This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution4.0 International License.

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschungin Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung derWissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht:Creative Commons Namensnennung 4.0 Lizenz.

PTERIDINE AND RIBOFLAVIN PATTERNS DURING REGENERATION IN TR1TURUS 2 8 5

Pteridine and Riboflavin Patterns During Tail Regeneration in Triturus Species and the Effects of Chloramphenicol, Isoxanthopterin

and Reserpine NIKOS K O K O L I S , NIKOS M Y L O N A S , a n d IRMGARD ZIEGLER

Department of General Biology, University of Athens, Botanisches Institut der Technischen Universität, München, and Forschungsgruppe für Biochemie der Gesellschaft für Strahlen-

und Umweltforschung m.b.H.

(Z. Naturforsch. 27 b , 285—291 [1972]; received May 21, 1971, revised December 18, 1971)

In Triturus cristatus amputation causes the reappearance of larval tetrahydrobiopterin, thus raising the ratios tetrahydrobiopterin/isoxanthopterin and tetrahydrobiopterin/riboflavin from zero to values between 3 — 5. This increase first occurs in the remaining skin and in the eyes. The increase of both ratios in the regeneration bud, beginning with the 20 t h day after amputation, coincides with their drop in both other tissues. Chloramphenicol and isoxanthopterin both strongly inhibit the formation of a regeneration bud. They also block the increase of both ratios in the remaining skin and in the rudimental regenerate as well. Reserpine induces regenerative ability in Triturus vulgaris, which normally lacks this. It has a strong melanizing effect and, moreover, it causes an increase of both ratios in the regeneration bud and in the remaining skin.

I. Introduction

Prior investigations have shown that in Triturus alpestris and T. cristatus larval tetrahydrobiopterin disappears with metamorphosis but reappears in the regeneration blastema of the adult newt, whereas isoxanthopterin and riboflavin stay at about the same level1. Thus the ratios tetrahydrobiopterin/ isoxanthopterin (TH/IX) and tetrahydrobiopterin/ riboflavin (TH/RB), starting from zero in the adult animal's skin, rise to a level of 2,5 — 3 in the re-generation bud; both ratios drop off again with decrease in mitotic activity and proceeding ^dif -ferentiation of the regeneration blastema 1 .

To investigate the apparent close relationship be-tween the onset of the regeneration and the high TH/IX and TH/RB ratios further, the time course of the pteridine and riboflavin patterns in the re-generation blastema and in the remaining pteridine containing tissues (skin and eye) were studied in more detail.

Moreover, the action of chloramphenicol, iso-xanthopterin and reserpine, which we know to in-fluence protein synthesis, DNA activation or mito-tic activity, were studied with respect to bud for-mation and to the pteridine pattern.

Chloramphenicol is a well known inhibitor of protein synthesis. In bacterial cell free systems it affects the peptide bond synthesis

Requests for reprints should be sent to Frau Dr. I. ZIEGLER, T.U. München, Institut f. Botanik, D-8000 München 2, Arcisstr. 21.

stage (see 1. c . 2 ) ; it has, however, no effect on most animal cell free systems synthesizing proteins3, but it blocks processes involving cell differentiation, such as antibody synthesis and reticulocyte formation (see 1. c. 2 ) . At the moment inhibition of the interaction of ribosomes with tem-plate RNA 4 seems to be the most probable point of attack (see 1. c. 2' 5 ) . Wherever its point of action in protein synthesis sequence is, this drug is reported to block regeneration, for instance in partially hepatectomized animals 6, in adult newTt6 or in other systems such as in worms7. Only BIEBER and HITCHINGS 8 fail to find an inhibitory action when bathing amputated tadpoles of Rana pipiens in chloramphenicol.

Isoxanthopterin, wdiich is a typical end-product of pteridine metabolism (see 1. c . 9 ) was found to inhibit DNA and ribosomal RNA syn-thesis in developing eggs of the milkweed bug1 0 . The mode of the inhibitory action has not as yet been explained; it may be at least partially due to the formation of a complex between isoxanthopterin and DNA1 1 . As seen in the following paper 12, high mitotic activity in general seems to be connected with high TH/IX and TH/RB ratios. The absolute amount of tetrahydrobiopterin, indeed, is also high in protein synthesizing, non-proliferating tissues. But in these tissues, elevated levels of isoxanthopte-rin and/or riboflavin keep the ratios low. Thus iso-xanthopterin seems to be of special interest with respect to its action on bud formation and to the onset of tetrahydrobiopterin accumulation.

2 8 6 N. KOKOLIS, N. MYLONAS, AND I. ZIEGLER

Reserpine is known to interfere with binding of catecholamines in the sympathetic nervous system, thus exposing them to enzymatic degradation. This in turn results in the release of a feed-back inhibi-tion of the tyrosine-hydroxylase (see 1. c . 1 3 ) . The coenzyme of this first and rate limiting step of norepinephrine biosynthesis is known to be tetra-hydrobiopterin (se 1. c . 1 4 ) . Besides the release of endproduct inhibition, the regulation of catechol-amine synthesis is also effected by enzyme indu-tion 15. This mechanism is initiated by the applica-tion of large doses (5 mg/kg) reserpine16. Reser-pine, moreover, has been found to act in one more, very different way; it initiates DNA activation in T. cristatus and a concomitant increase in the ratio T H / I X 1 ' . In contrast to T. cristatus and T. alpe-stris,, T. vulgaris does not form a regeneration bud, nor does it increase its TH/IX and TH/RB ratios Moreover, the skin of T. vulgaris is characterized by scarce, contracted melanophores in the dermal layer and the lack of melanophores in the epidermis at all. The promoting effect of reserpine on DNA activation and tetrahydrobiopterin formation on one hand and the close relationship between tetra-hydrobiopterin and melanin synthesis 18 (see 1. c . 1 4 ) on the other prompted us to examine its effect on formation of a regeneration bud, the tetrahydro-biopterin level and on the status of the melanopho-res in this species.

II. Methods

The raising and amputation of T. cristatus and T. vulgaris were done as described in 1. c.1 . The injections were made intraperitoneally. Dosage and timing are given in "Results". Reserpine (serpasil) was from Ciba, the chloramphenicol used was Kemicetine succi-nate, Carlo Erba.

We thank Dr. PFLEIDERER (Konstanz) and Dr. REMBOLD (München) for the generous gift of iso-xanthopterin.

For extraction, the samples were ground in the Potter-Elvehjem with cold methanol, to which mer-captoethanol (0,25% final conc.) and NH4OH (up to pH 10) were added. The homogenate was centrifuged at 8000 g for 15 min and the supernatant was dried in the presence of P205 . All samples were adjusted to 0.5 ml with a 0.25% solution of mercaptoethanol. Ali-quots (between 50 — 200 /A, varying with the material) were chromatographed on Whatman I with butanol/ acetic acid/water ( 4 : 1 : 5), to which 0.25% mercapto-ethanol was added.

All operations were done in red safe light; the chro-matograms were run in the dark.

The identification of the different pteridines as well as of tetrahydrobiopterin is described in 1. c. 1. The quantitative determination of riboflavin was done by the growth test with Lactobacillus casei 19.

III. Results

The compounds identified in the skin of T. crista-tus and T. vulgaris were isoxanthopterin, pterin-carbonic acid(6), 2-amino-4-hydroxypteridine, bio-pterin, and riboflavin. In addition to these tetrahy-drobiopterin appeared under given experimental con-ditions. As pterincarbonic acid and 2-amino-4-hv-droxypteridine were present at a very minor, but constant level, only isoxanthopterin, tetrahydro-biopterin and riboflavin are to be consiered here.

Even though the absolute amounts of pteridines vary in the normal adult skin of the different ani-mals, the ratios TH/IX and TH/RB are constant and denote the stable ratios of the adult, non pro-liferating tissue, used as control in the following experiments.

A. The ratios TH/IX and TH/RB during tail regeneration in Triturus cristatus

Figs. 1 a and 1 b showT that the increase in the ratios TH/IX and TH/RB not only takes place in the regeneration bud, but in all tissues with un-conjugated pteridines. Furthermore it is clearly in-dicated that it first occurs in the remaining skin and that it then follows in the eyes. In the regenera-tion bud it does not start before the 20th day after amputation. The increase here immediately is fol-lowed by a marked drop of both ratios in the re-maining skin and in the eyes.

B. The action of drugs on regeneration and on the ratios TH/IX and TH/RB

a) C h l o r a m p h e n i c o l

The injections were made at a dose of 0,2 mg/g body weight at intervals of 6 hours. For the experi-ments the animals were divided into 3 groups, a? follows: In group I the animals were amputated, chloramphenicol was simultaneously administered, and the treatment was maintained until the end of the experimental period (20 days after amputa-tion). The other 2 groups (II and III) of the ampu-tated newts also received chloramphenicol, however

PTERIDINE AND RIBOFLAVIN PATTERNS DURING REGENERATION IN TR1TURUS 2 8 7

longitudinal growth of the regenerates (regenera-tion time 20 days) . Apart f rom the numerical data about the length of the regenerates, the general ap-pearance of the regeneration tails clearly indicated (without any statistical evaluation) the consider-able difference between the tails under normal con-ditions and those of animals treated with chlor-amphenicol at various times after amputation. There was no gross or microscopic evidence of blastema formation in the drug-treated animals, when chlor-amphenicol was administered immediately after amputation (time 0, group I ) , while the regenerates obtained when the application of chloramphenicol was started 3 days (group II) or 10 days (group III) after amputation were definitely smaller than those of the control.

ß ) The effect of chloramphenicol on pteridine and riboflavin pattern of tail regenerates and skin

After a regeneration time of 20 days, quantitative examinations showed significant variations in the ratios TH/ IX and TH/RB as chloramphenicol was applied at different times after amputation. These variations wrere found not only in the regeneration bud itself (Table 1) but also in the remaining skin of the animals (Table 2 ) .

The most striking effect occurred in group I. No blastema formation had taken place, and con-sequently no tetrahydrobiopterin wras to be detected. Also no tetrahydrobiopterin was found in the re-maining skin of the animals. The amount of tetra-hydrobiopterin in the regeneration bud and in the skin increases as the application of chlorampheni-col after amputation is deferred. The opposite ef-

Group Starting t ime o f chlor-amphenicol appl icat ion 0

Mean + S.E. o f regenerate [ m m ] e

T H d I X d R B d T H / I X T H / R B

I 0 0.00 a a a _ I I 3 1.15 ± .06 402 ± 18 382 ± 17 806 ± 31 1.31 ± .07 0.49 ± .03

I I I 10 3.05 4 - .09 585 ± 23 312 ± 15 497 ± 20 1.87 ± .12 1.17 ± .07 Control — 4.20 ± .05 _ b _ b _ b 2 . 8 7 b 3.00*

Table 1. Effect of chloramphenicol on longitudinal growth and on pteridine and riboflavin of tail regenerate of Triturus cri-status. Each group: 10 animals.

a There was no blastema formation at all in this group; consequently no determinations of pteridines and riboflavin were made. b Results from another serie of experiments, where attention only was drawn to the relative changes. See 1. c. 1. c Time after amputation in days, after which the animals were treated with chloramphenicol until the end of the experiments (20 days). For explanations see also text. d Fluorometer units in the same weight of regenerates. The deviations here are given in absolute values. The deviation in T H / I X and TH/RB represent the square deviations. e The difference in length between the controls and the drug treated animals was clear without need of any statistical evaluation. Abbreviation: TH = tetrahydrobiopterin;

IX = isoxanthopterin; RB = riboflavin.

days—

Fig. 1 a. Ratio tetrahydrobiopterin/isoxanthopterin after tail amputation in T. cristatus. square deviations.

days *•

Fig. 1 b. Ratio tetrahydrobiopterin/riboflavin after tail ampu-tation in T. cristatus. square deviations. regenera-

tion bud, remaining skin, eye.

the injections did not start with the day of ampu-tation, buth rather 3 and 10 days respectively after it.

a) The effect of chloramphenicol on the growth rate of the regeneration bud

Apart f rom its effect on the formation of the re-generation bud, chloramphenicol in the concentra-tions used showed no apparent toxic effect on the animals. Table 1 summarizes the data concerning

2 8 8 N. KOKOLIS, N. MYLONAS, AND I. ZIEGLER

Group Starting t ime o f T H d I X d R B d T H / I X T H / R B chloramphenicol application [day]

I 0 0 642 ± 23 762 ± 26 0 0 I I 3 90 + 5 405 ± 17 571 ± 22 0.22 ± .02 0.15 ± . 0 1

I I I 10 107 ± 8 232 ± 19 587 ± 20 0.46 ± . 0 5 0.18 ± .01 Control — —1> — b 0.55 b 0 . 2 1 b

Table 2. Changes of pteridines and riboflavin of the remaining skin during regeneration of Triturus cristatus under the in-fluence of chloramphenicol*. Each group: 10 animal. * For explanations (a — e) and abbreviations see Table 1.

feet is seen in the case of isoxanthopterin and ribo-flavin. In general their amount increases in the regeneration bud and in the skin the sooner the ap-plication of chloramphenicol is started after ampu-tation. Only the level of riboflavin in the skin shows no differences between group II and III.

Thus, as seen in Tables 1 and 2, the ratios T H / I X and TH/RB increase with the deferment of the starting time of chloramphenicol application, exhibiting their maximum value in group III. Both ratios strictly parallel the lengths of the regene-rates.

b) I s o x a n t h o p t e r i n

The injections were made at a dose of 3 jug/g body weight at intervals of 12 hours. The difference between groups I, II, and III with respect to the timing of injection corresponds to the experiments clone with chloramphenicol, with the exception that the injections in group III started with the 7 t h day after amputation and all animals were killed after 17 days.

a) Effect of isoxanthopterin on the growth rates of the regeneration bud

As does chloramphenicol, isoxanthopterin also exerts a strong inhibitory effect on the formation of the regeneration bud (Table 3 ) . The inhibition in-creases the earlier the injections were started after amputation.

ß) Effect of isoxanthopterin on pteridine and ribo-flavin pattern in tail regenerate and remaining skin

As seen in Table 3, the ratios T H / I X and TH/RB are strongly reduced both in the regeneration bud and in the remaining skin. This decrease is also here increasingly expressed the earlier the injec-tions started after amputation. Seemingly the per-centage of reduction in both ratios, compared with the control is expressed even more in the remaining skin than it is in the regeneration bud itself.

c) R e s e r p i n e

The injections were made at a dose of 10 jugfg body weight at intervals of 24 hours. This is double the amount needed for induction of the hydroxy-lating enzyme system16 (see introduction). Even though the regeneration time (17 days) was the same for all animals, the timing of injections was different in the groups I —VII. In group I the in-jections wTere started 12 days, in group 117 days pre-ceding the amputation and were stopped 24 hours before it. In groups III and IV, injections were started at the time of amputation and were continued for 16 days (group III) or 7 days (group IV) again. In groups V and VI injection of reserpine was started 5 days and 10 days respectively after amputation; thus the animals of group V received 12, those of group VI 7 injections. Group VII was run as a control.

Group Starting t ime Mean of T H / I X T H / R B of isoxanth. regenerate Regenerate Skin Regenerate Skin appl icat ion 0 [ m m ] e

I 0 0.5 ± . 0 1 0.37 ± . 0 2 9 0.28 ± .039 0.47 ± .037 0.25 ± . 0 3 6 I I 3 0.6 ± . 0 1 0.67 ± . 0 5 0 0.32 ± .032 0.93 ± .045 0.23 ± .022

I I I 7 0.8 ± . 0 1 0.73 ± . 0 5 2 0.69 ± .071 0.81 ± .058 0.45 ± . 0 4 3 Control 3.5 ± . 0 5 1.14 ± .071 1.05 ± . 0 8 0 1.45 ± .096 1.25 ± . 1 0 1

Table 3. Changes of pteridines and riboflavin of the regenerates and of the remaining skin, during regeneration of T. cristatus under the influence of isoxanthoperin. Each group: 10 animals. For explanations (a — e) see Table 1.

PTERIDINE AND RIBOFLAVIN PATTERNS DURING REGENERATION IN TR1TURUS 2 8 9

G r o u p a ' b Animals T H / I X T H / R B Longitudinal Regenerate Skin Regenerate Skin growth (mm)

I 10 1.30 ± . 0 8 1.22 ± . 0 8 1.08 ± .07 4.06 ± . 3 4 3.2 - 4.0 I I 9 1.12 ± . 0 8 1.06 ± . 0 7 0.99 ± . 0 7 3.11 ± .32 2.9 - 3.3

I V 9 1.13 ± . 0 7 1.28 ± . 1 0 1.25 ± . 0 8 1.76 ± . 1 6 3 . 0 - 3 . 5 V 5 1.63 ± . 1 1 1 . 4 3 — .09 1.46 ± . 1 0 2.05 - . 15 3.7 - 4.5

V I 8 1.81 ± . 1 1 1.75 ± . 1 3 1.70 ± . 1 1 2.36 ± . 1 3 3 . 9 - 5 , 0 Control 8 0 0 0 0 1.0 - 1.2

Table 4. Effect of reserpine on longitudinal growth and on pteridines and riboflavin of tail regenerate and remaining skin in Triturus vulgaris. aMortality in group III 100% (see also text) ; b Explanations see text.

a) The effect of reserpine on mortality, growth rate and pigmentation in Triturus vulgaris

In contrast to the other two drugs, reserpine had an apparent toxic effect on the living animal. As seen in Table 4, especially the animals of group III proved to be very susceptible and died at the end of the experiment. The animals of those groups without lethal effect showed strong irritability. Furthermore, Table 4 shows that in T. vulgaris, which normally does not regenerate, the formation of a regeneration bud is promoted under the in-fluence of reserpine. In contrast to normal regene-ration buds of T. cristatus or T. alpestris, how-ever, it is bent upward at its tip. The wound healing zone and very tiny regeneration onset of T. vulga-ris is normally slightly greyish and does not show any melanophores in the epidermis, whereas the re-generation bud of the drug treated animals is stuf-fed with them (Figs. 2 a*, 2 b) . Besides this, reser-pine influences the state of the melanophores: 8 hours after a single injection the melanophores at the tip of the regenerating tail are expanded. Its influence, however, decreases with the age of the tissue: in the proximal direction contracted and ex-panded melanophores are mixed and finally those of the remaining skin are not at all influenced by the drug.

Although quantitative determinations of melanin were not made here, the complete absence of mela-nization in the epidermal layer before and its marked load with melanin granules after reserpine application suggest that at least in the epidermis new melanin synthesis had occurred. In the dermal layer expansion of the melanophores, which nor-mally are contracted, may participate in the general appearance of increased melanization. ß) The effect of reserpine on pteridine and ribo-

flavin pattern in the tail regenerate and in the remaining skin in Triturus vulgaris

* Fig. 2 a and b see Table page 290 a.

The most striking effect is that in all drug treated animals tetrahydrobiopterin is found in the regene-ration bud and in the remaining skin. Thereby the ratios TH/IX and TH/RB in the regeneration bud increase with the deferment of starting time of the drug application after amputation; they reach their highest values in group VI, where the application started 10 days after amputation. This does not ac-count for the remaining skin, where the drug ap-plication preceding the amputation causes the highest levels of tetrahydrobiopterin (Table 4 ) .

Besides tetrahydrobiopter in a y e l l o w f luoresc ing substance appeared to a minor extent in the skin of the drug treated animals . It seems to b e a degradat ion product , caused by ac id i c chromatography . It is neither r ibof lavin (or any of its derivatives l ike F M N or F A D ) nor it is identical with 7 ,8 -d ihydro -6 - lac toy lpter id ine ( sep iapter in ) , i sosepiapter in or 7 ,8 -d ihydro-6 - lactoy l -lumazine. It is not a growth factor f o r Crithidia (see 1. c . 9 ) . Its chemical nature has still to b e identif ied.

Discussion

The time course of tetrahydrobiopterin synthesis shows that the initiating event, the amputation, first affects the pteridine metabolism of the remaining skin. The subsequent appearance of tetrahydro-biopterin in the regeneration bud coincides with its decline in the remaining skin. Therefore one tends to assume that it is synthesized in the remaining skin and then transported into the regeneration bud. This view is supported by the fact that no in-crease in degradation products, e. g. isoxanthopte-rin, goes along with the decline of tetrahydro-biopterin and that changes in the ratio RB/IX do not take place. Furthermore, the action of reser-pine, administered before amputation, causes the highest levels of tetrahydrobiopterin in the re-maining skin. This may be due to the fact that the drug causes tetrahydrobiopterin synthesis immedia-tely after amputation, however, it cannot be trans-

2 9 0 N. KOKOLIS, N. MYLONAS, AND I. ZIEGLER

ported quickly. In contrast, later application of re-serpine may cause the newly formed tetrahydro-biopterin to be immediately transported into the growing bud.

The period of intensive tetrahydrobiopterin syn-thesis in the remaining skin in turn coincides with the time of DNA synthesis, wThich starts after 5 days in the limbs of amputated newts 20 and in the liver after partial hepatectomy 21.

Histochemical 22 and biochemical 23>24 analyses have shown that intensive DNA and RNA synthesis as well as ribosomal protein synthesis are important metabolic activities in dedifferentiating tissues. This agrees with the above results: both drugs which interfere with these processes also inhibit the for-mation of a regeneration bud. In parallel, the level of tetrahydrobiopterin is kept low. In contrast, re-serpine, which activates DNA 1 7 and increases the regenerative ability, also does improve tetrahydro-biopterin accumulation.

Thus two main problems arise. The first is the question of wdiether the drugs affect the synthesis of tetrahydrobiopterin from its purin precursor (see I . e . 9 ' 1 4 ) , or whether they mainly influence its further metabolism. The second question is, whether the basic events of blastema formation, which are DNA, RNA and ribosomal protein synthesis on the one hand, and, a high value of TH/IX and TH/RB ratios on the other, have any causative connection, or whether they are parallel phenomena initiated by one common event.

As to the first question, a possible inhibition of tetrahydrobiopterin synthesis by chloramphenicol and isoxanthopterin and its enhancement by reser-pine respectively, has to be examined by applica-tion of guanosine-14C-phosphate (see 1. c. 9 ' 1 4 ) . By an additional effect, chloramphenicol may prevent the formation of folic acid reductase, which is to some degree active in the reduction of 7,8-dihyro-biopetrin 14, to form the tetrahydrocompound; in-duction of folic acid reductase has already been shown to be blocked in S. faecium by chloramphe-nicol 25. As we know, not only isoxanthopterin but also lumazines, including the lumazine derivative riboflavin, are endproducts of pteridine meta-bolism (see 1. c. 9) : tetrahydrobiopterin is converted by the action of a pterin deaminase and by xanthin-oxidase into lumazines 26. Table 1 and 2 show that chloramphenicol, for instance, causes a marked in-crease in isoxanthopterin as wTell as in riboflavin,

indicating an enhanced catabolism of tetrahydro-biopterin under the influence of this drug. HALDAR and F R E E M A N 2 7 showed that the N A D H oxidizing activity is reduced by chloramphenicol, and it was found that twro different sites are attached to the enzyme molecule, one for pterin and another for N A D H 28. Thus it seems not unlikely that an inhi-bition of N A D H oxidation capacity enhances its pteridine metabolizing activity.

As to the second question, the action of tetra-hydrobiopterin as a cofactor in hydroxylation reac-tions (see 1. c . 1 4 ) does not seem to be involved here. It should be emphasized that in tissues with high protein synthesis levels (and a high rate of phenylalanine hydroxylation) the amount of tetra-hydrobiopterin is high indeed, but the ratios TH/ IX and TH/RB remain low due to the high levels of both products of pteridine metabolism 17. There-fore tetrahydrobiopterin and, respectively, the ratios TH/IX and TH/RB may act in some additional way, controlling mitotic activity and blastema formation. The only indication up to know we have for a direct causal connection between DNA and RNA activa-tion and pterins is the observation that isoxanthop-terin inhibits DNA and ribosomal RNA synthesis 10

and forms an unstable association with DNA 1 1 . The strong inhibition of blastema formation by iso-xanthopterin found here and its suppressing effect on tumor growth 12 favors the idea that neoplastic growth and low content in metabolites of tetra-hydrobiopterin may be directly connected. The sup-pression of a high TH/IX ratio by application of isoxanthopterin in turn may exert an amplifying action.

The lack of regenerative ability in the scarcely melanized T. vulgaris, in contrast to the well established regeneration ability in both melanized species T. cristatus and T. alpestris on one hand and the coupled enhancement of increase in mela-nization and regeneration by reserpine on the other emphasize the close connection of rapid cell proliferation and melanization. The failure of BIBER and HITCHING8 to find an inhibitory action of chloramphenicol on regeneration in tadpoles may be connected with this, as at this stage tetrahydro-biopterin is still present in large amounts1 and melanization is going on. The action of reserpine on melanization observed here may be explained to some degree by its dispersing action on melanine granules, rendering them more accessible to the

N. KOKOLIS, N. MYLONAS. and I. ZIEGLER, Pteridine and Riboflavin Patterns During Tail Regeneration in Triturus Species and the Effects of Chloramphenicol, Isoxanthopterin and Reserpine (page 285)

dermal layer

epidermal layer

dermal epidermal

Fig. 2 a. Melanization of the wound healing zone in normal Fig. 2 b. Melanization of the regeneration bud in Triturus Triturus vulgaris. vulgaris, under the influence of reserpine (Group VI ) . Magni-

fied: x 132.

Zeilschrift für Naturforschung 27 b ,page 290 a.

PTERIDINE AND RIBOFLAVIN PATTERNS DURING REGENERATION IN TR1TURUS 2 9 1

tyrosine hydroxylation cofactor tetrahydrobiopte-rin 24 and thus activating melanin synthesis 9 . How-ever, melanization seems to be more generally con-nected with celUproliferation; the melanomas, espe-cially those caused by hybridization of Xiphopho-rus species (see I.e. 3 0 ) , seem to be a most suitable

1 N . KOKOLIS U. I . ZIEGLER, Z . N a t u r f o r s c h . 2 3 b , 8 6 0 [ 1 9 6 8 ] .

2 R . SCHWEET u . R . H E I N T Z , A n n . R e v . B i o c h e m . 3 5 , 7 2 3 [1966] .

3 A . WEISBERGER U. S . W O L F E , F e d e r a t . P r o c . 2 3 , 9 7 6 [ 1 9 6 4 ] ,

4 S . W O L F E u . A . WEISBERGER, P r o c . na t . A c a d . S e i . U S A 5 3 , 9 9 1 [1965 ] .

5 K . M O L D A V E , A n n . R e v . B i o c h e m . 3 4 , 4 1 9 [ 1 9 6 5 ] . 6 B . M . B U R N E T u . R . A . LIVERSAGE, A m e r . Z o o l o g i s t 4 ,

427 [1964] . 7 R. J. Goss , Principles of regeneration, Acad. Press, Lon-

don 1969. 8 S . BIEBER U. G . H . HITCHINGS, C a n c e r R e s . 1 9 , 1 1 2 [ 1 9 5 9 ] . 9 I. ZIEGLER, Ergebn. Physiol. , biol. Chem. exp. Pharmakol.

56, 1 [ 1965 ] . 1 0 S . E . HARRIS U. H . S . FORREST, P r o c . nat . A c a d . S e i . U S A

58, 89 [1967 ] . 1 1 J . SMITH u . H . S . FORREST, N a t u r e [ L o n d o n ] 2 2 9 , 2 1 6

[ 1 9 7 1 ] . 1 2 N . KOKOLIS U. I . ZIEGLER, Z . N a t u r f o r s c h . 2 7 b , 2 9 2 [ 1 9 7 2 ] . 13 J. AXELROD, Science [Washington] 173, 598 [1971] . 1 4 S . K A U F M A N , A n n . R e v . B i o c h e m . 3 6 , 1 7 1 [ 1 9 6 7 ] . 1 5 M . L E V I T T , S . SPECTOR, A . SJOERDSMA U. S . UDENFRIED,

J. Pharmacol , exp. Therapeut. 1 4 8 , 1 [1965] . 1 6 H . T H O E N E N , R . A . M Ü L L E R U. J . AXELROD, N a t u r e [ L o n -

don] 2 2 1 , 1 2 6 4 [1969] .

tool for further investigations of the connection between tetrahydrobiopterin metabolism, melanin synthesis and cell proliferation.

One of us (N. K.) is indebted to H u m b o l d t -Stiftung for a grant and generous help.

1 7 N . KOKOLIS U. M . ISSIDORIDES, E x p . C e l l R e s . 6 5 , 1 8 6 [ 1 9 7 1 ] .

1 8 I . ZIEGLER, Z . N a t u r f o r s c h . 1 8 b , 5 5 1 [ 1 9 6 3 ] . 19 D i f co Manual, 9TH Edition, D i f c o Laboratories, Detroit

1 9 5 3 . 20 E. D. HAY U. D. A . FISCHMAN, Development. Biol. 3, 26

[ 1 9 6 1 ] . 2 1 R . A . M C D O N A L D , T . GUINEY U. R . T A N K , P r o c . S o c . e x p .

B i o l . M e d . 1 1 1 , 2 7 7 [ 1 9 6 2 ] , 22 E. D. HAY, Regeneration, Ed. HOLT, Rinehart u. Winston.

New York 1966. 23 E. D. HAY, Regeneration, Ronald Press Co., New York

1 9 6 2 . 24 H. J. ANTON, Regeneration in animals and related

problems. North Holland Publ. Co., Amsterdam 1965. 25 A . BLOCH, Biochim. biophysica Acta [Amsterdam] 201,

3 2 3 [ 1 9 7 0 ] . 26 H. REMBOLD, Chemistry and Biology of Pteridines. Int.

Acad. Printing Co., Tokyo 1970. 2 7 D . H A L D A R U. K . B . FREEMAN, C a n a d . J . B i o c h e m . 4 6 , 1 0 0 9

[ 1 9 6 8 ] . 28 N. KOKOLIS, Comparat. Biochem. Physiol. 25, 683 [1968] . 2 9 A . B . LERNER U. J . D . CASE, J . I n v e s t . D e r m a t o l . 3 2 , 2 1 1

[ 1 9 5 9 ] . 3 0 F . ANDERS, Z b l . V e t . m e d . , B 1 5 , 2 9 [ 1 9 6 8 ] .