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UPD-galactose:Ceramide galactosyltransferase activity in dissociated cell cultures from brain hemispheres of newborn rats
Neuroscwnce Letters, 19 (1980) 109-114
109
© Elsevier/North-Holland Scientific Pubhshers Ltd.
UPD-GALACTOSE:CERAMIDE GALACTOSYLTRANSFERASE ACTIVITY IN D I S S O C I A T E D CELL C U L T U R E S F R O M B R A I N H E M I S P H E R E S OF N E W B O R N RATS
N.M. NESKOVIC and G. LABOURDETTE Centre de Neurochtmte du CNRS, 11 rue Humann, 67085 Strasbourg Cedex (France)
(Recewed April 21st, 1980) (Revised version recewed May 27th, 1980) (Accepted May 27th, 1980)
SUMMARY
Primary cultures enriched in oligodendroglial cells were prepared from dissociated newborn rat brain. Enzymatic study revealed the presence of high specific activities of UDP-galactose'ceramide galactosyltransferase (CGalT) and 2' ,3' -cyclic nucleotide-3 ' -phosphohydrolase (CNP). The specific activity of CGalT was twice as high as that of brain. The CGalT activity increased from 16 days in culture, reached a maximum at about 50 days and declined thereafter. The CNP activity reached a maximum after 60-70 days in culture and remained more or less stable in the following period. The results indicate that in oligodendroglial cells in vitro the regulation of CGalT activity proceeds in a manner similar to that of myelinating brain.
The nervous tissue cultures provide a valuable system for the studies of myelinogenesis in vitro [1-3, 12]. The galactolipid biosynthesis, one of the most prominent parameters of myelination, was observed in explants or reaggregate cultures of CNS, and the corresponding enzyme activities were shown to parallel the appearance of the myelinated axons [7, 10, 19]. Such systems faithfully reproduce, to a certain extent, the structural and biochemical events which occur in vivo. On the other hand, the heterogeneity of cell populations in culture of this type is, like in brain, a serious obstacle in studying the enzyme regulation of myelinogenesis at the cellular level. Due to the presence of neurons and the formation of myelin, these cultures do not permit us to discriminate between the overall control of myelination and the mechanisms which regulate the synthesis of proteins and enzymes specific to the myelin-forming oligodendroglia (OG).
110 In our laboratory we have recently obtained primary cultures of d~ssoclated cells from newborn rat brain which contain astroblasts and OG cells [9]. Due to the absence of neurons [18] the formation of myelin is not observed m these cultures; however, the maturation of OG occurs to some extent, as shown by the presence of W1 Wolfgram protein, myelin basic proteins and 2',3'-cyclic nucleotide-3'phosphodiesterase (EC 3.1.4.16; CNP) [9]. Therefore, this type of culture seems suitable for studies of OG maturation and of regulation of OG°speclfic enzymes and proteins, independently of the myelin formation. In the present paper we report the presence of UDP-galactose:ceramide galactosyltransferase (EC 2.4.1.45; CGalT) m such primary cultures. This transferase, which catalyzes the synthesis of cerebroside, the major glycolipid of myelin, is presumably an OG-specific enzyme [6, 13]. The time course of CGalT activity was determined and compared with that of CNP, another enzyme related to the activity of myelin-forming OG cells [16]. The cultures were prepared and grown as previously described [9], except that the trypsin treatment was omitted. For the enzyme assays cells from 2 - 4 culture dishes (10 cm diameter) were harvested by scraping with a rubber policeman, washed in cold Isotonic NaC1 solution and recovered by centrifuging. The pellets were dispersed in a small volume of 50 mM potassium phosphate buffer (pH 7.6) containing 50% (v/v) glycerol and sonically irradiated for 1 mm at 0°C (150 W MSE model sonicator with a 4 mm ntanium probe at 15% of the maximum intensity). The protein content of the homogenates so prepared averaged 6 mg/ml as determined by the method of Lowry et al. [11], with bovine serum albumin as a standard. Brains of Wistar albino rats were homogenized with 9 vol. of the buffer described above in a Potter-Elvehjem homogenizer at 0°C. The enzyme preparations were stored at - 2 0 ° C for several days. The assay of CGalT was performed as previously described [14] except that the dispersion of ceramide was prepared by heating at 100°C [15]. In each experiment at least two assays were run at different protein concentrations ranging from 160 to 280 #g/incubation mixture. To identify the labeled glycolipid formed with homogenates of cultured cells, lipids were extracted from 5 incubation mixtures and glycolipids separated by TLC on the borate-impregnated plates [13]. After scanning for radioactivity, only one labeled glycolipid, which co-chromatographed with reference cerebroside containing ~hydroxy-fatty acids, was detected. Determination of CNP activity m the homogenates treated with sodium deoxycholate [8] was carried out by the precipitation method [20]. The activity of CGalT determined as a function of time in culture is presented in Fig. lB. The data from several separate sets of cultures are given. Fairly reproducible results were observed with respect to the specific activity at a given age and to the pattern of development. CGalT activity could be detected after 16 days in culture; thereafter the enzyme activity increased up to the 50th day in culture and decreased rapidly until the 70th day. In one set of cultures which could be maintained beyond this period the low level o f CGalT activity remained stable up to the 90th
111
21 o)
=
1
.~
o
o
1
A
G 0
20
40
60
80
AGE (DAYS)
Fig 1. Specific actwity of CGalT as a function of age. Enzyme actw~t~es are gwen as nmol of galactose transferred per hour per mg of protein. A: CGalT m brain homogenates of rat. Abscissa, days after birth. B: CGalT in homogenates of cultured brain cells. Values from the separate sets of cultures are represented by different symbols Abscissa, days m culture.
day. To facilitate the comparison with in vivo evolution, the CGalT values in developing rat brain are given in Fig. 1A. As observed earlier [4, 5, 13] the specific activity of the enzyme reached the maximum after about 18 days of age and decreased rapidly to attain relatively low level after 30 days of age. Therefore, the peak of CGalT activity observed in our cultures was clearly retarded in comparison with the peak of CGalT in brain. This is in contrast with the results of explant cultures where the activities of the galactolipid-synthesizing enzymes could be detected in the first week of the culture [10]; also in reaggregated cell cultures the activity of cerebroside sulfotransferase was measurable after 12 days in vitro and the peak of activity was observed after about 21 days in culture, which closely resembles the situation in brain [19]. On the other hand, the 'retarding effect' was observed in the cultures of dissociated brain cells from 16-day-old mouse embryo, showing a maximum CGalT activity at about 40 days in culture [17]. The increase of CGalT activity observed between 16 and 50 days in culture could result from the proliferation of OG and/or from the increased synthesis of the enzyme within these cells. Although it was difficult to evaluate the relative numbers of OG and astrocytes in such cultures, the light microscopy and immunohistochemical observations indicated that the proportion of OG in the culture increased as a function of time (Labourdette, unpublished results). The finding of the maximum specific activity of CGalT twice as great as the corresponding activity in brain is in agreement with a relative enrichment of OG in
112 these types of primary cultures. As far as we are aware, such high specific activity of CGalT has not been previously reported in nerve cell cultures. The specific activity of CNP increased gradually up to about 50 days in culture and remained more or less stable thereafter, with only a slight decrease after the 60th day (Fig. 2B). As observed earlier [9], the developmental curve of CNP in cultures was comparable to that found in vivo (Fig. 2A; see also ref. 8). Although CGalT and CNP attained maximal activities approximately during the same period, the comparison of the two activities at different points of the developmental curves shows that the changes of CGalT proceeded at a more rapid rate: C N P / C G a l T specific activity ratio was greatest after 16 days in culture (220); it then decreased to attain the lowest value (70) at the 50th day, and then increased again (130 and 170 after 60 and 70 days in culture, respectively). Moreover, the results of Fig. 2 show that CNP activity was stable from the 50th-60th day in culture, i.e. in the period when CGalT activity decreased rapidly. As a constituent of OG membranes [16], CNP can be considered as a reliable indicator o f the relative number of OG cells in the cultures where myelin is absent. Therefore, the above results suggest that a loss of OG from the cultures did not occur to such an extent that it could account for the decrease of CGalT after the 50th day. If so, the evolution of CGalT observed in our cultures is due, at least in part, to the changes of the enzyme activity within OG cells. The finding of a high CGalT activity in the OG-enriched cultures, coupled with the previously reported presence of basic protein, Wl Wolfgram protein and CNP [9], show that OG in long-term dissociated cultures retain the capacity to synthetize at least 4 of their specific proteins. The observation that OG exhibits a characteristic development of the CGalT activity in the absence of the complete myelination
o) o s
o~ 200 E ..c
N loo
2: 0
0 20 '
40 °
6'0
80 '
AGE (DAYS)
F~g 2. Specific actw~ty of CNP as a function of age. Enzyme actw~tles are g~ven as ~tmol of adenosme-2'phosphate formed per hour per mg of protein A: CNP in brain homogenates of rat Abscissa, days after b~rth B' CNP m homogenates of cultured brain cells Values from the separate sets of cultures are represented by different symbols Abscissa, days m culture
113 s y s t e m , is a p h e n o m e n o n
n o t p r e v i o u s l y r e p o r t e d in tissue c u l t u r e s o f C N S a n d m a y
h a v e i m p o r t a n t r e p e r c u s s i o n s in s t u d y i n g t h e e n z y m e r e g u l a t i o n o f m y e l i n o g e n e s i s . ACKNOWLEDGEMENTS T h i s w o r k w a s s u p p o r t e d in p a r t b y g r a n t s f r o m C N R S . W e a r e m o s t g r a t e f u l to M i s s M . P e r r a u l t , D. B r e v i a n d R. R e e b f o r t h e i r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . W e t h a n k D r L . L . S a r l i e v e f o r c o m m u n i c a t i n g us his results p r i o r t o p u b l i c a t i o n . T h e a u t h o r s a r e C h a r g 6 s de R e c h e r c h e o f I N S E R M .
REFERENCES 1 Bornstein, M.B., Differentiation of cells in primary cultures, myellnatlon. In S. Fedoroff and L Hertz (Eds.), Cell, Tissue and Organ Cultures m Neurobiology, Academic Press, New York, 1977, pp. 141-146. 2 Bourre, J.M., Honegger, P., Daudu, O. and Matthieu, J.M., The hpld composition of rat brain aggregating cell cultures during development, Neurosci. Lett., II (1979) 275-278. 3 Bradbury, K. and Lumsden, C.E., The chemical composition of myelin in organ cultures of rat cerebellum, J. Neurochem., 32 (1979) 145-154. 4 Brenkert, A. and Radln, N.S., Synthesis of galactosyl ceramlde and glucosyl ceramlde by rat brain: assay procedures and changes with age, Brain Res., 36 (1972) 183-193. 5 Costantmo-Ceccarini, E. and Morell, P , Biosynthesis of brain sphingohpids and myelin accumulation in the mouse, Llplds, 7 (1972) 656-659. 6 Deshmukh, D S., Flyn, T.J. and Plermger, R A , The biosynthesis and concentration of galactosyl diglyceride in ghal and neuronal enriched fractions of actively myehnating rat brain, J. Neurochem., 22 (1974) 479-485. 7 Fry, J.M., Lehrer, G.M. and Bornsteln, M.B., Sulfatlde synthesis, inhibition by experimental allergic encephalomyehtls serum, Soence, 175 (1972) 192-194 8 Kurlhara, T., Nussbaum, J.L. and Mandel, P., 2',3'-Cyclic nucleotide 3'-phosphohydrolase m brains of mutant mice with deficient myelination, J. Neurochem., 17 (1970) 993-997. 9 Labourdette, G., Roussel, G., Ghandour, M S and Nussbaum, J.L., Cultures from rat brain hemispheres enriched in ohgodendrocyte-hke cells, Brain Res., 179 (1979) 199-203. l0 Latovltzkl, N. and Sflberberg, D.H., UDP-galactose:ceramlde galactosyltransferase and 2',3'-cychc nucleotlde 3'-phosphohydrolase activities in cultures of newborn rat cerebellum. Assocmtlon with myelinatlon and concurrent susceptibility to 5-bromodeoxyundlne, J Neurochem, 29 (1977) 611-614. ll Lowry, O H., Rosebrough, N.J., Farr, A.L. and Randall, R J , Protein measurement with the Fohn phenol reagent, J. biol. Chem., 193 (1951) 265-275 12 Matthieu, J.M., Honegger, P., Trapp, B.D., Cohen, S.R. and Webster, H. de F., Myehnation in rat brain aggregating cell cultures, Neurosoence, 3 (1978) 565-572. 13 Neskovlc, N.M., Sarlleve, L.L. and Mandel, P , Biosynthesis of glycollplds m myelin deficient mutants, brain glycosyl transferase m Jlmpy and Quaking mice, Brain Res., 42 (1972) 147-157 14 Neskovlc, N.M., Sarlieve, L.L. and Mandel, P., Purification and properties of UDP-galactose: ceramide galactosyltransferase from rat brain mlcrosomes, Blochim. blophys. Acta (Amst.), 334 (1974) 309-315. 15 Neskovlc, N.M., Mandel, P. and Gatt, S., UDP-galactose ceramlde galactosyltransferase. Kinetic properties and effect of detergents and phosphohpids on the partially purified enzyme of rat brain In
114
16 17
18 19 20
S Gatt, L Freysz and P. Mandel (Eds), Enzymes of Lipid Metabohsm, Plenum, New York, 1978, pp 613-630 Poduslo, S E., The isolation and characterization of a plasma membrane and a myelin fraction derived from ohgodendrogha of calf brain, J Neurochem , 25 (1975) 647-654 Sarheve, L L., Subba Rao, G., Campbell, G LeM and Plermger, R.A , Investigation of myehnation m vitro: biochemical and morphological changes m cultures of dissociated brain cells from embryonic mice, Brain Res , 189 (1980) 79-90 Sensenbrenner, M., Dlssooated brain cells m primary cultures In S Fedoroff and L Hertz (Eds), Cell, Tissue and Organ Cultures m Neuroblology, Academic Press, New York, 1977, pp 191-213 Sheppard, J R , Brus, D and Wehner, J M , Brain reaggregate cultures biochemical evidence for myehn membrane synthesis, J Neuroblol , 9 0978) 309-315 Sims, N.R. and Carnegie, P R , A rapid assay for 2',3'-cychc nucleotlde 3'-phosphohydrolase, J Neurochem , 27 0976) 769-772
109
© Elsevier/North-Holland Scientific Pubhshers Ltd.
UPD-GALACTOSE:CERAMIDE GALACTOSYLTRANSFERASE ACTIVITY IN D I S S O C I A T E D CELL C U L T U R E S F R O M B R A I N H E M I S P H E R E S OF N E W B O R N RATS
N.M. NESKOVIC and G. LABOURDETTE Centre de Neurochtmte du CNRS, 11 rue Humann, 67085 Strasbourg Cedex (France)
(Recewed April 21st, 1980) (Revised version recewed May 27th, 1980) (Accepted May 27th, 1980)
SUMMARY
Primary cultures enriched in oligodendroglial cells were prepared from dissociated newborn rat brain. Enzymatic study revealed the presence of high specific activities of UDP-galactose'ceramide galactosyltransferase (CGalT) and 2' ,3' -cyclic nucleotide-3 ' -phosphohydrolase (CNP). The specific activity of CGalT was twice as high as that of brain. The CGalT activity increased from 16 days in culture, reached a maximum at about 50 days and declined thereafter. The CNP activity reached a maximum after 60-70 days in culture and remained more or less stable in the following period. The results indicate that in oligodendroglial cells in vitro the regulation of CGalT activity proceeds in a manner similar to that of myelinating brain.
The nervous tissue cultures provide a valuable system for the studies of myelinogenesis in vitro [1-3, 12]. The galactolipid biosynthesis, one of the most prominent parameters of myelination, was observed in explants or reaggregate cultures of CNS, and the corresponding enzyme activities were shown to parallel the appearance of the myelinated axons [7, 10, 19]. Such systems faithfully reproduce, to a certain extent, the structural and biochemical events which occur in vivo. On the other hand, the heterogeneity of cell populations in culture of this type is, like in brain, a serious obstacle in studying the enzyme regulation of myelinogenesis at the cellular level. Due to the presence of neurons and the formation of myelin, these cultures do not permit us to discriminate between the overall control of myelination and the mechanisms which regulate the synthesis of proteins and enzymes specific to the myelin-forming oligodendroglia (OG).
110 In our laboratory we have recently obtained primary cultures of d~ssoclated cells from newborn rat brain which contain astroblasts and OG cells [9]. Due to the absence of neurons [18] the formation of myelin is not observed m these cultures; however, the maturation of OG occurs to some extent, as shown by the presence of W1 Wolfgram protein, myelin basic proteins and 2',3'-cyclic nucleotide-3'phosphodiesterase (EC 3.1.4.16; CNP) [9]. Therefore, this type of culture seems suitable for studies of OG maturation and of regulation of OG°speclfic enzymes and proteins, independently of the myelin formation. In the present paper we report the presence of UDP-galactose:ceramide galactosyltransferase (EC 2.4.1.45; CGalT) m such primary cultures. This transferase, which catalyzes the synthesis of cerebroside, the major glycolipid of myelin, is presumably an OG-specific enzyme [6, 13]. The time course of CGalT activity was determined and compared with that of CNP, another enzyme related to the activity of myelin-forming OG cells [16]. The cultures were prepared and grown as previously described [9], except that the trypsin treatment was omitted. For the enzyme assays cells from 2 - 4 culture dishes (10 cm diameter) were harvested by scraping with a rubber policeman, washed in cold Isotonic NaC1 solution and recovered by centrifuging. The pellets were dispersed in a small volume of 50 mM potassium phosphate buffer (pH 7.6) containing 50% (v/v) glycerol and sonically irradiated for 1 mm at 0°C (150 W MSE model sonicator with a 4 mm ntanium probe at 15% of the maximum intensity). The protein content of the homogenates so prepared averaged 6 mg/ml as determined by the method of Lowry et al. [11], with bovine serum albumin as a standard. Brains of Wistar albino rats were homogenized with 9 vol. of the buffer described above in a Potter-Elvehjem homogenizer at 0°C. The enzyme preparations were stored at - 2 0 ° C for several days. The assay of CGalT was performed as previously described [14] except that the dispersion of ceramide was prepared by heating at 100°C [15]. In each experiment at least two assays were run at different protein concentrations ranging from 160 to 280 #g/incubation mixture. To identify the labeled glycolipid formed with homogenates of cultured cells, lipids were extracted from 5 incubation mixtures and glycolipids separated by TLC on the borate-impregnated plates [13]. After scanning for radioactivity, only one labeled glycolipid, which co-chromatographed with reference cerebroside containing ~hydroxy-fatty acids, was detected. Determination of CNP activity m the homogenates treated with sodium deoxycholate [8] was carried out by the precipitation method [20]. The activity of CGalT determined as a function of time in culture is presented in Fig. lB. The data from several separate sets of cultures are given. Fairly reproducible results were observed with respect to the specific activity at a given age and to the pattern of development. CGalT activity could be detected after 16 days in culture; thereafter the enzyme activity increased up to the 50th day in culture and decreased rapidly until the 70th day. In one set of cultures which could be maintained beyond this period the low level o f CGalT activity remained stable up to the 90th
111
21 o)
=
1
.~
o
o
1
A
G 0
20
40
60
80
AGE (DAYS)
Fig 1. Specific actwity of CGalT as a function of age. Enzyme actw~t~es are gwen as nmol of galactose transferred per hour per mg of protein. A: CGalT m brain homogenates of rat. Abscissa, days after birth. B: CGalT in homogenates of cultured brain cells. Values from the separate sets of cultures are represented by different symbols Abscissa, days m culture.
day. To facilitate the comparison with in vivo evolution, the CGalT values in developing rat brain are given in Fig. 1A. As observed earlier [4, 5, 13] the specific activity of the enzyme reached the maximum after about 18 days of age and decreased rapidly to attain relatively low level after 30 days of age. Therefore, the peak of CGalT activity observed in our cultures was clearly retarded in comparison with the peak of CGalT in brain. This is in contrast with the results of explant cultures where the activities of the galactolipid-synthesizing enzymes could be detected in the first week of the culture [10]; also in reaggregated cell cultures the activity of cerebroside sulfotransferase was measurable after 12 days in vitro and the peak of activity was observed after about 21 days in culture, which closely resembles the situation in brain [19]. On the other hand, the 'retarding effect' was observed in the cultures of dissociated brain cells from 16-day-old mouse embryo, showing a maximum CGalT activity at about 40 days in culture [17]. The increase of CGalT activity observed between 16 and 50 days in culture could result from the proliferation of OG and/or from the increased synthesis of the enzyme within these cells. Although it was difficult to evaluate the relative numbers of OG and astrocytes in such cultures, the light microscopy and immunohistochemical observations indicated that the proportion of OG in the culture increased as a function of time (Labourdette, unpublished results). The finding of the maximum specific activity of CGalT twice as great as the corresponding activity in brain is in agreement with a relative enrichment of OG in
112 these types of primary cultures. As far as we are aware, such high specific activity of CGalT has not been previously reported in nerve cell cultures. The specific activity of CNP increased gradually up to about 50 days in culture and remained more or less stable thereafter, with only a slight decrease after the 60th day (Fig. 2B). As observed earlier [9], the developmental curve of CNP in cultures was comparable to that found in vivo (Fig. 2A; see also ref. 8). Although CGalT and CNP attained maximal activities approximately during the same period, the comparison of the two activities at different points of the developmental curves shows that the changes of CGalT proceeded at a more rapid rate: C N P / C G a l T specific activity ratio was greatest after 16 days in culture (220); it then decreased to attain the lowest value (70) at the 50th day, and then increased again (130 and 170 after 60 and 70 days in culture, respectively). Moreover, the results of Fig. 2 show that CNP activity was stable from the 50th-60th day in culture, i.e. in the period when CGalT activity decreased rapidly. As a constituent of OG membranes [16], CNP can be considered as a reliable indicator o f the relative number of OG cells in the cultures where myelin is absent. Therefore, the above results suggest that a loss of OG from the cultures did not occur to such an extent that it could account for the decrease of CGalT after the 50th day. If so, the evolution of CGalT observed in our cultures is due, at least in part, to the changes of the enzyme activity within OG cells. The finding of a high CGalT activity in the OG-enriched cultures, coupled with the previously reported presence of basic protein, Wl Wolfgram protein and CNP [9], show that OG in long-term dissociated cultures retain the capacity to synthetize at least 4 of their specific proteins. The observation that OG exhibits a characteristic development of the CGalT activity in the absence of the complete myelination
o) o s
o~ 200 E ..c
N loo
2: 0
0 20 '
40 °
6'0
80 '
AGE (DAYS)
F~g 2. Specific actw~ty of CNP as a function of age. Enzyme actw~tles are g~ven as ~tmol of adenosme-2'phosphate formed per hour per mg of protein A: CNP in brain homogenates of rat Abscissa, days after b~rth B' CNP m homogenates of cultured brain cells Values from the separate sets of cultures are represented by different symbols Abscissa, days m culture
113 s y s t e m , is a p h e n o m e n o n
n o t p r e v i o u s l y r e p o r t e d in tissue c u l t u r e s o f C N S a n d m a y
h a v e i m p o r t a n t r e p e r c u s s i o n s in s t u d y i n g t h e e n z y m e r e g u l a t i o n o f m y e l i n o g e n e s i s . ACKNOWLEDGEMENTS T h i s w o r k w a s s u p p o r t e d in p a r t b y g r a n t s f r o m C N R S . W e a r e m o s t g r a t e f u l to M i s s M . P e r r a u l t , D. B r e v i a n d R. R e e b f o r t h e i r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . W e t h a n k D r L . L . S a r l i e v e f o r c o m m u n i c a t i n g us his results p r i o r t o p u b l i c a t i o n . T h e a u t h o r s a r e C h a r g 6 s de R e c h e r c h e o f I N S E R M .
REFERENCES 1 Bornstein, M.B., Differentiation of cells in primary cultures, myellnatlon. In S. Fedoroff and L Hertz (Eds.), Cell, Tissue and Organ Cultures m Neurobiology, Academic Press, New York, 1977, pp. 141-146. 2 Bourre, J.M., Honegger, P., Daudu, O. and Matthieu, J.M., The hpld composition of rat brain aggregating cell cultures during development, Neurosci. Lett., II (1979) 275-278. 3 Bradbury, K. and Lumsden, C.E., The chemical composition of myelin in organ cultures of rat cerebellum, J. Neurochem., 32 (1979) 145-154. 4 Brenkert, A. and Radln, N.S., Synthesis of galactosyl ceramlde and glucosyl ceramlde by rat brain: assay procedures and changes with age, Brain Res., 36 (1972) 183-193. 5 Costantmo-Ceccarini, E. and Morell, P , Biosynthesis of brain sphingohpids and myelin accumulation in the mouse, Llplds, 7 (1972) 656-659. 6 Deshmukh, D S., Flyn, T.J. and Plermger, R A , The biosynthesis and concentration of galactosyl diglyceride in ghal and neuronal enriched fractions of actively myehnating rat brain, J. Neurochem., 22 (1974) 479-485. 7 Fry, J.M., Lehrer, G.M. and Bornsteln, M.B., Sulfatlde synthesis, inhibition by experimental allergic encephalomyehtls serum, Soence, 175 (1972) 192-194 8 Kurlhara, T., Nussbaum, J.L. and Mandel, P., 2',3'-Cyclic nucleotide 3'-phosphohydrolase m brains of mutant mice with deficient myelination, J. Neurochem., 17 (1970) 993-997. 9 Labourdette, G., Roussel, G., Ghandour, M S and Nussbaum, J.L., Cultures from rat brain hemispheres enriched in ohgodendrocyte-hke cells, Brain Res., 179 (1979) 199-203. l0 Latovltzkl, N. and Sflberberg, D.H., UDP-galactose:ceramlde galactosyltransferase and 2',3'-cychc nucleotlde 3'-phosphohydrolase activities in cultures of newborn rat cerebellum. Assocmtlon with myelinatlon and concurrent susceptibility to 5-bromodeoxyundlne, J Neurochem, 29 (1977) 611-614. ll Lowry, O H., Rosebrough, N.J., Farr, A.L. and Randall, R J , Protein measurement with the Fohn phenol reagent, J. biol. Chem., 193 (1951) 265-275 12 Matthieu, J.M., Honegger, P., Trapp, B.D., Cohen, S.R. and Webster, H. de F., Myehnation in rat brain aggregating cell cultures, Neurosoence, 3 (1978) 565-572. 13 Neskovlc, N.M., Sarlleve, L.L. and Mandel, P , Biosynthesis of glycollplds m myelin deficient mutants, brain glycosyl transferase m Jlmpy and Quaking mice, Brain Res., 42 (1972) 147-157 14 Neskovlc, N.M., Sarlieve, L.L. and Mandel, P., Purification and properties of UDP-galactose: ceramide galactosyltransferase from rat brain mlcrosomes, Blochim. blophys. Acta (Amst.), 334 (1974) 309-315. 15 Neskovlc, N.M., Mandel, P. and Gatt, S., UDP-galactose ceramlde galactosyltransferase. Kinetic properties and effect of detergents and phosphohpids on the partially purified enzyme of rat brain In
114
16 17
18 19 20
S Gatt, L Freysz and P. Mandel (Eds), Enzymes of Lipid Metabohsm, Plenum, New York, 1978, pp 613-630 Poduslo, S E., The isolation and characterization of a plasma membrane and a myelin fraction derived from ohgodendrogha of calf brain, J Neurochem , 25 (1975) 647-654 Sarheve, L L., Subba Rao, G., Campbell, G LeM and Plermger, R.A , Investigation of myehnation m vitro: biochemical and morphological changes m cultures of dissociated brain cells from embryonic mice, Brain Res , 189 (1980) 79-90 Sensenbrenner, M., Dlssooated brain cells m primary cultures In S Fedoroff and L Hertz (Eds), Cell, Tissue and Organ Cultures m Neuroblology, Academic Press, New York, 1977, pp 191-213 Sheppard, J R , Brus, D and Wehner, J M , Brain reaggregate cultures biochemical evidence for myehn membrane synthesis, J Neuroblol , 9 0978) 309-315 Sims, N.R. and Carnegie, P R , A rapid assay for 2',3'-cychc nucleotlde 3'-phosphohydrolase, J Neurochem , 27 0976) 769-772