Chronic ethanol effects on glycoconjugates and glycosyltransferases of rat brain

Alcohol, Vol. 11, No. 4, pp. 301-306, 1994 Copyright©1994ElsevierScienceLtd Printedin the USA.All rightsreserved 0741-8329/94$6.00 + .00

Pergamon 0741-8329(94)E0009-2

Chronic Ethanol Effects on Glycoconjugates and Glycosyltransferases of Rat Brain F A U S T A O M O D E O - S A L I ~ .1 A N D P A O L A P A L E S T I N I t

*Institute of General Physiology and Biochemistry, Faculty of Pharmacy, University of Milan, Italy t Study Center for the Functional Biochemistry of Brain Lipids, Department of Medical Chemistry and Biochemistry, University of Milan, Italy Received 24 M a y 1993; Accepted 16 December 1993 OMODEO-SALI~, M. F. AND P. PALESTINI. Chronic ethanol effects on glycoconjugates and glycosyltransferases of rat brain. ALCOHOL 11(4) 301-306, 1994.-We studied the effects of a four week administration of low doses of ethanol on glycoconjugates of the synaptosomal and microsomal fraction prepared from the brain of rats aged 2 and 7 months. Synaptosomes were the more sensitive to ethanol treatment. Total lipid bound sialic acid and neutral glycolipid and glycoprotein content were significantly reduced only in the synaptosomal fraction, with greater differences in the younger age, while glycoprotein sialic acid was not affected. None of the above differences were statistically significant in the microsomai fraction. Ganglioside pattern was altered only in the 2 month rats, showing a reduction of GM1 and GDla in the synaptosomal fraction and of GDla in the microsomal fraction. UDP-Gal : asialo-mucin galactosyltransferase, UDP-Gal : GlcCer galactosyltransferase, and UDP-Gal : GM2 galactosyltransferase activities were decreased and could account for the observed modifications in glycoconjugate content and distribution. Ethanol

Glicosphingolipids

Glycoproteins

Glycosyltransferases Synaptosomes

ACUTE and chronic ethanol administration have been shown to exert functional effects at the level of cell membranes of the central nervous system. In most of the experimental designs the effects of chronic ethanol consumption have been studied in animals given high doses of ethanol (leading to a blood concentration greater than 30 mM). Much of the attention has been focused on ethanol-induced changes in cholesterol concentration and glycerophospholipid pattern or fatty acid composition; these modifications may reflect an adaptive mechanism of the membrane to compensate for the disordering effect of alcohol. However, the results reported from differents investigations are often scattered and conflicting (4, 10,21,36,45); this might be due to different experimental designs (different membranes structures or animals of different strain or age or submitted to different conditions of ethanol administration). It has been suggested that acute ethanol administration promotes an enhancement of the enzymatic cleavage of sialic acid residues, possibly due to conformational changes of glycoconjugates (17,23); however, in spite

Microsomes

of the glycosphingolipid and glycoprotein role in neuronal membranes, little attention has been payed to the biochemical changes developed after a chronic ethanol exposure on their content and pattern as well as on the enzymatic activities involved in their synthesis. The neuronal membranes are the primary site of ethanors action. Therefore, the aim of this study was to investigate the effects of a 4-week administration of low doses of ethanol on glycoconjugates of rat brain synaptosomal and microsomal fraction. The subcellular fractions have been prepared from rats of two different ages and analyzed for a) the glycolipid and glycoprotein content, b) the ganglioside percentage distribution, and c) the activity of some lipid and protein glycosyltransferases. METHOD

Chemicals The reagents used (analytical grade) and HPLC columns were purchased from Merck GmbH (Darmstadt, Germany).

Requests for reprints should be addressed to Prof. Fausta Omodeo-Sal~, Istituto di Fisiologia Generale e Chimica Biologica, Facolt~ di Farmacia, via Trentacoste 2, 20134 Milano, Italy. 301

302

OMODEO-SALI~ AND PALESTIN1

ATP (sodium salt), Ouabain, was purchased from BoehringerMannheim. Ficoll, which was purchased from Pharmacia Fine Chem. (Uppsala,Sweden), was exhaustively dialyzed against water before use. UDP-(3H)Galactose (UDP-Gal,9.25 GBq/ mmol) and CMP-(~4C) N-acetylneuraminicacid (CMP-NeuAc, 9.69 GBq/mmol) were obtained from Amersham (UK). Bovine submaxillary mucin type I and serum albumin, cold UDP-Gal, CMP-NeuAc, and the glycosphingolipids GlcCer and GM2 were from Sigma Chemical Co.(St. Louis, MO).

Animals and Diets Male rats (Wistar strain, Charles River), at the time of the experiments aged 2 months and 7 months, were housed individually with free access to a rodent laboratory chow. Animals of each age were divided in two groups: the ethanol treated were allowed free access to drinking water containing 20°7o (v/v) ethanol, while the controls were pair fed with an equicaloric drinking water with sucrose substituted for ethanol. Three experiments were performed, eight rats being taken for each experiment. Food and water intake were monitored daily; the animals were mantalned on this regime for 4 weeks and fasted overnight with water or the alcoholic solution ad lib prior to sacrifice. Blood was collected for ethanol determination (Boehringer-Mannheim Test Combination).

Preparation of Subcellular Fractions All work was performed at 0-4°C. Rats were killed by decapitation and whole brains without pons, medulla oblongata, and cerebellum were freed from meninges, weighed, washed with cold homogenizing solution, and homogenized with 9 vol. of cold 0.32 M sucrose solution containing 1 mM K + phosphate buffer (pH.7.4) and 0.1 mM EDTA. The homogenization and the preparation of the crude mitochondrial fraction were accomplished as reported (26) using a Kontron Centrikon T 2070 ultracentrifuge and a rotor type T 35. The fraction enriched in nerve endings (synaptosomal fraction) was prepared from the crude mitochondrial fraction (26), using the above ultracentrifuge with rotor type SW28. The microsomal fraction (P3) was enriched in the light component by a centrifugation at 17.000 × g for 25 min and then pelleted by centrifugation at 105.000 × g for 1 h. The subcellular fractions were stored at - 8 0 ° C until further use.

(26,35). 2' ,3'-Cyclic nucleotide 3' phosphoidrolase (CNPase) was assayed in the synaptosomal fraction as measure of myelin contamination (44). For each enzyme the RSA, (relative specific activity) as referred to the starting homogenate, was calculated. Protein concentration was determined (22) with crystalline bovine serum albumine as a standard.

Biochemical Analyses Lipid andprotein analyses. Lipids were extracted and partitioned (39). Aliquots of the ganglioside extracts were used for the determination of lipid-bound sialic acid by a modification (25) of a reported procedure (40). Gangliosides were fractionated by HPLC, identified, and quantified (30), using standard gangliosides as reference compounds. Neutral glycolipid content was determined in the organic phase as neutral hexoses (46) after hydrolysis in 1 N HCI at 100°C for 4 h. In order to estimate the membrane glycoprotein content, the defatted residue was assayed for neutral sugars after 4 h hydrolysis in 1 N HCI at 100°C (46) and for sialic acid (43) after 1 h hydrolysis in 0.5 N HzSO, at 80°C and purification on a Dowex 2 x 8 column (40). Glycosyltransferase assays. Mucin was desialylated by mild hydrolysis in 0.1 M HESO4 (37). UDP-Gal : GM2-ganglioside (/~ 1-3) galactosyltransferase (GalTIII) was assayed in the crude mitochondrial fraction (P2 fraction) (2); CDP-cotine was added to the incubation mixture (31). UDPGal : asiatomucin transferase, CMP-NeuAc : asialo-mucin transferase, UDPGal : GlcCer (fl 1-4) galactosyltransferase (GalTII) were assayed in the microsomal fraction as reported (2,6,28). Incorporation of radioactivity into glycoproteins was determined in the pellets obtained by precipitation with 1007o TCA. Pellets were washed and centrifuged with the TCA solution twice, solubilized in 100/xl Soluene (Packard)at 50°C overnight and counted with a Packard Tricarb 1500 liquid Scintillation Analyzer. When using a glycolipid as acceptor, the enzymic reactions were stopped by the addition of 20 #l chloroform/methanol (2 : l, by vol.); the mixture was streaked on Whatman 3 MM filter paper and subjected overnight to descending chromatography with 1070 Na2B407. The labeled origin plus 3 cm was quantitated for radioactivity.

Statistics

Assays of Marker Enzymes

The statistical significances of differences in values between ethanol and control groups were determined by the Student's t-test.

Ouabain-sensitive Na*-K ~ ATPase, a marker for plasma membranes, and TPPase, a marker for intracellular membranes, particularly trans Golgi apparatus, were assayed, respectively, in the synaptosomal and microsomal fraction

The average food intake of animals supplemented with ethanol was lower than that of control rats, but the difference

RESULTS

TABLE 1 CONCENTRATION OF GLYCOCONJUGATESOF SYNAPTOSOMAL FRACTION FROM CONTROL AND ETHANOL-TREATEDRATS ~g/mg protein) Protein-Bound NeutralHexoses 2Months-control 2 Months-ethanol 7 Months-control 7 Months-ethanol

253 ± 41 175 ± 30* 370 = 50 320:1:43

Lipid-Bound NeutralHexoses 47.4 33.5 55.1 44.5

± -r ± ±

5.1 5.0~ 3.0 4.9*

Protein-Bound SiaticAcid 3.80 3.50 2.75 2.88

± ± ± ±

0.7 0.6 0.3 0.6

Lipid-Bound SialicAcid 14.7 10.5 14.5 11.6

± 1.0 ± 0.7* ~: 0.4 ± 0.5

Mean ± SD (n = 6). Statistical significances between ethanol and the same age group. *p < 0.05; tp < 0.02.

ETHANOL

EFFECTS

303

TABLE 2 CONCENTRATION OF GLYCOCONJUGATES OF MICROSOMAL FRACTION FROM CONTROL AND ETHANOL-TREATED RATS (#g/mg protein) Protein-Bound Neutral Hexoses 2 Months-control 2Months-ethanol 7 Months-control 7Months-ethanol

98 i00 67 66

Lipid-Bound Neutral Hexoses

± 17.0 ± 7.6 ± 7.6 ± 9.9

82 83 127 137

+ ± ± +

Protein-Bound Sialic Acid

12 10 15 20

5.4 5.3 6.9 6.3

± ± ± ±

Lipid-Bound Sialic Acid

0.5 0.6 1.7 0.2

18.7 16.3 20.1 20.9

± 1.8 _+ 0.8 ± 0.9 ± 1.I

Mean ± SD (n = 6). None of the differences between ethanol and control groups were statistically significant.

was not statistically significant. In the 2 a n d 7 m o n t h ethanolfed rats, the daily intake o f e t h a n o l was 10.12 + 0.77 a n d 5.57 + 0.81 g / k g b.wt., respectively. The m e a n b l o o d e t h a n o l c o n c e n t r a t i o n at the time o f sacrifice was 18.6 _+ 0.9 m M for 2 m o n t h rats a n d 13.3 + 0.4 m M for 7 m o n t h rats. T h e RSA values o f the m a r k e r enzymes showed in the syna p t o s o m a l fraction a n e n r i c h m e n t o f a b o u t threefold over total h o m o g e n a t e in Na+-K ÷ A T P a s e a n d a C N P a s e R S A o f a b o u t 0.5; T P P a s e , a Golgi m a r k e r enzyme, was enriched 2.5-fold in the m i c r o s o m a l fraction. R S A values were not significantly different a m o n g the different groups o f rats. The low cholesterol a n d p h o s p h o l i p i d c o n t e n t (data not shown) a n d the low c h o l e s t e r o l / p h o s p h o l i p i d m o l a r ratio (ranging from 0.45 to 0.55 in the four g r o u p s o f rats) in the synaptosomal fractions exclude significant c o n t a m i n a t i o n by myelin membranes. Analysis o f b r a i n m e m b r a n e s showed t h a t s y n a p t o s o m e s were m o r e sensitive t h a n m i c r o s o m e s to e t h a n o l treatment. S y n a p t o s o m a l l i p i d - b o u n d neutral hexoses were significantly decreased b o t h in the 2 m o n t h ( p < 0.02) a n d in the 7 m o n t h rats ( p < 0.05) (Table 1), while glycoprotein neutral hexoses a n d lipid b o u n d sialic acid showed significant reductions only

in the 2 m o n t h rats ( p < 0.05). In contrast, the c o n t e n t o f p r o t e i n - b o u n d sialic acid was not affected either in the 2 or 7 m o n t h rats. Similarly, n o effects were exerted by the e t h a n o l t r e a t m e n t o n the lipid a n d p r o t e i n - b o u n d neutral hexoses a n d sialic acid o f the m i c r o s o m a l fraction (Table 2). A f t e r chronic e t h a n o l c o n s u m p t i o n ganglioside p a t t e r n was affected in b o t h the s y n a p t o s o m a l a n d the microsomal fraction (Tables 3 a n d 4). In the 2 m o n t h group, the ganglioside c o n t e n t o f the synapt o s o m a l fractions showed the reduction o f G M I a n d G D l a gangliosides paralleled by their percentage decrease ( p < 0.05).In the 7 m o n t h group, none o f the differences were statistically significant. In the microsomal fraction o f 2 m o n t h rats ethanol t r e a t m e n t induced a significant reduction o f G D l a , a l t h o u g h the ganglioside p a t t e r n showed the reduction o f G D I a ( p < 0.05) a n d the increase o f G D l b ( p < 0.05) a n d G T l b ( p < 0.001). The percentage values in the 7 m o n t h rats were not significantly affected. A t b o t h ages, glycolipid synthesis was i m p a i r e d in the Gal TI1 activity (LacCer synthase) ( p < 0.05 a n d p < 0.02 for 2 a n d 7 m o n t h rats, respectively), while Gal T i l l activity (GM1 synthase) was affected only in the younger rats ( p < 0.05). Glycoprotein synthesis was impaired only in the 2 m o n t h rats in the U D P - G a l : asialo

TABLE 3 EFFECT OF CHRONIC ETHANOL TREATMENT ON THE SYNAPTOSOMAL GANGLIOSIDE COMPOSITION OF UNTREATED AND TREATED RATS 2 Months-Control #g

GM1 GDla GDIb GTIb GQIb

Ganglioside/ mg Protein

2.5 13.1 3.3 9.3 1.0

± ± + ± ±

0.08 0.6 0.5 0.8 0.2

2 Months-Ethanol

Ganglioside (Molar ~/0) 16.8 44.6 15.7 21.0 1.7

+ ± ± ± ±

0.5 2.2 2.2 1.9 0.3

#g Ganglioside/ mg Protein 1.6 8.4 3.9 7.4 1.0

7 Months-Control ttg Ganglioside/ mg Protein GM1 GDla GDlb GTlb GQIb

2.1 11.8 6.5 8.7 1.3

± + + + ±

0.3 0.7 0.8 0.4 0.2

0.08 0.5 0.4 0.7 0.3

15.0 40.0 18.8 23.6 2.5

± ± ± ± ±

0.8* 2.6* 2.2 2.4 0.7

7 Months-Ethanol

Ganglioside (Molar ~0) 14.5 41.0 22.3 20.0 2.2

± ± ± ± ±

Ganglioside (Molar *70)

± 1.5 _ 2.4 + 2.7 _+ 0.9 + 0.2

#g Ganglioside/ mg Protein 1.5 9.8 5.5 6.7 0.8

_+ 0.2 ± 0.3 + 0.4 + 0.6 ± 0.2

Ganglioside (Molar %) 13.0 42.1 23.9 19.2 1.8

_+ 1.7 + 1.3 _+ 1.9 ± 1.8 ± 0.4

Mean + SD of 4-6 determinations. Statistical significances between ethanol and the same age control group. *p < 0.02.

304

OMODEO-SALE AND PALESTINI

TABLE 4 EFFECT OF CHRONIC ETHANOL TREATMENT ON THE MICROSOMAL GANGLIOSIDE COMPOSITION OF UNTREATED AND TREATED RATS 2 Months-Control ~g Ganglioside/ mg Protein GM1 GDIa GDIb GTlb GQlb

3.6 17.4 5.0 10.4 1.3

± ± ± ± ±

2 Months-Ethanol

Ganglioside (Molar %l

0.3 0.7 0.7 0.3 0.5

19.4 46.5 13.5 18.6 1.7

t~gGanglioside/ mg Protein

+_ 1.5 ± 2.0 ± 1.9 ± 0.5 ± 0.7

3.0 13.7 5.4 10.3 1.1

± ± + ± ±

7 Months-Control /tg Ganglioside/ mg Protein GM1 GDIa GDIb GTIb GQIb

3.1 16.4 8.1 13.0 1.7

± ± ± ± ±

15.4 40.8 20.1 21.5 2.2

0.3 0.6* 0.5 0.6 0.5

18.4 42.1 16.7 21.0 1.7

± ± ± ± ±

1.7 1.8t 1.5I 1.35 0.8

7 Months-Ethanol

Ganglioside (Molar %)

0.2 0.6 0.4 0.4 0.5

Ganglioside (Molar %)

± ± ± ± ±

/~gGanglioside/ mg Protein

1.2 1.5 1.0 0.8 0.7

3.2 18.2 7,9 12.7 1.6

± ± ± ± ±

Ganglioside (Molar %)

0.4 0.8 0.5 0.5 0.6

15.2 43.6 19.0 20.3 1.9

± 1.1 _+ 2.0 ± 1.3 ± 0.9 ± 0.6

Mean ± SD of 4-6 determinations. Statistical significances between ethanol and same age control groups. *p < 0.02; 1"p < 0.05; ~p < 0.001.

mucin transferase ( p < 0.01); in contrast, the activity o f C M P - N e u A c : asialo m u c i n t r a n s f e r a s e in e t h a n o l treated rats did n o t s h o w any significant difference (Table 5). DISCUSSION In agreement with d a t a o b t a i n e d f r o m total b r a i n studies (1,24), control 7 m o n t h rats c o m p a r e d to the younger ones show a similar ganglioside c o n t e n t a n d a m o d i f i c a t i o n o f the ganglioside p a t t e r n . Feeding the rats with a nutritionally adequate diet s u p p l e m e n t e d with low doses o f ethanol for 4 weeks

results in alterations in glycoconjugate c o n t e n t a n d pattern o f brain as well as in i m p a i r m e n t o f s o m e glycosyltransferase activities involved in glycoconjugate b i o s y n t h e s i s . : S y n a p t o somes were m o r e affected t h a n m i c r o s o m e s in their g l y ~ n jugate content. The data show that the s y n a p t o s o m a l gaaglioside c o n t e n t was decreased in the e t h a n o l - t r e a t e d rats, b u t t h e differences were statistically significant only i n t h e 12 m o n t h animals. These data could be explained by the ethanol b l o o d c o n c e n t r a t i o n that was s i ~ f i c a n t t y h i g h e r : r a t h e younger rats; the lower alcohol c o n s u m p t i o n o f older animals is i n agreem e n t with data showing that alcohol c o n s u m p t i o n decreases

TABLE 5 GLYCOSYLTRANSFERASE ACTIVITIES IN MICROSOMAL FRACTION FROM THE BRAIN OF CHRONICALLY ETHANOL-TREATED AND UNTREATED RATS Enzymatic Activity (pmol/mg prot/30)

2 Months Control

2 Months Ethanol

7 Months Control

7 Months Ethanol

UDPGal : asialomucin Galactosyltransferase

798

± 46

619 ± 21"

809

± 40

753

± 30

CMPNeuAc : asialomucin sialyltransferase

267

± 15

262 ±

259

± 15

289

± 14

UDPGal : GlucCer Galactosyltransferase (GAL T II) UDPGal : GM2 Galactosyltransferase (GalTIII)

99.7 ±

8.0

8.0

80 +

2.0-~

60 ±

3.1

141.5 ± 10.0

104 ±

8.11"

142.5 +

6.0

44,9 +__

126

1.5~t

± 15.0

Gal T II activity was determined in the P2 fraction. Mean values + SD (n -- 6). Results are expressed as picomol of sugar incorporated into exogenous protein or lipid acceptor. *p < 0.01; tP < 0.05; ~;p < 0.02.

ETHANOL EFFECTS

305

with age (47); however, a greater sensitivity of the 2 month rats, compared to the older ones, cannot be excluded because it has been suggested that cell membranes, whose lipid composition undergoes substantial changes with age (3,30), could respond in different ways to ethanol treatment due to their structural features (45). Ethanol-induced decrement in gangliosides can occur from a decreased synthesis or an increased catabolism. It appears that in rats exposed to ethanol, a decrease in ganglioside as well in neutral glycolipid synthesis occurs; the decrement of synaptosomal lipid-bound sialic acid and neutral hexoses is accompanied by the impairment of Gal TII activity leading to the synthesis of lactosylceramide, precursor in glycolipid biosynthesis. Similarly, a decrease of neutral and acidic carbohydrates has been found in erythrocyte membranes from alcoholics (38). No differences in total ganglioside content were found by Harris et al. (10) in synaptosomal membranes of DBA/2 mice after administration of an ethanol-containing liquid diet. Moreover, differently from us, they found a significant decrease of cholesterol content in the ethanol-exposed animals. These contradictory findings could be due to the different experimental animals or to the different conditions of ethanol exposure, and outline that caution must be adopted in comparing results from different experimental designs. The synaptosomal ganglioside pattern is altered only in the 2 month rats, showing the decrease of GM1 and G D l a . The absolute amount of GM1, G D I a , and G T I b gangliosides is also decreased; the lower content of GM1 can be related to the lower activity of GalTIII synthase involved in its synthesis (15). In agreement with our data are data from other laboratories showing the lower developmental increase of GM1 in the offspring of ethanol-treated rats (9). The inhibitory effect of alcohol on GMI sialyltransferase activity (GDIa synthase) has been shown (29) and could be related to the lower content of G D I a and GTIb. However, attempts to correlate G D l a and GT1 b changes to their synthase activity were unsuccessful because, in agreement to other reports (16), this enzymatic activity was undetectable in the microsomes of 2 and 7 month animals. Some reports have suggested that gangliosides may be involved in the interaction of ethanol with the neuronal membrane at the membrane-water surface (12) and may modulate the sensitivity of the membrane to the effects of this drug (11). A role has been hypothesized for GM1 in introducing new binding sites at the surface of the membrane (19). Moreover, it has been suggested that tolerance to the perturbing effects of ethanol could be due to the decreased binding of ethanol to the membrane (34). In agreement with these hypotheses, a different amount of GM 1 ganglioside was found by Ullman et al. (41) in mice genetically different in sensitivity to ethanol hypnosis. A lower GM1 content was found in the whole brain and synaptosomal membranes from the cerebellum of the eth-

anol-insensitive rats (short sleep) compared to the ethanolsensitive ones (long sleep), suggesting that this ganglioside might be one of the several biochemical factors underlying ethanol sensitivity and playing a role in tolerance/dependence development. On the basis of these and our data, we cannot exclude, as suggested in acute ethanol intoxication (18), that beside impairing the biosynthesis of gangliosides, chronic ethanol treatment might also enhance their degradation. However, microsomes were not affected in their total ganglioside sialic acid. The tolerance to the effects of ethanol may be due to the structural differences between the synaptic membranes and the plasma membranes included in this fraction, probably derived from the neuronal soma as well from different types of glial cells. The decrease in glycoprotein neutral hexoses, shown in the synaptosomal fraction of young rats, can be related to the impairment of the UDPGal : asialo mucin transferase. However, no differences were found between control and treated animals in the glycoproteic sialic acid and in the CMPNeuAc : asialo mucin transferase activity. The different sensitivity of ganglioside and glycoprotein sialyltransferases to ethanol could support their belonging to different classes of enzymes (28). It is also well known that membrane-bound enzymes can be influenced by the lipid composition and by the physical properties of the membrane. In particular, it has been shown that the activity of different glycosyltransferases is modulated by phospholipids (13,34), whose distribution and fatty acid composition are affected by ethanol treatment (2, 7,20,27). In this study it is, thus, difficult to discriminate between the direct action of ethanol on the glycoprotein and glycolipid glycosyltransferase activities and the effects mediated by other lipid components that could be affected by ethanol treatment. In conclusion, we have shown that brain subcellular fractions may be affected in their glycoconjugate content and distribution by low doses of ethanol and differ in their sensitivity to alcohol. It is generally accepted that ethanol preferentially affects functions that involve synaptic transmission (5); our studies, showing significant effects in the synaptosomal fraction, suggest its vulnerability to ethanol damage. It is possible that the biochemical modifications induced by ethanol represent for synaptosomes, the more sensitive structures, an adaptive mechanism to resist its perturbing effects. Because gangliosides modulate many physiological processes (8,14,33), alcohol-induced modifications could affect important cellular functions and mediate some of the pharmacological effects of the drug. ACKNOWLEDGEMENT This work was supported by a research grant from the M.V.R.S.T. (60°70).

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