- Email: [email protected]
Effect of thyroxine on de novo pyrimidine biosynthesis in developing rat cerebellum
346
Brahz Research. I I 7 (1976) 346-350 ~(~ Elsevier/North-HollandBiomedicalPress, Amsterdam - Printed in The Netherlands
Effect of thyroxine on de novo pyrimidine biosynthesis in developing rat cerebellum
MORTON E. WEICHSEL, Jr. Department of Pediatrics, Harbor General Hospital Campus., UCLA School of Medicine, Torrance, Calif. 90509 ( U.S.A,.)
(Accepted August 13th, 1976)
Thyroid hormone has been shown recently to have a profound effect upon the developing nervous system. Using the developing rat cerebellum as a model, hyperthyroidism results in a premature termination of cell division2,7,11,16 as well as a shift to an earlier age in the developmental time course of DNA biosynthesis and activity of thymidine kinase 1~, an enzyme necessary for incorporation of thymidine into newly synthesized DNA. Conversely, hypothyroidism results in a delay in cerebetlai cell acquisitiona,7,s,11,~7 with a concomitant delay in the maximum activity of thymidine kinase. Whereas thymidine kinase is an enzyme representing metabolic pathways involved in salvage (reutilization) ofpyrimidine nucleosides for incorporation into newly formed nucleic acids, enzymes from the de novo pyrimidine biosynthetic pathway are also potential markers for cerebellar cell replication ~a with peak activities occurring near the time of maximum rate of cerebellar cell division xa. Because of the known effect of thyroxine on activity of thymidine kinase during cerebellar development in, the present study was designed to determine the developmental effect of thyroxine on aspartate transcarbamylase (ATC), the second enzyme in the de novo pyrimidine biosynthetic pathway. Although ATC may be subject to complex regulation, and is not the rate limiting enzyme of this reaction sequence, it is felt to be representative of the pathway because of its known association as a functional complex with carbamyl phosphate synthetase (CPS) and dihydroorotase (DHO), the first and third enzymes in the pathway 9. Litters of Wistar rats born within a 12 h period were reduced to a total of 8 pups/ day, regardless of sex. The day of birth was considered as day 0. Four pups from each litter were treated subcutaneously on day 0 and each day thereafter with 0.4/~g thyroxine/g body weight. The 4 remaining pups received an equal amount of saline. Dams were fed white rat growth chow obtained from Simonsen Laboratories, Gilroy, Calif. Litters were housed at room temperature, and sacrificed as previously described 16 in groups of 4-8 litters on day 2, 4, 6, 8, 10, 14 and 19 of life. After sacrifice, the cerebellum was separated from cerebrum and brain stem and was immediately weighed on a torsion balance and homogenized at 1 : 10 (w/v) in a cold homogenizing medium, using 20 up
347 and down movements of the homogenizer pestle. At age 2 and 4 days, 2 cerebella from treated animals and 2 cerebella from littermate controls were pooled to provide experimental data points. At other ages, data points were derived from single cerebella. Aliquots from the crude homogenate were drawn for D N A determinations at ages 2, 4 and 19 days. Remaining homogenate was then centrifuged at 35,000 × g at 5 °C for 20 min. The assay for A T C activity and determination of protein content were performed on the supernatant solution. All of the measurable A T C activity was recovered in the supernatant fraction. Homogenizing medium consisted of sucrose (0.25 M), potassium phosphate p H 7.4 (0.1 M), magnesium-ATP (0.015 M MgSO40.01 M ATP), dithiothreitol 1 m M , and glutamine (3 m M ) , as designed for the assay of thymidine kinase and other enzymes involved in salvage and de novo pyrimidine biosynthesis. Aspartate transcarbamylase was assayed by a modification of the method described by Bethell et al. 4. The enzyme reaction mixture contained 0.05 ml N a - H e p e s p H 7.4 (0.5 M), 0.10 ml K-aspartate, p H 7.4 (0.1 M), 0~05 ml [14C]carbamyl phosphate (0.517 #Ci/#mole), 0.20 ml tissue homogenate supernatant, and 0.10 ml homogenizing medium. The final volume was 0.5 ml with concentrations of 0.05 M Na-Hepes. 0.02 M K-aspartate, 0.15 M sucrose,0.06 M potassium phosphate, 6 m M ATP, 9 m M MgSO4, and 380 # M [14C]carbamyl phosphate. Tubes were incubated at 37 °C for 5 min, and the reaction stopped by adding 0.1 ml of 50 % trichloroacetic acid and heating at 100 °C for 3 rain. Tubes were then cooled to room temperature and shaken on a vortex mixer after 3 separate additions of a small quantity of powdered dry ice. An aliquot of 0.4 ml was placed in a counting vial TABLE I Measurements of development in thyroxine treated rat pups and controls
Mean value 4- S.E.M. In parentheses are given the number of values for each calculation. Age (days)
2 4 6 8 10 14 19
Treatment
Body wt. (g)
Cerebellar wt. (rag)
CerebellarDNA (~g/cerebellum)
Cerebellar ATC activity (nmole/ min/mg prot.)
Control Treated Control Treated Control Treated Control Treated Control Treated Control Treated Control Treated
(33) 8.8 4- 0.2 (39) 8.8 4- 0.1 (37)11.64- 0.2 (36)10.74- 0.2* (23)15.2+ 0.3 (26)14.2± 0.3* (24)21.64- 0.5 (23)18.84- 0.5* (19)22.5i 0.5 (17)18.84- 0.5* (24)35.94- 0.8 (27)26.34- 0.6* (21)48.54- 0.7 (18)27.84- 1.0"
(32) 18.1 ± 0.3 (15) 68 ± 2 (31) 19.0 4- 0.3 (15) 72 4- 3 (30) 25.9 4- 0.4 (16)1164- 3 (31) 27.5 4- 0.5 (16)140± 5* (18) 39.9 4- 1.3 - (18) 43.3 4- 0.8 (18) 66.5 4- 1.2 - (18) 67.4 4- 1.2 (16) 88.6 4- 1.8 - (15) 83.0 4- 1.2" (18)145.1 4- 3.0 - (18)119.7 4- 1.8" (17)184.6 4- 2.4 (16)1458 q- 25 (15)128.3 4- 2.2" (14) 975 4- 27*
(16)3.84- 0.17 05)3.9 ± 0.14 (16)5.44- 0.10 (16)5.6 4- 0.13 (18)4.8 ± 0.08 (18)5.1 4- 0.14 (18)6.6 ± 0.15 (18)6.0 4- 0.13" (16)5.9 4- 0.15 (15)5.0 4- 0.10" (18)2.7 4- 0.05 (18)2.2 ± 0.04* (16)2.0 4- 0.07 (14)1.6 4- 0.06*
* Indicates statistical significance between treated and control groups by t-test (P < 0.05).
348 with TRIPOP liquid scintillation solution a. Radioactivity was measured on a scintillation spectrophotometer. Protein was measured on samples of supernatant solution using the Oyama and Eagle modification12 of the method of Lowry et al. t0. DNA was determined on samples of crude tissue homogenate using the modification by Giles and Myers6 of the method of Burton 5. Statistical analysis was applied to data points for treated and control animals at each specific age by use of the t-test. All data are presented in Table I. The effect of thyroxine on body weight and cerebellar weight was similar to that previously described 16 with the first significant deficit in body weight in treated animals occurring at 4 days, and the first significant deficit in cerebellar weight occurring by age 10 days. A significant enhancement of cerebellar DNA synthesis in treated animals was noted on day 4 (120~ of control) and a deficit noted on day 19 (67 ~/ of control), confirming previously established experimental observations16. The developmental curve for aspartate transcarbamylase activity in cerebella of control animals peaks around 9 days and falls to 29 ~o of maximum activity by day 19 as described previously by Weichsel et al.lS. In treated animals, a significant decrease in aspartate transcarbamylase activity was noted on days 8-19 (maximum deficit 81 ~ of control on day 14). In contrast to the early enhancement of thymidine kinase activity by thyroxine, no significant difference in ATC activity was noted prior to day 8. The transient decrease in day 6 values for both treated and control animals reflects variation inherent in studying animals of different ages on separate days. Pyrimidine nucleotide precursors of nucleic acids may be synthesized by a de novo pathway which utilizes the simple substrates CO2, ATP and glutamine and results in formation of uridylic acid. Pyrimidine nucleotides may also be generated via salvage pathways whereby preformed nucleosides are enzymatically phosphorylated. Subsequent to its formation by either pathway, uridylic acid is converted into several pyrimidine nucleotides by sequences of enzymatic reactions which appear to be under complex metabolic control 9. Along one such sequence is the interconversion pathway enzyme thymidylate synthetase which is necessary for conversion of uridylic acid to thymidylic acid, thus providing a mechanism by which the de novo pathway may provide thymidine triphosphate for incorporation into DNA. The de novo pathway enzyme aspartate transcarbamylase was the subject of this study because of the profound effect of thyroxine previously noted on cerebellar thymidine kinase 16, as well as the effect noted on the salvage pathway enzyme uridine kinasO 9 and the interconversion pathway enzyme thymidylatesynthetase (B.R. Clark, personal communication) during the early postnatal acceleration and deceleration phase of cerebellar DNA synthesis. The suppression of cerebellar aspartate transcarbamylase activity after day 6 in thyroxine treated animals was similar to that of thymidine kinase 16, uridine kinase 19 and thymidylate synthetase (B. R. Clark, personal communication). However, the lack of a significant effect of thyroxine on ATC during the first week of cerebellar ontogenesis when the hormone is known to enhance DNA synthesis16(Table I) suggests
349 that during early development under the influence of thyroxine, de novo pathway enzymes for pyrimidine nucleotide biosynthesis are not influenced in a manner similar to reutilization enzymes. Studies utilizing a variety of model systems are inconclusive as to the differential contribution and control mechanisms for formation of pyrimidine nucleotides by de novo and salvage pathways prior to their incorporation into newly synthesized nucleic acidsL Although activity of the salvage pathway enzyme thymidine kinase appears to correlate with the rate of cerebellar D N A biosynthesis 1~, there is less certainty concerning the role played by the de novo pathway during mammalian cell division. Roux et al. 14, using isoproteranol to stimulate cell division in isolated mouse salivary gland, have found an early increase in de novo pathway enzymes, and have speculated that it is the de novo pathway which supplies the pyrimidine nucleotides for the R N A synthesis which may be required for subsequent synthesis of DNA. Our data showing a lack of effect on the de novo pathway enzyme aspartate transcarbamylase during early thyroxine enhancement of cerebellar cell division do not support such an early role for the de novo pathway, although the possibility remains that an early significant elevation in de novo pathway enzyme activity could be undetectable due to lack of sensitivity of the assay and lack of synchronized cell division in such an in vivo system. The mechanisms by which thyroid hormones regulate protein synthesis remain poorly understood. The present data compared with previous findings 16 suggests that a given hormone may not affect all metabolic pathways involved in nucleic acid biosynthesis in a similar manner. These results support the possibility that in a developing organ, the sensitivity of its enzyme systems to thyroid hormone may depend on the developmental state of the cells in that organ. Additionally, such findings support the possibility suggested by Tata 15 that the final expression of a hormone involved in growth and development may result from a cooperative action generated by simultaneous or sequential but independent interactions of a hormone with more than one nuclear or extranuclear site. The author gratefully acknowledges the technical assistance of Shirley M. Guo, M.S. This research was supported by Harbor General Hospital General Research Support Grant GR-2076 and by National Institute of Health Grant HD-09277-02 from the U.S. Public Health Service. 1 Anderson, L. W. and McClure, W. O., An improved scintillation cocktail of high-solubilizing power, Analyt. Biochem., 51 (1973) 173-179. 2 Bahtzs, R., Kov~tcks,S., Cocks, W. A., Johnson, A. L. and Eayrs, J. T., Effect of thyroid hormone on biochemicalmaturation of rat brain: postnatal cell formation, Brain Research, 25 (1971)555-570. 3 Bal~izs,R., Biochemicaleffects of thyroid hormones in the developing brain. In D. C. Pease (Ed.), Cellular Aspects of Neural Growth and Differentiation, UCLA Forum Med. Sci., Univ. of Calif. Press, Los Angeles, Calif., 1971, pp. 273-320. 4 Bethell, M. R., Smith, K. E., White, J. S. and Jones, M. E., Carbamyl phosphate: an aliosteric substrate for aspartate transcarbamylase of Escherichia coli, Proc. nat. Acad. Sci. (Wash.), 60 (1968) 1442-1449.
350 5 Burton, K., A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid, Biochem. J., 62 (1956) 315-323. 6 Giles, K. W. and Myers, A., An improved diphenylamine method for the estimation of deoxyribonucleic acid, Nature (Lond.), 206 (1965) 93. 7 Gourdon, J., Clos, J., Coste, C., Dainat, J. and Legrand, J., Comparative effects of hypothyroidism, hyperthyroidism, and undernutrition on the protein and nucleic acid contents of the cerebellum in the young rat, J. Neurochem., 21 (1973) 861-871. 8 Hamburgh, M., Mendoza, L. A., Burkhard, J. F. and Weil, F., In D. C. Pease (Ed.), Cellular Aspects of Neural Growth and Differentiation, UCLA Forum Med. Sci., Univ. of Calif. Press, Los Aogeles, Calif., 1971, pp. 321-328. 9 Levine, R., Hoogenraad, N. J. and Kretchmer, N., A review: biological and clinical aspects of pyrimidine metabolism, Pediat. Res., 8 (1974) 724-734. 10 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 11 Nicholson, J. L. and Altman, J., The effects of early hypo- and hyperthyroidism on the development of rat cerebellar cortex. 1. Cell proliferation and differentiation, Brain Research, 44 (1972) 13-23. 12 Oyama, V. I. and Eagle, H., Measurement of cell growth in tissue culture with a phenol reagent (Folin-Cicaltreau), Proc. Soc. exp. Biol. ( N. Y.), 91 (1956) 305-307. 13 Patel, A. J., Bal~.zs, R. and Johnson, A. L., Effect of undernutrition on cell formation in the rat brain, J. Neurochem., 20 (1973) 1151-1165. 14 Roux, J. M., Hoogenraad, N. J. and Kretchmer, N., Biosynthesis of pyrimidine nucleotides in mouse salivary glands stimulated with isoproteranol, J. biol. Chem., 248 (1973) 1196-1202. 15 Tata, J. R., How specific are nuclear "receptors" for thyroid hormones? Nature (Lond.), 257 (1975) 18-23. 16 Weichsel, Jr., M. E., Effect of thyroxine on D N A synthesis and thymidine kinase activity during cerebellar development, Brain Research, 78 (1974) 455-465. 17 Weichsel, Jr., M. E. and Dawson, L., Effects of hypothyroidism and undernutrition on D N A content and thymidine kinase activity during cerebeltar development in the rat, J. Neurochem., 26 (1976) 675-681. 18 Weichsel, Jr., M. E., Hoogenraad, N. J., Levine, R. L. and Kretchmer, N., Pyrimidine biosynthesis during development of the cerebellum, Pediat. Res., 6 (1972) 682-686. 19 Weichsel, Jr., M. E., Thyroxine effect upon activity of uridine kinase in developing rat cerebellum, Biol. Neonat. (Basel), In press.
Brahz Research. I I 7 (1976) 346-350 ~(~ Elsevier/North-HollandBiomedicalPress, Amsterdam - Printed in The Netherlands
Effect of thyroxine on de novo pyrimidine biosynthesis in developing rat cerebellum
MORTON E. WEICHSEL, Jr. Department of Pediatrics, Harbor General Hospital Campus., UCLA School of Medicine, Torrance, Calif. 90509 ( U.S.A,.)
(Accepted August 13th, 1976)
Thyroid hormone has been shown recently to have a profound effect upon the developing nervous system. Using the developing rat cerebellum as a model, hyperthyroidism results in a premature termination of cell division2,7,11,16 as well as a shift to an earlier age in the developmental time course of DNA biosynthesis and activity of thymidine kinase 1~, an enzyme necessary for incorporation of thymidine into newly synthesized DNA. Conversely, hypothyroidism results in a delay in cerebetlai cell acquisitiona,7,s,11,~7 with a concomitant delay in the maximum activity of thymidine kinase. Whereas thymidine kinase is an enzyme representing metabolic pathways involved in salvage (reutilization) ofpyrimidine nucleosides for incorporation into newly formed nucleic acids, enzymes from the de novo pyrimidine biosynthetic pathway are also potential markers for cerebellar cell replication ~a with peak activities occurring near the time of maximum rate of cerebellar cell division xa. Because of the known effect of thyroxine on activity of thymidine kinase during cerebellar development in, the present study was designed to determine the developmental effect of thyroxine on aspartate transcarbamylase (ATC), the second enzyme in the de novo pyrimidine biosynthetic pathway. Although ATC may be subject to complex regulation, and is not the rate limiting enzyme of this reaction sequence, it is felt to be representative of the pathway because of its known association as a functional complex with carbamyl phosphate synthetase (CPS) and dihydroorotase (DHO), the first and third enzymes in the pathway 9. Litters of Wistar rats born within a 12 h period were reduced to a total of 8 pups/ day, regardless of sex. The day of birth was considered as day 0. Four pups from each litter were treated subcutaneously on day 0 and each day thereafter with 0.4/~g thyroxine/g body weight. The 4 remaining pups received an equal amount of saline. Dams were fed white rat growth chow obtained from Simonsen Laboratories, Gilroy, Calif. Litters were housed at room temperature, and sacrificed as previously described 16 in groups of 4-8 litters on day 2, 4, 6, 8, 10, 14 and 19 of life. After sacrifice, the cerebellum was separated from cerebrum and brain stem and was immediately weighed on a torsion balance and homogenized at 1 : 10 (w/v) in a cold homogenizing medium, using 20 up
347 and down movements of the homogenizer pestle. At age 2 and 4 days, 2 cerebella from treated animals and 2 cerebella from littermate controls were pooled to provide experimental data points. At other ages, data points were derived from single cerebella. Aliquots from the crude homogenate were drawn for D N A determinations at ages 2, 4 and 19 days. Remaining homogenate was then centrifuged at 35,000 × g at 5 °C for 20 min. The assay for A T C activity and determination of protein content were performed on the supernatant solution. All of the measurable A T C activity was recovered in the supernatant fraction. Homogenizing medium consisted of sucrose (0.25 M), potassium phosphate p H 7.4 (0.1 M), magnesium-ATP (0.015 M MgSO40.01 M ATP), dithiothreitol 1 m M , and glutamine (3 m M ) , as designed for the assay of thymidine kinase and other enzymes involved in salvage and de novo pyrimidine biosynthesis. Aspartate transcarbamylase was assayed by a modification of the method described by Bethell et al. 4. The enzyme reaction mixture contained 0.05 ml N a - H e p e s p H 7.4 (0.5 M), 0.10 ml K-aspartate, p H 7.4 (0.1 M), 0~05 ml [14C]carbamyl phosphate (0.517 #Ci/#mole), 0.20 ml tissue homogenate supernatant, and 0.10 ml homogenizing medium. The final volume was 0.5 ml with concentrations of 0.05 M Na-Hepes. 0.02 M K-aspartate, 0.15 M sucrose,0.06 M potassium phosphate, 6 m M ATP, 9 m M MgSO4, and 380 # M [14C]carbamyl phosphate. Tubes were incubated at 37 °C for 5 min, and the reaction stopped by adding 0.1 ml of 50 % trichloroacetic acid and heating at 100 °C for 3 rain. Tubes were then cooled to room temperature and shaken on a vortex mixer after 3 separate additions of a small quantity of powdered dry ice. An aliquot of 0.4 ml was placed in a counting vial TABLE I Measurements of development in thyroxine treated rat pups and controls
Mean value 4- S.E.M. In parentheses are given the number of values for each calculation. Age (days)
2 4 6 8 10 14 19
Treatment
Body wt. (g)
Cerebellar wt. (rag)
CerebellarDNA (~g/cerebellum)
Cerebellar ATC activity (nmole/ min/mg prot.)
Control Treated Control Treated Control Treated Control Treated Control Treated Control Treated Control Treated
(33) 8.8 4- 0.2 (39) 8.8 4- 0.1 (37)11.64- 0.2 (36)10.74- 0.2* (23)15.2+ 0.3 (26)14.2± 0.3* (24)21.64- 0.5 (23)18.84- 0.5* (19)22.5i 0.5 (17)18.84- 0.5* (24)35.94- 0.8 (27)26.34- 0.6* (21)48.54- 0.7 (18)27.84- 1.0"
(32) 18.1 ± 0.3 (15) 68 ± 2 (31) 19.0 4- 0.3 (15) 72 4- 3 (30) 25.9 4- 0.4 (16)1164- 3 (31) 27.5 4- 0.5 (16)140± 5* (18) 39.9 4- 1.3 - (18) 43.3 4- 0.8 (18) 66.5 4- 1.2 - (18) 67.4 4- 1.2 (16) 88.6 4- 1.8 - (15) 83.0 4- 1.2" (18)145.1 4- 3.0 - (18)119.7 4- 1.8" (17)184.6 4- 2.4 (16)1458 q- 25 (15)128.3 4- 2.2" (14) 975 4- 27*
(16)3.84- 0.17 05)3.9 ± 0.14 (16)5.44- 0.10 (16)5.6 4- 0.13 (18)4.8 ± 0.08 (18)5.1 4- 0.14 (18)6.6 ± 0.15 (18)6.0 4- 0.13" (16)5.9 4- 0.15 (15)5.0 4- 0.10" (18)2.7 4- 0.05 (18)2.2 ± 0.04* (16)2.0 4- 0.07 (14)1.6 4- 0.06*
* Indicates statistical significance between treated and control groups by t-test (P < 0.05).
348 with TRIPOP liquid scintillation solution a. Radioactivity was measured on a scintillation spectrophotometer. Protein was measured on samples of supernatant solution using the Oyama and Eagle modification12 of the method of Lowry et al. t0. DNA was determined on samples of crude tissue homogenate using the modification by Giles and Myers6 of the method of Burton 5. Statistical analysis was applied to data points for treated and control animals at each specific age by use of the t-test. All data are presented in Table I. The effect of thyroxine on body weight and cerebellar weight was similar to that previously described 16 with the first significant deficit in body weight in treated animals occurring at 4 days, and the first significant deficit in cerebellar weight occurring by age 10 days. A significant enhancement of cerebellar DNA synthesis in treated animals was noted on day 4 (120~ of control) and a deficit noted on day 19 (67 ~/ of control), confirming previously established experimental observations16. The developmental curve for aspartate transcarbamylase activity in cerebella of control animals peaks around 9 days and falls to 29 ~o of maximum activity by day 19 as described previously by Weichsel et al.lS. In treated animals, a significant decrease in aspartate transcarbamylase activity was noted on days 8-19 (maximum deficit 81 ~ of control on day 14). In contrast to the early enhancement of thymidine kinase activity by thyroxine, no significant difference in ATC activity was noted prior to day 8. The transient decrease in day 6 values for both treated and control animals reflects variation inherent in studying animals of different ages on separate days. Pyrimidine nucleotide precursors of nucleic acids may be synthesized by a de novo pathway which utilizes the simple substrates CO2, ATP and glutamine and results in formation of uridylic acid. Pyrimidine nucleotides may also be generated via salvage pathways whereby preformed nucleosides are enzymatically phosphorylated. Subsequent to its formation by either pathway, uridylic acid is converted into several pyrimidine nucleotides by sequences of enzymatic reactions which appear to be under complex metabolic control 9. Along one such sequence is the interconversion pathway enzyme thymidylate synthetase which is necessary for conversion of uridylic acid to thymidylic acid, thus providing a mechanism by which the de novo pathway may provide thymidine triphosphate for incorporation into DNA. The de novo pathway enzyme aspartate transcarbamylase was the subject of this study because of the profound effect of thyroxine previously noted on cerebellar thymidine kinase 16, as well as the effect noted on the salvage pathway enzyme uridine kinasO 9 and the interconversion pathway enzyme thymidylatesynthetase (B.R. Clark, personal communication) during the early postnatal acceleration and deceleration phase of cerebellar DNA synthesis. The suppression of cerebellar aspartate transcarbamylase activity after day 6 in thyroxine treated animals was similar to that of thymidine kinase 16, uridine kinase 19 and thymidylate synthetase (B. R. Clark, personal communication). However, the lack of a significant effect of thyroxine on ATC during the first week of cerebellar ontogenesis when the hormone is known to enhance DNA synthesis16(Table I) suggests
349 that during early development under the influence of thyroxine, de novo pathway enzymes for pyrimidine nucleotide biosynthesis are not influenced in a manner similar to reutilization enzymes. Studies utilizing a variety of model systems are inconclusive as to the differential contribution and control mechanisms for formation of pyrimidine nucleotides by de novo and salvage pathways prior to their incorporation into newly synthesized nucleic acidsL Although activity of the salvage pathway enzyme thymidine kinase appears to correlate with the rate of cerebellar D N A biosynthesis 1~, there is less certainty concerning the role played by the de novo pathway during mammalian cell division. Roux et al. 14, using isoproteranol to stimulate cell division in isolated mouse salivary gland, have found an early increase in de novo pathway enzymes, and have speculated that it is the de novo pathway which supplies the pyrimidine nucleotides for the R N A synthesis which may be required for subsequent synthesis of DNA. Our data showing a lack of effect on the de novo pathway enzyme aspartate transcarbamylase during early thyroxine enhancement of cerebellar cell division do not support such an early role for the de novo pathway, although the possibility remains that an early significant elevation in de novo pathway enzyme activity could be undetectable due to lack of sensitivity of the assay and lack of synchronized cell division in such an in vivo system. The mechanisms by which thyroid hormones regulate protein synthesis remain poorly understood. The present data compared with previous findings 16 suggests that a given hormone may not affect all metabolic pathways involved in nucleic acid biosynthesis in a similar manner. These results support the possibility that in a developing organ, the sensitivity of its enzyme systems to thyroid hormone may depend on the developmental state of the cells in that organ. Additionally, such findings support the possibility suggested by Tata 15 that the final expression of a hormone involved in growth and development may result from a cooperative action generated by simultaneous or sequential but independent interactions of a hormone with more than one nuclear or extranuclear site. The author gratefully acknowledges the technical assistance of Shirley M. Guo, M.S. This research was supported by Harbor General Hospital General Research Support Grant GR-2076 and by National Institute of Health Grant HD-09277-02 from the U.S. Public Health Service. 1 Anderson, L. W. and McClure, W. O., An improved scintillation cocktail of high-solubilizing power, Analyt. Biochem., 51 (1973) 173-179. 2 Bahtzs, R., Kov~tcks,S., Cocks, W. A., Johnson, A. L. and Eayrs, J. T., Effect of thyroid hormone on biochemicalmaturation of rat brain: postnatal cell formation, Brain Research, 25 (1971)555-570. 3 Bal~izs,R., Biochemicaleffects of thyroid hormones in the developing brain. In D. C. Pease (Ed.), Cellular Aspects of Neural Growth and Differentiation, UCLA Forum Med. Sci., Univ. of Calif. Press, Los Angeles, Calif., 1971, pp. 273-320. 4 Bethell, M. R., Smith, K. E., White, J. S. and Jones, M. E., Carbamyl phosphate: an aliosteric substrate for aspartate transcarbamylase of Escherichia coli, Proc. nat. Acad. Sci. (Wash.), 60 (1968) 1442-1449.
350 5 Burton, K., A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid, Biochem. J., 62 (1956) 315-323. 6 Giles, K. W. and Myers, A., An improved diphenylamine method for the estimation of deoxyribonucleic acid, Nature (Lond.), 206 (1965) 93. 7 Gourdon, J., Clos, J., Coste, C., Dainat, J. and Legrand, J., Comparative effects of hypothyroidism, hyperthyroidism, and undernutrition on the protein and nucleic acid contents of the cerebellum in the young rat, J. Neurochem., 21 (1973) 861-871. 8 Hamburgh, M., Mendoza, L. A., Burkhard, J. F. and Weil, F., In D. C. Pease (Ed.), Cellular Aspects of Neural Growth and Differentiation, UCLA Forum Med. Sci., Univ. of Calif. Press, Los Aogeles, Calif., 1971, pp. 321-328. 9 Levine, R., Hoogenraad, N. J. and Kretchmer, N., A review: biological and clinical aspects of pyrimidine metabolism, Pediat. Res., 8 (1974) 724-734. 10 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 11 Nicholson, J. L. and Altman, J., The effects of early hypo- and hyperthyroidism on the development of rat cerebellar cortex. 1. Cell proliferation and differentiation, Brain Research, 44 (1972) 13-23. 12 Oyama, V. I. and Eagle, H., Measurement of cell growth in tissue culture with a phenol reagent (Folin-Cicaltreau), Proc. Soc. exp. Biol. ( N. Y.), 91 (1956) 305-307. 13 Patel, A. J., Bal~.zs, R. and Johnson, A. L., Effect of undernutrition on cell formation in the rat brain, J. Neurochem., 20 (1973) 1151-1165. 14 Roux, J. M., Hoogenraad, N. J. and Kretchmer, N., Biosynthesis of pyrimidine nucleotides in mouse salivary glands stimulated with isoproteranol, J. biol. Chem., 248 (1973) 1196-1202. 15 Tata, J. R., How specific are nuclear "receptors" for thyroid hormones? Nature (Lond.), 257 (1975) 18-23. 16 Weichsel, Jr., M. E., Effect of thyroxine on D N A synthesis and thymidine kinase activity during cerebellar development, Brain Research, 78 (1974) 455-465. 17 Weichsel, Jr., M. E. and Dawson, L., Effects of hypothyroidism and undernutrition on D N A content and thymidine kinase activity during cerebeltar development in the rat, J. Neurochem., 26 (1976) 675-681. 18 Weichsel, Jr., M. E., Hoogenraad, N. J., Levine, R. L. and Kretchmer, N., Pyrimidine biosynthesis during development of the cerebellum, Pediat. Res., 6 (1972) 682-686. 19 Weichsel, Jr., M. E., Thyroxine effect upon activity of uridine kinase in developing rat cerebellum, Biol. Neonat. (Basel), In press.