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CIrcadian rhythmicity in cultured liver cells II. Re-induction of rhythmicity in tyrosine aminotransferase activity
ht. J. Biochem.,
[Scientechnica
1973, 4 59x-595.
CIRCADIAN
RHYTHMICITY
II. RE-INDUCTION
(Publishers) Ltd.]
IN CULTURED
OF RHYTHMICITY
AMINOTRANSFERASE RUDIGER I. Zoologischa
591
LIVER
CELLS
IN TYROSINE
ACTIVITY
HARDELAND
Inatitut, univcrsitiit
cm&al,
Germany
(Racciwd 7 March, 1973) ABsTRAcr In aspension cultures of rat liver cells, the circadian rhythm of tyroaine aminotransferase activity disappears in the course of 2 weeks in constant light. 2. Even after 3 weeks under these conditions, the rhythm can be m-induced by a single I.
~-hour paiod of darkness. 3. The increase in enzyme activity after the dark puke can bc completely blocked by cyclohcximide or pactamycin and partially inhibited by a-amanitine. 4. The possible role of post-transcriptional regulation in the elevation of enzyme activity isdiscus&.
SUSPENSIONcultures of hepatocytes from young rats can be used as a system for studying circadian rhythms in u&o. Until now, the following functions tested revealed a rhythmicity : tyrosine aminotransferase activity, protein synthesis (Hardeland, ( IgTS), 0, consumption (Langner and Rensing, evidence, x972), and, with preliminary nuclear size (Wassmann, 1975). Among these, only the rhythm of tyrosine aminotransferase has been shown to persist under constant lighting conditions. From the freerunning behaviour of this oscillation, it must be concluded that the rhythm is self-sustained and not induced by the light. In this paper further evidence will be presented for the circadian nature of the rhythm. In particular it will be shown that a rhythm damped-out under unfavourablc conditions can be reinduced. Moreover, the requirements of RNA and protein synthesis for the reinduction of the rhythm will be analysed. MATERIALS
AND
METHODS
Liva cell mspensions were prepared and cultured as described in the preceding paper (Hat&land, 1973). Bcforc subjecting the cells to constant light, the cultures had been kept for at lcast 6 we& under a light-dark cycle of I 2 : 12 . . . .l.yrosme ammotranstcrase activlty was hours.
measured according to Diamondstone (1966) and incorporation of L-[4,5-*H]leucine as in the preccdin~ paper. Pactamycin and a-amanitine kre gc&&s gifts to o& laboratory by Dr.
For&en, The Upjohn Company, m, Dr. Th. Wicland, Heidelberg.
and
RESULTS
from a light-dark regimen to constant light, the rhythm of tyrosine aminotransferase activity persists for several days (cf. Hardeland, 1973). In the course of about 2 weeks, however, the amplitude of the oscillation declines and the culture finally becomes completely ax-rhythmic. Such a loss of rhythmicity after a longer exposure to constant light is a well-known phenomenon and means no objection against the selfsustained nature of the oscillation. In principle, the disappearance of a rhythm can have two different reasons. Either the circadian oscillators within the cells are damped out, or the oscillations persist, but the different cells desynchronize with respect to each other. For the hepatocyte suspensions the first possibility seems to be right, since enzyme activity does not stabilize at the mean value of the oscillation, as should be expected in a case of desynchronization, but rather declines concomitantlv I with the decrease in After
a transition
Id. J. Biochm.
HARDELAND
592
amplitude to a lower level, which is in the range of the minima of the oscillatory state. This low level of enzyme activity in arrhythmic cultures is shown in the left part of Fig. I. The loss of rhythxnicity is, however, not irreversible. Even after 3 weeks in constant
values measured are even somewhat above those found under normal light-dark conditions. Until now, changes in light intensity have been the only means by which oscillations could be re-induced. Tryptophan, which is
90 80 70
40 30 20 10 0
6
12
18 lima
0
6
12
18
0
6
of dry (hours)
FIG. 1 .-Re-induction of rhythmicity in tyrosine aminotraosferasc activity by a dark pulse. After 3 weeks in constant light, one culture (0) was exposed to a I -hour period of darkness, and another culture (b) was first incubated with tryptophan in a final concentration of 2 x IO-~ M and then exposed to the dark pulse. The vertical lines represent the thmfold standard error. The shaded areas indicate the dark pulses. light, a ~-hour period of darkness is sufficient for a re-induction of the oscillation (Fig. I a). Together with the’ reappearance of the rhythm, a marked increase in average enzyme activity can be observed. The mean
required for the rhythmicity in vivo (Wurtman, Shoemaker, and Larin, 1g68), proved to be completely ineffective in the cell suspension (Fig. rb). Also in this culture, however, a dark pulse of I hour resulted in an
RE-INDUCTION
1973:4
OF CIRCADIAN
immediate
increase in enzyme activity. Likewise, additions of hyclrocortisone or insulin did not effect the reappearance of the oscillation, as far as this has been pursued. Nonetheiess, these hormones proved to be good inducers of enzyme activity also in this system (Fig. 2). This result demonstrates that the re-initiation of rhythmicity CaMOt be 120
g
110
;
100
; : $
90
r
R?lYTHMS
593
de rzovosynthesis of enzyme protein. The inhibition of protein synthesis in the cell culture by cycloheximide is shown in Fig. 4. It should be noted that in the untreated culture protein synthesis increases after the dark pulse ; as under normal light-dark conditions (cf. Hardeland, I 973), the increase is limited, but statistically significant. In principle, the increase of a+ novo synthais of tyrosine aminotransfemse could be caused by stimulation of either transcripor a post-transcriptional step. As a
80
: $
70
0
3
8
9 12 time(hours)
15
18
21
24
0
i s.
i2
Time (hourI)
FIG. a.-Induction of tyroslnc aminotransferase in arrhythmic cell cultures by hydrocortisone and by insulin. -, Controls; -- -, incubation with 5 mu. insulin per ml. ; . . ,, incubation with 104 M hydrocortisone hydrogensuccinate. At the time of addition of the hormones, the culture had been kept for 3 weeks in constant light. Statistics as in Fig. 1.
FIG, 3.-The effects of cyclohexlmide and pactamycin on the re-induction of the rhythm of tyrosine amiuotransferaae after a ~-hour period of darkness. -, Controls (oscillation initiated as described under Fig. I);- - -,addition of 10 pg. cycloheximide per ml. at the end of the dark pI&e..., addition of I 5 erg. pactamyein per ml. at the end of the dark pulse.
brought about simply by an increase in enzyme activity. On the other hand, the elevation of enzyme
distinction between these possibilities could perhaps be made by inhibition of mFWA synthesis, hepatocytes were incubated with a-amanitine. This antibiotic is a strong in-
activity after a dark pulse can be completely suppressed by inhibition of protein synthesis. Roth cycloheximide and pactamycin are effective to the same extent (Fig. 3). Therefore, it must be concluded that the increase in enzyme activity, which is accompanying the reappearance of the rhythm, is due to
hibitor of RNA polymerase II, but has no direct effect on RNA polymemse I (review: Wieland, 1972). As can be seen from Fig.5, the increase in enzyme activity after a dark pulse can be blocked by prolonged treatment with a-amanitine. However, an incubation
Int. J. Biochm.
HARDELAND
594
with the antibiotic for only 3 hours results at most in a gradual inhibition. After this short period of incubation, the values obtained with a-amanitine are not statistically different from the controls, but they are significantly higher than the values at the end of the dark pulse. This finding could be interpreted in different ways: on the one hand, there could
0
3
6
9
12
Ttme (hourrj
DISCUSSION The re-inducibility of the rhythm by a short alteration in the lighting conditions can be regarded as a further confirmation of the conclusion that the rhythm is self-sustained. However, its persistence over many periods requires a repeated stimulation by light-dark changes, or a transition to the non-oscillatory
0
s
Tmw(how)
FIG. 4.-Effects of a dark pulse and of cycloheximide on protein synthesis. The incorporation of [*Hjleucine was measured simultaneously in the same culture that had been used for the experimcnts shown in Fig. 3. -, Without cycloheximide; - - -, with cycloheximide.
FIG. 5.-The effect of a-am&tine on the reinduction of the rhythm of tyrosine aminotransferase after a ~-hour period of darkness. -, Controls (cf. Figs. I, 3) ; - - -, addition of I pg. a-amanitinc per ml. at the end of the dark pulse.
be a lag in the inhibition of RNA polymerase by a-amanitine; from all what we know about this agent, however, such an explanation does not seem to be very likely. On the other hand, enzyme activity could have been stimulated in the absence of mRNA production. This would imply the existence of a post-transcriptional control mechanism. In the latter case an elevation of enzyme activity would be possible, as long as the specific mRNA is available. A few hours later, however, when the sources of mRNA are exhausted, translation would cease and enzyme activity would decline owing to the rapid degradation of this enzyme.
state will occur. It has been shown in these experiments that the system can alternately exist in an oscillatory or in a non-oscillatory state and that transitions in both directions are possible. This resembles very much the occurrence of rhythmicity and arrhythmicity in the circadian oscillator of Drosophila (cf. Zimmerman, I#$; Winfii, x97X). In the cell suspension, the oscillator not only controls the temporal pattern, but also the average value of enzyme activity. From the experiments with the antibiotics, it may be supposed that the oscillator influences the level of tyrosine aminotransferase by the regulation of post-transcriptional steps. The
RE-INDUCTION OF CIFtCADL4NRHYTHMS
‘97324
involvement of transcriptional control still remains hypothetical, since the decrease in enzyme activity after prolonged treatment with sr-amanitine does not necessarily imply that an enhanced ‘production of specific mRNA is required. If the enzyme could be stimulated at translational level-which would be in line with the results on treatment with insulin (Hardeland, rg73)-enzyme synthesis would cease after some hours of regardless of incubation with a -amanitine, whether transcription was normally stimulated by the circadian oscillator or not. This interpretation presumes, of course, that the specific mRNA is produced to some extent at any time of day. SUMMARY I. Hepatocytes were isolated from livers of young rats by treatment with tetraphenylboron. After at least 6 weeks under a lightdark cycle of 12 : 12 hours, during which a rhythm in tyrosine aminotransferase activity could be observed, the cultures were transferred to constant light. Although enzyme activity continued to oscillate for several days, the rhythmicity disappeared in the course of about 2 weeks, Concomitantly a decline in average enzyme activity could be observed. 2. Even after 3 weeks in constant light, the rhythmicity could be re-induced by a x-hour period of darkness. The reappearance of the rhythm was accompanied by an increase in enzyme activity. 3. Tryptophan, hydrocortisone, and insulin were ineffective in re-inducing the rhythmicity, although the hormones caused a marked elevation of tyrosine aminotransferase activity. 4. The increase in enzyme activity after the dark pulse could be completely blocked
595
by cycioheximide or pactamycin. a-Amanitine, however, proved to be inhibitory only after a treatment for longer than 3 hours. This result could indicate the involvement of post-transcriptional regulation. ACKNOWLEDGEMENT This work was supported
Forachungsgcmeinschaft.
by the Deutsche
REFERENCES DXAMO~ONE, T. I. ( Ig66), ‘ Aasay of tyrosine transaminase activity by convemion of
phydroxyphenylpyruvate to phydroxybenzaldehyde ‘, Anolyt. Biochm.? 16, ?g+pr . HARDELAND, R. (x973), ‘ &cadum rhythmicity in cultured liver cells. I. Rhythms in tyrosine aminotransferase activity and inducibility and in SH-leucinc incorporation ‘, Znt. J. Biochm.,
6 581-590. Ii., and FCENSING,L. (Ig72), ‘ Circadian rhythm of oxygen consumption in rat liver suspension culture: changes of pattern ‘, <. Natwfwsch., zq, I 117-1 I 18. WKBMNN, G. (x973), personal communication. WIELAND, TH. (tg72), ‘ Struktur und Wirkung da- Amatoxine ‘, &tzuwkm&fh, sg, 22523x. WINFREE, A. T. (x97x), ‘Corkscrews and singula&es in fruit&s: l-esming behaviour of the circadian e&&on rhythm ‘, in Biochmmm Washington (ed. Menaker), pp. 81-9. National Academy of Sciences. WURTMAN,R. J., SHOEMAKER,W. J., and LARPI, F. (Ig68), ‘ Mechanisms of the daily rhythm in
LANGNER,
hepatic tyrosinc transam inase activity: roie of dietary tryptophan ‘, Pm. natn. Acad. Sci.,
w,
U.S.A., 5g,%o-8o7. W. F. ( Ig6g), ‘ On the absence of circadian rhythmicity in Drosophila pseudoobscwa pupae ‘, Bioi. Bull. mar. biol. Lab., Wooa Ho&, 136,494-500.
Key Word Z&x: eirea+ar+ enzyme z;z:_ti
.
a-Amanitine,
cell
culture,
induction, hydrocartiso?e, MNbgic((s, rhythm, me
[Scientechnica
1973, 4 59x-595.
CIRCADIAN
RHYTHMICITY
II. RE-INDUCTION
(Publishers) Ltd.]
IN CULTURED
OF RHYTHMICITY
AMINOTRANSFERASE RUDIGER I. Zoologischa
591
LIVER
CELLS
IN TYROSINE
ACTIVITY
HARDELAND
Inatitut, univcrsitiit
cm&al,
Germany
(Racciwd 7 March, 1973) ABsTRAcr In aspension cultures of rat liver cells, the circadian rhythm of tyroaine aminotransferase activity disappears in the course of 2 weeks in constant light. 2. Even after 3 weeks under these conditions, the rhythm can be m-induced by a single I.
~-hour paiod of darkness. 3. The increase in enzyme activity after the dark puke can bc completely blocked by cyclohcximide or pactamycin and partially inhibited by a-amanitine. 4. The possible role of post-transcriptional regulation in the elevation of enzyme activity isdiscus&.
SUSPENSIONcultures of hepatocytes from young rats can be used as a system for studying circadian rhythms in u&o. Until now, the following functions tested revealed a rhythmicity : tyrosine aminotransferase activity, protein synthesis (Hardeland, ( IgTS), 0, consumption (Langner and Rensing, evidence, x972), and, with preliminary nuclear size (Wassmann, 1975). Among these, only the rhythm of tyrosine aminotransferase has been shown to persist under constant lighting conditions. From the freerunning behaviour of this oscillation, it must be concluded that the rhythm is self-sustained and not induced by the light. In this paper further evidence will be presented for the circadian nature of the rhythm. In particular it will be shown that a rhythm damped-out under unfavourablc conditions can be reinduced. Moreover, the requirements of RNA and protein synthesis for the reinduction of the rhythm will be analysed. MATERIALS
AND
METHODS
Liva cell mspensions were prepared and cultured as described in the preceding paper (Hat&land, 1973). Bcforc subjecting the cells to constant light, the cultures had been kept for at lcast 6 we& under a light-dark cycle of I 2 : 12 . . . .l.yrosme ammotranstcrase activlty was hours.
measured according to Diamondstone (1966) and incorporation of L-[4,5-*H]leucine as in the preccdin~ paper. Pactamycin and a-amanitine kre gc&&s gifts to o& laboratory by Dr.
For&en, The Upjohn Company, m, Dr. Th. Wicland, Heidelberg.
and
RESULTS
from a light-dark regimen to constant light, the rhythm of tyrosine aminotransferase activity persists for several days (cf. Hardeland, 1973). In the course of about 2 weeks, however, the amplitude of the oscillation declines and the culture finally becomes completely ax-rhythmic. Such a loss of rhythmicity after a longer exposure to constant light is a well-known phenomenon and means no objection against the selfsustained nature of the oscillation. In principle, the disappearance of a rhythm can have two different reasons. Either the circadian oscillators within the cells are damped out, or the oscillations persist, but the different cells desynchronize with respect to each other. For the hepatocyte suspensions the first possibility seems to be right, since enzyme activity does not stabilize at the mean value of the oscillation, as should be expected in a case of desynchronization, but rather declines concomitantlv I with the decrease in After
a transition
Id. J. Biochm.
HARDELAND
592
amplitude to a lower level, which is in the range of the minima of the oscillatory state. This low level of enzyme activity in arrhythmic cultures is shown in the left part of Fig. I. The loss of rhythxnicity is, however, not irreversible. Even after 3 weeks in constant
values measured are even somewhat above those found under normal light-dark conditions. Until now, changes in light intensity have been the only means by which oscillations could be re-induced. Tryptophan, which is
90 80 70
40 30 20 10 0
6
12
18 lima
0
6
12
18
0
6
of dry (hours)
FIG. 1 .-Re-induction of rhythmicity in tyrosine aminotraosferasc activity by a dark pulse. After 3 weeks in constant light, one culture (0) was exposed to a I -hour period of darkness, and another culture (b) was first incubated with tryptophan in a final concentration of 2 x IO-~ M and then exposed to the dark pulse. The vertical lines represent the thmfold standard error. The shaded areas indicate the dark pulses. light, a ~-hour period of darkness is sufficient for a re-induction of the oscillation (Fig. I a). Together with the’ reappearance of the rhythm, a marked increase in average enzyme activity can be observed. The mean
required for the rhythmicity in vivo (Wurtman, Shoemaker, and Larin, 1g68), proved to be completely ineffective in the cell suspension (Fig. rb). Also in this culture, however, a dark pulse of I hour resulted in an
RE-INDUCTION
1973:4
OF CIRCADIAN
immediate
increase in enzyme activity. Likewise, additions of hyclrocortisone or insulin did not effect the reappearance of the oscillation, as far as this has been pursued. Nonetheiess, these hormones proved to be good inducers of enzyme activity also in this system (Fig. 2). This result demonstrates that the re-initiation of rhythmicity CaMOt be 120
g
110
;
100
; : $
90
r
R?lYTHMS
593
de rzovosynthesis of enzyme protein. The inhibition of protein synthesis in the cell culture by cycloheximide is shown in Fig. 4. It should be noted that in the untreated culture protein synthesis increases after the dark pulse ; as under normal light-dark conditions (cf. Hardeland, I 973), the increase is limited, but statistically significant. In principle, the increase of a+ novo synthais of tyrosine aminotransfemse could be caused by stimulation of either transcripor a post-transcriptional step. As a
80
: $
70
0
3
8
9 12 time(hours)
15
18
21
24
0
i s.
i2
Time (hourI)
FIG. a.-Induction of tyroslnc aminotransferase in arrhythmic cell cultures by hydrocortisone and by insulin. -, Controls; -- -, incubation with 5 mu. insulin per ml. ; . . ,, incubation with 104 M hydrocortisone hydrogensuccinate. At the time of addition of the hormones, the culture had been kept for 3 weeks in constant light. Statistics as in Fig. 1.
FIG, 3.-The effects of cyclohexlmide and pactamycin on the re-induction of the rhythm of tyrosine amiuotransferaae after a ~-hour period of darkness. -, Controls (oscillation initiated as described under Fig. I);- - -,addition of 10 pg. cycloheximide per ml. at the end of the dark pI&e..., addition of I 5 erg. pactamyein per ml. at the end of the dark pulse.
brought about simply by an increase in enzyme activity. On the other hand, the elevation of enzyme
distinction between these possibilities could perhaps be made by inhibition of mFWA synthesis, hepatocytes were incubated with a-amanitine. This antibiotic is a strong in-
activity after a dark pulse can be completely suppressed by inhibition of protein synthesis. Roth cycloheximide and pactamycin are effective to the same extent (Fig. 3). Therefore, it must be concluded that the increase in enzyme activity, which is accompanying the reappearance of the rhythm, is due to
hibitor of RNA polymerase II, but has no direct effect on RNA polymemse I (review: Wieland, 1972). As can be seen from Fig.5, the increase in enzyme activity after a dark pulse can be blocked by prolonged treatment with a-amanitine. However, an incubation
Int. J. Biochm.
HARDELAND
594
with the antibiotic for only 3 hours results at most in a gradual inhibition. After this short period of incubation, the values obtained with a-amanitine are not statistically different from the controls, but they are significantly higher than the values at the end of the dark pulse. This finding could be interpreted in different ways: on the one hand, there could
0
3
6
9
12
Ttme (hourrj
DISCUSSION The re-inducibility of the rhythm by a short alteration in the lighting conditions can be regarded as a further confirmation of the conclusion that the rhythm is self-sustained. However, its persistence over many periods requires a repeated stimulation by light-dark changes, or a transition to the non-oscillatory
0
s
Tmw(how)
FIG. 4.-Effects of a dark pulse and of cycloheximide on protein synthesis. The incorporation of [*Hjleucine was measured simultaneously in the same culture that had been used for the experimcnts shown in Fig. 3. -, Without cycloheximide; - - -, with cycloheximide.
FIG. 5.-The effect of a-am&tine on the reinduction of the rhythm of tyrosine aminotransferase after a ~-hour period of darkness. -, Controls (cf. Figs. I, 3) ; - - -, addition of I pg. a-amanitinc per ml. at the end of the dark pulse.
be a lag in the inhibition of RNA polymerase by a-amanitine; from all what we know about this agent, however, such an explanation does not seem to be very likely. On the other hand, enzyme activity could have been stimulated in the absence of mRNA production. This would imply the existence of a post-transcriptional control mechanism. In the latter case an elevation of enzyme activity would be possible, as long as the specific mRNA is available. A few hours later, however, when the sources of mRNA are exhausted, translation would cease and enzyme activity would decline owing to the rapid degradation of this enzyme.
state will occur. It has been shown in these experiments that the system can alternately exist in an oscillatory or in a non-oscillatory state and that transitions in both directions are possible. This resembles very much the occurrence of rhythmicity and arrhythmicity in the circadian oscillator of Drosophila (cf. Zimmerman, I#$; Winfii, x97X). In the cell suspension, the oscillator not only controls the temporal pattern, but also the average value of enzyme activity. From the experiments with the antibiotics, it may be supposed that the oscillator influences the level of tyrosine aminotransferase by the regulation of post-transcriptional steps. The
RE-INDUCTION OF CIFtCADL4NRHYTHMS
‘97324
involvement of transcriptional control still remains hypothetical, since the decrease in enzyme activity after prolonged treatment with sr-amanitine does not necessarily imply that an enhanced ‘production of specific mRNA is required. If the enzyme could be stimulated at translational level-which would be in line with the results on treatment with insulin (Hardeland, rg73)-enzyme synthesis would cease after some hours of regardless of incubation with a -amanitine, whether transcription was normally stimulated by the circadian oscillator or not. This interpretation presumes, of course, that the specific mRNA is produced to some extent at any time of day. SUMMARY I. Hepatocytes were isolated from livers of young rats by treatment with tetraphenylboron. After at least 6 weeks under a lightdark cycle of 12 : 12 hours, during which a rhythm in tyrosine aminotransferase activity could be observed, the cultures were transferred to constant light. Although enzyme activity continued to oscillate for several days, the rhythmicity disappeared in the course of about 2 weeks, Concomitantly a decline in average enzyme activity could be observed. 2. Even after 3 weeks in constant light, the rhythmicity could be re-induced by a x-hour period of darkness. The reappearance of the rhythm was accompanied by an increase in enzyme activity. 3. Tryptophan, hydrocortisone, and insulin were ineffective in re-inducing the rhythmicity, although the hormones caused a marked elevation of tyrosine aminotransferase activity. 4. The increase in enzyme activity after the dark pulse could be completely blocked
595
by cycioheximide or pactamycin. a-Amanitine, however, proved to be inhibitory only after a treatment for longer than 3 hours. This result could indicate the involvement of post-transcriptional regulation. ACKNOWLEDGEMENT This work was supported
Forachungsgcmeinschaft.
by the Deutsche
REFERENCES DXAMO~ONE, T. I. ( Ig66), ‘ Aasay of tyrosine transaminase activity by convemion of
phydroxyphenylpyruvate to phydroxybenzaldehyde ‘, Anolyt. Biochm.? 16, ?g+pr . HARDELAND, R. (x973), ‘ &cadum rhythmicity in cultured liver cells. I. Rhythms in tyrosine aminotransferase activity and inducibility and in SH-leucinc incorporation ‘, Znt. J. Biochm.,
6 581-590. Ii., and FCENSING,L. (Ig72), ‘ Circadian rhythm of oxygen consumption in rat liver suspension culture: changes of pattern ‘, <. Natwfwsch., zq, I 117-1 I 18. WKBMNN, G. (x973), personal communication. WIELAND, TH. (tg72), ‘ Struktur und Wirkung da- Amatoxine ‘, &tzuwkm&fh, sg, 22523x. WINFREE, A. T. (x97x), ‘Corkscrews and singula&es in fruit&s: l-esming behaviour of the circadian e&&on rhythm ‘, in Biochmmm Washington (ed. Menaker), pp. 81-9. National Academy of Sciences. WURTMAN,R. J., SHOEMAKER,W. J., and LARPI, F. (Ig68), ‘ Mechanisms of the daily rhythm in
LANGNER,
hepatic tyrosinc transam inase activity: roie of dietary tryptophan ‘, Pm. natn. Acad. Sci.,
w,
U.S.A., 5g,%o-8o7. W. F. ( Ig6g), ‘ On the absence of circadian rhythmicity in Drosophila pseudoobscwa pupae ‘, Bioi. Bull. mar. biol. Lab., Wooa Ho&, 136,494-500.
Key Word Z&x: eirea+ar+ enzyme z;z:_ti
.
a-Amanitine,
cell
culture,
induction, hydrocartiso?e, MNbgic((s, rhythm, me