- Email: [email protected]
Effects of proflavine on ultrastructure and RNA and protein synthesis in the liver of rats
Q 1963 by Academic Experimental
Press Inc.
Cell Research
505
50, SOS-51 4 (196X)
EFFECTS OF PROFLAVINE ON ULTRASTRUCTURE AND RNA AND PROTEIN SYNTHESIS IN THE LIVER OF RATS U. STENRAM and R. WILL&N Department
of Pathology,
University
Received June
of Uppsala,
Sweden
19, 1967
WHES studying
the effects of various substances on the ultrastructure of the rat liver cell, we found profound changes in the nucleoli following proflavine administration. This substance is known to interfere with the ribonucleic acid (RSA) synthesis. A combined study of the effects of proflavine on the RNA and protein synthesis and the ultrastructure of the rat liver cell was therefore considered to be of interest.
MATERIAL
AND
METHODS
Preliminary experiments were performed to determine the dose, if any, that provoked changes in the ultrastructure of rat liver cells in vivo. Five mg proflavine per rat was found to be suitable, though the animals succumbedwithin 23 h. One mg had no effect. In the main experiment 17 male white rats (Sprague-Dawley strain) were used, each weighing about 45 g. Having been fed on a protein-free diet [la] for four days and starved for one day, each rat, then weighing about 36 g, was subcutaneously given 5 mg proflavine N.F. (Nutritional Biochemicals, USA), dissolved in 1 ml distilled water, or 1 ml 0.9 per cent NaCl at B-B.30 a.m. Exactly 1 h later, the rats were intraperitoneally given 5 ,UC3H-cytidine (Schwarz BioResearch, USA) or 5 pc 3H-L-leucine per g body weight (New Engl. Nuclear Corp., USA). The rats were sacrified 20 min or 3 h later. One of the intended 3 h animals, treated with proflavine, died spontaneously and was excluded. The other animals were in fairly good condition at sacrifice. Liver slices were fixed in 1 per cent 0~0, for electron microscopy and Carnoy’s fluid Nr. II for optical microscopy and radioautography [16]. The remainder of the livers was frozen on dry ice. RNA was prepared, fractionated in ultracentrifuge and analyzed for ultraviolet absorption and radioactivity as described previously [18] with a later modification [17]. Protein was prepared essentially according to the methods of Simkin and Work [lo] and Munro ef al. [5] from the livers of the animals given leucine. While thawing, the liver tissue was pressedas rapidly as possible through a plastic sieve with holes with a diameter of 1 mm. About 0.3 g of liver was homogenized in a microhomogenizer Experimental
Cell Research
50
506
U. Stenram
and R. Will-h
(Type Nelco. Measuring and Scientific Equipment Ltd, London, England) at 0°C for 1 min in 4 ml of a 0.01 M sodium phosphate buffer, pH 7.4, with 0.01 M NaCl and 0.015 M MgCl,. An equal volume of 20 per cent (w/v) trichloroacetic acid (TCA) was added. After centrifugation for IO min at 2700 g, the precipitate was dissolved in 4 ml N NaOH at 70°C. After cooling, IO mg of unlabelled leucine was added, the mixture was stirred, and 4 ml 36.3 per cent TCA was added. The precipitate was recovered by centrifugation and washed once with IO per cent TCA and then heated at 90°C for 15 min in 10 per cent TCA to remove nucleic acids. After cooling, the precipitate was washed once with 10 per cent TCA, once with 95 per cent ale., once with absolute alcohol, once with a 3 : I alcohol-chloroform mixture, then heated to 60°C with alcohol-ether (3 : I), washed once with ether and allowed to dry. The sampleswere then treated according to Mahin and Lofberg [4] in the following way. About 20 mg dry protein, determined with one decimal, was weighed into glass vials. To each sample, 0.2 ml 70 per cent perchloric acid and 0.4 ml 30 per cent hydrogen peroxide were added, and the caps tightly screwed. The samples were heated to 80°C for 3 h. After cooling, 15.5 ml was added of a mixture of 5.5 ml cellosolve (ethylene glycol monoethylether), 10 ml toluene and 0.06 g PPO. The radioactivity (counts/min/mg dry protein) was determined in duplicate in a CM-25 liquid scintillation counter, Tracerlab. USA. For each group of rats the mean, the standard error and the standard deviation (s) of the mean were calculated from the mean of the individual animals. One specimenwas lost. Grain counts were performed in the radioautographs of the rats given 3H-leucine. 30 random square fields were counted from each animal, 3 controls and 5 given proflavine. For each group of the animals the mean, the standard error and the standard deviation (s) of the mean were calculated from the figures of the individual animals. For further details seeStenram [13].
OBSERVATION Optical microscopy suggested a decrease in nucleolar size in the rats killed 4 h after administration of proflavine. The nucleoli retained their intense basophilia. Radioautography showed heavy labelling of nuclear and, at 3 h, also of cytoplasmic RNA in the controls given 3H-cytidine. The heaviest labelling was found over nucleoli. The proflavine-treated rats showed roughly the same labelling at 20 min as the controls. At 3 h there was less labelling over nucleoli and cytoplasm compared to the controls (Figs l-4). This was seen not only in the livers but also in some other organs examined, the pancreas, kidneys, adrenals and peripheral ganglion cells. There were no discernible differences in labelling among the animals given 3H-leucine, which were all killed 3 h after administration of the radioactive substance (Figs 5, 6). The result of the grain counts is given in Table 1. Electron microscopy of the animals killed 4 h after the administration of Experimental
Cell
Research
50
Proflavine
507
and rat liver cells
Figs l-6.-Radioautographs of liver sections. Figs 1-4 of animals given SH-cytidine 20 min (Figs 1 and 2) and 3 h (Figs 3 and 4) before death, Figs 5 and 6 of animals given 8H-leucine 3 h before death. Figs 1, 3 and 5 are from control animals, Figs 2, 4 and 6 from animals given proflavine. x 1575. Figs
is the same labelling
in both
control
Figs 3 and 4.-There
1 and 2.-There
is less labelling
nucleoli
and cytoplasm
Figs 5 and 6.-There
is the same labelling
in both
control
over
and treated
animals.
in the treated
and treated
animal.
animal.
proflavine showed, compared to the controls, pronounced changes in the nucleoli. These had decreased in size, had coalesced and showed only small areas of low contrast. The filamentous and granule-rich components were well discernible and often showed a tendency to segregation (Figs 7-9). Experimental
Cell Research
50
508
l-J. Stem-am and R. WillCn
Figs
7-lO.-Electron
Fig.
7.-Control
Experimental
micrographs showing
the normal
Cell Research
50
of livers. appearance
0~0,
fixation.
Uranyl
of the liver
cell.
acetate-lead x 30,500.
citrate
staining.
Proflauine
Fig. 8.-Proflavine x 30,500.
treatment
showing
and rat liver cells
coalescence
of nucleolus
509
and segregation
Experimental
of its components.
Cell Research
SO
510
Fig.
U. Stenram
9.-Proflavine
treatment.
A nucleolus
and R. Will&
is seen in higher
magnification.
x 56,000.
The rats killed 1 h and 20 min after the administration of proflavine showed no obvious nucleolar changes. With the alterations of the 4 h rats in mind, however, a slight coalescence of the nucleoli could be discerned. There were no other definite differences between controls and treated animals. However, the nucleoplasm sometimes appeared to be less homogeneous and the endoplasmic sacs to be slightly dilated in the latter. In all Experimental
Cell Research
50
Proflavine
Fig. lO.-Tangential view cytoplasm and the nuclear
of nucleus membrane
511
and rat liver cells
showing close with its pores.
association x 46,000.
between
animals a rather close relationship was seen between cytoplasm and the nuclear membrane (Fig. 10).
polyribosomes
polyribosomes
in the
in the
Biochemistry The incorporation of 3H-cytidine into RNA is depicted in Fig. 11. There is a definite difference only in the 3 h animals, where the proflavine-treated rats show little or no labelling at the 29 and 18 S ribosomal peaks. Determination of protein synthesis showed a nonsignificant decrease in the proflavinetreated animals (Table 1). Experimental
Cell Research
50
U. Stenram
512 1. Protein
TABLE M,
the
mean;
s, the
synthesis
and R. Wi2lkn after 3H-leucine
standard deviation of the mean; explanation, see under Material
administration
n, the number and Methods.
of animals.
;I1
Radioautography, grain counts Biochemical analysis, cpm/mg dry There
are no statistical
658&
differences
271
7150*394
protein between
further
Proflavine treatment
Controls Method
For
s
n
M
46
3
7.58?
684
3
GOlO&220
29
s
n
64
5
440
4
the groups.
Fig. Il.-Sedimentation patterns of RNA from the livers of rats labelled with 8H-cytidine. Faster moving components are to the left. Abscissa: Fraction number; ordinate: cpm, OD. 1(1(1, Control 20 min; 7, control 3 h; - - - pf 5 mg 20 min; . . . . . ., pf 5 mg 3 h. I
I
I
I
I
5
10
15
20
25
DISCUSSION
Proflavine was thus found to bring about a decrease in liver nucleolar size of rats and a concomitant decrease in RNA labelling of nucleoli and, to a large extent, also of the cytoplasm. At the ultrastructural level there was a coalescence of the nucleoli. The changes are very similar to those seen in rat liver [13, 141 and other cells (see Simard and Bernhard) [9] after administration of actinomycin D. The inhibitory effect of actinomycin on ribosomal RNA Experimental
Cell
Research
50
Proflavine
and rat liver cells
513
synthesis [6 and others] was also mimicked by proflavine, though the changes seem to set in more slowly with proflavine and may not be so specific. The apparently close association observed between polyribosomes and the nuclear membrane and its pores (Fig. 10) is interesting as there is bioc,hemical evidence that newly synthesized ribosomal subunit particles first go into the polyribosomes in the cytoplasm without passing the free ribosome pool
[l, 21. There are a few reports on proflavine in literature of interest for the present observations, but there does not seem to exist any correlated study of alterations in morphology and macromolecular synthesis. Proflavine has been shown to depress RNA and, to a lesser extent, protein synthesis in Bacillus subtilis [3] and in chick fibroblast cultures [7]. In our system we found heavy decrease of RNA and a slight, nonsignificant decrease of protein labelling. This points to a stability of the protein synthesizing mechanism of the rat liver cell, although RNA synthesis is depressed, which we also encountered in treatment with actinomycin D and fluorouracil [15]. Sirlin et al. [ 1 l] found a total inhibition of the nucleolar RNA synthesis in the salivary gland of the chironimid Smittia parthenogenetica but only a partial inhibition of chromosomal RNA synthesis. Observations on cytoplasmic RNA were not reported. At the ultrastructural level nucleolar segregation, apparently similar to the type described here, has been reported in rat embryonic cells in vitro [8, 91. \Ve did not find the advanced nuclear and cytoplasmic lesions described by these authors in cells that may not always have been very viable at these late observation times.
SUMMARY
The effect of proflavine on the liver cells of rats was examined. The nucleoli decreased in size. Under the electron microscope, a coalescence of the nucleoli and a segregation of their ultrastructural components was seen. Radioautography showed decreased RNA labelling over nucleoli and cytoplasm. Biochemical analysis of RNA synthesis indicated decreased synthesis of ribosomal RNA. Protein synthesis showed no significant changes. The alterations brought about by proflavine are similar to those caused by actinomycin D. The investigation was supported by grants from the Swedish Medical Research Council (Project K67-12X-623-03), the Swedish Cancer Society (Project 67:31) and the Medical Faculty of Uppsala. Experimenfal
Cell Research
50
514
C. Stenram
and R. Wille’n
REFERENCES
1. BACH, M. and JOHNSO~L,II. G., Nature 209, 893 (1966). 2. GIRARD, M., LATHAM, H., PISNMAS, S. and DARNELL, J. E., J. Mol. Biol. 11, 187 (1965). 3. HURWITZ, J., FURTH, J. J., MALAMY, M. and ALEXANDER, M., Proc. Mall Acad. Sci. Wash. 48, 1222 (1962). 4. MAIIIN, D. and LOFBERG, R., Anal. Biochem. 16, 500 (1966). 5. MUNRO, H. N., CHISHOLM, J. and NAISMITI~, D. J., Brif. J. Nutr. 16, 245 (1962). 6. PERRY, R., Nat! Cancer Inst. Monogr. 18, 325 (1965). 7. SCHOLTISSEK, C. and ROTT, R., Nature 204, 39 (1964). 8. SIMARD, R., Cancer Res. 26, 2316 (1966). 9. SIMARD, R. and BERSHARD, W., Int. J. Cancer 1, 463 (1966). 10. SIMKIN, J. L. and WORK, T. S., Biochem. J. 65, 307 (1957). 11. SIRLIN, J. L., TASDLER, C. J. and JACOB, .J., Erpfl Cell Res. 31, 611 (1963). 12. STENRAM, U., Acta And. 26, 350 (1956). 13. -Exptl Cell Res. 36. 242 (1964). 14. -Z. ke/Iforsch. 65, ill (1965). Nat1 Cancer Inst. Monoor. 23. 379 (1966) 15. __ 16. STENRAM, U. and WILL~N, RI, Carker Rks. 28, 765 (1966). 17. -Z. Zellforsch. In press. 18. WILL~N, R. and STENRAM, U., Arch. Riochem. 119, 501 (1967).
Experimental
Cell Research
50
Press Inc.
Cell Research
505
50, SOS-51 4 (196X)
EFFECTS OF PROFLAVINE ON ULTRASTRUCTURE AND RNA AND PROTEIN SYNTHESIS IN THE LIVER OF RATS U. STENRAM and R. WILL&N Department
of Pathology,
University
Received June
of Uppsala,
Sweden
19, 1967
WHES studying
the effects of various substances on the ultrastructure of the rat liver cell, we found profound changes in the nucleoli following proflavine administration. This substance is known to interfere with the ribonucleic acid (RSA) synthesis. A combined study of the effects of proflavine on the RNA and protein synthesis and the ultrastructure of the rat liver cell was therefore considered to be of interest.
MATERIAL
AND
METHODS
Preliminary experiments were performed to determine the dose, if any, that provoked changes in the ultrastructure of rat liver cells in vivo. Five mg proflavine per rat was found to be suitable, though the animals succumbedwithin 23 h. One mg had no effect. In the main experiment 17 male white rats (Sprague-Dawley strain) were used, each weighing about 45 g. Having been fed on a protein-free diet [la] for four days and starved for one day, each rat, then weighing about 36 g, was subcutaneously given 5 mg proflavine N.F. (Nutritional Biochemicals, USA), dissolved in 1 ml distilled water, or 1 ml 0.9 per cent NaCl at B-B.30 a.m. Exactly 1 h later, the rats were intraperitoneally given 5 ,UC3H-cytidine (Schwarz BioResearch, USA) or 5 pc 3H-L-leucine per g body weight (New Engl. Nuclear Corp., USA). The rats were sacrified 20 min or 3 h later. One of the intended 3 h animals, treated with proflavine, died spontaneously and was excluded. The other animals were in fairly good condition at sacrifice. Liver slices were fixed in 1 per cent 0~0, for electron microscopy and Carnoy’s fluid Nr. II for optical microscopy and radioautography [16]. The remainder of the livers was frozen on dry ice. RNA was prepared, fractionated in ultracentrifuge and analyzed for ultraviolet absorption and radioactivity as described previously [18] with a later modification [17]. Protein was prepared essentially according to the methods of Simkin and Work [lo] and Munro ef al. [5] from the livers of the animals given leucine. While thawing, the liver tissue was pressedas rapidly as possible through a plastic sieve with holes with a diameter of 1 mm. About 0.3 g of liver was homogenized in a microhomogenizer Experimental
Cell Research
50
506
U. Stenram
and R. Will-h
(Type Nelco. Measuring and Scientific Equipment Ltd, London, England) at 0°C for 1 min in 4 ml of a 0.01 M sodium phosphate buffer, pH 7.4, with 0.01 M NaCl and 0.015 M MgCl,. An equal volume of 20 per cent (w/v) trichloroacetic acid (TCA) was added. After centrifugation for IO min at 2700 g, the precipitate was dissolved in 4 ml N NaOH at 70°C. After cooling, IO mg of unlabelled leucine was added, the mixture was stirred, and 4 ml 36.3 per cent TCA was added. The precipitate was recovered by centrifugation and washed once with IO per cent TCA and then heated at 90°C for 15 min in 10 per cent TCA to remove nucleic acids. After cooling, the precipitate was washed once with 10 per cent TCA, once with 95 per cent ale., once with absolute alcohol, once with a 3 : I alcohol-chloroform mixture, then heated to 60°C with alcohol-ether (3 : I), washed once with ether and allowed to dry. The sampleswere then treated according to Mahin and Lofberg [4] in the following way. About 20 mg dry protein, determined with one decimal, was weighed into glass vials. To each sample, 0.2 ml 70 per cent perchloric acid and 0.4 ml 30 per cent hydrogen peroxide were added, and the caps tightly screwed. The samples were heated to 80°C for 3 h. After cooling, 15.5 ml was added of a mixture of 5.5 ml cellosolve (ethylene glycol monoethylether), 10 ml toluene and 0.06 g PPO. The radioactivity (counts/min/mg dry protein) was determined in duplicate in a CM-25 liquid scintillation counter, Tracerlab. USA. For each group of rats the mean, the standard error and the standard deviation (s) of the mean were calculated from the mean of the individual animals. One specimenwas lost. Grain counts were performed in the radioautographs of the rats given 3H-leucine. 30 random square fields were counted from each animal, 3 controls and 5 given proflavine. For each group of the animals the mean, the standard error and the standard deviation (s) of the mean were calculated from the figures of the individual animals. For further details seeStenram [13].
OBSERVATION Optical microscopy suggested a decrease in nucleolar size in the rats killed 4 h after administration of proflavine. The nucleoli retained their intense basophilia. Radioautography showed heavy labelling of nuclear and, at 3 h, also of cytoplasmic RNA in the controls given 3H-cytidine. The heaviest labelling was found over nucleoli. The proflavine-treated rats showed roughly the same labelling at 20 min as the controls. At 3 h there was less labelling over nucleoli and cytoplasm compared to the controls (Figs l-4). This was seen not only in the livers but also in some other organs examined, the pancreas, kidneys, adrenals and peripheral ganglion cells. There were no discernible differences in labelling among the animals given 3H-leucine, which were all killed 3 h after administration of the radioactive substance (Figs 5, 6). The result of the grain counts is given in Table 1. Electron microscopy of the animals killed 4 h after the administration of Experimental
Cell
Research
50
Proflavine
507
and rat liver cells
Figs l-6.-Radioautographs of liver sections. Figs 1-4 of animals given SH-cytidine 20 min (Figs 1 and 2) and 3 h (Figs 3 and 4) before death, Figs 5 and 6 of animals given 8H-leucine 3 h before death. Figs 1, 3 and 5 are from control animals, Figs 2, 4 and 6 from animals given proflavine. x 1575. Figs
is the same labelling
in both
control
Figs 3 and 4.-There
1 and 2.-There
is less labelling
nucleoli
and cytoplasm
Figs 5 and 6.-There
is the same labelling
in both
control
over
and treated
animals.
in the treated
and treated
animal.
animal.
proflavine showed, compared to the controls, pronounced changes in the nucleoli. These had decreased in size, had coalesced and showed only small areas of low contrast. The filamentous and granule-rich components were well discernible and often showed a tendency to segregation (Figs 7-9). Experimental
Cell Research
50
508
l-J. Stem-am and R. WillCn
Figs
7-lO.-Electron
Fig.
7.-Control
Experimental
micrographs showing
the normal
Cell Research
50
of livers. appearance
0~0,
fixation.
Uranyl
of the liver
cell.
acetate-lead x 30,500.
citrate
staining.
Proflauine
Fig. 8.-Proflavine x 30,500.
treatment
showing
and rat liver cells
coalescence
of nucleolus
509
and segregation
Experimental
of its components.
Cell Research
SO
510
Fig.
U. Stenram
9.-Proflavine
treatment.
A nucleolus
and R. Will&
is seen in higher
magnification.
x 56,000.
The rats killed 1 h and 20 min after the administration of proflavine showed no obvious nucleolar changes. With the alterations of the 4 h rats in mind, however, a slight coalescence of the nucleoli could be discerned. There were no other definite differences between controls and treated animals. However, the nucleoplasm sometimes appeared to be less homogeneous and the endoplasmic sacs to be slightly dilated in the latter. In all Experimental
Cell Research
50
Proflavine
Fig. lO.-Tangential view cytoplasm and the nuclear
of nucleus membrane
511
and rat liver cells
showing close with its pores.
association x 46,000.
between
animals a rather close relationship was seen between cytoplasm and the nuclear membrane (Fig. 10).
polyribosomes
polyribosomes
in the
in the
Biochemistry The incorporation of 3H-cytidine into RNA is depicted in Fig. 11. There is a definite difference only in the 3 h animals, where the proflavine-treated rats show little or no labelling at the 29 and 18 S ribosomal peaks. Determination of protein synthesis showed a nonsignificant decrease in the proflavinetreated animals (Table 1). Experimental
Cell Research
50
U. Stenram
512 1. Protein
TABLE M,
the
mean;
s, the
synthesis
and R. Wi2lkn after 3H-leucine
standard deviation of the mean; explanation, see under Material
administration
n, the number and Methods.
of animals.
;I1
Radioautography, grain counts Biochemical analysis, cpm/mg dry There
are no statistical
658&
differences
271
7150*394
protein between
further
Proflavine treatment
Controls Method
For
s
n
M
46
3
7.58?
684
3
GOlO&220
29
s
n
64
5
440
4
the groups.
Fig. Il.-Sedimentation patterns of RNA from the livers of rats labelled with 8H-cytidine. Faster moving components are to the left. Abscissa: Fraction number; ordinate: cpm, OD. 1(1(1, Control 20 min; 7, control 3 h; - - - pf 5 mg 20 min; . . . . . ., pf 5 mg 3 h. I
I
I
I
I
5
10
15
20
25
DISCUSSION
Proflavine was thus found to bring about a decrease in liver nucleolar size of rats and a concomitant decrease in RNA labelling of nucleoli and, to a large extent, also of the cytoplasm. At the ultrastructural level there was a coalescence of the nucleoli. The changes are very similar to those seen in rat liver [13, 141 and other cells (see Simard and Bernhard) [9] after administration of actinomycin D. The inhibitory effect of actinomycin on ribosomal RNA Experimental
Cell
Research
50
Proflavine
and rat liver cells
513
synthesis [6 and others] was also mimicked by proflavine, though the changes seem to set in more slowly with proflavine and may not be so specific. The apparently close association observed between polyribosomes and the nuclear membrane and its pores (Fig. 10) is interesting as there is bioc,hemical evidence that newly synthesized ribosomal subunit particles first go into the polyribosomes in the cytoplasm without passing the free ribosome pool
[l, 21. There are a few reports on proflavine in literature of interest for the present observations, but there does not seem to exist any correlated study of alterations in morphology and macromolecular synthesis. Proflavine has been shown to depress RNA and, to a lesser extent, protein synthesis in Bacillus subtilis [3] and in chick fibroblast cultures [7]. In our system we found heavy decrease of RNA and a slight, nonsignificant decrease of protein labelling. This points to a stability of the protein synthesizing mechanism of the rat liver cell, although RNA synthesis is depressed, which we also encountered in treatment with actinomycin D and fluorouracil [15]. Sirlin et al. [ 1 l] found a total inhibition of the nucleolar RNA synthesis in the salivary gland of the chironimid Smittia parthenogenetica but only a partial inhibition of chromosomal RNA synthesis. Observations on cytoplasmic RNA were not reported. At the ultrastructural level nucleolar segregation, apparently similar to the type described here, has been reported in rat embryonic cells in vitro [8, 91. \Ve did not find the advanced nuclear and cytoplasmic lesions described by these authors in cells that may not always have been very viable at these late observation times.
SUMMARY
The effect of proflavine on the liver cells of rats was examined. The nucleoli decreased in size. Under the electron microscope, a coalescence of the nucleoli and a segregation of their ultrastructural components was seen. Radioautography showed decreased RNA labelling over nucleoli and cytoplasm. Biochemical analysis of RNA synthesis indicated decreased synthesis of ribosomal RNA. Protein synthesis showed no significant changes. The alterations brought about by proflavine are similar to those caused by actinomycin D. The investigation was supported by grants from the Swedish Medical Research Council (Project K67-12X-623-03), the Swedish Cancer Society (Project 67:31) and the Medical Faculty of Uppsala. Experimenfal
Cell Research
50
514
C. Stenram
and R. Wille’n
REFERENCES
1. BACH, M. and JOHNSO~L,II. G., Nature 209, 893 (1966). 2. GIRARD, M., LATHAM, H., PISNMAS, S. and DARNELL, J. E., J. Mol. Biol. 11, 187 (1965). 3. HURWITZ, J., FURTH, J. J., MALAMY, M. and ALEXANDER, M., Proc. Mall Acad. Sci. Wash. 48, 1222 (1962). 4. MAIIIN, D. and LOFBERG, R., Anal. Biochem. 16, 500 (1966). 5. MUNRO, H. N., CHISHOLM, J. and NAISMITI~, D. J., Brif. J. Nutr. 16, 245 (1962). 6. PERRY, R., Nat! Cancer Inst. Monogr. 18, 325 (1965). 7. SCHOLTISSEK, C. and ROTT, R., Nature 204, 39 (1964). 8. SIMARD, R., Cancer Res. 26, 2316 (1966). 9. SIMARD, R. and BERSHARD, W., Int. J. Cancer 1, 463 (1966). 10. SIMKIN, J. L. and WORK, T. S., Biochem. J. 65, 307 (1957). 11. SIRLIN, J. L., TASDLER, C. J. and JACOB, .J., Erpfl Cell Res. 31, 611 (1963). 12. STENRAM, U., Acta And. 26, 350 (1956). 13. -Exptl Cell Res. 36. 242 (1964). 14. -Z. ke/Iforsch. 65, ill (1965). Nat1 Cancer Inst. Monoor. 23. 379 (1966) 15. __ 16. STENRAM, U. and WILL~N, RI, Carker Rks. 28, 765 (1966). 17. -Z. Zellforsch. In press. 18. WILL~N, R. and STENRAM, U., Arch. Riochem. 119, 501 (1967).
Experimental
Cell Research
50