in AD or in HD patients. this would present the most likely basis for the observed diminution of activity. Simultaneously. The destructive effect of GAPDH inhibition in the brain was substantiated by experiments in vivo. it was hypothesized previously that GAPDH : neuronal protein interaction would inhibit its glycolytic activity causing a signi®cant de®cit in energy production. In contrast. 1999. 1994. 1998). no measurable change was observed in crude AD or in crude HD cell sonicates. Accordingly. 1995. As GAPDH is an essential glycolytic enzyme. Ishitani and Chuang 1996. Ishitani et al. tissue transglutaminase may cross-link GAPDH with proteins produced from CAG expansions (Gentile et al. 1995. as de®ned by antigenic analysis. To our knowledge. Goldberg et al. (b) nuclear and (c) perinuclear. 1997). Our results suggest that GAPDH glycolytic activity may be reduced in the cytoplasm of AD cells resulting in decreased energy production. resulted in the development of excitotoxic lesions consistent with those found in HD (Matthews et al. 2. 1996). post-translational mechanisms may exist in AD or in HD cells that inhibit GAPDH glycolytic activity. Different isomers of GAPDH may exist in mammalian cells (Ryzlak and Pietruszko 1988. It is conceivable that this observation may be related to the putative GAPDH nuclear function of apoptosis. Busciglio and Yankner 1995. Apoptosis has been regarded as the mode of cell death in AD (Cotman et al. 1995. Journal of Neurochemistry. 442±449 . 1995. 1998) resulting in decreased GAPDH glycolytic activity (Cooper et al. Lassmann et al. (a) Postnuclear. 1999) as well as in unstressed HD ®broblasts (Cooper et al. 1996. In the latter analysis. Epner and Coffey 1996. Two scenarios may provide potential explanations for this post-translational effect: (1) inhibition of GAPDH dehydrogenase activity by subcellular protein interactions. previous studies demonstrate GAPDH : neuronal protein binding. 6 Subcellular expression of the GAPDH gene in AMC. A basic mechanism underlying its role in programmed cell death q 2001 International Society for Neurochemistry. this is the ®rst suggestion that subcellular. 1995. it was discovered that GAPDH glycolytic activity was decreased in metabolically stressed HD cells (Cooper et al. Intra-striatal administration of iodoacetate. Immunoblot analysis and subcellular fractionation were performed as described. However. Moreover. Alternatively. there does not appear to be any change of GAPDH primary protein structure in AD or in HD cells. In contrast. Zhang et al. Recent studies demonstrate that GAPDH may be involved in neuronal apoptosis (Sunaga et al. This idea is supported by in vivo data which demonstrated that glucose utilization is decreased by 20±40% in both AD and HD brain tissue (Meier-Ruge and Bertoni-Freddari 1996). This would greatly contribute or possibly even initiate the disease process (Barinaga 1996. 1997). Alzheimer's and Huntington's disease 447 Fig. 1998). Lubec et al. Roses et al. Saunders et al. Discussion The results presented in this study demonstrate a signi®cant decrease in intracellular GAPDH glycolytic activity in AD and HD cells which is independent of any change in GAPDH gene expression.GAPDH. or (2) the presence of different GAPDH isomers in AD or in HD cells. Roses 1996. Zeitlin et al.

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