(SANITIZED)UNCLASSIFIED FOREIGN-LANGUAGE BROCHURE ON INSTITUTE OF BIOLOGY(SANITIZED)

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FOLIA BIOLOGICA (PRAHA)
Me)1cay1lapoan)oe naaalfue aacypuua.ioe Ceskoslovenska biologie it Ceslcoslovenslc(i
7nikrobiologie
Pe ;>; a u it it o it it a n x O it it e r ii n:
AKageNnm H. MaJIei1 (i' i niIciii pegaBTOp), B. Bplllaxcuufi, M. TauItei , qa.-KOpp. 1ICAH
(1). rep tim, aia;jeMUK O. IIpORei[, 10. Alagypa, aHaAeMIIK C. 1IpaT, B. Pocugiuui (Cel;p. pCd.
Koaneinn), A. Llepamiti, H. IIITepgJIb.
I lepelioJl,ei xa pyccenli ftal,!B: jog. ;A-p IIInpoBa, ua aor'JInfic- ih nahnn: 1-p Pnikeeona, iia HIe-
Megimii Ilal>uc: n-p ccafirab
II! leTCtl BriOaOrii'CCKiiM IIIICTIITyTOYu Llexoc:louauKOii AKade uni xayic ii II;3 aTeJIbCTBe 'ICAII.
111X01TInT 6 pas B l'0;~. 110 ,r,i[1Id Ol I Iiexa Wt 1 r0;k I IC 60.-, gena o;kuoro i10Mep:I How 10.-.
A;lpec pe;iaxguu: Buoaoru'iecKliit IIHCTHTyT rICAII, IIa gfII91111ITII 2, llpara XIX. 3aKnai,i:
ApTitn, CMe=irn 30, llpara II, rloxocaonannn,
FOLIA BIOLOGICA (PRAHA)
International Edition of the Journals Oeskoslovensled biologic and Ceslcoslovenskci
ini/crobiologie
Academician I. Malek (Chief Editor), L. Nrny, M. Hayek, Corresponding Member of the Czechoslovak
Academy of Science F. Hercik, Academician O. Jirovec, J. Macura, Academician S. Prat, 13. Rosicky
(Editorial Secretary), J. ~terzl, V. Vrsansky.
Translations into Russian: Dr Schierova, into English: Dr Ridesova, into German: Dr Feigel.
Issued by Biologicky ustav i eskoslovensko akademie v6d at Nakladatelstvi Os. akademie ved. Yearly
subscription (6 numbers) Kcs 60. Single number Kcs 10. Address: Biologicky ustav OSAV, Na evicisti 2,
Praha XIX. Orders: Artia, Smecky 30, Praha If, Czechoslovakia.
FOLIA BIOLOGICA (PRAHA)
Internationale Ausgabe der Zeitschriften Ceslcoslovens/cci biologic and Ceslcoslovenska
7nikrobiologie
Akademiernitglied I. Malek (leitender Redakteur), L. Oerny, M. Hayek, korresp. Mitgl. d. Os. Akadernie
d. bliss. F. Hercik, Akademiemitglied O. Jirovec, J. Macura, Akademiernitglied S. Prat,
B. Rosicky (Redaktions-Sekretbr), J. Sterzl, V. Vrsansky.
Die Uborsetzungen besorgt Doz. Dr A. Schierova fiir die russischen, Dr A. Ridesova fiir die
englischen and Dr T. Feigel fiir die deutschen Artikel.
Herausgeber: Biologicky ustav OeskoslovenskC akademie v6d durch Vermittlung des Nakladatelstvi Os.
akademie ved. 6 Lieferungen jahrlich. Abonnementpreis 60 Kcs, Preis der Einzelnummer 10 Kes.
Anschrift der Redaktion: Biologicky ustav OSAV, Na evicisti 2, Praha XIX. Zu beziehen durch: Artia,
Smecky 30, Praha II, Oeskoslovensko.
Fol. biol. (Praha) pp. 1-64
Praha, 25. III. 1957
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FOLIA BIOLOGICA
The Production of Antibodies by Isolated Spleen Cells Following
Contact with an Antigen in vitro
J. STERZL
Institute of Biology, Czechoslovak Academy of Science, Department of Microbiology, Praha
Received September 19, 1956
The latest communications dealing with the possibility of antibody formation
in tissue cultures to which an antigen has been added in vitro show that antibodies
are not formed under these conditions (Parker 1937, Salle and McOmie 1937, Selmar
1944, Fastier 1948). Positive antibody formation in tissue cultures can be demon-
strated only if the tissue used for culturing is taken from an animal which has been
immunised in vivo (Meyer and Loewenthal 1927, Parker 1937, Fagraeus 1948a,
Ranney and London 1951, Thorbecke and Keuning 1953, Tanaka 1953, Stavitsky
1955, Askonas and Humphrey 1955). These results give evidence, as concluded by
Parker (1937), that the first phase of the reaction to an antigen takes place only
under the conditions of the organism.
It was assumed by the author that the non-specific mobilisation reaction which
occurs in the organism following administration of an antigen, directly participated
in antibody formation. This reaction is displayed both in biochemical and physiolo-
gical changes (changes in the blood sugar level, temperature, the number of leuco-
cytes, etc.) and also in changes of a morphological character in the mesenchymal
tissue (Fagraeus 1948b, Marshall and White 1950, Makinodan et al. 1954).
The present communication attempts to establish to what degree antibody form-
ation is dependent on the non-specific metabolic and morphological reaction of the
organism following administration of the antigen. The metabolic and morphological
changes were produced by a different antigen (guinea-pig serum); after a given
time (24 to 96 hours), the spleen was removed and the spleen cells isolated and
mixed with the antigen (Salmonella paratyphi B) in vitro. The mixture of spleen
cells and antigen was then administered intraperitoneally to young rabbits. These
had already proved to be the best animals for the transfer of immunised tissues in
previous experiments, as very young animals do not respond to an antigen by anti-
body formation (8terzl 1955a). The same procedure was used with the controls, in
which the spleen cells of a normal, non-immunised animal were mixed with the
antigen.
A non-specific antigenic stimulus was produced by injecting rabbits (2-3 kg.) intravenously with
1 ml. guinea-pig serum 24-96 hours prior to killing, as described in the individual experiments.
The spleen of both immunised and normal rabbits was removed and prepared in a cooled room at 2? C.
The cells were expressed from the spleen capsule into a chilled phosphate-physiological saline solution
with 0.2% gelatine. The individual cells were freed by repeated sucking into a pipette. For further
washing, which was carried out three times in the same solution, only a homogenous suspension of cells
was used. The final dilution was made by adding 1 ml. of fluid (suspension of antigen, in the controls
physiological saline) to 0.05 g. splenic tissue. In every experiment the number of cells was determined
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'n a Barker chamber; a suspension of cells prepared in this way contained on an average 30-40 x 106
lymphoid cells/1 ml.
The antigen used was a heat-inactivated suspension of S. paratyphi B. The quantitative inter-rela-
tionship of spleen cells and antigen was one of the decisive factors for the experiment and is given in the
results. The mixture of spleen cells and antigen was incubated at 37 ?C and then injected intraperitoneally
in doses of 1 ml. in 5-day-old rabbits. The incubation time varied in the different experiments and
precise details are given in the tables; in most
cases the time of mixing was 10 minutes.
The times at which blood was collected from
the young rabbits by cardiac puncture are also
given in the tables. After collection, the sera
were stored at - 15? C and agglutination was
carried out in every group at the same time. In
cases where it was necessary to remove lipoid
substances, the sera were shaken out with chlo-
roform (Sterzl 1955). The agglutination specimens
were stored in a refrigerator and the results read
off after 4, 6 and 8 days.
Fig 1. Transfer of spleen cells of normal rabbit
(40 x 106 cells/I ml.) to two groups of young rabb-
its; mixed for 10 minutes: a) dashed - with anti-
gen S paratyphi B in concentration of 100 x 166
microorganisms/1 ml., b) full - with antigen 500 x
x 106 micro-organisms/I ml. x: age of rabbits in
days; days on which blood collected denoted. y: titre
of agglutinating antibodies.
Fig. 2. Isolated spleen cells (32 x 106/1 ml.) of
rabbit immunised intravenously 24 hours previously
with 1 ml. guinea-pig serum. The cells were mixed
in vitro with the antigen (106 micro-organisms/ lml.)
for 10 minutes. x and y: as in tab. 1.
The cells were irradiated in a dish placed in
ice, in a layer not exceeding 1 mm. Irradiation
was carried out using a Mikrometa apparatus
(AEG - 50-X-ray tube) under the following
conditions: focal distance 10 cm., Al 0.1, KV 50,
mA 6. With these constants the concentration
of irradiation is 430 r/10 seconds.
In the first experiments (~terzl 1955),
in which the cells of a normal animal
were mixed with an antigen and trans-
ferred intraperitoneally to young rabb-
its, no antibody formation was found.
In these experiments the amount of
antigen used was 109 bacterial cells in
1 ml. On reducing the amount of anti-
gen added to the cells in vitro, con-
clusive evidence of antibody formatiom
was found after a transfer to young
rabbits. The optimal relationship be-
tween antigen and cells was therefore
investigated. The results obtained hi-
therto show that the most satisfactory
proportion is 1-2 micro-organisms to
one spleen cell. A higher concentration
of the antigen inhibits antibody forma-
tion (fig. 1).
Antibody formation was found after mixing the antigen (S. paratyphi B.) with
spleen cells isolated from animals following non-specific stimulation with guinea-pig
serum. The experiments were carried out in 60 young rabbits from 10 litters. In all
groups where there had been antigenic stimulation by the administration of a foreign
serum 24-96 hours before removal of the spleen, followed by mixture of the spleen
cells with the antigen (S. paratyphi B) in vitro, antibodies which agglutinated the
specific antigen were found within a few days following intraperitoneal transfer to
young rabbits (fig. 2 and 3). No substantial increase in antibody formation was
found even when the cells were isolated from the spleen of an animal which had
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been immunised over a period of 14 days with five doses of guinea-pig serum
(fig. 4). This finding is of interest in view of the fact that during the first days after
non-specific immunisation an increasingly marked morphological reaction took place
in the spleen tissue in all experiments (an increase in the number of reticular cells,
plasmoblasts and plasmocytes and a transitory decrease in the lymphocytes - Holub
1957). Antibody formation was also demonstrated in experiments in which spleen
cells were isolated from an organism immunised in vivo with the specific antigen
Fig. 3. Isolated spleen cells (41 x 108/1 ml.) of rabbit
immunised intravenously 72 hours previously with
1 ml. guinea-pig serum. Incubated in vitro with antigen
(108 micro-organisms/1 ml.) for 10 minutes and trans-
ferred intraperitoneally to group of young rabbits. x and
y: as in tab. 1.
5 8 10 12 1/ 1? 0 25 JO 35
Fig. 4. Spleen cells were isolated from the spleen of
rabbit No. 43 (35 x 108/1 ml.), which had been im-
munised over a period of 14 days with five intravenous
doses of 1 ml. guinea-pig serum. These were mixed for
10 minutes in vitro with the antigen 108 micro-orga-
nisms/l ml.) and after incubation were transferred
intraperitoneally to a group of young rabbits.
(S. paratyphi B) and mixed with
the antigen in vitro (fig. 5).
21
56 810 1214 17
Fig 5. Isolated spleen cells (45 x 108/1 ml.)
from two rabbits immunised intrave-
nously 72 hours previously with 1 ml. anti-
gen S. paratyphi B (108 micro-organisms).
Half the cells transferred to young rabbits
A) only washed in physiological saline, B)
incubated, after washing, with antigen for
10 minutes (108 micro-organisms/1 ml.).
x and y: as in tab. 1.
In further experiments carried out in eight groups of young rabbits, spleen cells
from normal animals which were mixed with the optimal amount of antigen were
transferred to the young animals. In all these experiments also there was clear
evidence of the formation of agglutinating antibodies (fig. 6).
Since spleen cells prepared in the same way and mixed with the optimal amount
of antigen did not form antibodies when cultured in tissue cultures (Rychlikov&
and Rterzl 1957), it was necessary to consider whether the young rabbits did not
participate actively in the formation of antibodies. It was demonstrated, however
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Fig. 6. Isolated cells from normal rabbit (44 x 1081
ml.), mixed with antigen (108/1 ml.) for 10 minutes.
x and y: as in tab. 1.
Fig. 7. Isolated spleen cells of normal rabbit (39 x
x 106/1 inl.) and irradiated with 860 r (full), 1,000 r
(dashed) and 1,200 r (dash and dot), then mixed
with antigen (108 micro-organisms/1 ml.) for 10 min-
utes and injected intraperitoneally in young rabbits.
x and y: as in tab. 1.
Fig. S. Isolated spleen cells (43 X 1081 ml.). Half
the cells not irradiated (dashed line), half irradiated
with 1,200 r (full line). After 4 hours incubation at
37?C mixed with antigen (108 micro-organisms) and
injected intraperitoneally in young rabbits.
in eight groups (55 animals) that if the
isolated cells are irradiated, antibodies
were formed only in those animals to
which cells not damaged by irradiation
had been administered. The cells which
go on to form antibodies are either those
which have received only small doses
of irradiation (fig. 7) or non-irradiated
cells (fig. 8). Doses of 1,000 and 1,200r
completely inhibit antibody formation.
The possibility of the active partici-
pation of the young animals in antibody
formation is likewise not supported by
the following finding: After transfer of
the cells, an initial phase of rapid de-
velopment of antibodies occurs, which
can be inhibited by irradiating the cells.
The second phase of the antibody re-
action is the active response of the
young animals to the antigen which is
transferred together with the cells, and
coincides chronologically with the de-
velopment of antibodies which takes
place when only the antigen is admin-
istered to young animals (20th-30th
day of life). In the transfer of cells
together with the antigen, therefore, the
question is not one of induction of the
active response of the young animals to
the antigen, as in that case active anti-
body formation would have to com-
mence shortly after transfer of the cells.
Nor could it be demonstrated that the
administration of a foreign antigen in
any way accelerated the response of
the young animals to the antigen. This
was demonstrated by an experiment
in which a different antigen (guinea-
pig serum) was first injected and the
antigen S. paratyphi B. was injected
seven days later. The formation of
antibodies was in no way speeded up
following this measure, as compared
with the controls (fig. 9). The author
regards the organism of the young
rabbits to which the spleen cells are
transferred, together with the antigen,
as passive, as a suitable culture medium
encouraging the development of the
cells and their complex biochemical
processes which participate in the for-
mation of the antibodies.
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It was also wished to determine whether active processes take place in spleen
cells in the course of short-term incubation of the antigen with the cells in vitro.
It is possible that the antigen and spleen cells are simply transferred into a suitable
environment and that the actual reaction of antibody formation takes place only
within the young animal. There is evidence for this possibility in the fact that if
spleen cells from a nor-
mal adult rabbit were in-
jected in young rabbits
and the antigen was not
injected until 24, 48 and
72 hours later (intraperi-
toneally), an antibody re-
action also occurred (fig.
10). This would indicate
that the transferred spleen
cells survive in the young
rabbit and that contact of
the antigen with them is
also possible in vivo. Anti-
body formation was also
demonstrated in cases in
which the spleen cells
Fig. 9. From a group of six young rabbits, four injected on the fifth
day of life with guinea-pig serum. Dash-dot: 2 ml. guinea-pig serum
intraperitoneally. Dashed: 3 ml. guinea-pig serum. Full: controls
without serum. Antigen (108 micro-organisms/1 ml.) administered by
intracardiac injection on 12th day of life. x and y: as in tab. 1.
were first injected intraperitoneally and the antigen was then injected into the blood
stream (intracardially - fig. 11). This experiment also indicates that cells transferred
intraperitoneally do not remain only locally, but that they find their way into the
internal organs of the animal, as demonstrated by Holub (1957).
An attempt was made to demonstrate the significance of the time for which the
cells are in contact with the antigen in vitro by washing out the antigen after incu-
r
I ' I /
4 I I 1 4' ~/ \I //
6 7 9 11 13 15 18 21 24
Fig. 10. Isolated spleen cells (28 x 106/1 ml.) injected
intraperitoneally in young rabbits. Full: cells mixed im-
mediately with 1 ml. antigen. Dashed: 1 ml. antigen in-
jected intraperitoneally 24 hours after injection of spleen
cells. Dash-dot: 1 ml antigen injected intraperitoneally
72 hours after injections of spleen cells. Dotted: spleen cells
killed by heating to 56? C for 30 minutes and injected in
young rabbits after being mixed with antigen. Concentra-
tion of antigen: 108 micro-organisms/ 1 ml. x and y: as
in tab. 1
bation together with the cells in vitro. In these experiments the cells were transferred
to four groups of young animals (21 in all). It was seen that washing out of the
superfluous antigen did not destroy the ability of the transferred cells to form anti-
bodies (fig. 12). The amount of antigen remaining in the cells was determined,
following destruction of the cells by freezing and thawing, by immunising adult
animals. In adult animals the amount of antigen added to the cells (108 micro-organ-
isms) gives an antibody titre of 1 :512-1,024. Following immunisation with
destroyed cells, the antibody titre in rabbits averaged 1 : 32-64. Cells were trans-
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Fig. 11. Isolated spleen cells of rabbit immunised 72
hours previously with guinea-pig serum. After washing,
the cells were suspended in physiological saline (35 x
x 106 micro-organisms/1 ml.) and 1 ml. injected intra-
peritoneally. The antigen (108 micro-organisms) was
then injected into the blood stream by the intracardiac
route. x and y: as in tab. 1.
1281
8 1012 14 F
Fig. 12. Spleen cells isolated from normal rabbit (39 x
x 106 micro-organisms/1 ml.) incubated together with
antigen (108 micro-organisms/l ml.) for two hours at
37? C. After incubation superfluous antigen washed out
with gelat. physiological saline and cells injected intra-
peritoneally in doses of 1 ml. in group of young rabbits.
Part of the cells destroyed by freezing and thawing to
determine the amount of antigen in the cells. x and
y: as in tab. 1.
ferred to two groups of young rabb-
its (13 in all); half of these cells had
been incubated, together with the
antigen, in a thermostat and half in
a refrigerator. Following incubation
the cells were washed and it was
found that the cells incubated to-
gether with the antigen in the refri-
gerator did not form antibodies.
Although it is not possible to con-
clude from these experiments that
the initial biochemical processes of
antibody formation take place al-
ready in vitro, the results permit the
conclusion that in vitro the cells bind
the effective amount of antigen,
which is only part of the total a-
mount added.
Antibody formation was obtained
on isolated spleen cells by mixing
them in the optimal proportion with
the antigen in vitro and transferring
them after incubation intraperitone-
ally to young rabbits. At the time
of our first communication on the
successful formation of antibodies
by isolated spleen cells (~;terzl and
Hrubesova 1955b), an extensive work
by Harris et al. was published (1955),
preceded by a preliminary commu-
nication (1954). Harris et al. suc-
ceeded in obtaining antibody forma-
tion on isolated cells of the lymphatic
glands, mixed with an antigen and
transferred to animals which had
undergone X-ray irradiation. Al-
though a different form of experi-
ment and a different antigen were
used, the results are basically the
same. Roberts and Dixon on the
other hand (1955), were unable to
demonstrate antibodies by mixing
the cells with the antigen in vitro and transferring the mixture to animals irradiated
with X-rays. They obtained positive results only when the animals had been
immunised in vivo. Our results do not confirm their findings. In our view, the
main cause of their negative results is the use of a protein antigen, as the demon-
stration of formed antibodies. by precipitation is not a very sensitive method.
The main question is whether the formation of antibodies in a transfer of a mixture
of cells and antigen to young rabbits is not due to an active reaction on the part of
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the recipients. It was demonstrated that disorganisation of the vital processes of the
transferred cells-e. g. by irradiation-destroys the possibility of antibody formation
after their transfer to the young animals. This does not, of course, exclude the
possibility that irradiation destroys cell structures-e. g. mitochondria-which,
when transferred to young animals, induce a metabolic change which makes
the active reaction of the animals to the transferred antigen possible. This possibility
is not supported, however, by a further finding. Following transfer of the cells,
antibodies are formed in two chronologically separate phases. The first of these
depends on the function of the spleen cells, while the second is the actual active
response of the young rabbit to the antigen injected together with the cells. In the
experimental part it was shown that the active response is not speeded up in any
way as compared with injection of the antigen alone. If induction of a metabolic
state occurred, making a reaction to the antigen possible, then the persistent anti-
body level characteristic of active formation would be determined as the first
response, and not the typical curve of passive transfer. We regard the participation
of the young organism in the whole reaction as passive and simply as a suitable
medium for maintaining viability of the transferred cells and for the biochemical
processes essential in antibody formation.
Although in experiments with Rychlikova (1957) we were not successful in
demonstrating antibody formation in tissue culture, after the addition of antigen to
explanted tissue, it is concluded from these experiments that the chief difference as
compared with transfers to young rabbits lies in inadequate nutritional conditions
in the tissue cultures. We believe that improvement of the culture medium in tissue
cultures and dynamic conditions of cultivation will produce the same result-i. e.
antibody formation- as in a transfer to young rabbits.
It also remains to be explained why antibody formation by transferred cells is of
relatively short duration. Why does antibody formation decrease at a time when,
as has been demonstrated, the transferred cells still survive in the organism of the
young animal? It is possible that further generations of cells, which do not come
into contact with the antigen, do not form antibodies and that the metabolic change
(the production of antibodies) is not inherited by further generations of cells. In this
association it should also be borne in mind that this may be the manifestation of
a transplantation immunity response on the part of the recipient. Further experi-
ments are being carried out in an attempt to determine the basis of this phenomenon.
The finding that antibodies can be produced by isolated cells under suitable
conditions provides an answer to a number of questions. It defines the importance
.of neurohumoral factors, primarily in the formation of the most suitable metabolic
conditions of the environment and of the metabolic level of the cells. On the other
hand, these experiments exclude the direct causal participation of nervous factors
and others in antibody formation. They are also an experimental reply to present
discussion on the significance of reflex processes for the formation of antibodies.
The finding of antibody formation in isolated cells permits attention to be con-
centrated on the stage of their development in which antibodies cannot be demon-
strated serologically either in cells or in serum. We are now embarking on these
investigations by studying metabolic changes which take place following contact
between antigen and cells and by studying the influence of antimetabolites and
radiation on the antibody reaction at cell level. At the same time the question arises
as to whether it will prove possible to bring about antibody formation following the
mixing of cell particles with the antigen in vitro. We raise this question because
antibody formation was transferred from immunised animals to young rabbits by
isolated mit ochondria (~terzl and Hrubesova 1955a).
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Summary
Cells isolated from the spleen of an adult rabbit and mixed with an antigen in vitro
(S. paratyphi B.) form antibodies when injected intraperitoneally in 5-day-old
rabbits. At that age young rabbits do not respond to the transferred antigen by
antibody formation.
No differences were found in the degree of formation, whether the young rabbits
were injected with the cells of a normal rabbit, the spleen cells of a rabbit immunised
with a non-specific antigen (guinea-pig serum) or with the specific antigen (S. par(,,-
typhi B.), after mixing with the antigen.
Antibody formation takes place in the presence of the optimal quantitative
relationship when mixing the cells and antigen in vitro. In a corpuscular antigen,
two micro-organisms of S. paratyphi B are added to one spleen cell. Larger doses of
the antigen inhibit antibody formation.
If isolated cells are irradiated, a dose of 860 r does not inhibit antibody formation,
while doses of 1,000--1,200 completely inhibit it. The transferred spleen cells of an
adult rabbit do not induce an active response to the antigen in young rabbits. The
participation of the recipient (the young rabbit) in antibody formation is regarded
as passive, i. e. the animal is regarded as a suitable culture medium for the trans-
ferred cells.
Cells injected into the organism survive; antibody formation can be evoked by the
injection of the antigen in young rabbits 24 and 72 hours after the injection of
washed spleen cells alone. Antibodies are also formed if the cells are injected intra-
peritoneally and the antigen is injected into the blood stream.
On mixing the cells with the antigen at 37 ?C, the effective amount of antigen is
rapidly bound by the cells. The washing out of superfluous antigen following incu-
bation does not prevent the formation of antibodies.
A s k o n a s, 13. A., H u m p h r e y, J. H.: Antibody Formation in Slices of Granulomata
.Produced by Adjuvant. Biochem. J. 60 : X, 1955.
F a g r a e u s, A.: The Plasma Cellular Reaction and its Relation to the Formation of Anti-
bodies in vitro. J. Immunol. 58 : 1, 1948a.
F a g r a e u s, A.: Antibody Production in Relation to the Development of Plasma Cells.
Acta med. stand. Suppl. 204, 1948b.
F a s t i e r, L. B.: An Attempt to Produce Bacterial Agglutinins in vitro. J. Immunol. 60: 399,
1948.
H a r r i s, S., H a r r i s, F. N.: Studies on the Transfer of Lymph Node Cells. V. Transfer
of Cells Incubated in vitro with Suspensions of Shig. paradysenteriae. J. Immunol. 74 : 318,
1955.
H o 1 u b, M.: Kvantitativni zmeny lymfaticke tkane behem imunisace. Cs. morfologie 5 1957.
M a k i n o d a n, T., R u s h, R. F., W o l f e, H. R.: Precipitin Production in Chickens.
X. Cellular Changes in the Spleen During Antibody Production. J. Immunol. 72 : 39, 1954.
M a r s h a 11, A. H. A., W h i t e, R. G.: Reaction of the Reticular Tissue to Antigens.
Brit. J. Exp. Pathol. 31 : 157, 1950.
M e y e r, H., L o e w e n t h a 1, H.: Untersuchungen uber Anaphylaxie an Gewebekulturen.
Zschr. Immunitatsforsch. 54 : 420, 1927.
P a r k e r, R. C.: Studies on the Production of Antibodies in vitro. Science 85 : 292, 19:17.
R o b e r t s, J. C., D i x o n, J. F.: The Transfer of Lymph Node Cells in the Study of the
Immune Response to Foreign Proteins. J. Exp. Med. 102 : 379, 1955.
R y 0c h 1 i k o v a, M., S t e r z 1, J.: Pokusy o tvorbu protilatek v tkanove kultui'e. Cs. bio-
01 logic, 6, 1957 (in press).
R a n e y, H. M., L o n d o n, M.: Antibody Formation in Surviving Tissues. Fed. Proc.
10 : 562,1 1951.
S a 1 1 e, A. J., M c 0 rn i e, W. A.: Immunological Responses of Tissues Cultivated in vitro.
J. Immunol. 32 : 157, 1937.
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S e 1 m a r, E.: On the Formation of Bacterial Antibodies in Tissue Cultures. Acta patho
microbiol. stand. 21 : 517, 1944.
S t a v i t s k y, A. B.: In vitro Production of Diphtheria Antitoxin by Tissues of Immunized
Animals. I. Procedure and Evidence for General Nature of Phenomenon. J. Immunol. 75:214,
1955.
S t e r z 1, J.: Prukaz a biologicke vlastnosti prekursoru serovch protilatek. Cs. biologie
4 : 321, 1955.
t e r z 1, J.: The Demonstration and Biological Properties of the Tissue Precursor of Serum
Antibodies. Fol. biol. (Praha) 1 : 193, 1955.
t e r z 1, J., H r u b es o v a, M.: Pfenos tvorby protilatek nukleoproteidov~mi frakcemi
na neimunisovane prijemce. Cs. biologie 4 : 600, 1955.
t e r z 1, J., H r u b e s o v a, Nt.: The Transfer of Antibody Formation by Means of Nucleo-
protein Fractions to Non-immunized Recipients. Fol. biol. (Praha) 2 : 21, 1956a.
t e r z 1, J., H rub es o v a, M.: Tvorba protilatek - model adaptivni proteosynthesy.
Sjezd o bilkovinach 1. 12. 1955b. Cs. gastroenterol. 10 : 228, 1956b.
T a n a k a, A. H.: Studies of Antibody-producing Cells. II. The Agglutinin Formation in the
Bone-marrow of Rabbits. Jap. Journ. Bact. 8 : 193, 1953.
T h o r b e c k e, G. J., K e u n i n g, F. J.: Antibody Formation in vitro by Haemopoietic
Organs after Subcutaneous and Intravenous Immunization. J. Immunol. 70 : 129, 1953
O6pa3OBaHne aHTHTeii II3OJIIIpOBaHHbIM14 i{JieTifaMii Ce.ie3eHKH HOCJ1e
CMeHIeHHH C aHTHFeHOM in vitro
H. IIITEPIUJb
Pe31wge
H IeTKn, H3OJI1JPOBaHHble H3 CeJie3eHK11 B3pOCJIOrO Kpor1HKa n CMemaxllble c aHTH-
reHOM in vitro (S. paratyphi B) o6pa3y1oT allTIlTeiia 1PH BHpbICKHBaHHH B HOJIOCTb
6pIOmHHbl 55-JjHeBHbIM HpoJuHKaM. B ATOM Bo3pacTe KpoJlbgaTa He pearspyloT Ha
BBegenne aHTHreHa o6pa3oBaHIIeM aHTHTeJI.
He Ha6Jno)jaaioCb pa3JIH'3HI3 B o6pa3oBaHHH allTHTeJl B CJlygaax, KOrZja KpOJIbLIaTaM
BBOZjHJIHCb HOCJIe nx cMemelun C alTHreHOM: KJIeTKH HopMaJlbHOrO KpoJIHKa, KJIeTHF1
H3 ceJIe3eHKH Kporrnna, HMMyHH3IIponanHoro HeCneljH4)HwemiM allTHreHOM (CbIBO-
POTKOI3 MOpCKOI3 CBIIHKH) win ane cneI 1l w TecKHM allTHreHOM (S. paratyphi B).
06pa3OBaxne allTIlTeii O6yCJI0BJIeHO OHTHMaablibIM KOJIHLneCTBeHHbIM COOTHOHIe-
HneM CMeCH KJneTOK H aHTHreHa in vitro. V KopnyCHyJIIIpHoro aHTHreHa Mbl Ha
I KJ1eTKy CWJIe3eHKH np 16aBJifieM 2 MHKpo6oB S. paratyphi B. BOJiee 3HanHTeabnLIe
A03b1 axTHrexa IIOJjaB59IOT o6pa3oBaxne allTHTeJI.
Ecnn 143OJI11pOBaHHbie KJIeTHH o6JlyiIalOTCa 860 r, 3T0 He no aBJlaeT o6pa3oBaHHa
alTIlTeJI. 06JIyilenxe KJIeTOK 1000-1200 r HapymaeT o6pa3onaHHe allTHTeJl. Hepe-
Hecexne KJIeTO1 CeJIe3e1KH B3POCJ1OFO KpoJi1Ka He BEI3LIBaCT aKTHBxol peanljnii
Ha aIITHrex y KpoabgaT. Mbl pacCMaTPHBaeM T CTHe peulirHeHTa (MOJIogoro
SKHBOTHoro) B o6pa3oBaHHH allTIlTea KaR nacCHBHoe, - Kan yLIacTHe 6JlaronplllTHOII
KyJIbTHBarjHOHHOH CpC jbI JLJla HepeHOCHMbIX KJIeTOK.
BBeJjeHHble B OpraHH3M KJIeTKH BbnIHBaIOT: o6pa30BaHHe allTHTeJI MOIKHO Bbl3BaTb
1IyTeM . BIlpbICKnBaHHl MOJIOZjbIM }KHBOTHbIM aHTHreHa gepe3 24 H 72 naca HOCJIe
BBegeHna TOJIbKO IIPOMbITbIX KJIeTOK CeJe3eHKH. AlTHTena o6pa3yeTCa H TorAa,
CCJIH KJIeTKH BBOZj}T B HOJIOCTb 6pIOmnxbl, a aHTHrex - B KPOBHHOe pycno.
HpH cMe11HBaHHH HaeTOK c aHTnreHOM npii 37? C CBa3ETBaxxe 34J41eITHBHOro
KoJlrmecTBa axTHrexa KJIeTKaMH HPOHCXOJjHT 6bicTpo. BbIMbIBaHHe OCTaTKOB axTH-
rexa nOCJle 14HKy6aIjlu He HapymaeT O6pa30BaHHa alTHTeJl.
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FOLIA BIOLOGICA
The Significance of Staphylococcal a toxin and Leucocidin
J. JOHANOVSKY
Institute of Biology, Czechoslovak Academy of Science, Department of Microbiology, Praha
Staphylococcal a toxin is almost universally held to be the main factor of the
pathogenicity of staphylococci. This view is based on the similarity of pathological
changes evoked by the injection of toxin and of a living culture in experimental
animals (Burnet 1929), on the relationship between the formation of toxin and of the
pathogenicity for experimental animals (Christie, North and Parlin 1946, Selbie
and Simon 1952), on the parallelism of antitoxic immunity and resistance to experi-
mental infection (Ramon et al. 1936, Downie 1937) and on the presence of a toxin
in the large majority of pathogenic staphylococci of human origin (Schwabacher
et al. 1945, Marks 1952). In the preparation of staphylococcal anatoxin, the strains,
culture media and purification methods are also selected on the basis of the formation
of a toxin (Ramon 1.950, Vygodchikov 1950, Wittler and Pillemer 1948, Turpin
et al. 1954).
It is a striking fact, however, that staphylococcal a toxin haemolyses the erythro-
cytes and kills the leucocytes of various experimental animals, but not those of man,
or only to a very slight degree. Great interest was therefore roused by the discovery
of staphylococcal leucocidin, which differs from a toxin and is specific for human
leucocytes (Panton and Valentine 1932, Valentine 1936, Proom 1937).
An analysis of strains and antibody responses in a somewhat small number of
patients showed a closer relationship between the degree of severity of staphylococcal
infection and the formation of specific leucocidin than the formation of a toxin and
that resistance is associated rather with the presence of antileucocidin than of anti-
toxin.
The work since the communications of Valentine and Butler (1939) has neither
been repeated nor verified. This is partly due to the difficult and tedious method
used for demonstrating specific staphylococcal leucocidin, which consists in the
microscopic evaluation of the damage done to the leucocytes.
A method has been elaborated in our laboratory for the easy demonstration of
staphylococcal leucocidin, similar to that for differentiating live and dead bacteria
by staining (Malek 1954). The quantitative production of toxin and of specific
leucocidin was determined in several hundred staphylococcal strains of varying origin
and a comparison made with the pathological process.
Most of the staphylococcal strains were obtained from routine diagnostic laboratories. All the strains
formed coagulase and agglutinated plasma on a slide. Coagulase negative strains do not form leucocidin.
Toxin and leucocidin were obtained by the cellophane method (Johanovsky 1956). A series of parallel
and repeated production confirmed that this method gives even results in the same strain, with only
slight fluctuation in the titres.
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Specific leucocidin was titrated in the following manner: the same volume (0.1 ml.) of human blood
cells, collected into diluted heparin and washed three times in Tyrode solution, was added to different
dilutions of toxin (1 : 4-1 : 512). After on hour's incubation in a thermostat, 0.1 ml. of 1% Congo red
was added and after three minutes the same amount of 0.2% Nile blue. The blood was examined
without fixation with an objective No. 20 or 45. Live leucocytes are stained pale blue, dead leucocytes
reddish brown; with high concentrations of toxin the leucocytes disappear completely. The living and
dead leucocytes are always counted until an absolutely clear result has been obtained in a given specimen.
As a rule, two counts are sufficient, with determination of 20-25 leucocytes, as the limits of titration
are fairly well defined. A dilution in which most or all of the leucocytes have been killed is regarded as
a titre of leucocidin. Control preparations show over 90% living elements.
This method was tested for reliability and a quantitative comparison made with other methods for
determining the state of the leucocytes. In 120 titrations carried out in 25 specimens, concurring results,
or results differing only within the limits of one degree of dilution were obtained in 96%. This method
was also compared with determination of the vitality of leucocytes by means of a change in reduction
capacity (Jensen and Maaloe 1950), supravital staining of the nuclei (Kredel and Sant 1936) and staining
with methylene blue (tlman 1957). It was established quantitatively that on using this method the same
titres of leucocidin were found as on using the method of microscopic evaluation (Panton and Valentine
1932) and determination of the vitality of leucocytes according to their phagocytic capacity (Puchkov
and Titova 1952).
Antibodies against specific leucocidin were
determined as the dilution of serum neutralising
a working dose of leucocidin. Antigenic potency 30
was determined in vitro according to the ability i
of the antigen to become bound to the corres- 20
ponding antibody and in vivo according to the
development of antileucocidin, as in the previous
work with a toxin (Johanovsky 1956). Titration 10
of a toxin and antitoxin was carried out by the
usual haemolytic method in rabbit blood cells. 0 8 32 128 572
4 16 64 256
320 1280 5120
640 2560 10240
__ ~ =- Illillll~~~
0 320 1280 5120
80 640 2560 10240
0 320 1280 5120
80 640 2560 10240
Fig. 1. Titre of a toxin in strains of varying
origin: I. from pathological material, II. from
the throat and nose of patients with staphy-
lococcal infection, III. from healthy subjects.
x: titre of toxin, y: number of cases.
_ Ill lill ,
M
0 4 8 6 32 64 1286 512
Illlllll~llilllll~llll I'
8 32 128 512
4 16 64 256
Fig. 2. Titre of specific leucocidin in staphylococ-
cal strains of varying origin: I. from pathological
material, IA from surface pathological processes,
II. from the throat and nose of patients with
staphylococcal infection, III. from healthy sub-
jects. x: titre of leucocidin, y: number of cases.
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In staphylococcal strains which produced coagulase, ability to form a toxin and
specific leucocidin was subjected to a detailed quantitative analysis. The strains were
classified in a number of groups according to their origin: I. Strains isolated directly
from a pathological process, II. Strains from patients with a staphylococcal infection
but not from pathological foci (isolated from nose and throat), III. Strains from the
nose and throat of subjects without manifest staphylococcal infection. The result of
this comparison is shown in fig. 1, with reference to the production of a toxin in
a total of 542 strains. The graph shows the number of strains in every group which
attained the given titre of a toxin. It shows that there is no difference between the
groups in the distribution of toxigenicity of the strains; on the contrary, it would
appear that strains from pathological material produce rather less toxin.
The results of determination of specific leucocidin in 476 strains is shown in fig. 2.
For the sake of greater clarity, the strains from pathological material have been
divided into two groups, one from more severe processes (mastitis, phlegmon,
otitis, etc.), the other from surface processes (suppurating wounds, eczema, etc.).
In these an evident relationship was seen. The majority of strains from pathological
processes and patients with staphylococcal infections produce a considerable amount
of leucocidin, whereas the majority of strains from normal subjects produce very
little leucocidin.
A still more convincing result was obtained on dividing the strains from patholo-
gical material into groups, according to the individual morbid processes (tab. 1).
The relationship between the severity of the process and the titre of leucocidin is
marked. For example, there are statistically significant differences between groups
of strains from mastitis, abscesses and eczema (P < 0.001), strains from furunculosis
Table 1. Titres of Specific Leucocidin in Staphylococcal Strains Isolated From Various Pathological
Processes
Condition
No. of
Average
titre of
No. of cases
with titre
cases
leucocidin
0-16
32-64
128--256
256
Osteomyelitis
-_-
pyelonephritis
with metastases
5
294
-
-
2
3
Otitis
11
164
-
5
3
3
Tonsillitis
5
147
-
1
4
-
Mastitis
20
130
1
6
9
4
Phlegmon
7
128
1
1
3
2
Panaritium
4
128
--
1
3
-
Furunculosis
23
90
3
8
9
3
Puerperal
subfebrility
60
64
12
19
21
8
Pemphigus
neonatorum
9
55
1
6
2
-
Abscess,
secondary
suppuration
53
46
13
29
7
4
Eczema
41
37
12
20
9
-
Afebrile puerperium
52
11.6
38
9
5
1
Throat and nose
I
of healthy subjects
146
12
98
24
21
3
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.and eczema (P < 0.01) and strains from eczema and from the throat and nose of
normal subjects (P < 0.001), etc. Particular attention should be paid to the eighth
and penultimate groups; these are staphylococci cultured from the vagina in the
first days after delivery and are divided into two groups according to whether
a postpartial subfebrile condition of staphylococcal origin did, or did not, develop
(~ebek, Schubert and Johanovsky 1957). It is evidentjthat precisely those strains
which are capable of producing leucoci-
din evoked infection of the genital tract
with a febrile course (P < 0.001). 251
Fig. 3 gives the collective results
for all strains isolated from patients
with staphylococcal infection and from 20
healthy subjects. The typical course of
the two-peak curve in strains from
healthy subjects shows that the mate-
rial is not homogenous. Staphylococci
from healthy subjects have a leucocidin
i
d
r
s
titre < 1 : 16 in more than twu-th
of the cases, while the curve from the
ether strains shows the same course as
that of the strains isolatea from patho-
logical material, with the maximum
titre between 1 : 64 and 1 : 128. 0
The results justify the view being
taken that specific leucocidin plays an Fig. 3. Titres of specific leucocidinin 6staphylococ-
appreciable role in the pathogenesis calstrains isolated from patients with staphylococ-
of staphylococcal infections. We have cal infection and from healthy subjects. Strains
therefore dealt with it from the aspect from patients: full line. Strains from healthy sub-
of the preparation of toxoid. A brief jests: dashed line x: titre of leucocidin, y: number
account of the results so far obtained of cases in / .
is given below.
It was confirmed that specific leucocidin can be produced in semi-solid media of
varying composition, but higher titres (sometimes decuple) were obtained with
production on cellophane (Johanovsky 1956). Leucocidin is an antigenic substance;
it can be detoxicated with formol under similar conditions as with a toxin. With
suitable detoxication (0.2-0.3% formol after several days), antigenic loss does not
occur.
In immunisation in rabbits, following several injections of toxoid prepared from
strains with a high titre of leucocidin, antibodies develop which neutralise several
dozen to several hundred minimal doses of leucocidin. Varying amounts of antibodies
are also found together with antitoxins in the blood of patients with staphylococcal
infections or who are convalescing from such infections. Antileucocidins and anti-
toxins do not influence one another in their action on corresponding toxins. In the
absence of a standard serum it is suggested that the amount of antibodies required to
neutralise 100 minimal doses of leucocidin should be taken as one unit of antileucocidin.
The results show an unequivocal and statistically significant difference between
the titres of leucocidin in staphylococcal strains from normal human carriers and
strains from morbid processes. Elevated titres of leucocidin are also found in strains
from the throat and nose of patients with a staphylococcal infection; of these, it is
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already known from serological and phago-typization analyses that they belong
for the most part to the same strain as that in the actual infectious process (Hobbs,
Carrunthers and Gough 1947, Valentine and Hall-Smith 1952, Parker, Tomlinson
and Williams 1955). In addition, a marked relationship was demonstrated between
the titre of leucocidin and the degree of severity of the clinical condition. This
parallel is not, of course, absolute, but it is clearly marked and the differences found
are statistically highly significant. These results confirm the original findings of Panton
and Valentine (1932) in considerably greater detail and on far more extensive material.
It is also necessary to consider whether the method used can provide an answer to
the question of the pathogenic significance of staphylococcal leucocidin. Specific
staphylococcal leucocidin is a substance which is selective for human leucocytes.
Its action on the white blood cells of the rabbit, guinea-pig and rat is relatively
about five times weaker as compared with its action on human blood cells. For this
reason the significance of specific human leucocidin can only be evaluated by an
analysis of material from infections in man and not by animal experiments, in
which its action is not sufficiently manifest and is concealed by the toxic effect
of a toxin.
Staphylococcal a toxin plays a decisive role in experimental infection in animals.
It is also of unquestionable significance in acute hypertoxic staphylococcal sepsis
(Burnet and Kellaway 1930, Kleiger and Blair 1940), but its role in banal forms of
staphylococcal infection is not yet sufficiently clear. It was found that titres of a toxin
are substantially the same in strains isolated from patients and from healthy sub-
jects. This concurs with individual data in the literature on the low production
of a toxin in strains from clinically severe infections (Mlcochovd 1941, Blair 1939,
Lack and Wailling 1954) and with the communications of other authors (Stevans
and Cort 1926, Tager and Hales 1947), who were unable, in a large number of strains,
to find a connection between the amount of toxin and coagulase produced and
clinical pathogenicity. Contrary findings are not very convincing. Marks (1952) de-
termined only the extent of the haemolytic zone around grown colonies, Jackson and
Dowling (1955) assess the pathogenicity of a strain according to whether they
cultured it from the upper or lower part of the respiratory tract in patients with
poliomyelitis.
There are two possible ways of putting these results to practical use: 1. from the
aspect of diagnosis, i. e. of the differentiation between coagulase positive strains of
different pathogenicity, and 2. in preventive immunology. Increased production
of leucocidin indicates to a fair degree of certainty the infectious origin and thereby
probably the virulence of a given strain. Only 13 of the strains of infectious origin
had a leucocidin titre lower than 1 : 16. The finding of leucocidin titres in strains
isolated from healthy subjects can be explained, with reference to the two-peak
curve (fig. 3) on the assumption that the majority of "carrier" strains produce very
small amounts of leucocidin and that some of the subjects investigated are carriers
of extremely virulent staphylococci with the same properties as the strains isolated
directly from pathological processes. This assumption could be confirmed by
epidemiological observations, e. g. phage-typing. The finding that an elevated
leucocidin titre helps to identify carriers of dangerous virulent staphylococci among
hospital personnel, for example, would be of exceptional practical importance.
The second, and still more important question, is that of the utilization of these
findings in immunisation practice and in the preparation of immunological pre-
parations (toxoid, antiserum). Strains, media and methods of the purification and
evaluation of staphylococcal, preparations are at present selected on the basis of
a toxin production. In our opinion, the results described above show that specific
leucocidin merits the same degree of consideration.
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Summary
A method for the simple demonstration of leucocidin specific for human blood cells
was elaborated, by means of differential staining of killed and living leucocytes. The
titre of specific leucocidin was determined in 476 strains, and the titre of a toxin
in 542 strains, all of different origin.
In the case of a toxin there is no difference in the titre in strains of different origin
In the case of specific leucocidin, strains isolated from a pathological process form
far more leucocidin than strains isolated from healthy subjects. There is a statisti-
cally significant correlation between the titre of the leucocidin and the degree of
severity of the condition evoked by the strains in question. The characteristics of
the strains isolated from healty subjects show that this is a non-homogenous group,
which contains both banal carriers of only slightly pathogenic staphylococci and
also carriers of strains with the same properties as those isolated from infections.
These results demonstrate the possibility of diagnosing particularly virulent and
dangerous strains of staphylococci according to the production of leucocidin. When
preparing a toxoid and staphylococcal serum for therapeutic purposes, the strains
and also the methods should be selected not only according the titration of a toxin
but also according to the production of specific leucocidin.
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D o w n i e, A. W.: A Comparison of the Value of Heat-killed Vaccine and Toxoid as Immunis-
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H o b b s, B. C., C a r r u t h e r s, H. L., G o u g h, J.: Sycosis barbae: Serological Types
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II : 572, 1947.
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Ramon, G., R i c h o u, It., 1) j o u r i c h i t c It, M.: Sur le mechanisme de 1'immunit6
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Identification of Pathogenic Staphylococci. Brit. J. Exp. Path. 26 : 124, 1945.
S t e v a n s, F. A., C o r t, L.: Toxicity of Filtrates of Staphylococcus aureus from Human
Infections and from Normal Nasopharynx. Proc. Soc. Exp. Biol. Med. 24 : 392, 1926.
e b e k, V., S c h u b e r t, J., J o h a n o v s k y, J.: V~znam stafylokokove vaginalni
infekce pro vznik poporodni subfebrilie. Cs. gynaekologie 1957 (in press).
T a g e r, M., H a I e s, H. B.: Quantitative Coagulase and Toxin Production by Staphylo-
cocci in Relation to the Clinical Source of the Organism. Yale J. Biol. Med. 20 : 41, 1947.
Turpin, A., Relyveld, E. H., Pillet, J., Raynaud, M.: Purification de la
toxine et de l'anatoxine staphylococcique alpha. Ann. Inst. Pasteur 87 : 185, 1954.
V a l e n t i n e , F. C. 0.: On the Role of the Toxin in Staphylococcal Infections. Lancet
II : 526, 1936.
V a l e n t i n e, F. 0. C., B u t t e r, E.C. B.: Specific Immunity in Acute Staphylococcal
Osteomyelitis. Lancet II : 973, 1939.
V a l e n t i n e, F. C. 0., H a 1 1- S in i t h, S. P.: Superficial Staphylococcal Infections.
Lancet I : 351, 1952.
\V i t t 1 e r, R. G., P i. 1 1 e in e r, L.: The Immunochemistry of Toxins and Toxoids.
V. The Solubility of Staphylococcal Toxin in Methanolwater Mixtures under Controlled
Conditions of pH, Ionic Strength and Temperature. J. Biol. Chem. 23 : 174, 1918.
H y 11 is o a, H. B., T H T o B a, C. M.: Moljn Hlcaijlla McTo a ,jJIH n3yueunn iarOI iITapHOi3
aI;THBHOCTH JIeHuoITHTOB. ( ruauo.I. HCypHa.7 38 : 456, 1952.
B bI r o Jj q H it o n, F. B.: Misnpo6HOJIorna u HMMyHOJIOFHH CTa(HHJJOFiORhcoB b[x 3a0OJJeBaHIII.
MoCKBa 1950.
3HatIeH11Ie CTaf HJIoIco1 IfoBOro aJlb(a TOKCHHa H JIeRl{OL HAHHa
10. 13OFAHOBCHHp1
Pe3JO.,ne
BbiJi paapa66OTaH yJio6Hbiii MCTOJj Olipe iOJIeHHH CTa(1)HJIOnoICICoBOro -TeiluoTgii(HHa,
CIIeIlH(J)HTIecKoro ;17TH KpOBHH:6IX TeTleIL IIeJIOBena, - C IIOMOHWIO Jjll (~Opoiigi JIbuOIi
onpaciai >JHBbIX 11 y6HTbiX JIevIKOIjlTOB. THTp CHeIlH(j uecnOro JIenKoJJHJjnHa 6bIJ1
onpeiteneii y 476 KoaryJIa3a-H03HTTiBHbIX IIITaMMOB pa3JIH1IHOr0 HPOHCXOUu jeHIIH,
?rlTp auib(fa ToHCHHa - y 542 IHTaMMOB.
Y aJlb(a TOICCHHa HOT pa3HHljbi B THTPO V IHTaMMOB pa3JI111iHOrO HPOHCXo KJ OHHH.
y CIIeIjH(HueCKOI'o JIenKOIuHAHHa mTaMMbI, BbIJjeneHHble B Te1ICHHe HaT050PH1ICCnOro
Ilpouecca, o6paayI0T ropa3Jjo 6ownbme neHnouuAnHa, 1TeM IHTaMMbI, H3OJIHpOBaHHbIO
OT 3 (OpOBbIX JIHIj. CynlecTByeT CTaTHCTH1IeCKH 3Ha1IHMaH ISOpJOTIAIjHH MC)KJjy THTI)OM
JIeHnOIrHJjuHa H CepbO3HOCTLIO 3a6oJIOBaHHH, BbI3BaHHOrO JjaHHbIM HITaMMOM. XapaK-
TepHCTHKa HITaMMOB, BbIJ[CJICHHbIX OT 3JjOPOBbIX JIHIj, noKa3blBaeT, uTO pe11b IIJtOT
o HeoJjHOpOJjHOH rpvIIIIe, BKJIIotialOHjef Kan 6aHaJIbHOe HOCHTenbCTBO MaJIoIIaTO-
rCHHbIX CTa(HJIOKOKnOB, TaK It HOCHTenbCTBO IHTaMMOB TCX SICe Ka1IeeTB, Kan H HITaMMbI
HH(JeKi5HOHHOro HpOHCXO?ICJjeHHH.
3TH pO3yJIbTaTbI OTHpbIBaIOT BO3MOuJHOCTb JjnarHOCTHuecKoro OHpCJjCJIeHHH oco6o
BHpyJICHTHbIX H OHaenmx IHTaMMOB CTa(HJIOKOKKOB no o6pa3oBaHHIO neln o IJjHHa.
HpH HpHrOTOBJIeHHH BaHUHHbI H Jie1Ie6H0ii npOTHBOCTa(HJIOKOKKOBOH CbIBOpoTICH
Heo6XOJjuMo iTo j6HpaTb IHTaMMbI ii pa6o'nie IIPHOMbI He TWIbRO Ha oCHOBaHIiH
THTpaiuHH aJlb(a TOKCHHa, HO H no o6pa3oBaHHI0 CHeIjH()HgeCHoro JIeAROIAR AHHa.
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FOLIA BIOLOGICA
Metabolic Products During the Growth of Clostridum acetobutylicum
J. DYR and J. PROTIVA
Technical University, Department of Fermentation Chemistry and Technology, Praha
The process of acetone-butanol'fermentation brought about by Clostridium aceto-
butylicum has long been subjected to detailed study by microbiologists, fermentation
technologists and biochemists. In summing up the findings, however, it is seen that
the whole process, although technologically controlled to a certain extent, has not
been so thoroughly investigated from the microbiological and biochemical point of
view as many other fermentation processes. This is due mainly to the experimental
difficulties of fermentation on a laboratory scale and to its complexity, involving
qualitative changes in the organism in the course of fermentation.
After a great deal of research work on the general laws of living Nature, Soviet
investigators began to study the physiological development of microorganisms.
In this connection they tried to resolve several questions of successive stages of
acetonebutanol fermentation. This was chiefly done by Jerusalimsky (1946, 1951)
who dealt with the physiology of butyric acid bacteria and of Cl. acetobutylicum,
which produces neutral solvents.
So far nothing is known of the conditions causing the transition of a culture
of Cl. acetobutylicum from the first stage to the second stage of fermentation, or of the
causes of the changes in the physiological properties of the organism. In the world
literature, with the exception of Soviet literature, no particular attention has been
paid to this problem. The above mentioned communications of Jerusalimsky
explained these changes on the basis of individual development during fermentation
of the culture.
The subject of the present study is this transition from the first into the second
stage. At the same time attention is centred on the conditions and state of the
external environment and on the state of the culture producing fermentation.
In our experiments an industrial culture of Cl. acetobutylicum, strain Ca 3, isolated by Dyr in 1946,
was used. Stock cultures were prepared on a mixture of one part soil and one part sand. Before sealing,
the tubes were dried over calcium chloride; they were then sealed with paraffin wax and stored in
a refrigator at 4? C. Vegetative seed cultures were taken from the same stock for all experiments.
The experiments were carried out on a complex fluid medium, a modification of Speakman's mixture
of salts: K2HP04 0.1%; MgSO4. 71120 0.02%; FeSO4.71120 0.001%; MnSO4.41120 0.001%;
NaCl 0.001 %. To a 750 ml. solution of these salts 200 ml. of tuber water from 120 g. potatoes, 40 g.
glucose, 4 ml. yeast autolysate according to Weitzmann and 5 g. corn-steep (55% dry weight) were
added. The volume was made up to one litre.
The tuber water was prepared from potatoes in the following way: The juice of finely grated potatoes
was removed by pressing and the press cake washed with water and pressed repeatedly. The tuber water
and the washings were mixed. The starch was removed by centrifuging, the heat-precipitable proteins
by heating in a water-bath for 30 minutes and by centrifuging. The clear brown liquid was used for
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preparing the medium, the pH of which was adjusted to 6.0 before sterilization. The medium was
autoclaved under a pressure of one atmosphere for 30 minutes, then rapidly cooled by a stream of cold
water to 35 - 38? C and immediately inoculated from a 20-hour-old vegetative culture. The volume of the
inoculum amounted to 2% of the final volume of the medium.
A test-tube containing potato mash to which a small amount of soil and sand had been added was
used in preparing the inoculum. The test-tube (16 X 180 mm.) was subjected to heat shock for 120 sec-
onds, cooled and incubated at 37-38? C. Samples were taken during the fermentation by means of
a glass rod extending into the medium and with an overpressure of sterile carbon dioxide.
Butanol and ethanol were estimated by the method of Johnson (1932), acetone by the iodometric
method of Goodwin (1920). Butyric and acetic acid were determined in the alkaline residue following the
distillation of neutral products in the same way as butanol and ethanol but omitting the oxidation
process. A solution of 73.4 g. K2SO4 in 1000 ml. 5N H2S04 was added to the samples in the distillation
tube, instead of the oxidation agent. The distillation constants for butanol and ethanol were experi-
mentally determined for purposes of calculation by the method of Johnson (1932). Glucose was estimated
by the method of Shaffer-Hartmann (1921). The solids of the microorganisms were determined by centri-
fuging 25 ml. of the culture medium, washing with distilled water and drying at 105? C.
Titratable acidity was determined by titrating 10 ml. of the centrifuged medium with 0.1 N KOH,
with phenolphthalein as indicator.
pH values were measured by means of a quinhydrone electrode on a Multoscop II electronic pHmeter.
The redox potential was measured by means of a platinum electrode on a potentiometer, with a galvano-
meter as a zero indicator (L. Schildknecht, Zurich). A saturated calomel electrode served as the standard
electrode. The measurement was made at
30? C. When taken from the fermentation
vessel the samples to be measured were
already covered with a layer of compact
foam which prevented contact of the liquid
with the air.
Fig 1. Fermentation Curves for Cl. acetobutylicum, strain
Ca 3. Curves: 1 - gas, 2 - butanol, 3 - acidity, 4 - ace-
tone, 5 - glucose, 6 - ethanol. Ordinate: hours of
fermantation, abscissa: I - fermentation gases 1/1 me-
dium, II - acidity, III - glucose mg/ml., IV - neutral
solvents mg/ml.. 4
The experimental part of the pre-
sent work was commenced by ob-
serving the course of the fermenta-
tion curves for the culture of Ca3
grown on the medium described. It
was essential to find out whether
the course of cultivation and the
end-products were similar to those
with a natural medium. The results
of one of the experiments are given
in fig. 1. The 60-hour fermentation
time does not exceed the usual time
for laboratory-scale experiments
with small volumes. The curve for
titratable acidity, illustrating the
accumulation of organic acids, mostly
volatile, had a normal course with
two inflections. The total yield of
solvents in actual production is approximately 32%, calculated by the sugar fer-
mented. In our case, after correcting for the products introduced with the inoculum,
the yield amounted to 30 ? o, calculated by glucose fermented.
The positive results of this experiment enabled investigations to be carried out on
the differences in the metabolism of Cl. acetobutylicum in the first and second stages
of fermentation. These differences can be estimated only approximately, from the
course of the acidity curves and of those of neutral products.
From the differences in the results of analysis of the culture media before and after
incubation, information on the biological activity of the culture was obtained in
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Age of culture 10 hr.
Age of culture 24 hr.
Mg./ml.
before
after
difference
before
after
difference
incubation
incubation
incubation
incubation
Glucose
30.40
22.65
- 7.75
20.90
13.87
- 7.03
Acetic acid
2.44
2.19
- 0.25
2.14
2.58
+ 0.44
Butyric acid
0.62
2.35
+ 1.73
0.62
0.01
- 0.61
Butanol
0.18
0.80
+ 0.62
2.66
4.14
+ 1.48
Acetone
0.19
0.27
+ 0.08
0.90
1.35
+ 0.45
Ethanol
0.55
0.79
+ 0.24
0.92
1.74
+ 0.82
Acidity
2.80
-
-
3.25
-
-
Bact. solids
-
4.65
-
-
5,65
-
a very short time; in this way the factor of a continuous change of metabolism in the
transition from the first stage to the second stage was eliminated. A comparison of
the data for biochemical activity in the first and second stages is given in tab. 1.
From this table it follows that the cells of C. acetobutylicum differ at various stages
of fermentation in their metabolic products, especially in the proportion of butyric
and acetic acid.
The authors were greatly interested in the environmental conditions of the culture
during its development, which assimilates and changes the medium by means of its
metabolism. It was decided to influence or alter the individual variables of the
medium and thus ascertain the conditions which cause (or contribute to) transition
Fig. 2. Correlations between the environmental
conditions and the growth of Cl. acetobutylycum.
Curves: 1 - acidity, 2 - bacterial solids, 3 - pH,
4 - En (30?). Ordinate: hours of fermentation,
abscissa: I - bacterial solids mg/ml., II - acidity,
III - pH, IV - Eh (30?).
of the culture into the second, productive,
stage of fermentation.
A series of similar experiments was
undertaken in which a study was made of
the values of the redox potential and pH,
the formation of acids and the develop-
ment of bacterial substance in the first
and second stage. The inflection in the
curve of titratable acidity was taken as
indicating the transition to the second
stage of fermentation. Flasks, each con-
taining 1,500 ml. of fluid medium, were
inoculated with 30 ml. of a 20-hour vegeta-
tive inoculum. The titratable acidity, pH,
Eh and the bacterial dry matter were estim-
ated during fermentation. The course and
correlation of the curves shown in fig. 2 are
in full accord with the other parallel experi-
ments. In every case a change in titratable
activity occurred between the 12th and 16th
hour of fermentation. This is also in agreement with the pH curve, which, after an
initial fall, is maintained at the same value during the second stage. The Eh curve
falls rapidly during the first hours to negative values and reaches the minimum
before the acidity curve reaches its peak. In the subsequent course of fermentation
it rises only slowly. The course of the curve for bacterial dry matter is very interest-
ing. In every case it had a relatively sharp peak and always reached a maximum
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before the sharp change in the acidity curve. This means that the bacteria proliferate,
and the bacterial matter increases, only in the first stage of fermentation. No great
changes, capable of altering the physiological activity of the culture, occurred in the
other variables measured, apart from the curve for the solids and titratable acidity.
It was necessary to ascertain whether the transition of the cultures into the second
stage depends on the accumulation of a certain quantity of free acids or whether the
formation of neutral solvents begins, for reasons of energy, only when the culture
+3C0
+200
+ f00
0
-100
Fig. 3b. Formed acids neutralized by calcium
carbonate.
Fig. 3a and 3b. Effects of Bacterial Solids and Titratable Acidity on the Transition of Cl. acetobutylicuna into
the "reducing" Stage. Curves: I - acidity, 2 - bacterial solids, 3 - pH, 4 - Eh (30?), b - acetone. Ordinate:
hours of fermentation, abscissa: I - acetone and bacterial solids, mg/ml., II - acidity, III - pH, IV - Eli
(30?).
ceases to synthesize living matter and to proliferate. In order to do this the course
of the curves for titratable acidity and bacterial solids were dealt with separately in
the next experiments by neutralizing part of the organic acids formed in the early
hours of fermentation. In the first experiment sodium carbonate was used, which
was added three times during the first ten hours of fermentation. In further experi-
ments a slight excess of calcium carbonate was added, which takes up the fatty acids
being formed until approximately the tenth hour of fermentation. The results of both
experiments were indentical.
The results of the experiment with the neutralization of the acids being formed
by means of an excess of calcium carbonate are given in fig. 3a and 3b together with
the controls. In this experiment the formation of acetone was observed as the
typical metabolic product of the second stage, as shown above. In this experiment
a correlation was demonstrated between the value of titratable acidity, the growth
of the organism and acetone formation. In the control experiment, the formation
of acetone started when the amount of solids had reached a maximum, i. e. at the
time when volatile acids were still increasing in the medium. From this alone, it
could be deduced that the inflection in the acidity curve did not synchronize with
the transition phase. This deduction is supported by the correlation of these values
during fermentation, in the initial presence of calcium carbonate. In this case, the
break of titratable acidity occurred in about the 26th hour, whereas the curve for
solids reached its height between the 14th and 18th hour. Acetone formation, typical
for the second stage of fermentation, starts from the 14th hour and is obviously
dependent on the transition of the culture into the stationary stage.
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 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
In the present work the correlation between the growing bacterial matter of
Cl. acetobutylicum, the change in the reaction of the medium and the production
of oxidized and reduced forms of metabolites was investigated. Attempts were made
to determine the conditions required for the transition of cultures from the first
"acid" stage to the second productive stage, chiefly for large scale production.
From the biological activity of the culture in the first and second stage a picture
was obtained of the changes in the microorganic enzyme system during fermentation.
If acetic acid is taken as a butyric acid precursor (which has been demonstrated in
a number of papers, Wood, Brown and Werkmann 1945, Davies 1942), it is obvious
that the accumulating acetic acid is the principle metabolic product of the early
hours of fermentation. As late as the tenth hour the ratio of acetic acid to butyric
acid is in favour of acetic acid; at this time, however, acetic acid is rapidly converted
into butyric acid, as seen by the decrease in the total amount of acetic acid and the
considerable increase in butyric acid after the two-hour incubation of a concentrated
culture. The other path of acetic acid conversion, i. e. to acetone, is very slow at this
stage. Butanol and ethanol production, however, takes place to a certain extent
before the break in the acidity curve. In the second stage of fermentation, neutral
products, especially butanol, are formed in larger amounts. Consequently butyric
acid formation is more rapid than that of acetic acid. On the other hand acetic acid
accumulates offering a source for acetone or ethanol formation. It is evident that
acetone is the only characteristic product of the second stage. Ethanol and butanol
were produced even in the first "acid" stage of fermentation but only in small
amounts. The increasing production of acetone is the indication of the transition of
the culture from one stage to the other. Our experiments with the neutralization
of acids accumulating in the first stage show that the amount of free acids and
consequently, to a certain extent, even the change in pH values are not decisive for
the transition of the culture to the second stage. Changes in pH, if sufficiently great,
may exert a considerable influence on the rate of the individual partial reactions.
The changes actually measured in pH values cannot, however, account for the
appearance of the ability to form acetone in the second stage of fermentation. The
same applies to the redox potential; changes in this seem to be associated with the
rapid proliferation of the microorganisms, as suggested by Hewitt (1950). The redox
potential is, for strict anaerobes, rather a limiting factor in the early hours of growth
in a fresh culture medium. The cause of the change in metabolism is, therefore, not
the shift in the balance of enzymatic reactions converting the central metabolic
products of Cl. acetobutylicum (according to recent findings, acetic acid and its active
form) into butyric acid or acetoacetic acid. For this reason attention was directed
to the proliferation of the culture, where the course and, in particular, the termin-
ation of the logarithmic phase, was synchronous with the time of transition and the
commencement of acetone production.
In earlier work of Peterson and Fred (1932) and more recent work of Jerusalimsky
(1946) the curve of the number of microorganisms in the course of fermentation, was
plotted. Hitherto, however, the growth of micro-organisms has not been considered
in causal relationship to the stage of acetone-butanol fermentation.
Summary
Studies in the physiological activity of Cl. acetobutylicum in the first and second
stage of acetone-butanol fermentation showed acetone to be the product characteris-
ing the transition of the culture into the production stage.
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The transition into the second production stage follows the completion of prolif-
eration and the growth of cells in the culture, independent on the accumulation
of free acids in the medium.
The change in the curve for titratable acidity, generally associated with the
transition into the second stage, is, according to the experimental results presented,
due to a change in the physiological activity of the culture after the completion of its
growth and proliferation.
D a v i e s, B.: Studies on the Aceton-butyl alcohol Fermentation. 2. Intermediates in the
Fermentation of Glucose by Cl. acetohutylicum. 3. Potassium as an Essential Factor in the
Fermentation of Maize Meal by Cl. acetobutylicum. J. Biochem. 36 : 582, 1942.
D y r, J.: V~rroba organickych rozpustidel kvasnou cestou. Sbornik prednasok, V. Last, 59,
1954, Banska Stiavnica.
G o o d w i n, L.: Modification of the Messinger Method for Aceton Determination. J.Am. Chein.
Soc. 42 : 39, 1920.
H e w i t t, L. F.: Oxidation-reduction Potentials in Bacteriology and Biochemistry. Edin-
burgh 1950.
J o h n s o n, M. J.: Determination of Small Amounts of Ethyl- and Butylalcohol. Ind. Eng.
Chem. 4 : 20, 1932.
P e t e r s o it, W., F r e d, E. B.: The Butanol-Aceton Fermentation of Cornmash. Ind. Eng.
Chem. 24 : 237, 1932.
S h a I f e r, P. A., H a r t m a n n, A. F.: The Iodometric Determination of Copper and
Its Use in Sugar Analysis. 11. J. Biol. Chem. 45 : 365, 1921.
W o o d, H. G., B r o w n, B. W., NV e r k in a it n, C. H.: Mechanism of the Butylalcohol
Fermentation with Heavy Carbon Acetic and Butyric Acids and Acetone. Arch. Biocheln.
6:243, 1945.
H e p y c a Ji n nI c x n ii, H. A.: 0 (fin3uoJlornuecicnx CTajnnx B pa3BBTnn 6axTepnfl. Mu);po-
6nonorgx 15 : 406, 1946.
H e p y c a ji in ,u c in n ii, H. AT.: OHTOreHeTngecHoe pa3BnTnn xyn6Typrr MacJlnno);nc.ni,Tx 6au-
Tepnr3. Miii po6nonornx 20 : 204, 1951.
HpOAyHTbl o6MeHa Beu~ecTB B TegeHne pOCTa Clostridium acetobutylicum
H. JbIP in 10. HPOT14BA
Pe310.11C
B HacTORtge14 pa6OTe Mid necaeJjoBaJIH CooTHOITIeHMH MC51iJjy POCTOM H pa3MHO-
7ueH14eM MHKpo6a Clostridium acetobutylicum, 143MO110HH9MR yCJIOBHII cl)eJjbl
ii o6pa3oBaH11eM OKIICJIOHHbIX 14 BOCCTaHOBJTeHHbIX (1)OPM MCTa60aHTOB. Mm CTpe-
MHJIHCb Ol1PCjjCJTIITb yCJIOBHFI, Heodxo Mble inn ITepexo is KyJIbTypbI Cl. aceto-
hUtylicnm OT IIepBOH, (), KO BTOpOH, KB0CCTaHOBHTeJIb11OH1), (Tia3e aILCTO-
6yTaHoJloBoro 6POHF 01 HR.
Y13ym,1B C HOMO11jb10 KOHJJCHTPHPOBaHHOH B3BOCLI MHxpo6a 6HOXHMLIuCCKyJO
JlORTeJIbllO('Tb KyJIbTVpbl B pa3JI111HbIX (J)a3ax 61)o}TneHnn, Mbl TIWIy~IH.;IH Kap'ri1Ty
I13MCHCTll11.1 CITC'TCMbF C1'0 311311MOB. 143 pe3yJIbTaT0B 0111,TTOB OtIeBHjuIO, TITO B TCgCHHC
IICPBbIX ~TaCOB 6pO>IieHHH p03yJIbTaTOM O6MeHa BCHICCTB HBJIHCTCR I1pOiiiJ[e 11CCr0
HaKOTIJ1CHIIC y'KCyCHOH K11CJIOTbI. lloajiee, HO eIlte Jo uepexoia T;ylb'rvpbl I; (BOC-
('TaHOBHTCJTbHOR)) 4)a3e, yKeycliaR KHCJIOTa 6bICTp0 11peBpaIIjaeTCR B Ma(JIRIIyIO,
~ITO HPH Hai mix 011bTTax IIp0RBJIHJTOCb 3HatJ11TCJIbHOH y6bIJIbIO ylcycHOH KHC TOTbi
It yBeJIHTICHI1CM KOJIHileCTBa MacJIRHOH TCITCJIOTbI B Tcneune 2-naco6011 IIH1iy6aljnli
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 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
HOHueHTpIIpoBaHHOI3 B3BeCn 10-9aCOBOII KYJIbTypbl Mnnpo6a. BTOpon CII0CO6 npe-
BpalueHnH yKCyCHOI3 KIICJIOThI - ee npespalueHHe B aueTOH - ocylueCTBJIRJICfi
TOJIbKO BO BTOPOII 4)a3e OAHOBpeMeHHo c o6pa3oBaHIIeM 3Ha9IITeJlbllbIX KOJIWJOCTB
6yTaHOJIa II 3TaHOJIa. 113 pe3yJlbTaTa OIIbITOB BI,ITeHaeT, 9TO eJj$HCTBeHHbIM npo-
JjyKTOM, XapaiTepn3yloujrM o6MeH BeujeCTB y C1. acetobutylicum BO BTOPOH ()a3e
6po5KeHna, BBJIHeTcq aueTOH.
IIpi HeIITpaJTII3aIjun HIICJIOT, CKOIIJIHIOMHXC$ B Te9eHne IIepBOl (a3bI 6poxHeHHH,
MM lOJIytIIIJIH TaHHe B3auMOOTHomeHlr OT)je hHbIX ncciieZjyeMbIX BeJIIILInH, KOTOpbIO
JjoKa3bIBaIOT, 9TO upII9nHOII II3MeHeHIIa McTa6oJIIIBMa III lIepexojje KyJlbTyl)bi
BO BTOpyJO 4a3y 6pOH eHna He HBJlaeTCH IIpOCTOe 113MeHeHIIe CKOpOCTen II}OTeKaHnf
OTJjeJlbHMIX peaKunn npeBpaweHna 1jeHTpaTIbHOrO IIpOZjyKTa o6MeHa BeIueCTB
Cl. acetobutylicum (HO nOBeliInuM B3rJlajjaM, ycHyCHaa KIICJIOTa maH ee aKTHBHag
(I)opMa) B MacJIaHYiO IIJIIf aueTOyxcycnylO KHCJIOTy.
jHo3TOMy MbI cocpeJjoTOLIwnn CBoe BHIIManne Ha OCT pn pa3MHO?HeHne KyJlbTypbl;
TC9eHIIe n B oco6ennocTn OKOH9aHIIe JlorapH(mH IecHOII (2a3bl OTBegarIo (npII HaIHHx
onbITaX) BpeMOH11 nepexoZja OT OjLHOl (a3bl H Jjpyroli 11 HaLIaJIa o6pa3OBaHII9 aueTOxa.
Mbl noJIaraeM, 1ITO IIpl3ilnHoI3 HanonJlexna 3HepreTnqecnn 6eJjHbIX BemeCTB (oxncJIeH-
TIMe ()opMbl) B Tegenile nepBOK 4a3b1 6po5KeiiiH BBJIaeTCR noB ImeHIIOe ll0Tpe6JieHIIe
3Heprnn JT lI crHTe3a KJieTOiinoro BeIueCTBa, HOJI1TUeCTBO KOTOpOPO B Tegenile
HKic ioII>) c a3b1 6p0B eHn} B03paCTaeT Jlorapn4Mw1ecKl. ToJIbnO nOCJie oxoHUaHnl
pa3MH0?KeHna II pOCTa KYJIbTypbl HaCTyIIalOT n3MeHeHIIa B o6MeHe BeujeCTB H BTOpaa
c a3a 6p0xceHHa, XapanTepII3ylollja}Ca o6pa3OBaHIIeM BOCCTaHOBJIeHHbIX McTa6o-
JIIITOB, 3TIIJIOBOPO CrIIpTa II 6yTaHOaa.
1103TOMy I'JIaBHbIM yCJIOBneM yCIIeIHHOCTH aljeTOH-6yTaHOJIOBOr0 6poxceHlla MbI
C9IITaeM o6eciieLIeHne oIITUMaJlbHbIX YCJIOB1 ZLJIa 6bICTpOTO pa3BIITna KyTIbTypbl,
B oco6eHHocTn me oT6op aKTIIBHLIX, 6LICTpo pa3MHo Haloujnxca npOZjyHTIIBHNNX
IIITaMMOB C1. acetobutylicum.
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FOLIA BIOLOGICA
The Influence of Ions on the Formation of Protease by the Actinomyces
Streptomyces griseus
J. CHALOUPKA
Institute of Biology, Czechoslovak Academy of Science, Department of Microbiology, Praha
Ions are important regulators of metabolic processes. They may activate or
inhibit enzymes and influence permeability, etc. Biologically, the most important
cations are K+, Mgt+, Ca2+ and Fe2+. Potassium and magnesium ions are essential
in the formation of protein and growth processes (Cannon et al. 1952, Frost and
Sandy 1953, Foster and Heiligman 1949, Friedman and Fox 1954, Webb 1948, 1949,
1953). Investigations were made to ascertain whether it is possible to influence
growth of a culture and the formation of protease by means of the various ions which
are a normal component of the nutrient medium.
The culture, method of cultivation and of measuring proteolytic activity in the culture fluid and the
dry matter have already been described in a previous communication (Chaloupka 1956a, b).
Culture media: Waksman medium B (WB, containing ions K+, Na+, Mgt+, Ca2+ Fe2+ and Zn2 ).
Standard Z media, composition: peptone 2%, glucose 2%, acid ammonium phosphate (separately
sterilised) 0.1%. Medium Z 1/2, containing 1.0% peptone, 1.5% glucose and 0.1% ammonium
phosphate. The nutrient media were sterilised twice at 110? C and the pH at the commencement of
.culturing was 7.1-7.3.
The ions under investigation were always used in the form of chlorides (with the exception of Fee}
where FeSO4 was used) and were always sterilised separately. The reagents were always analytically pure.
Methods. In long-term experiments, 48-hour-old cultures of actinomyces grown on a Z standard
medium were inoculated into a Z 1/2 medium containing the appropriate amount of ions, in amounts
of 1.0 ml./100 ml. nutrient medium in 500 ml. flasks. The flasks were then incubated on a shaker and the
dry substance and protease activity in the culture fluid and in the cells were determined at given
intervals.
In short-term experiments, a 48-hour-old culture grown on a Z medium was centrifuged, washed with
water and suspended under aseptic conditions in water or in a nutrient solution containing the requisite
ions. In order to ensure sterility, 300 units of penicillin G and 100 units of streptomycin per ml. were
added; enzyme production was not affected by the presence of the antibiotics. The flasks containing the
suspension of actinomyces mycelium were incubated for 24 hours on a shaker. Before incubation enzy-
matic activity was determined in the mycelium, after incubation in the supernatant fluid and in the
mycelium.
Determination of the activity of protease in the mycelium: 10 ml. mycelium suspension were centrifuged,
washed with distilled water and suspended in 0.5 ml. of 5. 10-3M solution of CaCl2 to stabilise the
enzyme. On the surface of the suspension 0.5 ml. toluene was added. The test-tubes were then stoppered
and placed in a thermostat at 28- 30? C. Autolysis took from 1- 3 days, according to the age of the
mycelium. Autolysis was always carried out in a number of parallel specimens, which were stopped at
given intervals. The toluene was carefully drawn off and the autolysate made up to the original volume,
or half the original volume with distilled water. Proteolytic activity was then measured in these speci-
mens by the usual method. The criterion for completion of autolysis was the reaching of constant protease
activity in the autolysate. When determining the increase in activity following 24 hours' incubation, the
values of activity in the supernatant fluid and in the mycelium were added, calculated to 1 xng. dry
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substance or 1 ml. suspension and the value of activity in the mycelium prior to incubation substracted.
The values of proteolytic activity are the averages of 4-8 determinations; the values of dry weight
are the averages of two specimens.
We first investigated the extent to which the ions being studied with reference to
their influence on the formation of protease, might influence the activity or stability
of protease and thus distort the results. K+ and Na+ had no effect on enzymatic
activity or stability, even in a concentration of 1 . 10-1 M. Mg2 } had no effect on
stability; in a concentration of 1 . 10-2M it reduced activity by about 17%, but
a concentration of 1 ..10-2 M had no effect. Ca2+ significantly stabilised the enzyme;
in a concentration of 10-2 M it reduced its activity by about 30%, but a concentration
of 1 . 10-2M had no effect. The ions Fee} and Zn2+ reduced activity and stability of
the enzyme, even in a concentration of 1 . 10-3 M.
When studying the influence of ions on the production of protease, a combination
of long-term experiments (five days) and short-term experiments (24 hours) was
selected. The results of the long-term experiments (figs. 1, 2 and 3) are expressed
in maximal activity (always reached on the fifth day) in relation to 1 mg. maximal
dry substance (two-day-old culture). The values of activity are the sum of activity
in the supernatant fluid and activity in the cells.
H2O -5 -4 -3
-2 -1
H2O -5 -4 -3 -2 -1
Fig. 1. Influence of Na+ on growth and forma- Fig. 2. Influence of K+ on growth and forma-
tion of protease. tion of protease.
x: log, of concentration of ion. y: dry substance in mg./l ml. (I); activity in a. 10-3 mEq. tyrosine
to 1 ml of the medium (II).
These results can only be in the nature of a guide, as considerable inactivation
of protease occurred in the course of the experiment. Of the ions under investigation,
the most significant in their influence were the potassium ions, which increased both
growth of the culture (dry substance) and also the formation of protease in con-
centrations when the maximal amount of dry substance had already been reached
and increased no further. The ions Zn2+ and Fe2+ did not affect formation of the
enzyme in concentrations lower than 1 . 10-3 M. In higher concentrations they
inactivated the enzyme.
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H2O -5 -4 -3 -2
Fig. 3. Influence of Mgta_ on growth and form-
ation of protease. x: log. of concentration of
ion. y: dry substance in mg./ml. (I); activity in
a. 10-3 mEq. tyrosine to 1 ml of the medium (II).
I I i W
No K Co My H2 0 DNP
Fig. 4. Influence of ions on formation of prote-
ase in distilled water. Black columns: activity
in cells; white columns: activity in supernatant
fluid. y: activity in a. 10-3 mEq. tyrosine to 1 mg.
dry substance.
M9 H2O
Fig. 5. Influence of ions on formation of pro- Fig. 6. Influence of K+ on short-term formation
tease in presence of nutrients. y: activity of protease in the presence of peptone and pep-
expressed in a . 10-3 mEq. tyrosine to 1 mg. tone + glucose. P: peptone; G: glucose; K: K--
dry substance is the sum of activity in the cells ions. y: activity expressed in a . 10-3 xnEq. tyro-
and in the supernatant fluid, sine to 1 mg. dry substance is the sum of activity
in the cells and in the supernatant fluid.
These long-term experiments were supplemented by short-term experiments,
in which the production of protease was studied during incubation in distilled water
(fig. 4) and in the presence of nutrients (fig. 5). In these experiments the concentration
of the ions was 5. 10-2M. The results are expressed as the increase in activity per
1 mg. dry substance. In incubation in distilled water (fig. 4), the decrease in dry
substance was the same in every case, but the production of protease was stimulated
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by Na+ and K+ by approximately 100% as compared with the controls. The effect
of the calcium ions can be explained by their stabilising action; magnesium ions had
no effect. In the presence of nutrients, the production of protease was stimulated
only by potassium ions, which increased the enzyme content by more than 100 0 ,
as compared with the controls. In these short-term experiments magnesium ions did
not increase enzyme production.
It is evident from all the above experiments that potassium ions have the most
profound effect on growth and on the formation of protease by the actinomyces, as
they increased both growth and production of the enzyme. It i s a well-known fact
that K+ interferes very significantly in the metabolism of sugars by stimulating
some of the processes associated with aerobic glycolysis (Ashford and Dixon 1935,
Farmer and Jones 1942, Buchanan et al. 1949, Hastings et al. 1952, Rothstein and
Demis 1953). An investigation was therefore made in our experiments (fig. 6) to
determine whether potassium ions interfere in and specifically and directly influence
proteosynthesis, or whether they act indirectly via glucose metabolism. The myce-
lium of Streptomyces griseus was therefore incubated, partly in a mixture of glucose
and peptone and partly in a peptone solution only, always in parallel specimens with
or without KC1 in a concentration of 5. 10-2M. The potassium ions significantly
stimulated the formation of protease only in the presence of glucose.
The formation of protease by Gram-positive and Gram-negative micro-organisms
is stimulated by ions, in particular by Ca2+ and Mg2+ and in the case of Bacillus
subtilis by Mn2+ (Merill and Clark 1928, Haines 1931, Stockton and Wyss 1946).
The action of calcium (Gorini 1951) consists chiefly in stabilising the enzyme rather
than in increasing its production. In our own experiments, Mg2 + ions alone had
a weak stimulating effect or no effect on the formation of protease by the actinomyces
Streptomyces griseus. Potassium ions, on the other hand, had a very marked influence
on growth of the culture and on the formation of protease. Their stimulating effects
on the amount of dry substance was manifested in a substantially lower concentra-
tion than their action on formation of the enzyme. The literature contains many data
on the action of potassium ions on metabolism, many of which have no bearing on
one another or are at variance with one another. K+ is also an essential biogenic
element for Streptomyces griseus (Acker and Lechevalier 1954). Its action interferes
in practically all metabolic processes. It influences the assimilation of amino acids
and proteosynthesis (Cannon et al. 1952, Frost and Sandy 1953, Davies et al. 1953
Folbergova 1955), fixation of free nitrogen (Fedorov 1950) and the formation of
purines (Friedman and Fox 1954). A lack of potassium causes morphological
changes in the nuclear apparatus (Fink 1950). The primary action of potassium,
however, probably consists in its influence on sugar metabolism (Farmer and Jones
1942, Barinova 1948, Hofmann and Schenck 1950, Sen and Sankhala 1953). K'
penetrates the cells of micro-organisms only in the presence of hexose, in contrast
to Na+ (Leibowitz and Kupermintz 1942, Cowie et al. 1949) and is probably bound
in the cells as a hexosophosphate salt (Roberts et al. 1949). It is an activator of
phosphorylation enzymes (Boyer et al. 1942, Bergmann et al. 1954), mediates the
formation of metaphosphate in yeasts (Schmidt et al. 1949) and is essential in the
synthesis of glutathione from y-glutamylcysteine and glycine combined with
a shift of cP from ATP (Snoke et al. 1953).
Our experiments investigating the influence of K+ in the presence of glucose and
peptone on the formation of protease provide evidence in support of the conception
that the action of potassium on proteosynthesis is associated with its influence on
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sugar metabolism. Potassium ions increased production of the enzyme almost
always only in the presence of glucose (fig. 6). On the basis of experiments investigating
the influence of ions on the formation of protease without the presence of external
nutrients (fig. 4), it is possible, however, to take the view that this mechanism is not
of necessity the only one. Since in this case production of the enzyme was also
stimulated by Na+, whereas Mg2+ and probably Ca2+ did not affect it, it appears
possible that in this case the action of the ions primarily took the form of influencing
the permeability of the cells. This is borne out by the fact that in these experiments
increased secretion of the enzyme into the medium occurred chiefly as a result of the
influence of Na+. It is possible that more intense diffusion of the enzyme from the
cells caused a fall in its level in the cells and thus indirectly made further synthesis
possible.
Summary
A study was made of the influence of various ions on growth and on the formation
of protease by the actinomyces Streptomyces griseus.
Growth of the culture, determined by the amount of dry substance, and production
of the enzyme are significantly stimulated by K+ ions. The concentration of K+
required for producing an increase in dry substance is lower than the concentration
required for affecting the production of protease.
The action of potassium ions probably consists in the way in which they influence
sugar metabolism, as their action is dependent on the presence of glucose, in addition
to a source of amino acids. Under the conditions of short-term production of the
enzyme without the presence of external nutrients, Na+ increases the formation of
protease as well as K+. In this case it is possible that the action of both ions is related
to the way in which they influence permeability.
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griseus for Growth and Candicidin Production. Appl. Microbiol. 2 : 157, 1954.
A s h f o r d, C. A., D i x o n, K. C.: The Effect of Potassium on the Glucolysis of Brain
Tissue with Reference to the Pasteur Effect. Biochem. J. 29 : 157, 1935.
Bergmann, E. D., Littauer, U. Z., VoIcani, B. C.: Formationofd-3-phospho-
glyceric Acid from Pentoses by Escherichia coli. Biochem. J. 56 : 174, 1954.
B o y e r, P. D., L a r d y, H. H., P h ill i p s, P. H.: The Role of Potassium in Muscle
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Buchanan, J. M., Hastings, A. B., Nesbett, F. B.: The Effect of the Ionic
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180 : 435, 1949.
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richia coli. I. Permeability to Sodium and Potassium Ions. J. Cellular Comp. Physiol.
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mulation of Glutamine Acid and Lysine by Bacteria and Yeast. Biochem. J. 54 : 430, 1953.
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150 : 768, 1942.
F o 1 b e r g o v a, J.: Vliv iontu na tvorbu lipasy v tkanoqch rezech pankreatn. Diplomova
prate, Praha 1955.
F i n k, H.: Experimental Investigations of the Effect of a Nutrient Salt Deficiency on Mitosis
in the Root Tips of Vicia faba. Chromosoma 3 : 510, 1950.
F o s t e r, J. W., II e i 1 i g m a n, F.: Mineral Deficiency in Complex Organic Media as
Limiting Factor in the Sporulation of Aerobic Bacilli. J. Bact. 57, 613, 1949.
F r i e d m a n, S., F o x, C h. L., J r.: The Relation of Potassium to Metabolism and Purine
Synthesis in Escherichia coli. J. Bacteriol. 68 : 186, 1954.
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F r o s t, D. V., S a n d y, H. R.: Effect of Mineral Deficiences on Amino Acid Utilization.
Critical Role of Potassium and Phosphorus. Proc. Soc. Exp. Biol. Med. 83 : 102, 1953.
G o r i n i, L., C r e v i e r, M.: Le comportement de la proteinase endocellulaire de Micro-
coccus lysodeikticus au tours de la lyse de cet organisme par lysozyme. Biochim. Biophys.
Acta 7 : 291, 1951.
H a i n e s, R. B.: The Formation of Bacterial Proteases, Especially in Synthetic Media.
Biochem. J. 25 : 1851, 1931.
Hastings, A. B., Feng, Ch. T., Nesbett, F. B., Sinex, R. M.: Carbo-
hydrate Metabolism in Rat Liver Slices. I. The Effect of Cations in the Media. J. Biol. Chem.
194:69, 1952.
H o f f m a n n, E., S c h e n k, H.: Ober den Einfluss des Kalium-lons auf Bildung and
Aktivitat der Fermente bei Hefen and Scbimmelpilzen. Biochem. Ztschr. 321 : 98, 1950.
C h a 1 o u p k a, J.: Proteolyticke enzymy aktinomycety Streptomyces griseus II. Vliv
povahy a koncentrace dusiku na sekreci proteasy. Cs. mikrobiologie I : 32, 1956a.
C h a 1 o u p k a, J.: III. Kratkodoba produkce enzymu. Cs. mikrobiologie 1 : 241, 1956b.
L e i b o w i t z, J., K u p e r m i n t z, N.: Potassium in Bacterial Fermentation. Nature
150: 233, 1942.
M e r i 1 1, A. T., C 1 a r k, M. W.: Some Conditions Affecting the Production of Gelatinase
by Proteus Bact. J. Bacteriol. 15 : 267, 1928.
R o b e r t s, R. B., R o b e r t s, I. Z., C o w i e, D. B.: Potassium Metabolism in
Escherichia coli. II. Metabolism in the Presence of Carbohydrates and their Metabolic
Derivates. J. Cellular Comp. Physiol. 34 : 259, 1949.
R o t h s t e i n, A., D e m i s, C.: The Relation of the Cell Surface to Metabolism. The
Stimulation of Fermentation by Extracellular Potassium. Arcb. Biochem. Biophys. 44 : 18
1953.
S e n, H. D., S a n k h a 1 a, R. H.: Citric Acid Production of Aspergillus fumaricus.
Intern. Sugar. J. 55 : 273, 1953.
S c h m i d t , G., H e c h t , L., T h a n n h a u e r, S. J.: Effect of Potassium Ions on the
Absorption of Orthophosphate and the Formation of Metaphosphate by Baker's Yeast. J. Biol.
Chem. 178 : 733, 1949.
S n o k e, J. E., Y a n a r i, S., B l o c k, K.: Synthesis of Glutathione from y-glutamyl-
cystein. J. Biol. Chem. 201 : 573, 1953.
Stockton, J. R., Wyss, A.: Proteinase Production by Bacillus subtilis. J. Bact.
52 : 227, 1946.
W a k s m a n, S. A.: The Actinomycetes. Waltham 1950.
W e b b, M.: Influence of Magnesium on Cell Division I. Growth of Clostridium welchii in
Complex Media Deficient in Magnesium. J. Gen. Microbiol. 2 : 275, 1948.
W e b b, M.: II. The Effect of Magnesium on Growth and Cell Division of Various Bacterial
Species in Complex Media. J. Gen. Microbiol. 3 : 410, 1949.
W e b b, M.: Effect of Magnesium on Cellular Division in Bacteria. Science 118 : 607, 1953.
B a p H H 0 B a, C. A.: BJIHHHHe KanHH H MarHHH Ha pa3BHTHe Rhizopus nigricans if Ha ero
cnoco6HOCTb x KHCJIOToo6pa3oBallulo. MHKpo6HoccorHH 17 : 10, 1948.
0 e j[ o p o B, M. B.: BJiHaHHe oTj(eTbnbIx HHTaTeJIbHbIx ajieueHTOB Ha (HHKCa[UHIO a3oTa aTMO-
Cceprl a3oTo6aKTepoM. )JAH CCCP 72 : 157, 1950.
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BJIHHHHe HOHOB Ha O6pa3OBaHHe HpOTea3bl JIy'lHCTbJM rpf6HOM
Streptomyces griseus
C 1OMO]TlLI0 JjJ[HTCnbHblx a IipaTKOCp09HbIX o1IbITOB MbI 14CCJIeJI,OBa7IH BJIHHHHe
pa3JI14tIHbIX HOHOB Ha pOCT H o6pa3oBaane I1poTea3bl y alT11HOM14 eTa StreptomyceS
griSeUS. MbI OTMe 4a21H yBe24lIgeHne aiTHBHOCT11 npOTea3bl HaK B F JIbTHBaUnoHHoli
Slcvl JOCT1, Tau 11 B MIIUCJIJI114 JIy9IICTOro rpn6Ka, pa3pynlan ero nyTeM CTepHJIbHOr0
aBTOJI143a B HpncyTCTBHl IIOHOB Ca2?-, iejCTBVIOHZnx Hai C.Ta611JI143aTOp 3H3IMa.
Ilpn HpaTKOCp09HbIX oIIbITax MbI IjeHTplOyrnpOBa2IH n OTMbIBaJIH 48-iJacOBbnf
M1IjeJ1Jlnii n HHHy6HpoBa2ln ero B BOjje HJI1 B nnTaTeJlbHOM paCTBOpe Ha HaiiajHCe
B np1CYTCTBHH ]IeHHIunJIJI1IHa H CTpenTOMHTjnna, KOTOpble HC BJIHHJIl Ha o6pa30-
BaHue 3H3UMa, HO Ilpejjynpexcjja2I4 B03MO1CHOCTb 3apaaceHHH.
MU H3ytIa2TH BJIHHHne Na+, K+, Mgt+, Fee 1- 11 Zn2+. IlanOJJee ;3uau11TeJ1b11oe
BJ11HHIIC oua3blBaJIH HOHbI Ha.InI, KOTOpbIC 3aMCTHO CTHMyJInpoBaJla uaIC pOCT,
Talc H HpOjjylci IIIO IlpoTea3bl, - 14 B np1ICyTCTBI11 IIHTaTCJIbHbIX BCIIjeCTB, u 6e3 Hllx
(HpH KpaTHOCI)0=IHOM olibITe). Ila poCT BJIHHJla y?He ropa3jjo 6oJZee H113Kall KOHIjeTI-
Tpauiig HOHOB, LICM Ha o6pa3oBalne 3H311MOB. CTHMyJInpylonjee ZjeICTBHe K+ Ha o6pa-
30BaHHe IIpOTea3bI CBH3aHo, BepofTHO, c ero BJInHHHeM Ha McTa6oJI113M yr2IeBO,jjOB,
Tau Hai OHO npOHBJIHCTCH TOJIb1O B IIpHCyTCTB1IH rJIIOH03bI (HapHjjy C ICTO1IH14ICOM
aMHHOHHCJIOT). B yCJIOBHBX I{paTKOCpOqHOrO CTHMyJInpoBaHHH npo7jyK1jn14 3H3HMa,
6e3 Ilpn6aBJIeHnH nnTaTeJIbHbIX BeIUecTB, o6pa3oBaHHe l1 OTCa3bI nOBb]maeTCH,
HpoMe K+, H IIOjj BJIHHHIIeM Na+. ,CIICTBHC 060HX 110ITOB 06yCJIOBJICHO B 3TOM
c2IyL1ae, BepOHTHO, nX BJIlHHHCM Ha Hp0HHIjaeMOCTb. ITOIIbI Mgt+ HO oua3blBaiOT
CHOJIbHO-Hn6yjjb CVIIjeCTBCHHOrO BJIHHHHH Ha o6pa3oBaHHe 3H31Ma, IIOHbI F02+
n Zn2+ B HOHIjeHTpaljlH dunce I . 10-3 M He 1Me1OT BJIHHHHH, a B 6oilee BbICOHHX
ICOHIjeHTpaijnnx nHaKTIIBHpvIOT IIpOTea3V.
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FOLIA BIOLOGICA
Photoreactivation of Bacteria Irradiated with X-rays
F. HERINK
Institute of Biophysics, Czechoslovak Academy of Science, Brno
Photoreactivation is a phenomenon in which organisms irradiated by UV light
suffer less damage if sufficiently exposed to visible light. Prat (1936), in a detailed
paper, drew attention to the antagonistic effects of various types of radiation
(v. also Prat 1950). More recently, Kelner (1949) observed photoreactivation in spores
of Streptomyces griseu8 inactivated by UV radiation. Almost at the same time
Dulbecco (1949) observed photoreactivation in bacteriophage of the group Esche-
richia coli which had undergone UV irradiation. He did not succeed, however, in
ascertaining whether phage irradiated by X-rays was capable of photoreactivation.
It was not until later that Dulbecco (1950) and in particular Watson (1950) found
that bacteriophage irradiated by X-rays can be photoreactivated. The degree of
reactivation, however, is small.
Latarjet (1951) observed that induction to phage formation caused by X-ray
irradiation can be reversed by photorectivation. The bells of B. megatherium (lyso-
genic strain) easily release phage following irradiation with a dose of 25,000 rep
(wave-length 0.9 A, 33kV). If, after irradiation, they are sufficiently exposed to the
light of a 500 W lamp (10-30 mins.) induction does not occur, phage does not
appear and the bacteria continue to grow, although at a slower rate.
The present experiments were aimed at ascertaining whether it is possible to
photoreactivate bacteria in which division has been halted by X-rays. It was evident
beforehand that the degree of photoreactivation would not be high, as the processes
which develop under the influence of ionizing radiation are completely different from
those which develop under the action of UV radiation. The starting point was the
assumption that ionizing radiation does not only evoke ionization in living matter,
but also excitation (Hereik 1956). It is probable that damage to cells which develops
on the basis of excitation could be reversed by the later action of visible light,
i. e. photoreactivation. Support for this view is found in the communication of Watson
(1950), who states that about half the energy of X-rays is dissipated in the form of
excitation, so that photoreactivation can only take place in those phage particles
which have been damaged by excitation. In the same way it may be expected that
the cells of bacteria which have been damaged by excitation in the course of
absorption of X-ray irradiation can be reactivated by visible. light. It would thus be
possible to differentiate between the effect of ionizing and excitation processes in
elementary processes which develop in living matter irradiated by X-rays.
Bacteria of Escherichia coli (strains 5k and 3c) were transferred from a solid medium to broth and
diluted to a strength of 7,000-8,000 micro-organisms/1 ml. A few ml. of this suspension were poured
into a Petri dish with nutrient agar, left for two minutes and then poured off. After drying, the surface
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 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
of the dish was irradiated with X-rays and photoreactivation then carried out in half the dishes. The
results were read off after 24 hours' incubation at 37? C. Survival was determined according to the
number of fully grown colonies on the irradiated and non-irradiated surface. Immediately after irradia-
tion with the X-ray tube, the controls were placed for 24 hours in the dark at 37? C. The possible effect
of reactivating light was excluded by exposing the entire surface of the Petri dish, i. e. including those
bacteria which had not been irradiated by X-ray and which served as controls.
Some of the experimental series were irradiated with a Siegbahn-Hadding tube, which was continually
exhausted by a rotary pump. A molybdenum cathode was used and the voltage was 18.5 kV at 10 mA,
without a filter except the foil on the window (Al 0.04 mm.). Irradiation was carried out at
a range of 4.7 cm., with a dose of 184 r/min.-1. The second part of the experimental series was irradiated
with a Chaoul tube at a voltage of 56 kV, 3 1nA, 0.15 mm Cu. The distance was 1.58 cm. and the diameter
of the tubus 1.5 cm., with a dose of 864 r/min.--1. The tubus touched the surface of the agar.
The doses were measured by the compensation method with a Wulf one-filament electrometer and
a Taylor-Stoneburner chamber with an diaphragm measuring 0.42 cm. in diameter. The reliability of the
method was tested by the uranium standard. A more detailed description of the method is given in an
earlier communication (Hercik 1948).
Photoreactivation was evoked either by irradiation with a 100 W incandescent lamp at a distance
of 50 cm. for 60-120 minutes or with a UV burner, the radiation of which was filtered through the glass
lid of the Petri dish (distance 72 cm., time of irradiation 30 or 60 minutes). Glass 2 mm. thick allows
sufficient passage for rays longer than 340 mu (Meyer-Seitz 1942, p. 127). In the case of a tungsten
filament lamp, emission is recognisable only with a wave-length of 400 m,u, with a maximum of 700 mh
(Withrow 1956, p. 159). As will be seen later, there is no difference between the reactivating effect of
a tungsten filament lamp and that of a mercury vapour lamp (filtered through glass). It may therefore
be assumed that in our case the active wave-length was 400 mp.
The significance of the results was evaluated by the t-test.
Using very soft X-ray radiation (1.8.5 kV), reactivation experiments were carried
out with one hour's irradiation with a 100 W lamp (distance 50 cm.) and doses
of 920-5,520 r in E. coli (strain 3c). The results are given in tab. 1. For every dose,
500-700 irradiated colonies were read off. If differences with P < 0.05 are taken as
significant, the table shows that very soft radiation can be somewhat reactivated by
Table 1. Photoreactivation of the Effect of Very Soft X-ray Radiation in E. coli, Strain 3c. X-ray
radiation: 18 kV, 10 mA, 184 r. rn 1, Reactivated for 60 rains. by exposure to a 100 W lamp at 50 cm.
Dose
Without
Photoreactivated
r
photoreactivation
by light
Difference
P
920
0.94 0.95
+ 0.01
0.4-0.3
1840
0.88 0.92
+ 0.04
0.2-0.1
3680
0.75 0.79
+ 0.04
0.1
5520
0.62 0.69
+ 0.07
Ke nepe-
Bapena, B OHMTHOI4 errje He HaCTyHHJI MaKCIMyM pacnaAa.
PHC. 4a - JIIOMUHaJIOBbII3 HaPH03, 46 - HOHTpOJIb. JIHJI(aTHYeCKHe (oJIJIHKyJIu Yepe3 17 gaCOB
noene o6TIy'eHuH. B cene3eHKe HapHOT11314pOBaHHOA NIMIUH Bee erne HaXOAHTCH K.TIeTOYHbAe o6JIOMKn,
B KOHTpoJIbHOI3 OHH yHfe 3a He6orIbrniMn uciJIno'euuiMu nepeBapeHbI.
Pnc. 5a - 3c up nepeA o6 iiygenneM, 56 - 3c np nocme o6Jlygerrna. Hpacuaa nyJrbna Yepe3 12 Bacon
noc.Ie o6nygeHna. llpn npBMenellnn 3nipa nepeA o6JIy IeuHeM HOTIngeCTBO spHTpo6JlaeTOB 6raBaeT
3HagiTenbno 6ojibine.
Ta6a. VI.
Pnc. 6a - xJlopaarHApaT, cepnH A, 66 - xnropaarnApaT, cepnH B. I{pacuas( nyJibna gepe3
16 gaeoB nocJie o6Jlygenna. B Cepnn A TOJ16HO oTAeJlbrrble 3pH1Tpo6JiacTb1, B Cepnn B - MHOH'OCTBO
3pHTpo6JIaCTOB reMOIjHTo6JIaCTOB H 6a3o(n.nbHbix peTnKy.TlapHbIX KJIeTOK.
Pne. 7. 3()np nepeA o6JIygeHneT. RpaeHan nyJlbna qepe3 5 gaCOB nociie o6JIy'CHHH. HpoMe
apFTpo6JIaCT0B Ha6JIIOAaIOTCH MHoroYHCTIeHHble 6a3o( nJIbHble peTHKy.rInpIlbre IiJIeTKu n reMO-
UHTo6JIaCTbI.
PHC. 8. 3 inp nepeA o6JlygeHueni. Rpacnaa ny.Bbna Yepe3 12 gacoB noc.ne o6JIygeHHa. MHoro-
gnCJIennbre 6a3o(nJlmHbre 3p1ITpo6TlaeTb1. FeaolriTo6TIaCTb1 OTCyTCTBy1OT.
PHC. 9. XJlopa.nrHApaT, cepnH B. Hpacrraa nyJrbna Yepe3 16 'iaeoB noc.iie o6.Tlygenna. Ppynna
reMOUHTo6JIaCTOB C Hy3bIpbHoo6pa3HbiMn gjpaMH H 6a3o(iJIbHuJX 3piTpo6JIaCTOB.
PHC. 10. JIIOMHHaaI. Rpacuaa nyJlbna Yepe3 12 YaCOB nOCJIe o6JIy ieHna. Oar rpaayTiOno33a
C MH0rOquCJIeHHLJMH Mn3JIOIfl4TaMH H McTaMHBJIoUiTaMH.
Pnc. 1-6: yneJIngenne 375x, pnc. 7-10: yBeJingenne 950x.
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 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
Increased Tolerance of Heterografts in Newborn Birds
V. HAAKOVA and M. HA8EK
Institute of Biology, Czechoslovak Academy of Science, Department of Experimental Biology and
Genetics, Praha
The aim of the present work was to ascertain how heterografts survive in newly
hatched birds and whether the simultaneous injection of isolated tissue cells from the
donor would have an effect on the survival of the grafts. In order that the most
effective cell suspension should be used, a suspension of spleen cells or bone marrow
was taken for the injections, as a large part of these could proliferate futher in the
recipient.
Skin grafting: A testing system which had proved satisfactory for grafting from old donors was used.
Skin from the wattle and legs of an adult fowl was transferred to Leghorn white chicks and Peking
ducklings within 20 hours after they had hatched out. This skin is the most satisfactory for transplant-
ation purposes as it is thinner and more homogenous than the feathered skin of a fully grown donor.
The graft does not wrinkle and it is sufficient to use collodion only, without suturing. The finally healed
transplant differs considerably in appearance from the normal skin so that healing and the survival of
the graft on the donor can be observed without any difficulty. When evaluating the life of the graft, the
number of days following transplantation on which the first macroscopic signs of regression in the
primarily healed graft are given (change of colour, drying of the surface or prominence of the graft and
inflammatory reaction) and the day on which the whole graft falls off.
Preparation and injection of isolated cells: Spleen tissue cooled in an ice bath was removed from the
capsule and homogenised by hand in a glass homogenizer or pressed through a platinum mesh, mixed
with the same volume of Krebs-Ringer phosphate. After centrifuging for 3-5 minutes at approxi-
mately 70 G, the sediment was suspended in Krebs-Ringer phosphate containing 10 i. units of heparin
per ml. After 10 minutes the suspension was injected intravenously into birds which had hatched out not
more than 20 hours previously, in amounts of 0.1-0.4 ml. containing a known number of cells, which
had been determined in a Barker chamber. Bone marrow was collected, immediately after killing the
donor, into sterile Krebs-Ringer phosphate with heparin (10 i. units/1 ml.), suspended by a hypodermic
syringe, gently centrifuged so as to separate the fat and injected.
Determination of blood chimera: the 2.5-3 % blood cell suspensions were tested by means of hen
antisera against guinea-fowl and turkey blood cells in titres of 1:64 and 1 : 128 and of duck antiserum
against goose blood cells (1 : 256). The tests were carried out in agglutination test-tubes. The blood cells,
together with the antiserum, were read off after 15 minutes at room temperature, then gently centrifuged
and again read off; they were then shaken for 30 minutes and read off a third time. In every case a micro-
scopic control was carried out.
Immunisation: Ducks were immunised at the age of exactly eight weeks, with four doses of 1.5 ml.
goose blood on alternate days (collected on the fifth day after the last injection) and reimmunisation was
carried out at 13 weeks with four doses of 4 ml. goose blood administered intravenously on alternate
days. The blood was collected on the fifth day.
Agglutination tests: The reaction of two drops of serum stored at - 20 ?C and mixed and diluted after
thawing, and of one drop of a 50 % suspension of erythrocytes washed three times in physiological
saline was read off after 60 minutes at room temperature. Incomplete antibodies were determined by the
reaction in 20 % equine albumin (Dunsford et al. 1953).
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No. of bird
First
macroscopic
signs
of destruction
(days)
Total
destruction
(days)
2517
8
17
2518
8
13
2519
8
17
2520
8
15
2521
8
13
2522
8
13
2523
8
13
2524
9
13
2525
8
15
2526
8
13
670
10
13
674
10
13
676
9
10
675
9
10
668
9
10
No.
of
bird
I
No. of cells
injected
First
macroscopic
sins of
destruction
(days)
Total
dest
ruc-
Lion
(days)
401
124 x 10, i. v.
10
18
402
124 X 106 i. v.
8
10
403
124 x 106 i. v.
8
10
405
124 X 106 i. v.
20
26
407
124 X 106 i. V.
20
22
413
124 x 106 i. v.
8
chick died
404
124 x 106 i. v.
8
14
part s. c.
409
124 x 106 i. V.
8
10
411
124x106i.v.
15
17
416
124x106i.v.
11
12
408
62 x 106 i. V.
10
20
418
186x106s.c.
12
14
412
155 x 106 s. c.
18
20
415
93x106s.c.
8
18
Tab. Ia. Survival of Turkey Grafts made to Tab. lb. Survival of Turkey Grafts
Chicks within 20 Hours after Hatching in One-day-old Chicks with Simultaneous
Injection of Suspension of Isolated Turkey
Spleen Cells
No. of
bird
First
macroscopic
signs of
destruction
(days)
Total
destruction
(days)
2501
10
15
2502
8
13
2503
8
13
2504
8
10
2505
8
13
2506
8
13
2507
12
17
2508
8
13
2509
8
13
2510
8
15
2511
8
15
2512
8
13
2513
8
12
2514
10
13
2515
8
13
2516
8
13
No
First
Total
No.
.
o
of
macroscopic
destruc-
of
injected
l
signs of
tion
bird
ce
ls
destruction
(days)
)
(days)
1701
152x106i.v.
18
26
bone marrow
1703
152 x 106 i. v.
15
26
1702
95 x 106 i. v.
7
12
1705
190x106i.v.
10
14
1708
114x106s.c.
7
14
(spleen)
Tab. 2b. Survival of Guinea-fowl Grafts
in One-day-old Chicks with Simultaneous
Tab. 2a. Survival of Guinea-fowl Grafts Trans- Injection of Bone Marrow or Suspension of
ferred to Chicks within 20 Hours after Hatching Guinea-fowl Spleen Cells
Heterografts from the wattle of an adult turkey and a fully grown guinea-fowl,
made within 20 hours of hatching, in chicks begin to show macroscopically evident signs
of regression on an average between the eighth and ninth day (turkey grafts: 8.53 +
? 3 .0.1919; guinea-fowl grafts: 8.3 + 3.0.284). If donor blood cells are injected
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at the same time, destruction begins on an average between the eleventh and twelth
day (turkey grafts: 11.7 ? 3.1.23; guinea-fowl grafts: 11.4 + 3.2.20). The
difference between the group which received the injection and those which did not
cannot be demonstrated statistically, or it is on the border of demonstrability
(turkey grafts: t = 1.395, P > 0.1 < 0.2; guinea-fowl grafts: t = 2.091,
P < 0.05 > 0.02). Tabs. 1 and 2 give the day on which the first macroscopic signs
of regression were observed (inflammatory reaction, drying of the surface, change
in colour) and the day on which the residue of the graft fell off. Fig. 1 gives a com-
parison of the length of life of the grafts (the values marked are the averages between
the day when the first signs of regression were observed and the day on which the
graft actually fell off).
The results of transplantation in newly hatched ducklings show that regression
of normal skin heterografts from the goose leg begins between the 10th and 11th day
No. of bird
No. of injected cells
First macroscopic
of destruction
(days)
Total
destruction
(days)
1014
-
15
17
1016
-
9
11
1021
-
9
11
1022
-
9
11
1009 = 254
46 x 106 i. v.
32
42
1013 = 253
92 x 106 i. v. + 184 x 106 s. C.
29
42
1018 = 255
92 x 106 i. v. + 184 x 106 s. c.
29
42
1015
69 x 106 i. v. + 184 x 106 s. c.
29
42
1019 = 256
46 x 106 i. v.
29
42
1025
46 x 106 i. v.
11
15
1026 = 258
46 X 106 i. v. + 20 x 106 s. c.
21
30
Tab. 3. Survival of Goose Skin Grafts on Newly Hatched Ducks following Injection of Isolated Goose
Spleen Cells. Transplantation from Goose's Leg carried out on 1st Day
Titre
of bird
No
.
After 1st immunisation
After reimmunisation
In protein medium
254
0
0
0
253
0
0
0
255
4
2
0
256
0
0
0
258
2
2
4
Controls:
233
16
64
234
128
512
235
16
128
236
32
256
Tab. 4. Results of Immunisation with Goose Blood in Ducks following Injections of Isolated Goose
Spleen Cells
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(10.5 ? 3. 1.16). The injection of isolated goose spleen cells postponed the
commencement of regression to the 25th day (24.9 ? 3.2.47). The influence of
the injection is therefore highly significant (t = 5.27, P < 0.001).
Ducks to which spleen cells are administered after hatching also tolerate blood cell
antigens. At the age of eight weeks, when immunised with goose blood, they do not
respond by forming antibodies (tab. 4), not even incomplete antibodies. When these
ducks and a series of control
ducks were given hetero-
survive longer in the experi-
mental birds than in the
controls.
In chicks and ducklings
which had received inject-
20[ ions, erythrocytes of the
Fig. 1. Comparison of the Survival of Homografts and Hetero-
grafts in Newly Hatched Chicks and Ducklings x: time in weeks
following grafting, y: percentage of surviving grafts. a = survival
of hetero rafts from turke wattle in newl hatched chicks
donor species were not found
in a specimen of peripheral
blood after 7-21 days.
g y y
b =survival of heterografts from guinea-fowl wattle in newly Discussion
hatched chicks, c = survival of heterografts from goose's leg in
newly hatched ducklings, A, B, C = survival of same heterografts
with simultaneous injection of tissue cells, r = curve of survival It follows from the above
of normal homografts in newly hatched chicks (Cannon, Weber results that heterografts
and Longmire 1954), s = survival of homografts in newly hatched
ducklings (Haskova 1957). transferred to newly hatched
chicks and ducks never sur-
vive permanently, as some
homografts do. A comparison is made in fig. 1. Whereas some homografts in one-
day-old chicks (Cannon, Weber and Longmire 1954) and all homografts in newborn
ducks (Haskova 1957) still survived in the fifth week, none of the heterografts
observed still survived at that period.
The results likewise show that the period when the organism is capable of adapting
itself to foreign cells depends on the degree of taxonomic difference of the donor and
recipient. In newly hatched chicks to which heterografts were transferred, prolonged
survival was never found in spite of the fact that some birds were still in the adaptive
period for homografts. Homografts take and continue to survive in all ducks, but
with heterografts the same does not occur.
Following simultaneous injections, in which entrance of the antigen into the
recipient is more rapid, slight, statistically non-significant prolongation of the
average period of survival of grafts is found. A few individual birds, however, show
slight signs of tolerance (chicks 405 and 407).This trace is fully confirmed in ducks,
in which duration of the adaptive period is longer (Haskova 1957). Following the
simultaneous injections, the survival of goose heterografts is statistically highly
significantly prolonged. A general comparison shows that the adaptive period and its
culmination have no absolute delimitation but that they involve a developmental
process of the cells which participate in immunity response, which is dependent on
the conditions under which it takes place. The quantitative aspect plays a part not
only in the degree of immunological tolerance, but probably also in circumscribing
the period in which adaptation to foreign cells can still be evoked.
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In no bird which had received an injection of spleen or bone marrow cells was even
a transitory appearance of an interspecific blood chimera observed. An intraspecific
blood chimera in the adaptive period develops by the natural route in bovine twins
(Owen 1945), sheep (Stormont et al. 1953) and man (Dunsford et al. 1953) and has
been produced experimentally by intravenous injections in rat embryos (Ripley 1953,
cited by Owen 1954) and by embryonal parabiosis in chicks (Billingham et al. 1956).
A similar blood chimera has also been demonstrated following lethal irradiation
(Ford et al. 1956, Lindsley et al. 1955). The development of an interspecific blood
chimera in the adaptive period has been demonstrated only after embryonal para-
biosis between turkey and hen (Hraba 1956) and following irradiation a rat blood
chimera was obtained in mice (Ford et al. 1956, Nowel et al. 1956, Makinodan 1956)
and a hen blood chimera in turkeys (Hasek). It is possible that in order to produce
a heterologous blo od chimera. it will be necessary in our experimental conditions
to damage, at least partially, the haemopoietic cells of the recipient. This question
will have to be resolved in further experiments. Nevertheless, the injection of
a suspension of spleen cells produces a total suppression of agglutinin formation.
Summary
In contrast to homografts, skin heterografts never survive on newly hatched chicks
and ducks. Direct transfer, therefore, does not lead to immunological tolerance of
their antigens. If newly hatched birds are injected with spleen or bone marrow cells
from the same donor, the survival of goose heterografts on ducks shows statistic-
cally significant prolongation. In chicks, the time of survival of guinea-fowl hetero-
grafts is not significantly prolonged.
A blood chimera was not found in birds injected with heterologous spleen orbone
marrow cells, but the suppression of the formation of immune agglutinins in ducks
injected with goose spleen cells is highly significant.
The communication discusses the limits of the adaptive period with reference to
varyingly distant foreign cells and.
B i l l i n g h am, R. E., Brent, L., Medawar, P. B.: Quantitative Studies on
Tissue Transplantation Immunity. III. Actively Acquired Tolerance. Philosoph. Trans-
actions Royal Soc., London, ser. B., 239 : 357, 1956.
C a n n o n, J. A., W e b e r, R. A., L o n g m i r e, W. P. J r.: Factors Influencing the
Survival of Successful Skin Homografts in the Chicken. I. Effects of Varying Age of Donor
and Recipient. Ann. Surg. 139 : 468, 1954.
Dunsford, I., Bowley, C. C., Hutchinson, A. M., Thompson, J. S.,
Sanger, R., Race, R. R.: A Human Blood-group Chimera. Brit. Med. J. 2 : 81, 1953.
Ford, C. A., Hamerton, J. L., Barnes, D. W. H., Loutit, J. F.: Cyto-
logical Identification of Radiation-chimaeras. Nature 177 : 452, 1956.
H as e k, M.: The Influence of Intra-embryonal Injection of Foreign Blood on the Formation
of Antibodies. II. Observation of Reactivity in Ducks, Geese and Guinea-fowl. Fol. biol.
(Praha) 2 : 48, 1956.
H a s k o v a, V.: Adaptivni etapa k cizorodym antigen4m v ontogenesi kachen. Cs. biologie
6, 1957 (in press).
H r a b a, T.: Immunological Behaviour of Embryonal Parabionts between Turkey and Hen.
Fol. biol. (Praha) 2 : 165, 1956.
L i n d s 1 e y, D. L., 0 d e 11, T. T. J r., T a u s c h e, F. G.: Implantation of Functional
Erythropoietic Elements Following Total-body Irradiation. Proc. Soc. Exp. Biol. Med.
90 : 512, 1955.
M a k i n o d a n T a k a s h is Circulating Rat Cells in Lethally Irradiated Mice Protected
with Rat Bone Marrow. Proc. Soc. Exp. Biol. Med. 92 : 174, 1956.
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 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
Nowell, P.C., Cole, L. J., llabermeyer, J. G., Roan, P. L.: Growthand
Continued Function of Rat Marrow Cells in X-radiated Mice. Cancer Res. 16 : 258, 1956.
0 w e n, R. D.: Transplantation Bull. 1 : 83, 1954.
S t o r m o n t, C., W e i r, W. C., L a n e, L. L.: Erythrocyte Mosaicism in a Pair of
Sheep Twins. Science 118 : 695, 1953.
IIOBbIHIeHne COBMeCT14MOCTH reTepOTpaHCHJIaHTaTOB y HOBOpOMgeHHbIX
MHBOTHbIX
FeTepoTpaldnJIaHTaTbI 143 cKJIaJjKH KO?KH B3pOCJIOi HHJjenRH Him ci{JIaJjKH KO?KH
B3pOCJIOfl uecapKH, LlepeCa?KeHHbie AbIHJIHTaM B Te13eHIIe nepBblX 20 iIacoB HoCJIe
BbIKJICBbIBaHHH, HaLIHHaIOT HpOHBJI}ITb 3aMOTHLIe H MaKpocnounLIecnn IIp13HaKH
JjeCTpyKI[HH B Cpe;lHeM Me?KJjy 8-M H 9-M JjHeM (HHJjIOIneLIbll TpaHCIIJIaHTaTbI:
8,53 ? 3 . 0,1919; TpancnJiaHTaTbI OT uecapoK: 8,3 ? 3 . 0,284). Hpll oJjnoBpeMeH-
HOM BHpbICKHBaHHH KJIeTOK CeJIe3eHKH JjoHOpa MaKpocnonHgCCKH 3aMOTHan JjeCTpyK-
HHH HaLlnHaeTCH B CPCJjHeM MenK y 11-M IT 12-M JjHeM (TpaHCHJiaHTaTbI OT HHJje1KH:
11,7 ? 3 . 1,23; TpaHCIIJiaHTaTbI OT uecapKH: 11,4 ? 3 . 2,20). Pa3HHua pe3yJIb-
TaTOB CO BHpbICKHBaHHFIMH H 6e3 HHX CTaTHCTHgecKH He JjoKa3yeMa I' JIH Haxo-
JjHTCH Ha rpaHnuax JjoHa3yeMOCTH (TpaHCHJIaHTaTbI OT IIHJjIOIHHH: t = 1,395,
P > 0,1 < 0,2; TpancHJIaHTaTbI OT uecapKH: t = 2,091, P < 0,05 > 0,02). Ha
Ta6JI. 1 H 2 HpHBOJjiTCH JjeHb HponBJTeHHH nepBbiX MaKpocnonHLIecuHX HPH3HaKOB
JjeCTpyHwHH H JjeHb, norJja CJIyljHBaJICH OCTaTOK TpaxcHJlaHTaTa. Ha rpa4nne I
Mbd cpaBHHBaeM noxa3aTer[H AJIHTeJlbxocTH BbI?KHBaHHFI TpaxcHJlaHTaTOB (HaHeceH-
Hble Ho1a3aTeffl HPeICTaBJIHIOT epeJjHee apH(IMeTHLIec.Koe Men iy AHeM HoHBJIelnn
nepBLIX Hp13Ha1OB JjeCTpyiIuHH H J[HeM OTnaUeHnri TpancnJlaHTaTa). Pe3yJIbTaTbl
OIIbITOB TpaHCHJIaHTaIlnn Y CBe?KeBbIJIyHHBHIHXCH TIT nonaabIBaIOT, LITO HOpMaJIbIlble
reTepOTpaHCHJIaHTaTbl KO?Kn C Horn HaLIHHaioT pa3pymmaTbC9 Me?KT,jy 10-M H 11-M
JweM (10,5 ? 3 . 1,16). OUHano BnpbICHnIjaHnn KJIeTOK, 1130JI14POBaHHbIX H3 rVCHHOH
ceJIe3eHK14, OTOJZBHraJH1 Haxiajio JjeCTPyRUHH Ha 25-bIII JjeHb (24,9 ? 3 . 2,47).
TaHHM O6pa3OM, BJIH}IHHe HH'beHUHH OKa3bABaeTCF1 BbICOKO CHFHH4IHKaHTHIIM
(t = 5,27, P < 0,001).
Y VTOK, KOTOPbIM HOUR) BLIKJIeBbIBaHH}i BBOJjHJIHCb KJIOTKH CeJIe3eHKH, npo-
FIBJIHeTCH C6JIH?KeHHe H HO OTHOIHeHHIO K allTHFeHaM HpOBHHbIX Te.ieu. B BO3paCTe
8 HeJjeJlb yTKH UPH HMMVHH3aUHH FyCHHOH KPOBbIO He pearnpyioT HH 06pa3OBaHHeM
aHTHTeJI, HIT o6pa3OBaHHeM HeHOJIHbIX alTHTeii (Ta6Jl. 4). 3TIIM yTKaM H cepHH
KOHTpOJIbHbIX YTOK MbI V?KC BO B3pOCJIOH CTaJjHH CHOBa nepeCa?KHBaJIH TpaHcnJIaHTaT
C ryCHHOH Horn. OJjHano OH HH B OJjHOM CIVLiae He Bbl?KHBaJI JjOJIbIHC, LLCM B KOHTpoJIe.
B OTJIHLIHe OT rOMOTpaHCHJIaHTaTOB KO?KHble reTepoTpaHcn aaHTaTbI V CBC?KeBbIJIV-
nHBIHHXCH IjbIHJIHT H yTFIT HnKorJja VCTOHLIHBO He HpTHHMaIOTCH. Ferro CBe"rKe-
BbIJIynHBHIHMCII YTFITaM OJjHOBpeMCHHO C IlepocaJjKOH BHpbICHVTb KJIeTKH cc Ie3eHKH
HJIH KOCTHOFO MO3ra Toro ?Re JjOHOpa, TO Y YTOK B aJjaHTnBHOM 3Talie (FaTnKOBa 1957)
MO?KHO 3HagllTeJIbHO IIPOJjJIHTb BbI?KHBaHHe reTepOTpaHCnnIaHTaTa.
MbI He Ha6JlloJjannr KpOBHHOH XnMepbI V ?KHBOTHbIX, KOTOpbIM Ji,eniaJlllCb BnpbICKH-
BaHHH. B CTaTbe 06Cy?KJjaIOTCH rpaHHljbl aJjaIITHBHOFO aTana Ji)I}I pa3JIH9HbIX LIV?Ke-
POJjHbIX ICJieTOK H BCTpegiaeMOCTb KPOBFIHOFO X4MepI3Ma, C03JjaIOlIjeFOCH B TegenHe
aJjanTHBHoro 3Tara HJIH B pe3VJIbTaTe CMepTeJIbHOFO o6J[VLIeHn9 JIygaMH X.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
FOLIA BIOLOGICA
K BOrlpocy ReJieHHR ngep ripH perexepai uu cxeJieTHOH
mbini obi xpornxa
B. HYRA
Boeauo-McAHunncxan aIaAeMHn R. E. rlypKne, xaceApa 6nonorHn, rpaAeu KpanoBe
B JInTepaType no BonpocaM KJleTOtIHOro aeneHHH npI3 pereHepauni Mbiing HJIII IIpI3
3M6pn0HaJIbH0M MHOreHe3e BCTpeilaeTCf 11 TanOi3 B3rjla, qTO MblmeilHble aapa
JjeJIHTCH HyTeM aMHT03a HnH c parMe1TauHll, H TaKoB, tlTO OHII aenaTCH nyTOM MnT03a.
ABTOpbI, KOTOpble OrHCbIBaIOT cJIyLIaI3 M11T03a, HJIn HOJIaraIOT, MTO MHTO3 BCTpetIaeTCfi
TOJIbI{O B paHH1IX CTaaHHX 3M6p11oHaJlbHoro Ml4oreHe3a, Him aonycnaiOT ero H B 6onee
H03AHHX CTaaHHX y MaJIO TIH((epeHIZnpoBaHHbIx 3JIeMeHTOB. 3aBap3BH (1938a, 6)
H XeilcHHa (1948), HCCneAOnanmHe perenepaI;HIo MbIlHIL, Haxoa1Jll MlIT03 TOJIbRO
B MHo6JlacTax, Toraa KaK B 6oJIee an44epeHiAHpoBaHHbIX 3JIeMeHTaX BCTpei1aJIH
TOJIbHO aMIIT03.
MbI HCCJIeaOBaJIH THn KJIeTo 1HOrO aeiieHHH IIpl pereHepagnri CKeJIOTHbIX Mblmu
y KpOJIHKOB H HaXOAHn1 Han MIITO3, TaK H aMHT03. Ha?KabIH H3 3TIIX cHOCO6oB
aeJIeHHH HMeeT CBOe McCTO 11 CBOH nepnoa HaH6OnbmerI BcTpeilaeMocTH. B CBoeH
pa6oTe mm aaeM IIOIIbITHY O6 b}CHeHHH 3TOr0 gBJIeHHH Ha OCHOBe Ha6J1loaeHHU, LITO
B perenepHpyI0IIIei3 TKaHH npoTenaeT I;eJIbiii pnA 6HoxHMn'Iecxnx npOIjeCCOB, pa3-
JIHgHblx HO cHJle II Ka1OCTBy B Teilexne pa3JIHLIHblx nepHOaoB. IlMnyJlbc0M 1JIFI 3Toro
Hamero npeanOJIOH{eHnn UOCJIYH{HJIH HOBeinmie pa60TM, HOCBHn;eHHble 611OxHMHH
perenepalHH.
Mamepua.a u .ntiemotu)ca
Y 36 KpowiHKOB *iacTh IeTbipexrnaBOii MbIIHgbI 6eApa BhIpe3anacb Tax, -ITo6bI 6plomllo MbamIIubI
He 6ar io IIOJIHOCTMO nepece*ieHO. Llepe3 onpeAeiienHbie HpoMeHCyTxn BpeMeHH (co 2-ro no 130-bifi
J[enb) KpOJIHHH y6HBa.Incb, H *IaCTb paHbI B MbIHIne (J)HKCHpOBaJIaCb (J)opMannHOM, fI(HAKOCTbIO
Helly, Bouin-a HJr I Zenker-a. Cpe3ia B 7-10,U TOJUHHHOi olpaiiiHBanncb mene3HbIM reMaTOKCH-
JIHHOM raft eHrafiHa c 30311HOM, c noMOHIbio peaKllHn no (Denbreny, no van Gieson-y n no McTOAy
HanneHrafiMa.
B HepBble aHH HOCne onepaiuH Hpeo6JlaaaloT H3MeneHHH ae311HTerpHpylowero
xapaKTepa. B HepeceLIennbix BOJIOKHax Hapa 6bInaI0T pacnoJio?Kexbl IIeHTpaJIbTO
H npeaCTaBJnIIOTCH 6onee 0BaJlbnbIMH, KpynxblMH H CBeTJIbIMH. Yx{e Ha 5-bifi-6-oi3
AeHE nocne orepauHH MOHCHO BHaeTb HHTeHCHBHOe aMHTOT111qeCKOe aeneHHe 3THx
Haep: BHYTpH napa o6pa3yeTCH nepenoHKa, pa3aenHIOHZafi ero Ha LBe npH6nH3H-
TenbHO Oa1HaKOBbIX gIacTH. IeneHHe HpOTel{aeT, HOBHAHMOMy, OxIeHb 6bicTpo,
an KaK BCTpe1alOTCf1 Izenble uenot1HH onenb 6JHBKo apyr K apyry pac11ono>KeHHb1X
Haep. 3T11 CHOnneHHH Haxo)HTCH 11pellMyll;eCTBeHHO B HepeceLIennblx BOJIOKHaX,
HO BCTpena1OTCH H B HepaHenbIX, HeHOCPeaCTBeHHO B6nn311 panbl. geimmHeca Hapa
BCTpe~la1OTCH H B 3Ha=IHTenbHO Ae3HHTerpnpoBannbix Bonoxxax (pnc. 1). HapymeH-
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
Hble BOJIOKHa no ICOHIjaM pacHllpfIIOTCf1, it jje.THI1jHecn HJjpa o6pa3yIOT :3 ecb Ijejlble
rpynllbl (pHC. 2). HpoMe jjer1eHHH HepeIIOHKOH BCTpC9aIOTCH H CJIy1al HOpMaJIbH01'o,
ItnnaCCH9ecKOPO aM14T03a (pHC. 3). B 3TOT IIepHojj Ham He yJjaBaJIOCb HaIT11 till OJjJIOH
M11TOTB9CCKOH ((H1'ypbI - Hp1 3Ha9HTeJIbHOM, HaIC 6bIJIo y?Ke yna3auo, KOJIn Iee1Be
pa3JI11PHbIX (I)OpM aM11TO3a B 3T14X HpenapaTaX.
C LulTOJTorngeCKOH TWIN11 3pCHHH HHTepeceH nepnoj ~Jepe3 9 jjlleH iiocile OlIepaiuin.
3Jjecb B03HIIOaIOT MbIIHCi1Hble II09uH, coiep?uanjiie MCJIHO3epHHCTyI0 CapHoIIJTa3My'
H rpyIijibl HJjep. IIeROTOpble 1,13 HHX IIpOHBJIHIOT HeCOMHCHHbIC ltpH3HaKH 1103HN0111)-
BCHnH tiepe3 CTaJjnio nepelloBRu. BJjpa 6bIBaIOT 60JjHbl XpoMaTHH0MHpaCllo.Jlaral0'I'(-11
B HCCKOJIbKux CJIOHX Jjpyr Hai j[pyroM. H0aTOMy Iia q)OTorpa(j)I1H (pHC. 4) 01111 II l)G,'(-
CTaBJ1HIOTCB 6oJiee TeMHbIMH. N 06011M Hot1ICaM Ha 3TOM CHHMlie JIpuJleraloT M11TOT11-
t1eCKH jeJIHII[Hecn MHO6JIaeTbI, ICOTOpLIC, HI0B1T 1IMOMy, OTI1OiIKOBaJIHcb 01' MIX.
MHUpOOOTOrpa(lnu pHC. 5 noua3bIBaeT HC6OJIbI1IyI0 rpynny cBo60T It() JTeHaHIHR
RJIeTOK HeHOCpej(CTBeHHO no C0ceJLCTBy OT HapyHleHHol'o BOJIOKHa. Tpu MHO6JTaCTa
CHHTbI B pa3JIH9HbIX Oa3aX M14T03a. C.TaJjHH M14TO3a MbJHIe9HbIX 3JICM0HTOB - MHO-
6JIaCTOB - HOHa3bIBaIO'C H CJIeJjyionjue CHHMKH (pHC. 6, 7, 8 H 9) Tan, liaoo Ml,T Hamjin1
IIX B IlpenapaTaX, CJjeJIaHHbIX Ha 9-bin JjeHb IIOCJIe onepanjn11. BO BCCX CJIy-iaux
MHTO3 6JIIOJjaJICl B CBo60AHbIX ICJIeTKax, MHO6JIacTax, It TOJIbKO OJ(TIH pa3 (HC. 10)
mbi HamJIH M14T03 Ha 1{0HIJC MbIIHeiiiiorO BOJTOHHa. Ile HCICJII09eHo 0JpiaHO, 'ITO 3TO
6bJJI M14TO3 B TOJIbKO qTO OTJjeJIHBHIeMCf MHo6JIaCTe, TCM 6ojiee, 9TO BOJIOKHO 136JI13I1
aToro o6pa3oBaHHH 6bIJI0 Cpe3aH0 HaleKOCb. IIO H B 3TOT HeJHOjj BCTpe9aIOTCH Hjjpa,
H Rauce HApa MH06JIaCTOB, KOTOpbI0 j(CJIHTCH neperopo uoi . MHo6JIacTbT c aMHTOT14-
9ee u JjeJIHHj1MHCH IIIpaMH 6bIBaIOT 6oJIee KpynHble, CpaBHIITe.nT HO j1JI11HIIble,
a B HCKOTOpbIX CJIV'Iaax MO?KHO 1'OBOpITb H 0 HpuMIITHBHbIX, T. 0. 0 IBVHJjepI1blX
MHOCHMHJIaCTaX (pHC. 11). IIOCnOJIbKy MOHMO CyJjHTb Ha OCHOBaHHH HamuX CTaT1l-
9ecuux H306pa?KeHHH, THH ICJICTOMHOI'O jjeJIeBHH oKa3bMBaeT BJIHHHHe Ha J aJIbHeiiIHHO
Cyjjb6bI Mno6JIaCTOB. EcJIH }IJjp0 jjeJIHTCH MHTOTngecKH, TO B03HIIKSaIOT OHrTTb-TaHl4
MHO6JIaeTbl. Hoene aMHTOT119ecKoro jjeJIeHHH HJjpa CJICJjyCT He pa3JjeJII0111le M-011
HJICTKH, a B03H14KaeT MHOrOTIJjepHOO o6pa3oBaHHe - MHOCHMHJIaCT - a 1103JLHee
MHOTOHJ(epHOe MbIIHe9HOe BOJIOKHO.
B 6o.lee II03;1HbIX CTaJjufX, - Ha 20-bIH JjeHb nOCJIC oIlepalUHH, - MOHMO BHJjOTb
yuco HOBOo6pa3OBaHHb1e MbIIIIe9Hble BOJIOKHa. OH14 HPOXOjjHT BOJI11006pa3HO. B HX
CapuonJla3Me y?Ke HMCIOTCH Kau Muo4u6piijui I, TaIZ YT nonepe911arI HOJIOCaTOCTb.
HpOMe BOJIOKOH BCTpeMaOTCH He3Ha914TejIbHOe KOJIH9OCTB0 M1406JIaCTOB 11 MilO-
CHMHJIaCTOB. B Hep14M11314H, OKyTbIBaIoiiAeM BOJIOHHa, BCTpetaIOTCH 3peJTble 4iH6po-
I(1ITbI. HOHIjbi HOBOO6pa3OBaHHbIX MbIHIe9HbIX BOJIOKOH, BpaCTaloIIj1TX B COeJj11HH-
TeJ1bITyIo TKaHb, HOCTeHeHHO pacmHpflOTCfl H yTpa911BaIOT CB01o CTpyiTypy. B IX
OJ[IIOpO,T];HOM 14JIll MCJIKO3epHHCTOM COJjep?KHMOM HaXOJjnTCrI rpyHllbT Hy3blpbuO-
o6pa3HbIX H>_jep, B IjeHTPC HOTOpbIX 9aCTO MONIHO BHJjeTb neperopOJj1ly (pHC. 12).
(.JIy9aeB MHT03a Mbl B 3TOT Ileptio j He BCTpC9aJIH.
14epea 60JIee HpOJjOJI?KHTeJ1bIble HpoMe?KyTKH BpeMexfl noene oHepaunu (40-130
jjHeHT) Ha6JTlo;jaeTCH aBaJIorH'iHaH H Bee 60JIee HpiI6JIH?KaIOHjaHCH K HopMe KapT1lHa.
OJjHauo BOJIOKHa 6bIBaIOT oupy?Keubl coeJjlHHTeJIbHO)%I THaBblo, 'ITO IIpOTI1B0pe9H'I'
Ha6JIIOJjeH14HM Rovati (1953), CTyRHTCICOI'o H CTpHraiOBOii (1951). Pa3pacTaHHe
COeJj1IH14T0JIbHOH TKaHH H HenoJIHOc B03MeIljOBHe MbI1110MI iX BOJIOKOH IIpl IIainlx
OIIbITHx MbI O67,HCHHeM TCM, 9TO mbi yjjaJIHJIH cpaBHHTCJIbIlo 6OJlhulylo 9aCTb Mb111110h
(BCCOM ;to 3,5 I').
J(ucIYccil.1
IIpH pereriepaljiu MbininbI aMHTO3 HpOACTaBJIHCT IIpe06JTaJjal0Hj1lii CIi0006 fI;(ep-
Horo JjeJIeHHH. 0 wand B onpeJjeJIeIIHbIH nepuoJj pereHepalUHH (Ha 9-Nfi jjellb HOCJIe
onepaitHH) MbI HaXOJIHJIH MHOHZOCTBO M14TOTH9edKHX (Hryp, JIOKaJIH3OBaIIBbIX Tau
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
'ITO 1O1MH HOBO3MOHCHO COMHeBaTbCII B HX y3KOH CBII3n C jjeJIHIuHMHCn aMHTOTHLIeCKII
nupaM1 MbIHIOLIHbIX no'qeK. H3y'IeHHe Halnero MaTepnaiia noKa3blBaeT, LITO MHTOTH-
LIecnoe jjeJleHne 1POHCXOjLHT H B TaKHX }I pax, KOTOpbIO B03HHKJIII nyTeM aMHT03a, -
BOnPOKn YTBep?KAOHHHM HeKOTOpbIX IIITOreHeTHKOB, KOTOPbIC He j[oIIycnaIOT TaKOH
BO3MOIKHOCTH.
MbI IIOIIbTTaJIHCb BbIHCHHTb IlpniIHHbI IqepejjoBannn aMHTOTngeCKOFO 14 MHTOTn-
qecHoro jje;IeHHH. Han6owIee upneMJreMoi H3 pnjja pa6oqnx rHnOTe3 HaM Ka}KeTCFI
TaKoe o6'bnCHeHne LIepe;kOBaHHn 3TIIX jLBYX THROB KJIeTOLIHOro j[eJIeHHH, KOTOpoe
HCXOA14T 143 BbIpa3HTeJIbHbIX 6HoxumH IeCKIIX H3MeHeHHII B TeMeHI1e pa3JIn'HbIX i a3
perenepaunn. HCCJiejjyn perenepaunio negIenH, Lang (1952) jjona3a.i, 'ITO jje3oKCH-
pII6onynJleoTnjubl o6pa3yIOTCFI TOJIbKO npH AeJleHH1 nJIeTOK. Tsuboi C CoTp. (1954)
He3aBnCnMO OT JIaHra, no Ha.TaKOM 1Ee MaTepnaiie H npaKTnuecKH npn Tex we
yCJIOBHHX OnbITa jjona3aJIH 3aBHCHMOCTb jje3onCnpn60HynJIenHOBbIX KHCJIOT (RPHK)
OT THna KJIeTOiIHOPO ueiieHHn. OHn yTBepwjjaiOT, KaK H JIaHr, LITO APHK CIIHTeTH-
3npY1OTCH TOJIbKO npH MuTOTngIeCKOM ueJIeHHH. MbI xce I oJlaraeM, iITO aMHTOTH-
xIecKoe geJleHne B nePBbIH HepnOjj 3a7KHBJIOHH1I oCylueCTBJrneTCH IOTOMy, LITO TKaHb
B 3TOT nepnojj 6biBaeT 6oraTa 'KPH KHCJIOTaMH. HoCJIe HCTOIIjennx 3anacoB APHK
B pe3yJIbTaTe ycnJieHHoro aMITOTH'IecKoro jjeJleHH1 HaCTynaeT nepHOjj MHTO3a,
B TeTrenne KoTOporo TKaxb CHOBa o6oraiijaeTCn RPH KHCJIOTaMH. Ham B3rJIlj[,
OIIHpaIOIun13ca npeuEAe Bcero Ha pa60TbI Ho 611oxHMnH, COOTBeTCTByeT H OTKpbITIIHM
LMHTpHeBOII (1954), KOTOpan IIPOH3BOjjHJla FHCTOx1MI'qeCK14e HCCJIe1LOBaHHH COjjep-
1KaHHn JIPHR ripe perenepaunn norlepe*IHoiiowrocaTbfx MbIIHU Y Kpblcbr. OHa npH-
BOj[1T jjaHHbie, ITO cojjepu anue JiPHK Ha'I1HaOT IIaj[aTb y1Ke B Te'enue nepBblX
24 LIaCOB nocJie oriepaunn, Torjja Kan Ha 6-oII n 7-oII geHb (T. e. B nepnojj, norjja
HrnaTbeBa (1954), icc iejjOBaBman perenepauHiO cKenneTHbix MbIIHU Y KpbICbI, naXo-
jjHJla MHTO3bI) - cojjep}Kanne L PHR pe3Ko noBTIInaeTCg. TaKHM O6pa3OM, H 3Ta
pa6oTa nojjTBep}KjjaeT npaBIIJibHOCTb Hamero npeunoJTOHCeHnn.
1. HpH perenepaunn CKeJIeTHblx MbIHIIj KpornHKa upeo6JlajjaeT aMnT03. MHT03
Ho1iBJIHeTCH B nepHOjj Han6oJlbmero pa3BHTHn MHO6JIaCTPI IeCInoIT (Ia3bl, - B HalueM
Onb1Te Ha 9-bIII jjeab 1OCJIe onepaunu.
2. HpH MHTOTHiIOCKOM jjeJIeHHH MHo6JIaCTOB BO3HHKaIOT OnHTb-TaKH MHo6JIaCTbI,
HO nOCJIe aMHTO3a MHO6JIaCTbI j lloepeHunpyIOTCH jjajlee 14 143 HHX BO3HHKaIOT
Mb CHMHJIaCTbI.
3. B BO1POCe nepejjoBannn aMHTO3a H M11TO3a aBTOP 3aluimaeT B3rJIHjj, iITO
B pe3yJIbTaTe 6bICTPO OCylueCTBJInIOIuerOCn aMHTOT1I IeCKOrO jjenleHHa nc epnblBaeTCn
3anaC jje3oKcupH60HyKJIeuHOBbIX KHCJIOT (APHK), nocae hero KJIeTK1 jjeJIHTCH
MHTOTHLIeCKH. Tan 3aHac jPHK jjOHOJIHfieTCn jjO Tpe6yeMoro ypOBHfI, Kan BbITeKaeT
143 pa6oT HexoTOpblx aBTOpoB, KOTOpble jjona3aJIn, 'ITO npH MHT03e OCYIueCTBJIHeTCI
C14HTe3 ,1PI1 K. Ho HcTegeHHH nepHOjja MHTO3a CHOBa 6bIJIO HaLljjeno aMHTOT11 ecnOe
jjeJIenne njjep.
4. T1epejjoBaHne aMITOTHilecKoro H MHTOTIIMeCKOro jjeJlennll 3T0 06bI'IHO& 1IBJIeHiie
HpH perenepaunn Mbunubl. BbIJi nalljjeH aMnT03, Hpe)jllleCTBOBaBIHHIr MHTO3y HpH
jjeJleHnH Ojjnoro H Toro lice njjpa, XOT1I 4opMaJlbnble reHeTHKH He jjonycxaiOT TaKOII
BO3MOIKHOCTH B HHTepecax TeopHH HHjjHBHjjyaJlbHOCTH XPOMOCOM.
(Ta6.2. VII, VIII)
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
(M H T p n e B a, E. B.: Pacnpege.nenne nyxnenaonbIX RHCJIOT B BOJIOI Hax cxeneTnoii Mycxy-
aaTypbt npn perenepagnn. AAH CCCP 98 : 653, 1954.
3 a B a p 3 n H, A. A.: 0 perenepagnn Mbimenabix aneMen'OB y Allolobophora caliginosa.
Apx. anaT., rncT. H aM6p. 29 : 342, 1938a.
3 a B a p 3 n n, A. A.: 0 perenepaiInn coMaTw iecxnx Mbrmg y pbi6 (Coitus cottus). Apx. anal.,
rncT. H aM6p. 29 : 353, 19386.
14 r n a T b e B a, 3. H.: BOCCTanoBJlenne nanepBagnn noBpexcueanou MaImgbI y ne1OTOpbix
MneEOHHTaioignx M14BOTnbIX. Bonpocbi BoccTanoBnennn opranoB n Txaner3 noanoHonnbIx
3HHBOTnbIx (cTp. 256). Mocxna 1954.
'C T Y A H T c x n ii, A. H. is C T p is r a n 0 B a, A. P.: BoccTanoBiTe.nbnbie iipogeccbi
B cxeneTHOii Mycxy.naType. MocHBa 1951.
X e ii c n H a, B. H.: Perenepagnx coMaTngecxois Mycxy.naTypbt y HOCTncTbix pb16. C6opnnx
cTaTeii naMHTn axag. A. A. 3aBapanna (FTp. 147). Mocxna 1948.
L a n g, K.: Lokalisation der Fermente and Stoffwechselprozesse in den cinzelnen Zellbestand-
teilen and deren Trennung (Mikroskopische and chemische Organisation der Zelle). 2. Collo-
quium der Deutschen Gesellschaft fur physiologische Chemie. Berlin 1952.
R o v a t i, L., C a s t o 1 d i, G.: Sulla rigenerazione muscolare post-traumatica. Ricerche
sperimentali. Ann. ital. chirurg. 30 : 719, 1953.
Tsuboi, K. K., Yokohama, H. 0., Stowell, R. E., Wilson, M. E.:
The Chemical Composition of Regenerating Mouse Liver. Arch. Biochem. 48 : 272, 1954.
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K BORPOCY CTpyKTypbl BHpyca Ta6auxoH M03aHE H
B. BbICTPI411HIIf
BnpycoJTorHgecKHFI HHCTHTYT T-ICAH, BpaTHCJIasa
IIoemynu/co e peaaxz#uzo 24 X 1956
r10BepXHOCTHaH H BHYTpeHHHH CTpyKTypy BIIpyCHbMX tIaCTHA Ta6agHOI3 MO3alHH
(Aailee BTM) HBJIHeTCH IIpeAMeTOM yaae cpaBHIITeJIbHO o61IIIIpHbIx HCCJIeLOBaHIIIT.
B nOCJIegHee BpeMn, Hanp. Schramm (1955) Ha6JIIogaJI B aJIKaJIIIgeCKOi3 cpeAe
pacnaA LIacnu BTM Ha TOHHHe Kpynfo1IKH, AHaMeTpoM B 1.50 A, H TOJIMIIHOII
B 50-100 A, C OTBepCTHeM B IzeHTpe, AHaMeTpoM OKOJIO 34 A. C TOMOHZbIO 3JIeK-
T OHHOI3 MHKpO( OTOrpa( HH eMy yAaJIOCb Taic a CHHTb BOJIOKHa pn60HVKJIenHOBOIT
HHCJIOTbI, TOJIII;IIHOH npH6JI143HTeJIbHO B 34 A. OH IIOJlaraeT, 1T0 Ha6JHOAaBIuHecH
HM KpyHfotIKH 61IBaIOT y HHaHTIIBHPOBaHHbIX gaCTRA HaHH3aHbI Ha BOJIOKHO pII6o-
HyWJIeHHOBOII HHCJIOTbI. CyxoB (1953), IICXOAH, McMAy IIpo1IHM, H 143 pa6oT l epiIHKa
{1953) OTHOCIITeJIbHO pacc ria6JIeHHH CTpyKTypy 6aKTepno4 ara nog Aei3CTBneM co-
oTBeTCTBenHblx noBOpxnocTHo-anTHBHnIx BeIIzeCTB, noJlaraeT, TITO eMy ygarLoch,
o6pa6aTbIBan LIaCTHIJbI BTM 3THJIOBbIM CIIHpTaM, cgenaTb HarrIHAHOH IIX CIIHpaJIb-
HYIO CTPYKTYPY?
Watson (1954) H Franklin H Klug (1955) HccJTegyIOT CTpyKTypy BTM c noMOIIgbIo
pa36opa gn4 panruionnoro pHCyHKa peHTreHOBCHKX Jlygeii. B cBOeJ4 nocJlegneci
pa6oTe (Franklin, . Klug 1956) 3TH HccJIeAoBaTeJIH IIpHXOAHT H 3aKJIIOileHHIO, 1ITO
HO nOBepXHOCTH gaCTIIIU BTM BHHTOO6pa3Ho nPOXOAHT aEeJio6on (H COOTBeTCTBeHHaI
rpaHb), OTBegaionuie CIIHpaTIH rJIaBHOIi 6eJIKOBOIl IZeRK C nOg'beMOM B 23 A. Lriy6HHa
BHHTOBOII Hape3KH 61IBaeT onOJTo 30 A. OTHOCIITerIbHO rpann BIIHTOBOII Hape3KH
aBTOpbI nOJlaraloT, iITO OHa npOXOAIIT He HenpepbIBHO, a COCTOHT H3 paga IIgymix
gpyr 3a gpyroM OTpOCTKOB, OTXOAHMHX OT OTAeJIbnbIX 3BexbeB r4IaBHOI3 cnnpaJlb-
HoIi Fenn.
OnnpanCb Ha yKa3aHHble pa60TbI FepLIIIKa H CyxOBa, MbI xoTeJIH IIOIIbITaTbCH
paccJia6HTb CTpyKTypy IIanno en BTM, o6pa6aTLIBa} HX COOTBeTCTBeHHbIMH XIIMH-
KaJIuHMH, II cgeJlaTb H3o6pa?KeHHH nogo6HOI3 AHCCOIj1HpOBaHHOI3 CTpyKTypb C HO-
MOIIZbIO 3JIOKTpOHHOFO MIIKpocxoua (npIILIeM Torga HaM en;e He 6bIJIH H3BeCTHhI
pa60Tbi Franklin-a H Klug-a (1956).
MM JOJIb3OBaJIHCb nypH(JIHKaTOM BTM, npnrOTOBJIeHHbIM C IIOMOll Mo HOM6nHHpoBaHHOH Tex-
HHKn nypH(m;aAHR 1YTeM ocaHigeHnfl cepHOKHCJIbIM aMMOHHeM n CKOpOCTHorO geHTpn(I)yrnpo-
BaHHH. 3a npeJIocTaBJienne ogHnIeHHhTx o6pa3IIoB aBTOp n3'bgBJIAeT 6JIaroJ[apHOCTb COTpyaHHHaM
uHCTHTyTa nHSi. Ip. ConOJIy n iHM. F. PyTTKaio-HeJ[eIIKOMy. LJIn npnrOTOBJIeHHH npenapaTa mm
o6pa6aTbxBaJIH BHPYC B TegeHne onpegeaeimoro BpeMean n npn onpeJ[eJIeHHOI3 TeMnepaType 3TH-
xaKHM-HH6yJ[b gpyrii BenlecTBOM), nOCJie qero pa3BOJ[HJIH o6pa3eJ
JIOBhIM CHHPTOM (HJIM we
,4Ba,1 I J[ecTnJIJlnpoBaHHoI3 BOJ(OH H HanocuJln MHKpoKanJIIo Ha KOJIJIOArIHHyIO uJIeHKy. BMICOxinHe
npenapaTbi HanbinnJHch xpoMOM IT nccJleJ[oBaJlncb C HOMOH[bIO 3JIeKTPOHHoro MHKpocnona cncTeMhl
Siegbahn-Schonander.
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Pe3y.Ilbmambl u o6cyarcaenue
fIpCABap1TeJlbubre OnbiTbi np1 KOMHaTHOII TCMI1CpaTVPC C IICI10JIb3OBaHIICM aue-
ToHa, 3(Hpa, 9TIMOBOFO CHIIpTa H MO9CBHHbl He AaJIH CyIUecTBeHHblX 113MeHeiiuH
B CTpy1TypC 9aCTIIij, XOTH y HeNoTopbIX npenapaTOB Mbi MOTJIII OTMCTHTb I13BCCT1IOC
VBeJInTIeHHe, (pa36yXa1IIe)) IIaJIo9eK nOA AeiiCTBIIeM aueTOHa II 3T11jIOBOP0 CHnpTa.
Pe3VJIbTaTbl, O KOTOpbIX MbI 3ACCb AOKJiaAbIBaeM, 6bTJi1i IIOJIy9erIbl IIYTOM O6pa6OTRII
3TIIJIOBI,IM CHHPTOM (KOHueIITpHpoBaHHbiii Hypll4HKaT BTM II 3TUJMOBbni C11npT
B COOTHOIneHHH 1 : 1) H HpH IIOBbIIICHIIOid TeMHepaType H 37 T. Pnc. 1 iIpejCTaBJIHeT
Ilypi4 a11aT BTM, HOTOpbil HCMCJLJICHHO IIoCJIC npH6aBJI0HHfl CHHpTa 6biJr pa3BCACH
Ao Heo6xojHmoH HoilueHTpaunh JILCCTHJIJInpOBalIHOH BOJLOH H HaHeCCn Iia 1iOJIJIojllfi-
HyIO HJIeHKy. IIa 1o9KII BTM CIAO He 113MOHnJIIICb. p] eo6X0AHMO OTMCTHTb HC6OJIhIHHC
CKOIIJIeHHH McHIKHX KpyrJIhIX 9aCTHI) HenOCPOACTBOHHO 110 COCCACTBV C HCKOTOpbIMII
IIano 1KaMn (plc. 2). Mbi nOJlaraeM, 9TO 3TO - i1 COp611pOBaiIHbIC 143 CPCAb1 He-
BIIpycHbie 6eJrKH, KOTOpble oTJILeJI1JIHCb OT HOBePXHOCTH 9aCT141j BTM. Pa~sMepbl
DTHX Me IKIIX InapHKOB 6bIBaIOT HpH6JH13HTeJIbHO HOCKOJIbKO ACCRTKOB A. 7 L lOCJ}e
24- iaCOBOI'o ACIiCTBnfl 3T1JIOBOFO c1114 Ta np1 37 ?C HOT enje CKOJlbHO-HH6y;[b
CVIIjeCTBCHHbIX n3MeHenHH, XOTH KOJIH9eCTBO OTAe.IHloll (IIXCa He60JlbIIIIIx 9aCTH1[
3Ha9HTe3bHO fOBbImaCTCH (plic. 3). Cpejii na2I09eK BTM 3aMeTHbI BbIHICOIIIICa1IHble
McJIKne IHaplHH, KOTOPbIO IIOJ1; Ae1CTBHCM 3THJIOBOTO ClllipTa, KaK KaaceTCH, VBCJIH9II-
BaloTCH. CJIeJ1,yIOIu1ii CHHMOK (pnc. 4) noKa3hIBaeT npenapaT noc2le 48-9acoBOro
AeiiCTBIIH 3Tn710BOF0 CHnpTa HpH 37 ?C. OT9eTJInBO 1314;010 yBC2T11M Hlle o61,6Ma
MHOPIIX IIa2Io'eK H HX pacnaA B nlapIIKII. MO}KHO OTMCTIITb Pa3JIIIMHbIC IIepeXOAHble
cTaAIIH. 09eBIIAHO, 9TO CHIIPT JeIICTByeT Ha 9acTHIIbi He OAIIHaHOBO: y He1OTOpbiX
iaCTIIX yBeini llU1CH TOJIbHO HX o6'beM H Iipu 3TOM CTaJIa HarJIHJjHOii IIpaBHJIbBI)CTI,
CTPyRTyPbl nonepxHOCTH, - Hollca (pHc. 4a); y ApyruX 3aMeTHa 6oneen03AHflfl
CTaJ(1In geiiCTBIIfl crilrpTa (pic. 46). MOCTaMII HacTynaeT IIOJIHbIII pacila;[ Ha cpaBHH-
Te2TbuO KPyIIHble KpyrJlbie o6pa3oBaHHx. HpOCTpaHCTBa Me}KAy Ha2Io9KaMH BTM
6b1Ba1OT IIOKPb1TbI 60Jlbm14M KoJIIIMCC'rHOM 3epHHCTOro MaTepuaJia o InapiiaMn pa3-
JIII9HbiX pa3MepoB.
IIpH coaeiyIOIueH cepuH OIIbITOB Mbr yCTaHOBIIJIH, 9TO 11 B KOHTPOJIbHOM IlpenapaTe)
6e3 CHnpTa, T. e. IIOABepraBIIICMCfl IICKJH09iTOJIbnO AeiCTBHI0 I101110IICHHOII TeMIlepa-
'rypbl B 37 ?C, 9epe3 44 naCa HaCTy1IIJIII 3aMeTHble H3MeueHHfl: JIHHe1HOe 60Jlee 9eM
TpoeHpaTH00 YAJIHHeHIIe na2I09CK. IIpH 3TOM OT'OTJIIIBO BbICTynIIJIa 60JIee HJIH MeneC
IIpaBHJIbHaH BOJIHHCTaH CTPyKTypa HX KpaeB, CBIIACTeJlbCTBy1OIIjaH O HCpaBHOMCPHOM
pa36yxaanl B IIIHp1Hy. PIIc. 5 IIoxa3bIBaCT 44-9acoBOe ACHCTBne CHHpTa HpH 37 ?(;,
a pnc. 6-44-9acoBoe AeKCTBne OAHOii TOJIbRO BOAbI (Tai a e npH 37 ?C). Ha 3TOM
nociieAHeM npenapaTe Mbi Ha6111ojaaiT pacnaA B MOJIKIIe, KpyrJIble 9aCTHI[b1 c 6o2iee
TeMHbIM MecTOM B I);OHTpC, HpCACTaBJIflIOIILHM, BO3MOIKHO, OTBepCTIle. Blom, rJIaBHOi'i
ocH pa36yxmiix HaJIO9eF (pHc. 5 II 6) OT'ICTJIIIBO BHAHa 6o2iee TCMHau no2Ioca. Tpy;J,Ho
cua3aTb, B Ka1Oii CTeneHH 3TO HBJIeHIIC MO?HHO OTIIecTII Ha C9eT CTpyi{Typbl 9aCT11 lj.
Mow-HO 6bi2i0 6b1 npCA1IOJIO}KIITb, 9TO UCHTpaJibHbie naCTII IICXOAHb1X 9aCTHu 3aiioJI-
HCHbI McHee HJIOTHOII MaCCOII HJIH COBceM IIVCTbie, 9TO HOTOM OCO6eHBO Bb1pa3IITCJ}6H0
HIpOHBJI}ICTC}I IIpii pa36yxaHIIH 9aCTIlu. B pa311CAeHHOM BoToii nypl(j)IIKaTC BTM,
KOTOpblH B TCIC1IIO IIpOAOJISH11TOJlbHOTO BPCMOHII CTOHJI B JICAIi1KC, Mid IIOCJIC 0601OA0-
CTOpOHHero HaIIbIJIeHHH MOTa2IJIOM HaHIJIII OTACJIIIBni1CCf 9aCTII1jbi pa3JlI9110II BCMII-
9HHbI, C OTBCJ)CTHHMH B ILCHTPC (pHc. 7). CJieAyeT OTMeTHTb,'ITO AO CIIX Hop HaM HIT Ha
OAHOM n3 upenapaTOB He y;AaBa2OCb Ha6J11oAaTb oHHCaHHble B JT1ITepaTypC (Schramm
1955) Bo2TOKHa pu60HyHJICHHOBOH KHCJIOTbI. i.I} AonOJIHeHna BbIIIIeH3JIONlCHHO1'O
IipHBOAIIM CIIje CHHMOK B BbICOKOH CTeHeHII 09III11CHHO17O nypn(HKaTa BTM (pHc. 8).
IJypI4IIKaT 6bIJI pa3BeACH ACCTHJIJIIIPOBaHHOH BOAOii n HeMCJ(JICHHO HaHCCCH Iia
KOJ1JIOAHHHy10 IIJIOHKy. Y 9aCTHIj He 6LIJIO 3aMeT110 HHKaKOH CTpyFTypbI.
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Mbi noJIaraeM, 'ITO HaM yJjaJIOCb, AeIICTByR 3TIIJIOBLIM CUHPTOM Hpn roBbIHIeHHOn
TeMnepaType Ha naJIOLIKn BTM, nolca3aTb, IqT0 OHH uOABepralOTCH pa36yxaHHlo
HepaBlloMepno, H 3TO JI OHBJIHeTCH B TOM, 'ITO cTpyKTypa naJIOLIeI CTaHOBHTCFI
BHAHMOI3. l lnprHa yBeJIHLIennblx gaCTIIA Ha pHC. 6 J[oCTHraeT npH6JIH3HTeJlbHO
Pnc. 7. HypuC HKaT BTM Hoene o601o9ocTopoHnero HanlaneanH xpOMOM.
700 A, T. e. OHa npn6JI143HTepJIbH0 B 4,5 pa3a 60JIbIHe HOpMaJIbHO1 nlnpnHbi naJrogei
BTM (KaK H3BeCTHO, 150 A). 0*-IeBHJ[HO, LITO 3Ty pa3HHIny CJIe;jyeT npnlHCaTb
BJIHHHHIO cnnpTa H HanbIJIeHHH. CTaBHIne BnAHMbIMli nonca (pHc. 4a) 6bIBaIOT
B nlnpnny oxoJIO 100 A. EcJrn npeAHOJio}KIITb, IqT0 H 3Ta BeJIHLInHa np116JIK31ITCJIbHO
B 4,5 pa3a 60Jlbnle HCT14HHOI3 (xOTH B HanpaBJreHHH npOAOJIbHOii OCn naJIO'KH Aell-
CTBHe aiiHoroiTa H BaHLIJIeHHH CKa3blBaeTCH, BepOHTHO, no ApyrOMy), - TO AJIH
W14pHHbI 3THX IIOHCOB nonry'aeTCH 3Ha'eHHe of oiio 22 A. B03MOHCHO no3TOMy,
IqT0 HaM yiaJiOCb CAeJIaTb HenOCpeACTBeHHO BHAHMLIMII BIITKII Ha IOBepXHOCTH
najiogen BTM, HaJi%iine KOTOpbIX KOCBeHHO JjOKa3aHO Franklin-oM H K1ug-or nyTeM
Jr(f) )paKI[noHuoro anaJrH3a c HOMOHjbIO peHTrexa. HeiipaBHnbHble OTpOCTKH 110
CTOPOHaM naJlo'eK BTM (pnc. 5 n 6) MoraH 616I npeACTaBJIHTb yBeJIH'eHHbie B He-
c1OJrbxo pa3 11OA jelCTBHeM cnnpTa, BOAbi 11 TenJIa H HepaBHOMepHO pearnpyioiuHe
Ha yCJIOBHH OnbITa gacTH 3BenbeB CHHpaJILHO CBepH TOI3 rjIaBHOi3 6eJIKOBOII IjenH,
143 KOTOpOII, HO MHeHHIO Franklin-a H Klug-a, COCTOHT rpaHb BHBTOBOII Hape3Kn
Ha HOBCPXHOCTH iiaJIo'eK BTM. OT'IeTIIBO BnAHaH nocpei[HHe narIo'KH (B HanpaB-
TIeHnH npOAOJIbHOIi ocli) noJioca H KpyrJlbie LIaCTHIAbi C OTBepCTHeM B i eHTpe TaK1Ke,
BepOHTHO, npeJjCTaBJIHIOT peaJlbHO CyMecTByi0Iljne CTpy1TypbI na1O'IeK. HyTeM
IIp16JIH3nTeJIbHOro H3MepeHn1 (TOiIHoe n3MepeHHe HeBO3MO?KHO) pa3MepOB OTBep-
CTHH B OTAeJIHIOnuHXCH iIaCTi4 ax, KaK 11 npOAOJrbHO1 n0JIOC16I Ha CHHMKaX 5 r3 6,
rIOJIy'alOTCH Be1H'IIHbI, KOTOpbie KOJIe6JIIOTCH OT HeCKOnb1HX AeCHTKOB npH6JIn314-
Te1IbHO Ao 200 A. MoanHO BnAeTb, LITO OTAennuBHIHecH LIaCTHI[bI H na1IOLIKH BTM
HenpaBHJILHO, HepaBHOMepHO OTBe'aIOT Ha AeHCTBHe rOBbIIHeHHOH TOMnepaTypbl
n CnnpTa. OAHano Mid nOJiaraeM, IqT0 HanHiine 3THX CTpyHTpp M05KHO CLIl4TaTb
AOKa3aTeJlbCTBOM TOTO, LITO H B IjeHTpe HCXOAHbIX, HeBHAOn3MeHeHHbIX 'IaCTMA Ha-
XOIj1TCH nojOCTb, KaK yTBep?KI(aeT, Hanp., Caspar (1956), KOTOpbIII Ha OCHOBaHHiI
oljeHKH 3KBaTOpnaJlbnoro paccerHHH peHTreHOBCKnX ayHeli OpneHTnpyJOluBM reJIeM
BTM, KaK ii paCYeTa ryCTOTbI 3JIeHTpOHOB yCTanoBHJI, 'ITO BHyTpH naJIotleK BTM
rlpOXOAHT noJTOCTb AHaMCTpOM B 19 A.
(Ta6ri. IX, X, XI, XII)
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TI 13 T e p a T y p a
C y x o a, E. C., H u H n~ o p 0 B a, F. C.: 0 cuupaneBff jnoN1 cTpoennn iaCTHIJ Brrpyca
Nao3auuw Ta6aFa. RAH CCCP 90 : 671, 1953.
C a s p a r, D. L. D.: Structure of Tobacco Mosaic Virus. Nature 177 : 928, 1956.
F r a n k 1 i n, H. E., K 1 u g, A.: The Splitting of Layer-Lines in X-Ray Fibre Diagrams
of Helical Structures: Application to Tobacco Mosaic Virus. Acta Cryst. 8 : 777, 1955.
F r a n k 1 i n, H. E., K 1 u g, A.: The Nature of the Helical Groove on the Tobacco Mosaic
Virus Particle. X-Ray Diffraction Studies. Biochim. Biophys. Acta 19 : 403, 1956.
H e r c i k, F.: Problem bakteriofaga. Praha 1953.
S c h r a m in, G.: f1ber die Struktar des Tabakmosaikvirus. III. Der Zerfall in alkalischer
Losung. Z. Naturforsch. 10b : 481, 1955.
W a t s o n, J. D.: The Structure of Tobacco Mosaic Virus. I. X-Ray Evidence of a Helical
Arrangement of Sub-Units around the Long-Axis. Biochim. Biophys. Acta 13 : 10, 1954.
On the Structure of Tobacco Mosaic Virus
V. BYSTRICKY
Summary
Particles of the tobacco mosaic virus which had been exposed for 48 hours to the
action of ethyl alcohol at a raised temperature of 37? C were studied by the'electron
microscope. On the surface regular bands, with an actual breadth of 22'A (calculated
from the swollen particles) were observed. The particles were several times enlarged
by the effect of the alcohol and heat. The structures observed are considered to be
identical with the surface structure of particles of the TMV (the screw-like spiral)
found by Franklin and Klug (1956). In preparations exposed to the action of alcohol
and also in control preparations, in which the particles of the TMV in bi-distilled
water were exposed only to the action of raised temperature, fragments of particles
with an opening were found, together with particles with a clearly discernible
central band running in the same direction as the main axis and other particles
with irregular processes protruding from the side. We are of the opinion that the
openings in the fragments, and also the less electron-scattering band, in preparations
of particles enlarged irregularly and several times, represent the hollow, internal part
of particles of the TMV, demonstrated by an indirect method by Caspar (1956).
The irregular processes might also represent enlarged sub-units of the main, twisted
protein chain, of which, according to Franklin and Klug, the edge of the spiral on the
surface structure of particles of the TMV is formed.
(Tables IX, X. XI, XII)
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RpaTjrne coo6uiieHHH Brief Reports Kurze Mitteilungen
The Question of the Conversion of Serum Globulin into Antibody
by means of the Influence of an Antigen
J. OTERZL and Z. TRNKA
Institute of Biology, Czechoslovak Academy of Science, Department of Microbiology, Praha
Although many experiments have been carried out with amino acids, using labelled isotopes, in which
it was found that antibody formation involves the formation of the protein molecule de novo (Gros,
Coursaget and Macheboeuf 1954, Green and Anker 1954), the possibility of the conversion of the complete
globulin molecule into a molecule of the character of an antibody as a result of the influence of the
antigen is still postulated. These conclusions are based primarily on the experiments of Pauling (1940,
1942) with antibody formation in vitro. It was not possible, however, to reproduce even these experi-
ments in vitro (Kuzin and Nevreava 1947, Hrubelova 1957).
Our own experiments demonstrated that the conversion of normal globulin into antibody by the
influence of the antigen is impossible not only in vitro, but also in the organism. The protein composition
of the serum was determined electrophoretically in newborn rabbits which had not yet commenced
sucking. All the serum protein fractions were determined in all sera collected immediately after birth
and also on subsequent days (fig. 1). These findings are in agreement with the findings of Brambell
y-globulin albumin y-globulin albumin
Fig 1. Electrophoresis of the sera of young rabbits collected immediately after birth. A, B, C, D:
different rabbits from the same litter.
(1951, 1954) that in rabbits the serum proteins are transmitted to the young by the mother during
uterine life. The injection of antigen in young rabbits in the period when their blood contains a sufficient
amount of y-globulin does not lead to antibody formation (fig. 2).
By injecting antigen into young rabbits whose serum contained normal y-globulin, it was demon-
strated that fully-formed globulins are not converted into protein molecules of the character of an
antibody by the influence of the antigen, even in vivo.
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1281
64
32
Fig. 2. Antibody formation in young rabbits following intraperitoneal injection of antigen S. pora-
typhi B (108 micro-organisms/ml)
Brambell, W. F. B., Hemmings, W. A., Henderson, M.: Antibodies and
Embryos. London 1951.
Brambell, B. F. W., Brierley, J., Halliday, B., Hemmings, W. A.:
Transference of Passive Immunity from Mother to Young. Lancet 266 : 964, 1954.
G r e e it, H., A n k e r, H. S.: On the Synthesis of Antibody Protein. Biochim. Biophys.
Acta 13 : 365, 1954.
G r o s, P., C o u r s a g e t, J., M a c h e b o e u f, M.: Becherches sur 1'existence de pre-
curseurs proteiques dans la formation des anticorps. Bull. soc. Chim. biol. 34 : 1070, 1954.
H r u b e s o v a, M.: Pfispevek k pokusurn o tvorbu protilatek in vitro. Cs. mikrobiol. 2 : 10,
1957.
P a u 1 i it g, L.: A Theory of the Structure and Process of Formation of Antibodies. J. Am.
Chem. Soc. 62 : 2643, 1940.
P a u l i n g, L., C a in p b e 1 1, D. A.: The Manufacture of Antibodies in vitro. J. Exp.
Med. 76 : 211, 1942.
K y 3 n it, A. M., H e n p e a n a, H. A.: K nonpocy o6pa3onannx aHTUTe.T in vitro. Bno-
xnMnH 12 : 49, 1947.
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Al. 1Ipaciunra u Al. Furi.2: PaHHHe I(HTOJIOFIPTeCI1 O H3MeHeHIIH JIHn[Oo1[HTapHOli Ta611. I.
II n[H3JIOHf"[HOII cocTaBHhIx HacTeH MIbIHIIIHOII
('eie3CHKH HOCJIe peHT1'e,HOBCIforo o6.iyieHHH.
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 Approved For Release 2008/04/10: CIA-RDP80T00246A002900500011-3
AI. /I pac.nuvsa a Ri. Tuna: Paaiiiie I;HTOJIOI'JIYeCUIIe IISMeHCHHH :HLM(j)OIu IrapHOii
H MH37IO11,JHO)3 COCTaBHLIS iiaCTeii M6111111HO1!
CediO3e1IHI! IIOC.:TP peHTI'eHOBC KOI'U OO.lV'1eII1IH
-t4 %e -% ski
5-117 ". ; ~:1 t
21
TeHCTVI 13 pHCyHIiaM CM. CTp. 118.
Approved For Release 2008/04/10: CIA-RDP80T00246A002900500011-3
 Approved For Release 2008/04/10: CIA-RDP80T00246A002900500011-3
:ti. ~pac.91/i1712 it At. I'11.1.1: ha11iiiie I~I1TO.'IOPHYCCH110 113110HCHHB ?I11M4)Of~HTRPHOI[ ~r t1OT. 111.
If MH3JIO1I HOH COCTBBHId\ '1ACTCM M1,1O111IN11H
(C:IC3CiIIUt 11OCJIC PCTITI'CIIOBC IO1'O o6):I3'4C711(9.
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 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
Al. llpac.tutru n 11. l'u.r.i: Pamtu e I I1TO.iorit~tec[tttc tta~mm~nrsr ,u[:~i~~~rn~uTap~toii 186.1. IV.
If ~rttaTtoiruFoii eoeTanithiX 'tacreii mimminoil
(T3(13e1IIII IIOG'n' penTrelroncttoro O(.I teui m.
41,
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 Approved For Release 2008/04/10: CIA-RDP80T00246A002900500011-3
I. 11puc16u+lua it i11. 1'm,, Paii"im i iiro;iorim,cialC II3MCIICHIIH 7HyH4IOIUHTapHON '1'u6.1. V.
it MH3JIOll IIOIi COCTaBHLIX HaCTCn MhHHHHOIi
CC;TI03CHIiH IIOCIC PCHTreHOBcICOI'O o6JIy9CHHft.
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 Approved For Release 2008/04/10: CIA-RDP80T00246A002900500011-3
111. TIpacrcuura it it. rii.i : Paaaae 1UHT0.norxvecHHe 113AIe[IeHlul AIIAI(olklrrapuoii 'fa6.1. VI.
11 AIH3J1oltJHOLI CO('TaBHIJX Ya('Teii AI61111H11ON
(',ed103e11ICH 110('.1e p('HTF ('HOBClcoro o6JlyYeit11B.
I e1:( I'61 1: p11CYI11ia01 l'D4. ('Tp. 118.
Approved For Release 2008/04/10: CIA-RDP80T00246A002900500011-3
 Approved For Release 2008/04/10: CIA-RDP80T00246A002900500011-3
B. IZyaaca: K Boupocy ;Ic:Ieiti10 r3Aep lIpit perenepau3nr eoe,3rr33uii Hhiiugbl I:pu.lnHa. Ta6;r. VII.
Pnc. 1. Mbiml];a HpoJlnHa Mepe3 6 AHeii nocJie paxennH. Ppynna nAep I:ax pe3y.IbTaT AeJIexnn
neperopoAHO I. - Pnc. 2. Mbimna IcpoJIHHa Mepe3 6 AHekl nocJie paxennH. HagaJlo o6pa3oBaHIIH
r1enogHn. B HOHIIO BOJIOHHa rpynila Tecxo npHJlera1oll;nx Apyr H Apyrv nAep. - Pnc. 3. Mbimga
HpOJIHHa Mepe3 6 AHei1 Hocaae paHeHnH. HopMaJIbHbILI aMHTO3 Ha nepncfiepnn MbIIHeMHoro BOJIOHHa.
PHC. 4. Maiuiika HpoJIHHa Mepe3 9 AHeii noeiie paHeHnH. K MbIIHeMHbIM no'HaM np3IJIeralOT MHO-
6JIaCTbi, B HOTOpbIX npOTeiiaeT npollecc MHT03a. --- Pnc. 5. Mbituna HpoJIHHa Mepe3 9 AHeii nocJIe
paHeHnH. MHTO3bI B Mbo6JIaCTax Ha I{OHIIaX HapVmeHHbIX MbIHIeMHbIX BOJIOHOH. - PHe. 6.
MbImtl,a iipoatma Mepe3 9 AHex nociie paHeHnH. MIT03bI B MHO6JIaCTOx.
PIle. 1-3: I)nHeaLInn (J)opMaJInxoM, oxpacxa no van Gieson-y.
Pnc. 4-6: (J)1HCaI(IH POpMaJIHHOM, OlipaCHa IHCJIe3HbIM reMaTOHCIIJInHOM HO ha i3OH ra1lHy.
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 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
B. IIy.uua: 1 HO Hpocy ;leJteH H o Il;yep I lplt pe re I lepa I k 1111 flies I el I I01i y L [.I1111 bI ICpO:ntua. ' I'attit. VIII.
PHC, 7, S. MbIHIL4a HpoJIIiIGa iepe3 9 ;I,He11 ]IOC.1e paneHLIH. lYMHTO3b1 B MHo6J1aCTuX. -- PHC. 9.
MbtmiLa hpoZHHa 'tepee 9 1,Hefi IIOCJIe paHeHHH, MIITO3 13 M31O6aaCTe. jJe.IeHIIe HApa IIVTeM BM1IT03a.
Pile. 10. MbILHua HpO.IIHHa 'Iepe3 9 ;Hell noc.ie paHeHHH, MIITO3 B M11p6JIi1CTI' HeiiOcpCACTBCHHO
110 COCeACTBy C MbiIHe'IHbiM BOJIOHHOM. - PHC. 11. MbHltl[a hpOJIHHa 9epe3 9 ;I,Het1 IIOCJ1O
paHeHHH. Ali nTOa B MHO6JIaCTe (06pa3oBaHHe HeperopoJAhIt B HApI'). Ilepexo;( H HpHMHTIi1IHOMy
(AByx'bHAepHOMy) CHMHJIacTy. - Pile. 12. MbiLHua HpoJ1Hi a 9epe:3 20 ;uHe1i HoeJIe paHeHHH. Pae-
IIIHpeHHaH 'IaCTb hoIIISB M1,i11Ie'I1101'O I1OJIORHa, CO;I,epihallia lI H;tpa C OT'ICTJI11BO 1311,1Hbi MII IIPpt'-
1'OpOAhaMIl.
I'll(-. 7-12: (J)IiiO UI 1133 )J)opMaJI11HOB, OhpaClel )I;e.-IC3Hb1?t reMtaTOW HJI1HIOJI Ito Paii;9'HraiiHy.
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 Approved For Release 2008/04/10: CIA-RDP80T00246A002900500011-3
11. Bwcmpu xuu: It BonpOCy cTpVITypTI Biipyca Ta6a I1IOii Mo3aimit:
PHC. 1. I1vp!(fimaT BTM, HCHOCpeJ[CTBCHHO HOCIIC HpH6aB.TICHHH CHHpTa pa3BCJ[CHHbrH ABaxcA6t
J ocTH1JIHpoBaIIHO1i BOAOH H HaHCCCHHlrYI Ha HJICHIiy.
PHC. 2. IIypH(HIHaT BTM Laic Ha puC. 1. OTJjC;ICHHC 6aLIaCTHLIx 6CJIHOB C IIOBCpXHOCTII iia:io'iei .
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 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
Piic. 3. I ly pii(In irr I3'11I uoc:ic 21-m ojmro t('i-i(i'mm :),rii.iosoro cimp-ra upl[ 37 ?(:.
I'uc. 8. I1a.1o'lim 1 I \I us ronrpu:u>iruru upt'uapa,ra.
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 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
Pnc. 4, a, 6. IIypH HIcaT BTM nocne 48-vacoBoro AelCTBnn aTnnosoro CnnpTa npn 37 C.
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 Approved For Release 2008/04/10: CIA-RDP80T00246A002900500011-3
PHC. 5. 44-iaeosoe ;LeHCTBHe )TH.IOBOI'O cHHpTa Ha 1IypH(~HICaT HIM Hpit 37? C. Ita AICrrax,
O6o3HageIl H bIX 1,rj)c,:m )H O-, HCII palnJ16HOe pa36yX9HHe I:pa1B II I( n ii; c r pP:1 1 II - o6O-
:311aYeHa BH YTI3eH Hflfl II0.T1OCa 11 IIa:!o91ia\.
P11C. 6. llyp1l(J)HK9T HTV1, pi18Be;(CBHbIFl jMGHjb[ ;Le(rIGI. 111pOB IIII I1 Hf>;U11i 11 1l( ;LHCpraLIHI1 iicH
?4'I- IM-0110n1y ;leii CT131110 TenlllepaTyph1 B 37? C. Tpe;IKamii C -~ (16U31Ia lP111,1 'ra RiI Htt' mime I111H,
lcax u Ha puc. 5. CTp'Jll:oii -> o6oana I('Hhi OT;keJ1116IH11CCH vacTII I.L C OTHCprTHe.NI a Iuvrrpe.
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 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500011-3
,terzl, J.: The Production of Antibodies by Isolated Spleen Cells Following Contact with
an Antigen in vitro (IIITep9JIb, fl.: 06pa3OBaHHe aHTHTeSI H3OJIHpOBaHHbIM11 HJIeTrcamH ce-
JleaeHHH nocJle cMeuleHun c allTHreHom in vitro) . . . . . . . . . . . . . . . . . . 1
Johanovsky, J.: The Significance of Staphylococcal a toxin and Leucocidin (floranoB-
cuu I, 10.: 3Ha`ieHne cTa[IHJIoHoHI{oro aJIbd)a TOHCnHa H JIe iKouu u1Ha) . . . . . . . . 10
Dyr, J. and Protiva, J.: Metabolic Products During the Growth of Clostridium aceto-
butylicum (JbIp, H. H HpOTHBa, 10.: Hpo yHTb& o6Mena BewecTB B TegeHne pocTa
Clostridium acetobutylicum) . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Chaloupka, J.: The Influence of Ions on the Formation of Protease by the Actinomyces
Streptomyces griseus (XaJioynHa, 10.: BJIHHHHe HOHOB Ha o6pa3oBaHHe npoTeaabl JIyun-
CTbIM rpn6HoM Streptomyces griseus) . . . . . . . . . . . . . . . . . . . . . . . 24
Her6ik, F.: Photoreactivation of Bacteria Irradiated with X-rays (I'epuHH, d).: (DOTO-
peaHTHBH3agmn 6aHTepHrl noc.ne peHTrenoncxoro o6JIygeHHn) . . . . . . . . . . . . 31
HpaCJInYHa, M. H FHJIJI, M.: PaHHHe gHTOJIorH9ecHae H3MenennH JIHM(JOHHTapHOrt n mn-
3JIOHAHOII cocTaBHblx uacTefl MbImHHOi ce2Ie3eHKH nocae peHTrenoncxoro o6JlykellHH
(Prasli6ka, M. and Hill, M.: Early Cytological Changes in the Lymphocyte and Myeloid
Components of Mouse Spleen Following X-ray Irradiation) . . . . . . . . . . . . . 37
Haskova, V. and Hasek, M.: Increased Tolerance of Heterografts in Newborn Birds
(FamlOBa, B. H Fanlea, M.: HOBbmIenne coBMecTHMOCTH reTepOTpancnJIaHTaTOB y HOBO-
pO:HJjeHHbIX }HHBOTHbIX) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
HyaSa, B.: K nonpocy geoeHHn Bgep npH pereaepagxn cxe.neTHOrl MbImgbl Hponnxa . . . . 55
BblcTpHUHH#, B.: H BOH OC cTpyHTypbl BHpyca Ta6auHoit Mo3aHHH (Bystricky, V.: On the
Structure of Tobacco Mosaic Virus) . . . . . . . . . . . . . . . . . . . . . . . 59
KpaTHHe coo6n[eHHn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
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