- Email: [email protected]
The histochemical demonstration of dehydrogenases in neuroglia
F. Wolfgram
and A. S. Rose
suspended cells can be made; for example, the specific gravity of the isolated rat liver cell lies between 1.116 and 1.128. An adjusted colloidal silica (density =1.18; pH -7.3) has been used repeatedly as the isopyknotic cushion in the stratification of the subcellular constituents of amphibian and mammalian liver and human tumor cells. The procedure has been to use the cushion with a gradient from 1.18 to 1.003, the latter being that of a physiological solution as Tyrode’s, thus providing for a range of closely graded densities for the cells. For subsequent studies, the stratified cells are removed from the medium by pipette and washed with physiological solution to remove the colloidal silica. While at present colloidal silica appears to be highly effective, the newer high molecular weight substances which meet the specified criteria may prove more useful. The data on problems of selecting suitable isopyknotic media will be published subsequently. Experiments are in progress to evaluate the use of colloidal silica and possibly other substances as suspending media for the ultracentrifugation of cellular homogenates. REFERENCE 1. HOLTER,
THE
H.
and
MBLLER,
M.,
HISTOCHEMICAL
Exptl.
Cell
Research
15, 631 (1958).
DEMONSTRATION
OF DEHYDROGENASES
IN NEUROGLIAl F. WOLFGRAM Division of Neurology, and Los Angeles
and A. S. ROSE
Department of Medicine, Veterans Administration Received
March
University of California at Los Angeles Center, Los Angeles, Calif., U.S.A. 17, 1959
T HE
neuroglia are the most numerous cells in the central nervous system, outnumbering the neurons by perhaps ten to one. Relatively little is known of their metabolism, due in large measure to the difficulty in separating them from the nervous elements. Neither tissue culture [19] nor homogenization and differential centrifugation [6] has as yet proven successful in producing quantities of intact adult neuroglia. Studies on neuroglial tumors have indicated a low rate of respiration [15]. Perhaps the first investigation of glial oxidative enzymes was that of Marinesco [9] who was unable to demonstrate indophenol oxidase (cytochrome oxidase) in neuroglia. Recently there have been a number of histochemical studies of succinic dehydrogenase in nervous tissue and all are in agreement that this enzyme could not be demonstrated in white matter [7, 10, 12, 17, 181. Friede [4] studied the histochemical development of the rat cerebellum and concluded that while succinic dehydrogenase can be demonstrated in the glia during active myelination it is not present in adult glia. Korey [6] found succinate to be a good substrate for the isolated neuroglia of lamb brain but did not 1 This Experimenfal
study
was
supported
Cell Research
17
in part
by
Grant
#202,
National
Multiple
Sclerosis
Society.
Histochemical
demonstration
of dehydrogenases
in neuroglia
527
extend this work to adult cells. Lowry [8] showed that glucose-6-phosphate dehydrogenase is higher in white matter than in gray matter. As there are large numbers of neuroglia, particularly oligodendrocytes, in white matter Pope [14] suggested that in these glia the hexose monophosphate shunt is in operation rather than the oxidation of glucose via dismutation and the glycolytic and tricarboxylic acid cycles. Succinic dehydrogenase is associated with mitochondria in both liver [5] and brain [l]. Tissue culture studies 1191 have demonstrated considerable numbers of mitochondria in the perinuclear cytoplasm of neuroglia and lesser numbers in the processes. If succinic dehydrogenase were absent from adult neuroglial mitochondria this would consitute a unique and interesting situation. However, the purpose of this report is to demonstrate that succinic dehydrogenase is present in adult neuroglia and that previous failures to demonstrate it were due to a technical difficulty rather than to its absence. The previous histochemical studies of succinic dehydrogenase in neuroglia utilized either neotetrazolium, triphenyltetrazolium or blue tetrazolium as the electron acceptor. These tetrazoles are reduced with some difficulty and although their reduction can be facilitated by the addition of methylene blue [2] or cyanide [16] they have been made obsolete by newer tetrazoles that are more readily reduced. Fox and Atkinson [3] have synthesized 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) and Pearson and Defendi [13] have utilized it for the histochemical demonstration of succinic dehydrogenase. They found that it was rapidly reduced and gave a sharp localization with a minimum of diffusion. Nachlas ef al. [II] have used Nitro Blue Tetrazolium (Nitro-BT) and claim it to be superior to INT in the ease of its reduction, its small particle size and its lack of lipid solubility. Methods.-Frozen sections of unfixed cerebrum, cerebellum and spinal cord of adult rats and cats were cut in the cryostat at 16 ,u, mounted on uncoated slides and dried in vacua at room temperature for ten minutes. They were then incubated in the substrate solutions at 25°C. When INT was used as the electron acceptor 30 to 60 minutes were sufficient to give a large number of reduced tetrazole particles in the white matter. With Nitro-BT an incubation period of from 8 to 12 hours was necessary. INT was subsituted for neotetrazolium in the solution of Rosa and Velardo [I61 which consists of 30 ml 0.1 molar phosphate buffer containing 0.1 per cent NaCN pH 8.2, 4 ml of 0.5 molar sodium succinate and 4 ml of 0.4 per cent INT. Nitro-BT was used as directed by Nachlas [Ill. This solution consists of equal parts of 0.2 molar phosphate buffer pH 7.6 and 0.2 molar sodium succinate. To IO ml of this is added 10 ml of H,O containing 10 mg Nitro-BT. In some experiments the Nitro-BT was used instead of INT in the solution of Rosa and Velardo. When lactic, malic or z-glycerophosphate dehydrogenases were being localized an equimolar amount of the particular substrate was substituted for the succinate. Resulfs.-When INT was used as the electron acceptor succinic, malic, lactic and a-glycerophosphate dehydrogenases were readily localized in the gray matter within 5 minutes and in the white matter within 30 minutes. In longitudinal sections of fiber tracts the blood vessels did not react intensely but were easily recognized as being stippled by fine tetrazole granules along their entire length. The cell bodies of the intrafascicular oligodendrocytes were outlined by the strong dehydrogenase reaction in the perinuclear cytoplasm (Fig. la). The localization of these granules coincided with the location of the perinuclear mitochondria as seen in oligodendroExperimental
Cell Resenrch
17
F. Wolfgram
528
Ezperimenlul
Cd
Research
17
and A. S. Rose
Histochemical
demonstration
of dehydrogenases
529
in neuroglia
cytes studied with phase contrast optics in tissue culture [ 191. Astrocytic mitochondria also showed dehydrogenase activity but the proper identification of these cells was somewhat difficult when INT was used because the nuclei could not be successfully counterstained (see below). The processes of neuroglia have been shown [19] to contain mitochondria both singly and in clusters which constitute the “gliosomes” of classical microscopy. Reduced tetrazole granules were rather heavily scattered throughout the white matter and while some of these were from axoplasmic mitochondria many of them were extraneural and localized the mitochondria in the glial processes. The extreme ease with which it is reduced is of great advantage in the use of INT but, as others have noted [li], the tetrazole granules tend to crystallize in the tissue sections. These crystals rapidly formed while the slides were being -photographed immediately after incubation (Fig. lb). However, the widespread localization of dehydrogenases in white matter with this tetrazole was easily demonstrated even though on a cytological level the granules often tended to form artifacts. When INT was used the neuroglial nuclei could not be counterstained for photography because the reduced tetrazole is soluble in lipid solvents and water soluble dyes stained the myelin and obscured the tetrazole granules. Nachlas [ll] has rated Nitro-BT as a more efficient electron acceptor than INT, but with the neuroglia the opposite was true. Regardless of whether Nitro-BT was used in the incubation solution of Nachlas or that of Rosa and Velardo, 8 to 12 hours were required to obtain a satisfactory number of tetrazole granules in the glial cytoplasm. However, the localization with Nitro-BT was very satisfactory in that the granules did not crystallize and were not soluble in lipid solvents. Thus the neuroglial nuclei were readily counterstained with hematoxylin and exact identification was made possible. The granules were mainly localized in the perinuclear cytoplasm. The precise location of the granules varied considerably, in some cells they surrounded the nuclei, in other cells they were seen at either or both ends of the nuclei (Figs. 2a to 21). With Kitro-BT reduced tetrazole granules were observed in the perinuclear cytoplasm of astrocytes, oligodendrocytes and microglia of adult rats and cats when any of the four dehydrogenase substrates were used. Summary.-Succinic, been histochemically
Succinic dehydrogenase
malic, demonstrated
lactic
localization
in
and the
a-glycerophosphate perinuclear cytoplasm
in longitudinal
dehydrogenases of the three
have types
sections of cat spinal cortl white matter.
l;ig. l.-Two oligodendrocytes with reduced INT granules in the cytoplasm. No counterstain. 700 :I Fig. ‘L.-Crystallization of reduced IKT granules in white matter photographed immediately after incubation. 430 x . Fig. 3.-Three oligodendrocytes (left and bottom) and a microglia (upper right). Nitro-BT granules are mainly in the perinuclear cytoplasm. Hematoxylin counterstain. 2800 Y . Fig. 4-Strong reaction surrounding a small astrocyte nucleus. Nitro-BT, hematoxylin. 2800 x . Fig. 5.-Two oligodendrocytes with granules extending into the cytoplasm of the processes. Nitro-UT, hematoxylin. 2800 x . Fig. 6.-Oligodendrocyte with granules at opposite ends of the nucleus. Nitro-BT, hematoxylin. 2800 ’ Fig. 7.-hstrocyte with granules at opposite ends of the nucleus. Nitro-BT, hematoxylin. 2800 x . 12ig. 8.-Oligodendrocyte with granules surrounding the nucleus. Nitro-BT, hematoxylin. 2800 x . Experimental
Cell
Research
17
530
Y. Kuriki
and R. Okazaki
of neuroglia in adult rats and cats. Previous failures by other investigators onstrate dehydrogenases in neuroglia were due to the lack of sufficiently tetrazolium salts. The authors wish to express their gratitude to Mr. P. Eugene assistance and to Mr. Philip Bleicher for the photography.
to demsensitive
Parks, Jr. for technical
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
BRODY, T. Ill. and BAIN, J. A., J. Biof. Chem. 195, 685 (1952). FARBER, E. and LOUVIERE, C. D., J. Hisfochem. Cytochem. 4, 347 (1956). Fox, S. W. and ATKINSON, E. H., J. Am. Chem. Sot. 72, 3629 (1950). FRIEDE, R., Arch. Psychiat. Neruenkr. 196, 196 (1957). HOGEBOOM, G. H., SCHNEIDER. W. C. and PALADE, G. E., J. Biol. Chem. 172, 619 (1948). KOREY, S. R., ORCHEN, M. and BROTZ, N., J. NeuropathoL Exptl. Neural. 17, 430 (1958). LEDUC, E. H. and WISLOCKI, G. B., J. Comp. Neural. 97, 241 (1952). LOWRY, 0. H., in Biochemistry of the Developing Nervous System, p. 350. H. Waelsch ed., Academic Press, New York 1955. MARISESCO, G., Compf. rend. Sot. biof. 82, 258 (1919). MUSTAKALLIO, K. K., Ann. med. exp. hiof. fenn. 32, 175 (1954). NACHLAS, M. N., Tsou, K. C., DESOUZA, E., CHENG, C. J. and SELIGMAN, A. M., J. Hisfochem. Cyfochem. 5, 420 (1957). PADYKULA, H. A., Am. J. Anaf. 91, 107 (1952). PEARSON, B. and DEFENDI, V., J. Histochem. Cyfochem. 2, 248 (1954). POPE, A., in Biology of Neuroglia, p. 211. W. F. Windle ed., Thomas, Springfield, Illinois, 1958. POPE, A., HESS, H. H. and ALLEN, J. N., in Ultrastructure and Cellular Chemistry of Nernous Tissue, p. 182. H. Waelsch ed., Hoeber-Harper, New York, 1957. ROSA, C. G. and VELARDO, J. T., J. Histochem. Cyfochem. 2, 110 (1954). RUTENBURG, A. M., WOLMAN, M. and SELIGMAN, A. M., J. Hisfochem. Cyfochem. 1, 66 (1953). SHIMIZU, N. and MORIKAWA, N., J. Hisfochem. Cyfochem. 5, 334 (1957). WOLFGRAM, F., and ROSE, A. S., J. Neuropafhol. Expfl. Neural. 16, 514 (1957).
DEOXYRIBOSIDIC
COMPOUNDS
MUTANT
OF ESCHER1CHIA
Y. KURIKI Biological
Institute,
Faculty
IN A THYMINE-REQUIRING COLIl
and R. OKAZAKI of Science,
Nagoya
University,
Nagoya,
Japan
Received March 21, 1959
IN
previous papers 19, 11, 121, it was shown that the major acid-soluble deoxyribosidic compound in a deoxyriboside-requiring strain of Lacfobacillus acidophilus is an unidentified thymidine compound (TDP-X2) and that this may serve as an inter-
1 Supported in part by a grant from the Rockefeller Foundation and a subsidy from the Tokai Gakujutsushoreikai. * Abbreviations: TMP, TDP, TTP, thymidine mono-, di- and triphosphate; DNA, deoxyribonucleic acid; dCMP, dCDP, dCTP, deoxycytidine mono-, di- and triphosphate; dAMP, deoxyadenosine monophosphate; dGMP, deoxyguanosine monophosphate; UMP, UDP, UTP; uridine mono-, di- and triphosphate; ADP, ATP, adenosine di- and triphosphate; GDP, GTP, guanosine di- and triphosphate. Experimental
Cell
Reesarch
17
and A. S. Rose
suspended cells can be made; for example, the specific gravity of the isolated rat liver cell lies between 1.116 and 1.128. An adjusted colloidal silica (density =1.18; pH -7.3) has been used repeatedly as the isopyknotic cushion in the stratification of the subcellular constituents of amphibian and mammalian liver and human tumor cells. The procedure has been to use the cushion with a gradient from 1.18 to 1.003, the latter being that of a physiological solution as Tyrode’s, thus providing for a range of closely graded densities for the cells. For subsequent studies, the stratified cells are removed from the medium by pipette and washed with physiological solution to remove the colloidal silica. While at present colloidal silica appears to be highly effective, the newer high molecular weight substances which meet the specified criteria may prove more useful. The data on problems of selecting suitable isopyknotic media will be published subsequently. Experiments are in progress to evaluate the use of colloidal silica and possibly other substances as suspending media for the ultracentrifugation of cellular homogenates. REFERENCE 1. HOLTER,
THE
H.
and
MBLLER,
M.,
HISTOCHEMICAL
Exptl.
Cell
Research
15, 631 (1958).
DEMONSTRATION
OF DEHYDROGENASES
IN NEUROGLIAl F. WOLFGRAM Division of Neurology, and Los Angeles
and A. S. ROSE
Department of Medicine, Veterans Administration Received
March
University of California at Los Angeles Center, Los Angeles, Calif., U.S.A. 17, 1959
T HE
neuroglia are the most numerous cells in the central nervous system, outnumbering the neurons by perhaps ten to one. Relatively little is known of their metabolism, due in large measure to the difficulty in separating them from the nervous elements. Neither tissue culture [19] nor homogenization and differential centrifugation [6] has as yet proven successful in producing quantities of intact adult neuroglia. Studies on neuroglial tumors have indicated a low rate of respiration [15]. Perhaps the first investigation of glial oxidative enzymes was that of Marinesco [9] who was unable to demonstrate indophenol oxidase (cytochrome oxidase) in neuroglia. Recently there have been a number of histochemical studies of succinic dehydrogenase in nervous tissue and all are in agreement that this enzyme could not be demonstrated in white matter [7, 10, 12, 17, 181. Friede [4] studied the histochemical development of the rat cerebellum and concluded that while succinic dehydrogenase can be demonstrated in the glia during active myelination it is not present in adult glia. Korey [6] found succinate to be a good substrate for the isolated neuroglia of lamb brain but did not 1 This Experimenfal
study
was
supported
Cell Research
17
in part
by
Grant
#202,
National
Multiple
Sclerosis
Society.
Histochemical
demonstration
of dehydrogenases
in neuroglia
527
extend this work to adult cells. Lowry [8] showed that glucose-6-phosphate dehydrogenase is higher in white matter than in gray matter. As there are large numbers of neuroglia, particularly oligodendrocytes, in white matter Pope [14] suggested that in these glia the hexose monophosphate shunt is in operation rather than the oxidation of glucose via dismutation and the glycolytic and tricarboxylic acid cycles. Succinic dehydrogenase is associated with mitochondria in both liver [5] and brain [l]. Tissue culture studies 1191 have demonstrated considerable numbers of mitochondria in the perinuclear cytoplasm of neuroglia and lesser numbers in the processes. If succinic dehydrogenase were absent from adult neuroglial mitochondria this would consitute a unique and interesting situation. However, the purpose of this report is to demonstrate that succinic dehydrogenase is present in adult neuroglia and that previous failures to demonstrate it were due to a technical difficulty rather than to its absence. The previous histochemical studies of succinic dehydrogenase in neuroglia utilized either neotetrazolium, triphenyltetrazolium or blue tetrazolium as the electron acceptor. These tetrazoles are reduced with some difficulty and although their reduction can be facilitated by the addition of methylene blue [2] or cyanide [16] they have been made obsolete by newer tetrazoles that are more readily reduced. Fox and Atkinson [3] have synthesized 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) and Pearson and Defendi [13] have utilized it for the histochemical demonstration of succinic dehydrogenase. They found that it was rapidly reduced and gave a sharp localization with a minimum of diffusion. Nachlas ef al. [II] have used Nitro Blue Tetrazolium (Nitro-BT) and claim it to be superior to INT in the ease of its reduction, its small particle size and its lack of lipid solubility. Methods.-Frozen sections of unfixed cerebrum, cerebellum and spinal cord of adult rats and cats were cut in the cryostat at 16 ,u, mounted on uncoated slides and dried in vacua at room temperature for ten minutes. They were then incubated in the substrate solutions at 25°C. When INT was used as the electron acceptor 30 to 60 minutes were sufficient to give a large number of reduced tetrazole particles in the white matter. With Nitro-BT an incubation period of from 8 to 12 hours was necessary. INT was subsituted for neotetrazolium in the solution of Rosa and Velardo [I61 which consists of 30 ml 0.1 molar phosphate buffer containing 0.1 per cent NaCN pH 8.2, 4 ml of 0.5 molar sodium succinate and 4 ml of 0.4 per cent INT. Nitro-BT was used as directed by Nachlas [Ill. This solution consists of equal parts of 0.2 molar phosphate buffer pH 7.6 and 0.2 molar sodium succinate. To IO ml of this is added 10 ml of H,O containing 10 mg Nitro-BT. In some experiments the Nitro-BT was used instead of INT in the solution of Rosa and Velardo. When lactic, malic or z-glycerophosphate dehydrogenases were being localized an equimolar amount of the particular substrate was substituted for the succinate. Resulfs.-When INT was used as the electron acceptor succinic, malic, lactic and a-glycerophosphate dehydrogenases were readily localized in the gray matter within 5 minutes and in the white matter within 30 minutes. In longitudinal sections of fiber tracts the blood vessels did not react intensely but were easily recognized as being stippled by fine tetrazole granules along their entire length. The cell bodies of the intrafascicular oligodendrocytes were outlined by the strong dehydrogenase reaction in the perinuclear cytoplasm (Fig. la). The localization of these granules coincided with the location of the perinuclear mitochondria as seen in oligodendroExperimental
Cell Resenrch
17
F. Wolfgram
528
Ezperimenlul
Cd
Research
17
and A. S. Rose
Histochemical
demonstration
of dehydrogenases
529
in neuroglia
cytes studied with phase contrast optics in tissue culture [ 191. Astrocytic mitochondria also showed dehydrogenase activity but the proper identification of these cells was somewhat difficult when INT was used because the nuclei could not be successfully counterstained (see below). The processes of neuroglia have been shown [19] to contain mitochondria both singly and in clusters which constitute the “gliosomes” of classical microscopy. Reduced tetrazole granules were rather heavily scattered throughout the white matter and while some of these were from axoplasmic mitochondria many of them were extraneural and localized the mitochondria in the glial processes. The extreme ease with which it is reduced is of great advantage in the use of INT but, as others have noted [li], the tetrazole granules tend to crystallize in the tissue sections. These crystals rapidly formed while the slides were being -photographed immediately after incubation (Fig. lb). However, the widespread localization of dehydrogenases in white matter with this tetrazole was easily demonstrated even though on a cytological level the granules often tended to form artifacts. When INT was used the neuroglial nuclei could not be counterstained for photography because the reduced tetrazole is soluble in lipid solvents and water soluble dyes stained the myelin and obscured the tetrazole granules. Nachlas [ll] has rated Nitro-BT as a more efficient electron acceptor than INT, but with the neuroglia the opposite was true. Regardless of whether Nitro-BT was used in the incubation solution of Nachlas or that of Rosa and Velardo, 8 to 12 hours were required to obtain a satisfactory number of tetrazole granules in the glial cytoplasm. However, the localization with Nitro-BT was very satisfactory in that the granules did not crystallize and were not soluble in lipid solvents. Thus the neuroglial nuclei were readily counterstained with hematoxylin and exact identification was made possible. The granules were mainly localized in the perinuclear cytoplasm. The precise location of the granules varied considerably, in some cells they surrounded the nuclei, in other cells they were seen at either or both ends of the nuclei (Figs. 2a to 21). With Kitro-BT reduced tetrazole granules were observed in the perinuclear cytoplasm of astrocytes, oligodendrocytes and microglia of adult rats and cats when any of the four dehydrogenase substrates were used. Summary.-Succinic, been histochemically
Succinic dehydrogenase
malic, demonstrated
lactic
localization
in
and the
a-glycerophosphate perinuclear cytoplasm
in longitudinal
dehydrogenases of the three
have types
sections of cat spinal cortl white matter.
l;ig. l.-Two oligodendrocytes with reduced INT granules in the cytoplasm. No counterstain. 700 :I Fig. ‘L.-Crystallization of reduced IKT granules in white matter photographed immediately after incubation. 430 x . Fig. 3.-Three oligodendrocytes (left and bottom) and a microglia (upper right). Nitro-BT granules are mainly in the perinuclear cytoplasm. Hematoxylin counterstain. 2800 Y . Fig. 4-Strong reaction surrounding a small astrocyte nucleus. Nitro-BT, hematoxylin. 2800 x . Fig. 5.-Two oligodendrocytes with granules extending into the cytoplasm of the processes. Nitro-UT, hematoxylin. 2800 x . Fig. 6.-Oligodendrocyte with granules at opposite ends of the nucleus. Nitro-BT, hematoxylin. 2800 ’ Fig. 7.-hstrocyte with granules at opposite ends of the nucleus. Nitro-BT, hematoxylin. 2800 x . 12ig. 8.-Oligodendrocyte with granules surrounding the nucleus. Nitro-BT, hematoxylin. 2800 x . Experimental
Cell
Research
17
530
Y. Kuriki
and R. Okazaki
of neuroglia in adult rats and cats. Previous failures by other investigators onstrate dehydrogenases in neuroglia were due to the lack of sufficiently tetrazolium salts. The authors wish to express their gratitude to Mr. P. Eugene assistance and to Mr. Philip Bleicher for the photography.
to demsensitive
Parks, Jr. for technical
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
BRODY, T. Ill. and BAIN, J. A., J. Biof. Chem. 195, 685 (1952). FARBER, E. and LOUVIERE, C. D., J. Hisfochem. Cytochem. 4, 347 (1956). Fox, S. W. and ATKINSON, E. H., J. Am. Chem. Sot. 72, 3629 (1950). FRIEDE, R., Arch. Psychiat. Neruenkr. 196, 196 (1957). HOGEBOOM, G. H., SCHNEIDER. W. C. and PALADE, G. E., J. Biol. Chem. 172, 619 (1948). KOREY, S. R., ORCHEN, M. and BROTZ, N., J. NeuropathoL Exptl. Neural. 17, 430 (1958). LEDUC, E. H. and WISLOCKI, G. B., J. Comp. Neural. 97, 241 (1952). LOWRY, 0. H., in Biochemistry of the Developing Nervous System, p. 350. H. Waelsch ed., Academic Press, New York 1955. MARISESCO, G., Compf. rend. Sot. biof. 82, 258 (1919). MUSTAKALLIO, K. K., Ann. med. exp. hiof. fenn. 32, 175 (1954). NACHLAS, M. N., Tsou, K. C., DESOUZA, E., CHENG, C. J. and SELIGMAN, A. M., J. Hisfochem. Cyfochem. 5, 420 (1957). PADYKULA, H. A., Am. J. Anaf. 91, 107 (1952). PEARSON, B. and DEFENDI, V., J. Histochem. Cyfochem. 2, 248 (1954). POPE, A., in Biology of Neuroglia, p. 211. W. F. Windle ed., Thomas, Springfield, Illinois, 1958. POPE, A., HESS, H. H. and ALLEN, J. N., in Ultrastructure and Cellular Chemistry of Nernous Tissue, p. 182. H. Waelsch ed., Hoeber-Harper, New York, 1957. ROSA, C. G. and VELARDO, J. T., J. Histochem. Cyfochem. 2, 110 (1954). RUTENBURG, A. M., WOLMAN, M. and SELIGMAN, A. M., J. Hisfochem. Cyfochem. 1, 66 (1953). SHIMIZU, N. and MORIKAWA, N., J. Hisfochem. Cyfochem. 5, 334 (1957). WOLFGRAM, F., and ROSE, A. S., J. Neuropafhol. Expfl. Neural. 16, 514 (1957).
DEOXYRIBOSIDIC
COMPOUNDS
MUTANT
OF ESCHER1CHIA
Y. KURIKI Biological
Institute,
Faculty
IN A THYMINE-REQUIRING COLIl
and R. OKAZAKI of Science,
Nagoya
University,
Nagoya,
Japan
Received March 21, 1959
IN
previous papers 19, 11, 121, it was shown that the major acid-soluble deoxyribosidic compound in a deoxyriboside-requiring strain of Lacfobacillus acidophilus is an unidentified thymidine compound (TDP-X2) and that this may serve as an inter-
1 Supported in part by a grant from the Rockefeller Foundation and a subsidy from the Tokai Gakujutsushoreikai. * Abbreviations: TMP, TDP, TTP, thymidine mono-, di- and triphosphate; DNA, deoxyribonucleic acid; dCMP, dCDP, dCTP, deoxycytidine mono-, di- and triphosphate; dAMP, deoxyadenosine monophosphate; dGMP, deoxyguanosine monophosphate; UMP, UDP, UTP; uridine mono-, di- and triphosphate; ADP, ATP, adenosine di- and triphosphate; GDP, GTP, guanosine di- and triphosphate. Experimental
Cell
Reesarch
17