Original Papers Dev. Neurosci. 2: 249-253 (1979)
Sulfatide Synthesis by Neural Cell Lines Nicholas IV. Seeds and Michael J. Marks Departments of Biochemistry, Biophysics and Genetics and Psychiatry, University of Colorado Medical Center, Denver. Colo.
Key Words. CNPase • Sulfatide • Schwannoma • G2(i glioma Abstract. Several neural cell lines were examined for their ability to synthesize sulfatide and 2',3'-cyclic nucleotide phosphohydrolase, biochemical components characteristic of myelin. The mouse glioma G 26 and the rat schwannoma TRM6B actively produced sulfatide, while the rat glioma Cfi was inactive, supporting the probable oligodendroglial origin of the G 2r>. In contrast, the CGcell line had a high level of 2'-3'-cyclic nucleotide phosphohydrolase activ ity, while TRM6B showed 30% and the Go« 75% lower activities. Thus, these two activities appear to be independently regulated.
16]. With the hope of being able to dissect some of the steps involved in myelination, we have examined several cells lines from tumors of mouse and rat nervous systems for their ability to express biochemical proper ties characteristic of myelin, namely, sulfa tide formation [9] and 2',3'-cyclic nucleotide phosphohydrolase (CNPase) [7].
Methods Cell cultures grown in basal Eagle’s medium plus 8% fetal bovine serum were incubated with Na-^SCA (final specific activity of 10 pCi/pmol) for 24hat37°C in a 5% CO. atmosphere. The radioactive medium was discarded and the cells rinsed twice with phos phate-buffered saline containing I mM CaCU. The
Downloaded by: King's College London 137.73.144.138 - 1/20/2019 6:50:22 PM
During the last decade clonal cell lines derived from spontaneous and chemically induced neural tumors have provided the neuroscientist with a better understanding of the cellular localization and molecular aspects of neural specific functions [10]. One of the more complex intercellular events in neural differentiation is the formation of myelin. The molecular basis of cell interactions that initiate the myelination process in both the central and peripheral nervous system are poorly understood. Although recent studies [20] with primary cultures of Schwann cells are most promising, unraveling the mecha nism of myelin formation is complicated in part by the heterogeneity of CNS cell cul tures required for in vitro myelination [5, 14,
Seeds/Marks
250
cells were scraped from the culture dishes in H .O and lyophilized. Following resuspension in 0 .1 ml H ;0 , the tissue was extracted twice with 2 ml of CHCI3: MeOH (2:1) containing carrier sulfatidc, and once with 1 ml of CHCIn:MeOH (1:1). The combined organic phases were washed with 1/12 vol 0.1 M KCI, I mM K.SO, followed by four washes with CHCl3:MeOH:0.1 M KCI (I m.'V/ K ;SO,) (3:48:47) to remove free ''SO , . The organic phase was evaporated under N - and resolved by thin-layer chromatography on Silica gel G in a C H C I,:M eO H :H ;0:N H ,OH (48:14:1 :l) sol vent. The unlabcled sulfatidc was identified by Ivapor prior to processing I-cm strips of the chromato grams for liquid scintillation counting. 2\3'-Cyclic nucleotide phosphohydrolase activity in the clonal cells was measured using a modification of the method of Prohaska el at. [13]. Subconfluent cells were harvested by scraping into 0.32 A7 sucrose followed by centrifuging. After centrifugation, the cells were homogenized by sonication (150,?, 5 min, Branson Sonificr VVIS5 equipped with the special micro-tip). Solubilization with deoxycholate (0.4%), a 10-min incubation (30 C, pH 7.2) with substrate 2',3'-cyclic AMP (7.5 mM ) and hydrolysis of the product, 2'-AMP with Escherichia coli alkaline phos phatase (pH 8.7) were as described except the buffers used were MES at pH 6.2 and glycine at 8.7. After hydrolysis of the 2'-AMP, the reaction was stopped by addition of HCIO, (final concentration 0.35 M). The detergent extracted with CHCla (100 pi) and phosphate was determined colorimetrically at 700 nm on the protein and detergent-free supernatant as the reduced phosphomolybdate complex. Protein was measured by the method of Lowry el al. [8],
cell lines. A thin-layer chromatograph of the lipid extract from the different cultured cells is shown in figure I. Sulfatide formation was found only in the schwannoma and the glioma G.,c. Although some radioactivity was associated with the CHCI3:MeOH extract of C(i and N18 cells, it did not comigrate with sulfatide. The small peak of radioactivity migrating behind the major sulfatide peak in the TRM6B extract may reflect a difference in acyl groups or be galactosyldiglyceride sul fate. A quantitative examination of sulfatide synthesis under conditions of linear 35SO, incorporation with time and cell number
Sulfatide
The cell lines chosen for study were the methylcholanthrene-induced rat astrocytoma C0 [2] and mouse glioma G.,0 [23], as well as an ethyl nitrosourea-induced mouse schwan noma TRM6B [4] and clone NI8 of the spon taneous mouse neuroblastoma C l300 [1, 15]. The incorporation of 35S-sulfate into galactocerebroside to form sulfatide was examined in logarithmically growing cultures of the four
Fig. 1. 3iS incorporation into sulfatide by clonal neural cells. Subconfluent cultures grown in basal Eagle's medium plus 8% fetal bovine serum w'ere incuated with Na*aiSO i (final specific activity of 10 pCi/ pmol) for 24 h at 37°C in a 5% CO- atmosphere. The chloroform-methanol lipid extract was resolved by thin-layer chromatography on Silica gel G and the unlabeled sulfatide was identified by I. vapor prior to processing I-cm strips of the chromatograms for liquid scintillation counting.
Downloaded by: King's College London 137.73.144.138 - 1/20/2019 6:50:22 PM
Results and Discussion
251
Sulfatide Synthesis by Neural Cell Lines
[ 12].
The presence of another activity, 2',3'cyclic nucleotide phosphohydrolase, was also examined in these same cell cultures. This enzyme appears early in myelination and has been found in high concentration in CNS white matter, isolated myelin and oligodendroglial cells; however, it has also been found in spleen, pancreas and erythrocyte mem branes, although at much lower specific ac tivity [for review see 17]. The rat glioma CG showed the highest level of CNPase (table 1); however, this activity was only 7% of that found in adult mouse brain tissue (3.9 pmol/ mg protein/min). CNPase activity in the CG glioma was shown previously by Zanetta et at. [21]. Also Sundarraj et at. [19] have dem onstrated the loss of CNPase from G 2gtumor cells during in vitro culture. Surprisingly, there was not a direct relationship between a cell's ability to synthesize sulfatide and its CNPase activity. This finding suggests that these two activities are not tightly linked functions in a given cell type and may be independently regulated, at least in these cells of neoplastic origin. These results provide confirmatory bio chemical evidence that the TRM6B cells, isolated from a trigeminal nerve tumor and histologically identified as a schwannoma [4], are of Schwann cell origin. In a similar man ner, these results are important in character-
Table I. Sulfatide and CNPase Neurotumor cell
Sulfatide synthesis nmol/mg protein/day
2',3'-CNPase pmol/mg protein/min
G sc glioma C„ glioma TRM6B schwannoma N,s neuroblastoma
0.120 0.004 0.052 0.006
0.064 0.241 0.174 0.030
Sulfatide synthesis was assessed by the incorpora tion of 31S from N a; 3;,SO, (10 pCi/mol) into sulfatide as described. The incorporation was a linear function of the time of incubation and the amount of cell protein. 2\3'-Cyclic nucleotide 3'-phosphohydrolase (2',3'-CNPase) activity was measured using a modifi cation of the method of Prohaska el at. [13]. The results are expressed as the mean of 10-12 incubations in two separate experiments; the standard errors were always less than 5%.
izing the G 2G tumor whose history is not totally clear. 30 years ago Zimmerman and Maier [23] identified a mixed tumor posses sing both oligodendroglioma and ependy moma components from which ‘pure' oligo dendroglioma and ependymoma sublines could be established [22]. The designation G 21i first appeared in a chapter by Sitgiura [18], however, there is no specific designation as to whether this glial tumor was of oligoden drocyte or ependymal cell origin. Our results would support the view that G 20 cells were of oligodendroglial origin. Since the comple tion of this study, a communication by Dawson et ai. [3] has independently demon strated sulfatide synthesis in the G 2G cells. The glioma G 2Gand schwannoma TRM6B cells should allow for future sutdies of regula tion as well as drug and hormonal influences on these biochemical markers related to myelin formation and the possible induction
Downloaded by: King's College London 137.73.144.138 - 1/20/2019 6:50:22 PM
showed (table I) that the G 2G glioma cells possessed a higher specific activity of incor poration than the schwannoma. It is impor tant to note that the rate of 35S 0 4 incorpora tion into sulfatide by the G 2G cells is very similar to that found for heterogeneous ex plant cultures of spinal cord [6] undergoing active myelination, and is greater than ten times the incorporation observed in isolated oligodendroglia preparations of bovine brain
of additional markers such as myelin basic protein. Thus far, basic protein has only been found in one rat tumor, RN-2 [11] and was not found in G.>g cells [19].
Acknowledgements We would like to thank Ms. S. Haffke and Ms. C. Martlila for their expert technical assistance and Dr. Kay Fields for her gift of the TRM6B schwan noma. M .J.M . was the recipient of a National Re search Service Award (NS-05131) and N.W.S. a Career Development Awardee (GM-40170). These studies were supported in part by grants from the USPHS, CA-15549 and NS-098IS.
References 1 Amano, T .; Richelson, E., and Nirenberg, M.: Neurotransmitter synthesis by neuroblastoma clones. Proc. natn. Acad. Sci. USA 69: 258-263 (1972). 2 Benda, P.; Lightbody, J.; Sato, G.; Levine, L., and Sweet, W.: Differentiated rat glial cell strain in tissue culture. Science 161: 370-371 (1968). 3 Dawson, G.; Sundarraj, N„ and Pfeiffer, S.: Syn thesis of myelin glycosphingolipids (galactosyl ceramidc and galactosyl (3-0-sulfate) ceramide (sulfatide) by cloned cell lines derived from mouse neurotumors. J. biol. Chem. 252:2777-2779 (1977). 4 Fields, K.; Gosling, C.; Megson, M., and Stern, P .: New cell surface antigens in rat defined by tumors of the nervous system. Proc. natn. Acad. Sci. USA 72: 1296-1300 (1975). 5 Fry, J.M .; Lehrer, G., and Bornstein, M.: Sulfa tide synthesis: inhibition by experimental allergic encephalomyelitis serum. Science 175: 192-194 (1972). 6 Fry, J.; Lehrer, G., and Bornstein, M.: Sulfatide synthesis: late inhibition in developing cultures of mouse spinal cord. J. Neurochem. 22: 459-460 (1974). 7 Kurihara, T. and Tsukada, J.: The regional and subcellular distribution of 2',3'-cyclic nucleotide 3'-phosphohydrolasc in the central nervous sys tem. J. Neurochem. 14: 1157-1174 (1967).
8 Lowry, O.; Rosebrough, W .; Farr, A., and Randall, R.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193: 265-275 (1951). 9 McKhann, G. and Ho, W.: The in vivo and in vino synthesis of sulphatides during development. J. Neurochem. 14: 717-724 ( 1967). 10 Nelson, P.G.: Neural and muscle cells in culture. Physiol. Rev. 55: 1-61 (1975). 11 Pfeiffer, S. and Wechsler, W.: Biochemically dif ferentiated neoplastic clone of Schwann cells. Proc. natn. Acad. Sci. USA 6S: 2885-2889 (1972). 12 Poduslo, S.; Miller, K., and McKhann, G.: Meta bolic properties of maintained oligodendroglia purified from brain. J. biol. Chem. 253: 1592-1597 (I97S). 13 Prohaska, J.; Clark, D., and Wells, W.: Improved rapidity and precision in the determination of brain 2',3'-cyclic nucleotide 3'-phosphohydrolase. Analyt. Biochem. 56: 275-282 (1973). 14 Schmidt, G.L.: Development of biochemical ac tivities associated with myelination in chick brain aggregate cultures. Brain Res. 87: 110-113 (1975). 15 Seeds, N.; Gilman, A.; Amano, T., and Nirenberg, M.: Regulation of axon formation by clonal lines of a neural tumor. Proc. natn. Acad. Sci. USA 60: 160-167 (1970). 16 Silverberg, D.; Benjamins, J.; Hcrschkowitz, N., and McKhann, G.: Incorporation of radioactive sulphate into sulphatide during myelination in cultures of rat cerebellum. J. Neurochem. 19: 11-18 (1972). 17 Sims, N.R. and Carnegie, P. R.: 2',3'-Cyclic nuclcotide-3'-phosphodiesterase; in Agranoff and Aprison, Advances in neurochemistry, vol 3, pp. 1-41 (Plenum Press, New York 1978). 18 Sugiura, K.: Tumor transplantation; in Gay, Methods in animal experimentation, vol. 2, pp. 171-222 (Academic Press, New York 1969). 19 Sundarraj, N.; Schachner, M., and Pfieffer, S.: Biochemically differentiated mouse glial lines carrying a nervous system specific cell surface antigen (N s-1). Proc. natn. Acad. Sci. USA 72: 1927-1931 (1975). 20 Wood, P. M. and Bunge, R .P .: Evidence that sensory axons are mitogenic for Schwann cells. Nature 256: 662-664 (1975). 21 Zanctta, J.P .; Benda, P.; Gornbos, G., and Morgan, I.: The presence of 2',3'-cyclic AMP 3'-phosphohydrolase in glial cells in tissue culture. J. Neurochem. 19: 881-883 (1972).
Downloaded by: King's College London 137.73.144.138 - 1/20/2019 6:50:22 PM
Seeds/Marks
252
25.1
Sulfatidc Synthesis by Neural Cell Lines
Dr. Nicholas Seeds, Associate Professor, Departments of Biochemistry, Biophysics anti Genetics, University of Colorado School of Medicine. Box Bi l l . 4200 East 9th Avenue, Denver, CO 80262 (USA)
Downloaded by: King's College London 137.73.144.138 - 1/20/2019 6:50:22 PM
22 Zimmerman, H.M.: The nature of gliomas as revealed by animal experimentation. Am. J. Path. 31: 1-29 (1955). 21 Zimmerman. M. and Maier, N.: Experimental brain tumors. Proc. N.V. Path. Soc.. 1948-1949, pp. 40-42.
Sulfatide Synthesis by Neural Cell Lines Nicholas IV. Seeds and Michael J. Marks Departments of Biochemistry, Biophysics and Genetics and Psychiatry, University of Colorado Medical Center, Denver. Colo.
Key Words. CNPase • Sulfatide • Schwannoma • G2(i glioma Abstract. Several neural cell lines were examined for their ability to synthesize sulfatide and 2',3'-cyclic nucleotide phosphohydrolase, biochemical components characteristic of myelin. The mouse glioma G 26 and the rat schwannoma TRM6B actively produced sulfatide, while the rat glioma Cfi was inactive, supporting the probable oligodendroglial origin of the G 2r>. In contrast, the CGcell line had a high level of 2'-3'-cyclic nucleotide phosphohydrolase activ ity, while TRM6B showed 30% and the Go« 75% lower activities. Thus, these two activities appear to be independently regulated.
16]. With the hope of being able to dissect some of the steps involved in myelination, we have examined several cells lines from tumors of mouse and rat nervous systems for their ability to express biochemical proper ties characteristic of myelin, namely, sulfa tide formation [9] and 2',3'-cyclic nucleotide phosphohydrolase (CNPase) [7].
Methods Cell cultures grown in basal Eagle’s medium plus 8% fetal bovine serum were incubated with Na-^SCA (final specific activity of 10 pCi/pmol) for 24hat37°C in a 5% CO. atmosphere. The radioactive medium was discarded and the cells rinsed twice with phos phate-buffered saline containing I mM CaCU. The
Downloaded by: King's College London 137.73.144.138 - 1/20/2019 6:50:22 PM
During the last decade clonal cell lines derived from spontaneous and chemically induced neural tumors have provided the neuroscientist with a better understanding of the cellular localization and molecular aspects of neural specific functions [10]. One of the more complex intercellular events in neural differentiation is the formation of myelin. The molecular basis of cell interactions that initiate the myelination process in both the central and peripheral nervous system are poorly understood. Although recent studies [20] with primary cultures of Schwann cells are most promising, unraveling the mecha nism of myelin formation is complicated in part by the heterogeneity of CNS cell cul tures required for in vitro myelination [5, 14,
Seeds/Marks
250
cells were scraped from the culture dishes in H .O and lyophilized. Following resuspension in 0 .1 ml H ;0 , the tissue was extracted twice with 2 ml of CHCI3: MeOH (2:1) containing carrier sulfatidc, and once with 1 ml of CHCIn:MeOH (1:1). The combined organic phases were washed with 1/12 vol 0.1 M KCI, I mM K.SO, followed by four washes with CHCl3:MeOH:0.1 M KCI (I m.'V/ K ;SO,) (3:48:47) to remove free ''SO , . The organic phase was evaporated under N - and resolved by thin-layer chromatography on Silica gel G in a C H C I,:M eO H :H ;0:N H ,OH (48:14:1 :l) sol vent. The unlabcled sulfatidc was identified by Ivapor prior to processing I-cm strips of the chromato grams for liquid scintillation counting. 2\3'-Cyclic nucleotide phosphohydrolase activity in the clonal cells was measured using a modification of the method of Prohaska el at. [13]. Subconfluent cells were harvested by scraping into 0.32 A7 sucrose followed by centrifuging. After centrifugation, the cells were homogenized by sonication (150,?, 5 min, Branson Sonificr VVIS5 equipped with the special micro-tip). Solubilization with deoxycholate (0.4%), a 10-min incubation (30 C, pH 7.2) with substrate 2',3'-cyclic AMP (7.5 mM ) and hydrolysis of the product, 2'-AMP with Escherichia coli alkaline phos phatase (pH 8.7) were as described except the buffers used were MES at pH 6.2 and glycine at 8.7. After hydrolysis of the 2'-AMP, the reaction was stopped by addition of HCIO, (final concentration 0.35 M). The detergent extracted with CHCla (100 pi) and phosphate was determined colorimetrically at 700 nm on the protein and detergent-free supernatant as the reduced phosphomolybdate complex. Protein was measured by the method of Lowry el al. [8],
cell lines. A thin-layer chromatograph of the lipid extract from the different cultured cells is shown in figure I. Sulfatide formation was found only in the schwannoma and the glioma G.,c. Although some radioactivity was associated with the CHCI3:MeOH extract of C(i and N18 cells, it did not comigrate with sulfatide. The small peak of radioactivity migrating behind the major sulfatide peak in the TRM6B extract may reflect a difference in acyl groups or be galactosyldiglyceride sul fate. A quantitative examination of sulfatide synthesis under conditions of linear 35SO, incorporation with time and cell number
Sulfatide
The cell lines chosen for study were the methylcholanthrene-induced rat astrocytoma C0 [2] and mouse glioma G.,0 [23], as well as an ethyl nitrosourea-induced mouse schwan noma TRM6B [4] and clone NI8 of the spon taneous mouse neuroblastoma C l300 [1, 15]. The incorporation of 35S-sulfate into galactocerebroside to form sulfatide was examined in logarithmically growing cultures of the four
Fig. 1. 3iS incorporation into sulfatide by clonal neural cells. Subconfluent cultures grown in basal Eagle's medium plus 8% fetal bovine serum w'ere incuated with Na*aiSO i (final specific activity of 10 pCi/ pmol) for 24 h at 37°C in a 5% CO- atmosphere. The chloroform-methanol lipid extract was resolved by thin-layer chromatography on Silica gel G and the unlabeled sulfatide was identified by I. vapor prior to processing I-cm strips of the chromatograms for liquid scintillation counting.
Downloaded by: King's College London 137.73.144.138 - 1/20/2019 6:50:22 PM
Results and Discussion
251
Sulfatide Synthesis by Neural Cell Lines
[ 12].
The presence of another activity, 2',3'cyclic nucleotide phosphohydrolase, was also examined in these same cell cultures. This enzyme appears early in myelination and has been found in high concentration in CNS white matter, isolated myelin and oligodendroglial cells; however, it has also been found in spleen, pancreas and erythrocyte mem branes, although at much lower specific ac tivity [for review see 17]. The rat glioma CG showed the highest level of CNPase (table 1); however, this activity was only 7% of that found in adult mouse brain tissue (3.9 pmol/ mg protein/min). CNPase activity in the CG glioma was shown previously by Zanetta et at. [21]. Also Sundarraj et at. [19] have dem onstrated the loss of CNPase from G 2gtumor cells during in vitro culture. Surprisingly, there was not a direct relationship between a cell's ability to synthesize sulfatide and its CNPase activity. This finding suggests that these two activities are not tightly linked functions in a given cell type and may be independently regulated, at least in these cells of neoplastic origin. These results provide confirmatory bio chemical evidence that the TRM6B cells, isolated from a trigeminal nerve tumor and histologically identified as a schwannoma [4], are of Schwann cell origin. In a similar man ner, these results are important in character-
Table I. Sulfatide and CNPase Neurotumor cell
Sulfatide synthesis nmol/mg protein/day
2',3'-CNPase pmol/mg protein/min
G sc glioma C„ glioma TRM6B schwannoma N,s neuroblastoma
0.120 0.004 0.052 0.006
0.064 0.241 0.174 0.030
Sulfatide synthesis was assessed by the incorpora tion of 31S from N a; 3;,SO, (10 pCi/mol) into sulfatide as described. The incorporation was a linear function of the time of incubation and the amount of cell protein. 2\3'-Cyclic nucleotide 3'-phosphohydrolase (2',3'-CNPase) activity was measured using a modifi cation of the method of Prohaska el at. [13]. The results are expressed as the mean of 10-12 incubations in two separate experiments; the standard errors were always less than 5%.
izing the G 2G tumor whose history is not totally clear. 30 years ago Zimmerman and Maier [23] identified a mixed tumor posses sing both oligodendroglioma and ependy moma components from which ‘pure' oligo dendroglioma and ependymoma sublines could be established [22]. The designation G 21i first appeared in a chapter by Sitgiura [18], however, there is no specific designation as to whether this glial tumor was of oligoden drocyte or ependymal cell origin. Our results would support the view that G 20 cells were of oligodendroglial origin. Since the comple tion of this study, a communication by Dawson et ai. [3] has independently demon strated sulfatide synthesis in the G 2G cells. The glioma G 2Gand schwannoma TRM6B cells should allow for future sutdies of regula tion as well as drug and hormonal influences on these biochemical markers related to myelin formation and the possible induction
Downloaded by: King's College London 137.73.144.138 - 1/20/2019 6:50:22 PM
showed (table I) that the G 2G glioma cells possessed a higher specific activity of incor poration than the schwannoma. It is impor tant to note that the rate of 35S 0 4 incorpora tion into sulfatide by the G 2G cells is very similar to that found for heterogeneous ex plant cultures of spinal cord [6] undergoing active myelination, and is greater than ten times the incorporation observed in isolated oligodendroglia preparations of bovine brain
of additional markers such as myelin basic protein. Thus far, basic protein has only been found in one rat tumor, RN-2 [11] and was not found in G.>g cells [19].
Acknowledgements We would like to thank Ms. S. Haffke and Ms. C. Martlila for their expert technical assistance and Dr. Kay Fields for her gift of the TRM6B schwan noma. M .J.M . was the recipient of a National Re search Service Award (NS-05131) and N.W.S. a Career Development Awardee (GM-40170). These studies were supported in part by grants from the USPHS, CA-15549 and NS-098IS.
References 1 Amano, T .; Richelson, E., and Nirenberg, M.: Neurotransmitter synthesis by neuroblastoma clones. Proc. natn. Acad. Sci. USA 69: 258-263 (1972). 2 Benda, P.; Lightbody, J.; Sato, G.; Levine, L., and Sweet, W.: Differentiated rat glial cell strain in tissue culture. Science 161: 370-371 (1968). 3 Dawson, G.; Sundarraj, N„ and Pfeiffer, S.: Syn thesis of myelin glycosphingolipids (galactosyl ceramidc and galactosyl (3-0-sulfate) ceramide (sulfatide) by cloned cell lines derived from mouse neurotumors. J. biol. Chem. 252:2777-2779 (1977). 4 Fields, K.; Gosling, C.; Megson, M., and Stern, P .: New cell surface antigens in rat defined by tumors of the nervous system. Proc. natn. Acad. Sci. USA 72: 1296-1300 (1975). 5 Fry, J.M .; Lehrer, G., and Bornstein, M.: Sulfa tide synthesis: inhibition by experimental allergic encephalomyelitis serum. Science 175: 192-194 (1972). 6 Fry, J.; Lehrer, G., and Bornstein, M.: Sulfatide synthesis: late inhibition in developing cultures of mouse spinal cord. J. Neurochem. 22: 459-460 (1974). 7 Kurihara, T. and Tsukada, J.: The regional and subcellular distribution of 2',3'-cyclic nucleotide 3'-phosphohydrolasc in the central nervous sys tem. J. Neurochem. 14: 1157-1174 (1967).
8 Lowry, O.; Rosebrough, W .; Farr, A., and Randall, R.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193: 265-275 (1951). 9 McKhann, G. and Ho, W.: The in vivo and in vino synthesis of sulphatides during development. J. Neurochem. 14: 717-724 ( 1967). 10 Nelson, P.G.: Neural and muscle cells in culture. Physiol. Rev. 55: 1-61 (1975). 11 Pfeiffer, S. and Wechsler, W.: Biochemically dif ferentiated neoplastic clone of Schwann cells. Proc. natn. Acad. Sci. USA 6S: 2885-2889 (1972). 12 Poduslo, S.; Miller, K., and McKhann, G.: Meta bolic properties of maintained oligodendroglia purified from brain. J. biol. Chem. 253: 1592-1597 (I97S). 13 Prohaska, J.; Clark, D., and Wells, W.: Improved rapidity and precision in the determination of brain 2',3'-cyclic nucleotide 3'-phosphohydrolase. Analyt. Biochem. 56: 275-282 (1973). 14 Schmidt, G.L.: Development of biochemical ac tivities associated with myelination in chick brain aggregate cultures. Brain Res. 87: 110-113 (1975). 15 Seeds, N.; Gilman, A.; Amano, T., and Nirenberg, M.: Regulation of axon formation by clonal lines of a neural tumor. Proc. natn. Acad. Sci. USA 60: 160-167 (1970). 16 Silverberg, D.; Benjamins, J.; Hcrschkowitz, N., and McKhann, G.: Incorporation of radioactive sulphate into sulphatide during myelination in cultures of rat cerebellum. J. Neurochem. 19: 11-18 (1972). 17 Sims, N.R. and Carnegie, P. R.: 2',3'-Cyclic nuclcotide-3'-phosphodiesterase; in Agranoff and Aprison, Advances in neurochemistry, vol 3, pp. 1-41 (Plenum Press, New York 1978). 18 Sugiura, K.: Tumor transplantation; in Gay, Methods in animal experimentation, vol. 2, pp. 171-222 (Academic Press, New York 1969). 19 Sundarraj, N.; Schachner, M., and Pfieffer, S.: Biochemically differentiated mouse glial lines carrying a nervous system specific cell surface antigen (N s-1). Proc. natn. Acad. Sci. USA 72: 1927-1931 (1975). 20 Wood, P. M. and Bunge, R .P .: Evidence that sensory axons are mitogenic for Schwann cells. Nature 256: 662-664 (1975). 21 Zanctta, J.P .; Benda, P.; Gornbos, G., and Morgan, I.: The presence of 2',3'-cyclic AMP 3'-phosphohydrolase in glial cells in tissue culture. J. Neurochem. 19: 881-883 (1972).
Downloaded by: King's College London 137.73.144.138 - 1/20/2019 6:50:22 PM
Seeds/Marks
252
25.1
Sulfatidc Synthesis by Neural Cell Lines
Dr. Nicholas Seeds, Associate Professor, Departments of Biochemistry, Biophysics anti Genetics, University of Colorado School of Medicine. Box Bi l l . 4200 East 9th Avenue, Denver, CO 80262 (USA)
Downloaded by: King's College London 137.73.144.138 - 1/20/2019 6:50:22 PM
22 Zimmerman, H.M.: The nature of gliomas as revealed by animal experimentation. Am. J. Path. 31: 1-29 (1955). 21 Zimmerman. M. and Maier, N.: Experimental brain tumors. Proc. N.V. Path. Soc.. 1948-1949, pp. 40-42.
