9

Growth experiments on tadpoles with cystine and auxine.'By

Bengt Bylven.

(Anatomical Department, Karolinian Institute. Stockholm, Sweden.)

(With II figures ill the text)

Introduction.The growth promoting properties of l-eystine have been described

by several authors. Osborne and Mendel found 1918 on rats, thatI-cystine was the first limiting amino acid when the percentage of proteinin the food mixture was reduced below the zone of entire adequacy.The same role of cystine has been stated concerning amino acids inmilk by Sherman and Merrill (1925), and by H. Beard (1926) incasein. Du Vigneaud, Dorfmann and Loring have demonstratedthe inactivity of d-cystine for growth purposes. Earlier experimentshave all been made on mice, and it was therefore my intention tocontrol the stated results on tadpoles. In addition to that, feedingexperiments have also been performed with auxine, the newly discoveredpro-ferment in Avena sprouts, void of chlorophyll, grown in a dark room.

Experiment.

At the beginning of the experiment the tadpoles had just beenhatched with a length of about 10 mm. I started with the followingseries and concentrations of the additional food-stuffs.

-i Number ofLabelled ,; Additional food-stuff i Concentration animals

_~ __. __ " .._. __~ .__.._!,. __. _,.. _.__ _. __ ._. l _------j'------ ------ -------·-_·····_-_··----··1--·----

Controls . 15Cystine A l.oystine 0·0066% (24. V.-I. VI.) ; 15

0·0010% (4. VI.-13. VI.) : 10 -Cystine B I-cystine O·0033% 15Cysteine C. cysteinehydrochloride: o· 0066% 15-Cysteine D . II. cysteinehydrochioride : 0·0033% 15Auxine "10" _' Avena sprouts 10/150 c.c, 15

1 Der Redaktion am 25.•Iuli 1933 zugegangen.Skandinav. Archiv. LXVII.

130 BENGT SYLVEN:

The tadpoles were all kept in glassbasins, each of them measuring150 c.c, water. As F. Bilski (1921) has stated, is growth influencedby the space of the accomodation in which the animals live. Theveracity of this data has been ascertained during the research of lastyear in Stockholm, that was performed on tadpoles by medicine graduatesSail strom, Turesson and Zander. In consideration of this factI have only kept three tadpoles in each basin throughout all series.By choosing specimens of different size to each basin it was also possibleto follow their individual growth. Careful regard was taken to the sameconditions of life in all basins, concerning water, temperature and light.'I'hey got fresh water every day and had the same temperature, about200 C. and the same light conditions. Their food consisted mainly ofcarbohydrates in the form of warfer and also of dried and crushedants-eggs, which furnished them with amino acids. The analysis of theants-eggs showed minute traces of cysteine after acid hydrolysis of thecontainedprotein, but as for cystine no positive reaction could be obtained.Warfer and ants-eggs were given in the same amount in all basins.In addition to this the concentrations previously mentioned of cystineand cysteinehydrochloride were given in four series.

Because the difficulties in the synthesis of auxine from humanurine I used Avena sprouts, that were grown in a dark room. Inorder to obtain a fairly constant concentration of auxine I used tofinely crush the very tips of a hundred sprouts every day with a knownvolume of water of about 200 C. and then to distribute the solutioninto the basins in the concentrations above. At the beginning I hadonly oneauxine series of ten Avena sprouts per basin, but later I startedtwo 'series with respectively five and one per basin.

Methods for measuring.Former experiments with tadpoles have .clearly shown the dis­

adv~ntage of the practised methodpf pouring the animals from theirrespective basins into a special glass-bowl, the bottom of which wasprovided with a millimetre scale of translucent glass. Several tadpoleshad after. some time their axial skeleton broken and were spoilt. Forthat reason I made scales of canon that was ruled in square millimetres.They were of the same size as the bottom of the basins and wereprotected against water with a celluloid film on both sides. Such ascale was lying in each basin throughout the experiment and was notremoved. When measuring the tadpoles I had only to draw off water,leaving sufficient for them to swim a little. Then the whole basinwas put on the objeot-tableof the camera in a dark room. I had aspecial electric source of light for uniform illumination of the object.

GROWTH EXPERIMENTS ON TADPOLES WITH CYSTINE AND AUXINE, 131

Table 1.

1"}!ean values of the various series of tadpoles at the dates of measurement.Figures are given in millimetre.

Dates 11 24. V.127. V. i31. v·1 1. VL/4. VI. , 6. VI.jlO. VI.113. VI,=~~,~~~=~, -~c~='T~~1----====='='-"=-i'==-I='-"==

... :110.3 12·2 14'~ - 17.2! 18·5 ! 20·8 23·2

. .. ; 10·3 12·9 14'0 15·1 ' 17·6 '21·4 25·310·3 12·3 15·3 17.3119.6 23·7 26·610·6 12·0 14·3 16·6 I 18·4 21·1 23·610·2 12·3 13·7 15·3 i 17·1 20·6 24·010,1 111.6 14·0 15·7 17·9 20·5 24·3.

I 12.3 14.4 15·4 17·1 19.915·6 18·3 21,4

Control ..Cystine A ..Cystine B ..Cysteinehydrochlor. CCysteinehydrochlor. DAuxine "10"

_Auxine "5".Auxine "1".

Lfl!1jlhi81/f123r--.---,--'--""----'--

27

25

15

2J

22

2/

2IJ

IP

/3

/7

/5

1/J

39'Cystine 8

4g·C,ySlineA

AuKine,'

Fig. 1.

Graphical representation of the values of table 1. .9*

132 BENGT SYLVEN:

The camera was of the stationary type with a vertical foot, that madeit possible to place both the objective and the ground-glass in a constantposition, that was maintained both in measuring and photographing.In that way the scale got the same relative size whichever methodI used, and therefore my given values have the same relative error.Every third day I measured each animal on the scale on the ground­glass and every ninth day I photographed them. With the length ofthe tadpoles is meant their whole length including their tales.

Discussion.In the course of the experiment some observations were made.

When studying the values from each measuring of the control series(they are not given here) one really understands what a difficult taskthe analysis of the growth curves must be. On the first day of measuringthere were tadpoles, the length of which differed in 0·1 mm. and afterthree days the difference between the same specimens was 0·9 mm.,after six days only 0·4 mm, The external factors were exactly alike,but their so called "internal factors" differed in some way. We canonly vary one component of the external complex and from suchexperiments make some conclusions, but the postulate is, that the inter-

26

15

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u12

11

20

19

/8

11

16

I,)

lJ

9 l-l-L...JL...J---l---l---l---l--L---L--L---L---L-.L-.L-.L-.L-L-L-l

1Jt1Ie10. 25 2£ 27 24 29. Jo JI 16 1 1 " 5 ti 7. 8 9. /0. I/. 12. lJ.

Fig. 2.Curves' showing the spreading of the. various control animals.

GROWTH EXPERIMENTS ON TADPOLES WITH CYSTINE AND AUXINE. 133

nal factor, that is a sort of integration of all the various factors insidean animal, must be identical in the experimental and in the controlanimals. We do not know how those external variations influence uponthe various internal factors. An attempt has been made in fig. 2 toshow those facts. The middle growth curve represents the averagevalues of the control series. The dotted line above is the curve of theanimal of maximum length in the same environment and the dottedline below is the curve of the animal of minimum length. If an idealcomparison should be made, I think, one must compare an experimentalanimal and a control tadpole of the same length for each day ofmeasurement. But the example given above inhibits that method, be­cause we do not know, if the change in the environment of an animalor of a plant has the same effect on the relative rate of growth at allstages of development of the organism. In the values of length thereare several, which lie with minor variations at the same point. I haverepresented their average values in fig. 2 with the middle dotted line

Lengllir:illl.:::::'III,.-,---,---,--_-r---,--,----,.----r----,-,---,---,---,,--,--,---,---,--....---,

12

I.

16

15

17

19

22

21

20

23

25

2~

16

JO

29 .

26

21

13

116//1~J.15. 16.11. 13.11. Jo. Jl IiI J. ~. 5. 6. 7. 8. !J. 10. 1112. 1J.

Fig. 3.Growth curves for single individuals from the series auxine "10" and "1".

Special interest should be paid to the sudden rise of the curves.

134 BENGT SYLVEN:

and have found that it pretty well follows that of the general meanvalue. Therefore this work is based on the mean values of all animals.of the various series with one exception, that will be mentioned below.

When I started, the cystine A series had a concentration of0·0066 per cent cystine. The curve shows after three days a suddenrise from 10·3 mm, to 12·9 mm. with an angle of 41°, but at thattime the tadpoles showed great mortality. During the next three daystwelve died and the mean value of the three remaining animals camenearer the normal line. Suspecting some intoxication from the cystinesolution I found a minute amount of hydrogen sulfide in the waterof the basins. I found on experiments with some fresh animals thattadpoles were very sensitive to its toxic action. Hydrogen sulfide mustcome from the cystine in solution, either by the decomposition ofcystine in the cells of the tadpoles or by some bacterious action.However that may be, I started a new set with reduced cystine con­centration of 0·0010 per cent, the curve of which has an angle of 49°on an average. The animals of the cystine B series showed a minormortality, because the smaller possibility of evolving the fatal dose ofhydrogen sulfide. Out of 15 tadpoles there were still 6 left after twentydays. The angle of cystine B is 39°. In those cases of intoxicationI have left some animals out from the average, that means, sometadpoles showed already at an early stage of development a slowlyfalling rate of growth until they died. It is evident that they werevictims to intoxication from hydrogen sulfide. Therefore the mean valueshave only been taken on animals without any sign to such a disease.

It may look rather curious that I used cysteine hydrochloride intwo series, because the first stage in the catabolism of cystine is con­version to cysteine. The evidence was shown by H. B. Lewis andD. A. Mc Ginty (1922). On the other hand the water used in thebasins contained traces of iron and this involves a cyclic process, whichends in the oxidation of all cysteine to cystine. Oxygen gas is theoxidizing agent. As a matter of fact, the growth curves of the seriesfed with cysteine hydrochloride do not show any accelerated rate ofgrowth. This furnishes evidence that cysteine is not a growth promotingamino acid, or that tadpoles are incapable of oxidizing cysteine tocystine. But there is a difference in the aspect of the curves of thecysteine series with the concentration of 0·0033 per cent. I cannotgive credence to the small rise of that curve in comparison with thenormal curve. The possibility always remains that cysteine has beenoxidized by air oxygen to cystine in the basins.

Auxine is a growth promoting hormone in plants. It is also foundin human urine in its greatest known concentration, which is the best

GROWTH EXPERIMENTS ON TADPOI,ES wrrn CYSTINE AND AUXINE. 135

material for its synthesis. It is very sensitive to light, that transformesit into an inactive auxine ester. J cannot join the opinion of Kogi(Utrecht) that there does not exist evidence that a plant ferment shouldbe active on an animal organism. The possibility remains thatour inactive au xin e ester could be reactivated by the fer­ments of the living cells. In fig. 3 are some typical values frommy auxine series represented. The tadpoles were given Avena sproutsfor their first time on the 31st of May. A general description of theauxine curves shall be given here.

1. Before Avena sprouts were given their mean value growthcurve is subnormal, the special curve of the maximum animal is seenon fig. 3, line 2. The average angle is about 27°.

2. After four days orin some cases six days the relative rateof growth of the tadpoles is greatly accelerated for a time of abouttwo days. The angle increases to values from 49° to 62°.

3. After this sudden increase the relative rate of growth diminishesbut is still standing on a higher level than before the time when auxinewas distributed.

This typical rise of the auxine series may be described as com­pensatory growth, or as an effect of auxine. But such a suddenacceleration is also seen on fig. 2 'on the subnormal control curve,from the 4 til to the 6th of June at the same time as most atixinetadpoles showed the same phenomenon. 1 am inclined to say thatthe plant hormone auxine in this experiment had no growth acceleratingproperties.

S11llUl1afY•

In consequence of those experiments I will furnish the following data.1. Cystine shows a definite property of increasing the rate of

growth of tadpoles.2. Concerning cysteine no such influence has been observed.3. As to auxine further experiments must be made before a sure

result can be obtained. I think the experiments ought to be performedon rats and pure auxine solutions by means of intraperitoneal injectionsshould be used.

* * *I wish to express my gratitude to Professor Dr. Gaston Backman

for many valuable suggestions and to the chief of the anatomical de­partment at Stockholm Professor Dr. Carl Hesser for his renderingpossible of this work.

1.36 BENG'l' SYJ,VEN: GROW'l'H EXPERIMENTS ON TADPOLES, l~C.

Literature.

1, Beard, H., Am. J. PhY8iol. FebI'. 1926. Vol. LXXV.2. Briggs, G. E., Proc. Roy. Soc. 8. B. Jan. 1928. Vol. CII.3. Crozier, J. W., J. General Phy8iol. Sept. 1926. Vol. X.4. von Hoess1in, H., Zeit8chr.J. Biol. 1930. Bd. XC.5. Hosoya, S., Jap. M. World. April 1926.6. Kogl, F., (Utrecht). Die Naturwi88enBchaften. Jan. 1933. Bd. XIII.7. Lewis, H. B. and Mc Ginty, D. A., J. Biol. Chem" Aug. 1922.

Vol. LIII.8. Lewis, H. B., Updegraff, H. and Me Ginty, D. A., Ibidem.

Febr, 1924. Vol. LIX.9. Ludwig, Biol. Zentralblatt. 1929. Bd. XCIX.

10. Michaelis, L., .J. Biol. Chem. Nov. 1929. Vol. LXXXIV.11. Osborne, Th. and Mendel, L., Ibidem. 1918. Vol. XXXIV.12. Sure, B., Ibidem. Sept. 1920. Vol. XLIII.13. Sure, B., Am. J. PhY8iol. June 1922.14. Tsukano, M., J. Biochem, July 1932. Vol. XV.15. du Vigneaud, Dorfmann and Loring, J. Bioi. Chem, Nov, 1932.

Vol. XCVIII.16. Voegtlin, C., Johnson, J. M. and Dyer, H. A., J. Pharmacol. and

Exper. Therap. 1926. Vol. XXVII and XXIX.17. Went, F. A. F. C., (Utrecht). Die NaturwiB8enBchajten. Jan. 1933.

Bd.VI.18. Woods, Ella, J. Biol. Ohem, Nov. 1925. Vol. LXVI.19. Yaoi, H., Jap, M.World.May 1926.20. Yaoi, H. and Hosoya, S., Ibidem. April 1926.