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

• Document Type: CREST
• Collection: General CIA Records
• Document Number (FOIA) /ESDN (CREST): CIA-RDP80T00246A002900500012-2
• Release Decision: RIPPUB
• Original Classification: K
• Document Page Count: 92
• Document Creation Date: December 21, 2016
• Sequence Number: 12
• Content Type: REPORT

Approved For Release 2008/04/10: CIA-RDP80T00246A002900500012-2
CESKOSLOVENSKA AKADEMIE V$D
ACADEMIA SCIENTIARUM BOHEMOSLOVENICA
FOLIA
BIOLOGICA
.M*KI? UAlI ISTYI
CSAV1
Fol. biol. (Praha) Tom. 2 - Fasc. 4 Praha 24. 10. 1956
Approved For Release 2008/04/10: CIA-RDP80T00246A002900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
F O L I A B I O L O G I C A (PRA RA)
Medtcayttayoattoe 113aa1tne a1cyyrta.2oe Ceslaoslovenska biologie ii Ce.ckoslovensl,;a
mikrobiologie
P(' I a 11 C II 011 ua 11 00 Jt .-1 (' r It t[:
A1aj[emm; 11. 1VIaJtej (r.uaOHI,!11 pedda11'rop), B. Bp1111110Nnll, M. 1'x111(001 , 4.L-Iiopp. tlCAII
(h. Pep'uui, a11ageMn11 O. Iipoaeq, 10. ilouypa, anaieMo C. IIpaT, 13. PocIII1lalii (00lIp. pe;d.
iioJIJIeI rIo), A. rlep ltl,l ii, I. IIITepggib.
Ilepenour,r lra py1 o1111ii 11:11111: T(o1[. ;1-p Ultlpoaa, 110 a11IJTIldc'II6 flahRL' A-l) 11111(0cona, IIa ue-
w IlIIuil 1011,111: A-p Paf ,in,
IIa it'reEl Boo.[01,11oeoIinmt 1Ii[CTI1Ty'1'Oo UexooJIOnal~l(OIl AKa~eMInI irl yi n 11:130'l'CJIbe.THO II(:1~I1.
131,1 XO3IIT 6 pill 1 1,'O,1. 11031 El [C, tr11t 1101111 Ha 1 ro;( I#'10 60.-, 1e1111 0311010 llo0epa 1#'10
A3Spec pepllltllll: UIIOJIOI'IIYeC1Mr1 13HOTIATyT 11CA11, [III ][n1l'IIIIIITIi 2, I11ara XIX. 3a11a:n,l:
ApTIU1, Coe'1 on 30, 111:1111 II, ' IC XOOJIOnaI1111.
F O L I A B I O L O G I C A (P R A H A)
International I,'di,ti.on of the Journals Ceskoslovenske biologic and Ceskoslovens?ku
mikrobiologic
Academician I. MAlek (Chief Editor), L. (Ferny, M. 11odek, Corresponding Dlemher of t1w Czechoslovak
Aeademv of Science F. ]Iercik, Academician 0. Jirovec, J. Rlacura, AI'ELdcmicion S. Prat, 13. Rosi 'IIy
(Editorial Secretary), J. Sterzl, V. Vrz;ansky.
Translations into Russian: Dr Schierovd, into English: Dr hide( oVa, into Uermall: Dr Tcigcl.
Issued by Biologicly Qsttty Ceskoslovenske akademie vlid at Nakladatelstvi Cs. akademie ved. Yearly
subscription (6 Iuunbers) Ki s 60. Single number Kcs 10. Address: Biologicky ilstav CSAV, Na cviciAti 2,
Praha NIX. Orders: Artia, Smecky 30, Praha II, Czechoslovakia.
FOLIA BIOLOGICA (PRAHA)
Internationale Ansgabe der Zeitschriften Ceskoslovens/,-a biologie nntl Ceskoslovert,ska
nti,lerobiologi.e
Akadetniemitglied I. Malek (leitender Redalaeur), L. Corny, M. Hawk, korresp. 1litgl. d. Cs. Akademie
d. Wish. F. Hercik, Akademiemitglied O. Jirovee, J. Maeura, Akademiemitglil d S. I'rAt, B. Rosiclly
(Itedaktions-Sekretilr). J. Sterzl, V. Vrsanslcy.
Die Ubersetznngen besorgt Doz. Dr A. SchierovA fur die russiscl(en, Dr A. RidesovA fur die
englischen Imd Dr T. Feigel film die doutselien Artikel.
Herausgeber: Biologieky ilstav Ceskoslovenske akademie ved durch Vermittlung des Nakladatelstvi Cs.
akademie v6d. 6 Lieferungen jahrlich. Abonnementpreis 60 .6s, Preis der Einzehuunlner 10 Kcs.
Anschrift der Redaktion: Biologicky ustav CSAV, Na evicisti 2, Praha XIX. Zu heziehen lurch: Art ill,
Smecky 30, Praha II, Ceskoslovensko.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
FOLIA BIOLOGICA
The Mechanism of the Biological Action of Radiation*)
F. HERUK
Institute of Biophysics, Czechoslovak Academy of Science, Brno
Received April 10, 1956
The aim of the present communication is to draw attention to certain features of
decisive importance in the primary effect of radiation, which have hitherto been
neglected.
The processes which start the long chain of reactions leading to the final effect
of ionizing radiation, begin with the following basic stages:
1. An elementary process,
2. A primary process,
3. An intermediary reaction,
which then leads to the visible reaction by which the action of the radiation is estim-
ated. By the elementary process we mean the interaction between the photon or
particle on the one hand and the living matter on the other. The elementary process,
therefore, corresponds to the excitation and ionization produced by the absorbed
photon or particle. This is followed by the primary reaction, which is of a chemical
nature, and which either spreads directly, as in the case of the direct action of
radiation, or is brought about indirectly by the action of radiation absorbed through
an aqueous medium (action of radicals). The intermediary reaction involves the
effect of radiation on certain important components of living matter, which play
a decisive role in the resultant visible reaction, namely, the specific action of radiation
on protein and nucleic acids.
1. The Elementary Process
The absorbed photons of hard radiation or swiftly moving particles impart conside-
rable momentum to electrons or, in rarer cases, to whole atoms. The energy of photons
and particles is in most cases greater than the binding energy of the electrons
and a large number of excitations and ionizations therefore occur. Excitation can
occur only where the energy of the photon or the particles is less than the ionizing
potential of the atom. These states of excitation and ionization are very varied and
can finally lead to chemical change.
The energy required for extracting an electron from the atom is termed the
binding energy and is equal to several eV. This energy depends on the electric charge
of the particles concerned and on their distance from one another. The potential
energy of particles with one elementary charge and lying 1 A apart equals 15 eV.
The minimal energy required for excitation of external electrons is a few eV, whereas
the energy required for excitation of internal electrons is considerable and equals the
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
binding energy of the electrons. Chemical bonds develop equal to several eV between
the atoms. The forces of cohesion between atoms and molecules in large aggregates
and in macromolecules are then weakened still further. The energy required for
excitation of the bodily motion of the atom is therefore less than 1 eV.
Excitation differs from ionization in that the excited electron possesses excess
kinetic energy and does not break loose from the atomic structure; the atom soon
changes over to its basic state, during which it either releases electromagnetic
radiation in the form of fluorescent radiation, or internal re-grouping occurs within
the atom, leading to a photochemical reaction. The excited external electrons
change the inter-atomic bonds and thus the arrangement of the atoms within the
molecule and thereby this photochemical reaction develops. In this way the action
of weak photons of visible and ultra-violet light brings about a change in the chromo-
phore groups in biologically important molecules, resulting in a specific photo-
chemical reaction. Such chromophore groups are formed by certain biologically
important colouring matters (chlorophyll) or by the purine and pyrimidine groups
of nucleoproteins, which have a characteristic band at about 2,600 A. These cir-
cumstances are important with regard to the action of radiation on protein. The
protein molecule is composed of polypeptide chains, linked in a number of places
by hydrogen bonds. The binding energy of these bonds is about 0.2 eV. Pauling
(v. Franck and Platzmann 1954) is of the opinioti that the simultaneous disruption
of these bonds in several places leads to denaturation of proteins.
Similar destruction was recently produced by Ephrussi-Taylor and Latarjet
(1955), on irradiating the transforming factor of the pneumococcus, which may be
considered as nucleic acid. After X-ray irradiation with 33 KV it was seen that the
molecule was destroyed by a single "hit". The authors concede that the energy was
dissipated, about 50% in ionization and 50% in excitation, one ionization being
equal to about 4.5 excitations of 5 eV each. On this basis, specific absorption in the
chromophore groups of nucleic acid is inevitable. Owing to the fact that by certain
mutations, the quantum yield with a photon of 5 eV is 100-1,000 times smaller
than for ionization, these two authors therefore assume that excitation participates
in about only 5% of the transmitted energy and that it can be ignored.
In my opinion this view is not correct, since the external action of ultra-violet
light on biological units cannot be compared as a matter of course with excitation,
which develops within the protein molecule simultaneously with ionization. This
view is also supported in a communication by Watson (1950) and by my own
experiments.
Watson succeeded in reactivating phage which had been inactivated by X-ray
irradiation. This reactivation was carried out by visible light. It is assumed that
bacteriophage can be reactivated because one half of the absorbed energy of the
X-rays is dissipated in the form of excitations and the second half in the form of
ionizations. A phage inactivated by excitation can be reactivated by visible light.
For the photoreactivation of X-ray damage by visible light, the following cir-
cumstances must be taken account: On irradiation with a wave-length greater than
0.4 A, the photon is completely transformed into a photoelectron. During the
Compton effect (A < 0.4 A), besides ionizations, weak photons develop and a large
number of excitations are also evident. It may therefore be expected that in radiation
produced by a tension lower than 30 kV, photoreactivation will be very small or
non-existent, whereas with radiation at 60 KV, photoreactivation increases. This
assumption has been confirmed in experiments, which are now being conducted
in our laboratory.
Using visible light we succeeded in photoreactivating non-lysogenic bacteria,
of Escherichia coli B, which had previously been irradiated by X-rays (56 KV). The,
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
degree of photoreactivation is naturally not so marked as with photore activation
of damage caused by irradiation with ultra-violet rays. This is due to the fact that
only a small percentage of bacteria are damaged by excitation, since in most cases
ionization also participates. Details will be given in a separate report. Similar results
in lysogenic bacteria were obtained by Latarjet (1951).
In my opinion, excitation, particularly in the case of hard radiation, cannot be ignored.
The energy required for excitation of the electrons may not amount to as much as 20 eV (Fano 1954).
This energy is always a few eV lower than the binding energy of the electrons. Excitation of the atom
can bring about changes in the molecule which, although they may not lead directly to inactivation, may
have an effect on late inactivation.
Bacq and Alexander (1955) cite the communication of Burton, Magee and Samuel, who take the
view that the free radicals H and OH develop only by dissociation of excited water molecules. It is an
interesting hypothesis, which assumes the development of two types of excited molecules, of which only
one is rich in energy and sufficiently stable for the radicals, developed by dissociation, to have sufficient
time to escape. It is again seen, however, that excitations are significant for the biological action of
radiation. In addition to the facts already mentioned, there is still the further possibility of a shift of
energy from one part of a macromolecule to another. These phenomena of energy migration are known to
exist in crystals (Riehl 1940) and were found by Wallenstein (1952), to exist in hydrocarbon molecules
also. In the case of large molecules, such as insulin, trypsin or chymotrypsin, Pollard assumes that
energy migration comes into play in the difference between molecular weight as determined by direct
action and by other methods. From this it is already evident that the processes which take place in
macromolecules after absorption of radiation are very complex and that it is not sufficient to interpret
them by a simplifying conception such as inactivation of a whole molecule by a single ionization.
As far as the action of actual ionization is concerned, these processes are suf-
ficiently well known and will be only touched on here. For ionization of an atom
there must be a sufficient amount of energy (in the form of absorbed photons or
colliding electrons) to overcome the ionizing potential of the atom. Ionizing poten-
tials have been exactly determined for only some elements and range within the
limits of 3.9 eV for CS and 24.6 eV for He (Franck and Platzmann 1954). Ionization
of a molecule, on the other hand, is relatively easier. In this case it is necessary to
increase the basic electronic state of the molecule. In stable molecules the ionizing
potential is between 10 and 16 eV. As already mentioned, in ionization electrons are
released which can again be caught up by positive ions. In this case, however, the
process is not one of simple supplementation, but the molecule becomes electrically
excited. Dissociation may occur or its oscillation energy may be increased. In this
connection Franck and Platzmann draw attention to one feature which very sharply
differentiates gaseous ionization from aqueous ionization; freshly formed ions in
aqueous solutions are very quickly hydrated, and a considerable amount of heat is
freed. All these facts lead to the conclusion, with which we agree that the experi-
mentally ascertained "sensitive volume" is not related to some specific part of the
molecule, but that it is the average of effective cross-sections for various types of
excitation and that this is modified still further by the corresponding probability
with which the given biological affect occurs.
A similar point of view was recently adopted by Volkova and Pasynsky (1955), who irradiated
protein solutions with ultra-violet and X-rays and found that the quantum yield required for the
denaturation of seralbumin in an aqueous solution was 7 x 10-4, whereas a 37 % dose of X-rays is 10?r.
With these doses of irradiation it was seen that amino nitrogen is more or less stable, so that only 2 - 3
peptide links are broken down in the protein molecule (molecular weight 70,000), corresponding to an
intensity of hydrolysis of 0.6%. Denaturation of protein following irradiation cannot therefore be ex-
plained by disruption of the peptide chains, but by re-grouping of the intramolecular bonds within the
protein molecule. This involves the hydrogen bonds already mentioned above. Pasynsky is of the opinion
that from 20 to 40 hydrogen bonds need to be destroyed in order to produce probable denaturation of the
protein molecule. He opposes the view, however, that the small probability of such simultaneous
destruction of the hydrogen bonds should be based on "hitting" a special centre in the molecule. He
points out that it is not correct to speak of an ionizing effect of X-rays in relation to the target theory,
since the passage of these photons through the molecule leads to a quantity of excitations of the molecules
that could possibly exceed the number of ionized molecules and be sufficient to bring about denaturation
of the protein molecules.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
There is some evidence that on the track of a swiftly-moving particle not only
excitations and ionizations are found but also active radicals, which develop as
a result of these elementary physical processes. From this it could be concluded that
in some cases further intermediary products following irradiation are also distri-
buted in column formation in the living matter for a small fraction of a second.
2. Primary Reaction
As already stated in the introduction, the elementary physical process gives rise
to a primary reaction, which covers the group of phenomena occurring in the irra-
diated area. The primary reaction, is caused either by direct action or indirectly
by radicals released in an aqueous medium. These theories, which formerly conflicted
with each other, are now regarded as being complementary.
As far as direct action is concerned, there are two things which should be very
carefully differentiated. Statistical fluctuations undoubtedly take place in the
incidence of elementary physical processes. This is due to the physical character of
the whole process and there can be no doubt that it is actually so. Nor can there be
any doubt that "sensitivity" is not evenly distributed in living matter. That was
demonstrated many years ago in our own experiments (Hercik 1939) and in those
of Petrova (1942). Using alpha radiation of varying strengths it was demonstrated
that the nucleus was considerably more sensitive than cytoplasm. It is therefore very
probable that discontinuous action occurs in living matter the sensitivity of which
is uneven. These facts should, however, be distinguished very carefully from a gener-
alisation such as the target hypothesis. According to this hypothesis, a sensitive
volume exists in the cell, in which the direct action of one or more ionizations causes
a change which leads to its destruction and thus to the death of the cell, or to
a profound functional or morphological change. The target theory is incorrect
mainly in both these assumptions, which are exclusively of a biological character.
As already stated, no objections can be raised to physical facts, but the conception of
a single sensitive volume for the whole cell is indubitably incorrect. It cannot be
assumed that there are parts of the cell with very low sensitivity and, on the other
hand, that it contains only one macromolecule, about 10-e cm. in diameter, which
decides on the life of the whole cell.. There is no doubt that various processes parti-
cipate in living systems, in which considerable amplification of the original stimulus
occurs, but the hypothetical assumption of a sensitive volume in the cell, limited to
a certain structure, is too improbable.
At the present time, the target theory is being subjected to criticism. Fano (1954) states, for example,
that many facts are accumulating which oppose this hypothesis. If the uneveness of biological effect
depends only on the chance localisation of the primary processes, then the correlation between the two
chance phenomena ought to have a definite geometrical structure, i. e., the trend of the curves expressing
the relation to the dose ought to be independent of the factors of the environment. Since this is the case,
an auxiliary hypothesis is required, which, according to Fano, detracts from the weight of the original
working hypothesis. Houtermans (1953) makes a critical analysis of the target hypothesis and points
out that the chief substantiation of this-the "single hit" curve-i., a formal phenomenon, from which
all the others are deduced. I once suggested that instead of the single sensitive volume the possibility of
sensitive sites in the cell should be taken into account (Her6ik 1946), These sites would change their
localisation according to the area where the most sensitive reactions to radiation are taking place.
As I see it today, however, this modification of the target theory actually changes its own basis, since it
denies the existence of a sensitive. volume. If the various objections to the target theory are taken into
account, the only fact which can finally be conceded is that the effective cross-section can be calculated
from the relatonship between the biological effect and the radiation dose. The values of the effective
average of different types of radiation and different objects have only a formal significance, which in
a certain way expresses the scnsitivityof a given object to radiation. They have a true significance only in
homogenous systems.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
This brings us to the question of whether the target theory also remains valid for
determining the size of single objects such as macromolecules, viruses, etc. Lea (194$)
verified the target theory when irradiating molecules of myosin and ribonuclease
and from the decrease in activity determined the inactivation volume and from
this the molecular weight. His results were in agreement with the known molecular
volume. Since then, many similar experiments have been carried out. Pollard (1955)
established a detailed table giving approximately 40 molecular volumes determined
by radiation in contrast to other methods. From these tables, it can be seen that
considerable differences exist between molecular weights determined by radiation
and other methods. It is not possible, therefore, to share Pollard's optimistic view
that this technique has promising prospects.
It cannot be said that the target and radical hypotheses complement each other in
any way. The hypothesis of radicals is based on the assumption that the action of
radiation on water leads to the development of the radicals OH, HO2 etc., in the
course of the primary reaction, and that these, in the presence of oxygen, are con-
verted into H2O2. The presence of these radicals changes according to the structure
of the environment. It is assumed that the above-mentioned radicals develop on
irradiation of pure water. Their existence with the exception of the radical OH
(Dainton 1948), has not yet been determined by a direct method.
In general, it may be said that a primary reaction may be seen in the disintegration
of water or of other simple molecules by radiation, which automatically follows the
process of elementary absorption in matter.
Densitity of ionizing radiation, such as alpha particals, produces such a concen-
tration of hydroxyl radicals on their track that a molar level is reached (Gray 1955).
In such a case the formation of H2O2 is more than usually accelerated. On the other
hand, around swiftly moving electrons radicals are formed in concentrations of
about 1,000 times smaller and there is therefore a preponderance of the reaction
H + OH = H2O, by which the formation of radicals is "stabilized" and peroxide
can again be formed. Radicals have hitherto not been reliably demonstrated, since
they are very short-lived, with the exception of the radical OH, found by Dainton
in 1948, so that in pulsed radiation sources they could be demonstrated by micro-
wave spectroscopy, as has already been done in the case of hydrogen atoms in
rradiated ice (Gray 1955).
It may be seen from the foregoing that oxygen is of great importance for irra-
diation. It would be a mistake, however, to evaluate the presence of oxygen only in
the light of the hypothesis of radicals. The role of oxygen in respiration and meta-
bolism should not be forgotten. In this connection Gray draws attention to the
work of Laser, who found that the radiosensitivity of bacteria ceases to be dependent
on the concentration of oxygen from the moment their enzyme system is damaged
by poison. The part played by oxygen in chromosome breaks is also very complex.
The concentration of oxygen not only has an influence on increased radiosensitivity,
but also on the restoration processes which develop in the later phase following
irradiation (Giles 1954).
It is not possible to deal here in detail with one question which is still a subject
of discussion, namely whether the final product of the primary biological reaction in
the presence of oxygen is hydrogen peroxide, which has been demonstrated in pure
water, or whether it is the radicals OH and HO2. It would appear necessary to differ-
entiate the final biological effect. Sufficient material has been collected in favour of
hydrogen peroxide, most of which concerns indirect evidence. It must, however, be
taken into account that hydrogen peroxide is very rapidly broken down by catalase.
Finally, I should, like to stress that all these reactions are of very short duration,
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
which leads us to the opinion that what we call the primary reaction must of
necessity take place within a fraction of a second.
3. The Intermediary Reaction
The primary action is followed by the intermediary reaction, which in a certain
sense is the most obscure part of the whole process. We should not fail to take into
account the great complexity of the reactions which take place in living matter
and to label one of them as the intermediary reaction following irradiation by penetrat-
ing rays would not be correct. It would seem more likely that qualitatively different
types of reactions take place, depending on the dose of radiation. In other words, a
small dose of radiation produces a qualitatively different reaction from a large dose.
Until recently, the majority of radiobiological reactions were tested on very rough indi-
cators, such as death of the cell, chromosome breaks, a decrease in metabolism, etc.
In some cases very large doses are required to produce the desired effect. It is known
that in order to kill certain Protozoa, doses of hundreds of thousands of r are
needed. On the other hand, however, a dose of only 30 r reduces growth of a root
after only a few days. A few hundred r cause destruction of the meristem and the
root dies in a few days. If a site only 1 cm. from the root tip is irradiated, a dose
of 200,000 r is required to reduce mitosis. This means that during 24 hours the
sensitivity of the root is changed by 1,000 times (Gray 1955). This demonstrates how
important it is to find the correct relationship between the dose and the sensitivity
of the object. I should like to draw attention to some circumstances which have
a significance for understanding the intermediary reaction in living systems. Latarjet
(1951) found that X-rays can evoke the formation of bacteriophage in lysogenic
strains. Recently the development of a lytic factor in a non-lysogenic strain of
Escherichia coli was evoked by supersonics (Hradecna) and later also by X-ray
irradiation of 60 kV (Hercik). Further details are given in separate reports. It has
already been known for some time that ultra-violet light has a similar action (Jacob
1950, Hradecna 1952). In Escherichia coli, ultra-violet radiation gives rise to the
formation of colicine (Lwoff 1953), an antibiotic produced by a number of strains of
intestinal bacteria. It is also interesting that hydrogen peroxide can evoke the
formation of phage in lysogenic bacteria and of colicine in colicinogenic bacteria.
The intensity of this phenomenon is influenced by the concentration of oxygen. In
summing up it may be said that the action of X-ray radiation or the direct influence
of hydrogen peroxide brings about changes in bacteria which lead to the formation
of bacteriophage (Lwoff). In this connection it should be pointed out that, according
to the latest research, the formation of bacteriophage is due to a deviation in nucleo-
protein metabolism. It follows from this that radiation or the action of hydrogen
peroxide can also bring about a similar deviation in nucleoprotein metabolism.
Yamafugi and Osawa (cit. by Lwoff 1953) achieved the formation of insect viruses
by the action of hydrogen peroxide and by the influence of other peroxides, such as
tertiolutyl. These peroxides, however, are known to be cancerogenic. We therefore
arrive at the conclusion that there is a whole series of physical or chemical factors
capable of producing a change in nucleoprotein metabolism, which leads to the
development of a substance termed a deviant, which in some cases has the character
of a virus and in others of a foreign protein.
It must be concluded from the above that quality of the damage caused by ionizing
radiation does actually depend on the radiation dose. With a small dose of radiation,
there can only be a slight deviation in nucleoprotein metabolism, which may be
manifested by a permanent, hereditary change. With larger doses, disintegration
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
_d-
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
of nucleoprotein takes place as a result of de-amination and dephosphorylation.
This does not mean, of course, that the reaction between radiation and the organism
takes place only on the nucleoprotein substrate. Other substances are also involved.
We believe that the nucleoproteins are the central axis, as it were, of all these
changes.
B a c q, Z. M., A I e x a n d e r, P.: Fundamentals of Radiobiology. London 1955.
B a r r o n, E. S. G.: The Effects of X-ray on Systems of Biological Importance. A. Hollaender,
Radiation Biology. I. New York, 1954.
D a i n t o n, F. S.: J. Phys. Colloid Chem. 52 : 490, 1948. Cited by Bacq-Alexander, 1955.
Ephrussi - Taylor, H., L a t a r j e t, R.: Inactivation, par les rayons X, d'un
facteur transformant du pneumococque. Biochim. Biophys. Acta 16 : 183, 1955.
F a n o, U.: Principles of Radiological Physics. A. Hollaender, Radiation Biology. I. New
York 1954.
F r a n c k, J., P 1 a t z m a n n, R.: Physical Principles Underlying Photochemical, Radia-
tion-chemical and Radiobiological Reactions. A. Hollaender, Radiation Biology. I. New
York 1954.
G i 1 e s, N. H.: A. Hollaender, Radiation Biology. II. New York 1954.
G r a y, L. H.: Biological Damage Resulting from Exposure to Ionising Radiation. Intern.
Conf. Atomic Energy, Geneva, P/899, 1955.
H e r e i k, F.: Vber die Wirkung der Alpha-Strahlen auf die Zelle mit besonderer Beruck-
sichtigung der Kernreaktion. Strahlentherapie 64 : 655, 1939.
H e r e i k, F.: Od atomu k zivotu. Praha 1946.
H o u t e r m a n s, T h.: Kritische Diskussion der Argumente fur die Interpretation der
Inaktivierung von Bakterien durch Strahlen als Eintreffervorgang. Strahlentherapie 92 : 423,
1953.
H r a d e 6 n a, Z.: ULinek ultrafialoveho svetla na bakterie Escherichia coli. Cs. biologic
1 : 348, 1952.
J a c o b, M. F.: Induction de la lyse et de la production de bacteriophages chez une Pseudo-
monas pyocyanea lysogene. C. R. Acad. Sci. 231 : 1585, 1950.
L a t a r j e t, R.: Photo-restauration apres irradiation X chez une bacterie lysogene. C. R. Acad.
Sci. 232 : 1713, 1951.
L a t a r j e t, R.: Induction par les rayons X, de la production d'un bacteriophage chez
B. megatherium lysogene. Ann. Inst. Pasteur 81 :389 , 1951.
L e a, D. E.: Actions of Radiation on Living Cells. Cambridge 1946.
L w o f f, A.: L'Induction. Ann. Inst. Pasteur 84 : 225, 1953.
P e t r o v a, J.: Uber die verschiedenen Wirkungen von Alpha-Strahlen auf Kern and Plasma
der Zelle. Beih. Botan. Zbl. 61 : 399, 1942.
P 1 a t z m a n n, R.: Subexcitation Electrons. Rad. Res. 2: 1, 1955.
P o 1 1 a r d, E. C. e t a I.: The Direct Action of Ionizing Radiation on Enzymes and
Antigens. Progress in Biophysics 5 : 72, 1955.
R i e h 1, N.: Die ,Energiewanderung" in Kristallen and Molekulkomplexen. Naturwissen-
schaften 28 : 601, 1940.
W a 1 1 e n s t e i n, M. e t a 1.: N i c k s o n, J. J., Symposium on Radiobiology. New
York, Wiley, 1952.
W a t s o n, J. D.: The Properties of X-ray inactivated Bacteriophage. J. Bact. 60 : 697, 1950.
Z i r k I e, R. E.: The Radiobiological Importance of Linear Energy Transfer. A. Hollaender,
Radiation Biology. I. New York 1954.
T p a g e B H a q, 3.: B.nnaxne ynbTpac HOJ eJioBbix nyneu Ha 6axTepnu Escherichia coli.
LIcn. EHonorHH 1 : 309, 1952.
B o n H o B a, M. C., 11 a c u H c x H #, A. P.: L ellcTBae yJITbpa isoneToBoro H peHTreaoscxoro
HanyveHHa Ha pacTBOpbI 6enxos. BnoxHniua 20 : 470, 1955.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
MexaHHBM 6noaorHgeCK0ro AeHcTBIIH o6JIy'JeHHH
(D. rEPLIHH
Hpoueccbl, IIpOTeKaIOH[He B ?KHBOM Be1I[CCTBe, 11OJL]3epraB11I0MCa 1(IiCTBHIO IIo111131I-
pyioII[erO o6JIy'IeHHH, MO?KIIO pa3j0JIHTb Ha: 3JIoMCHTapHb1B IIpouCCC, IIOpBII'IHyIO
peaxuwlo H HHTepMeJ(Hapxylo peaHIjHIO, IIOCT10 KOTOpOH TOJIbKO CJICJLyCT Co6CCBOHII()
BHJ 14MbI>'I 344CKT o6JIyWHHH. Y 3JIeMeHTapxoro iipouecca Ba?KHa He TOJIbKO IIOIIH-
3auHH, Ho II 3KCunTauHH, 3Ha'IeHH0 KOTOpoFi JLo CHx Hop HeclpaB0JRIIBO IICJLooueBH-
BaJIOCb. 0 HaJIH'IHH 6umorwIecHorO JLeliCTBHH 3KCIilTaUHH rOBOplIT H TOT (T)a1T, 1T()
B HeKOTOpbIX CJIy3aax yJLaBaiioCb HpOH3BCCTII 4)oTOpeaKTHBHaaUHlo 10fiCTB11H
peHTI'eHOBCKOrO 06JIy'IeHHH. Ho MHeIHHIO H0KOTOpbIX 14CCJIeJjoBaTeJIe1i, cBo6oJk}Ib10,
pajHKaJIbl H H 011 BO3HHKaI0T TOJIbKO B 1)03 JlbTaTC ijiccommitHH aKCUHTHpcBaHHbIx
MoJIeKyJI BOJjbI. 0co6o Ba?KHOe 3Ha40HHe 9KC9HTauHa HMeeT HIM B03,L011CTB14H o6Jly-
'1e11140M Ha 6eJiKII, KorIa HpO:IICTaIO1I[He (J0TOHbI MoryT Bb13BaTb 3KCILH'I'auI11O B Ha-
CTOJIbKO BbICOKOII CTOI1OHH, 4TO MoJICKyJia HMH J[0HaTypHpyeTC}.
B HOpBII9BOM IIpouecce ocBo6o?KJILaIOH[Heca paJ[HKaJIbI HMeJOT 6OJIbHI00 3IIawime,
1eM HpBMO13 yJlap. HpojjCTaBJIeBHe o KJIeTKe, KaK o OJ 14HOM, 110 BCCMy o67>0 My
OJ IIHaKOBO 'IyBCTBHTCJIbHOM TOM, IIOB144HMOMy, HCHpaBHJIbHO. 11pIlMCIICHIIC I'11llo-
Te3b1 MHHIeHH JjJia OHp0JLCJIOHHH pa3MCpOB MOJIOKyJI TO"tK0 CTpaJjaeT pa3JIH9ilbIMII
HCJLOCTaTKaMH. HeJIb3H CKa3aTb, 'ITO rHIIOT03a MIlmeHH H rHHOTC3a pajLHKaJ10B
lb JjOIIOJIHHIOT jlpyr jlpyra.
KaK-HI16y,
HHTepMe IIapHaH peaKuHH HanMeHee 113y'IeHa. BOCbMa Bep0iTIlo, 'ITO B 3aBIICL1-
MOCTH OT IILo3bl O6Jlyg0H14a B03IIIIKaiOT Ka'IeCTBOHHO pa3JIH4HbI0 THIIMI peaKI01H.
LJIH T0'30HIIH IIHTepMeJLBap1ioH peaKuHH 60JIbIUOC 311a'I0HH0 IIMOOT JjeBIIauua MCTa-
6oJiH:3Ma HyHJIeOIIpOTeHJjOB, Bo3HHKaloHlaa y 6aKTepBH HpH He6oJIbIHIIX JL03aX
peHTI'CHOBCICOFO 06JIy'I0HHa 11 11 HBOJjFIuaa K 06pa30BaHHIo JILITHYCCIiHX (j)aHTOp013.
IIpHBOjUTTCH HpIIMOpbI, B3HTble 113 Co6CTBeHIIbIX HCCJIeJ(OBanhl. YKa3b1BaeTCB Ha T0,
'IT`) JI143OreHHi1H 6aKTepHO4)ar BO3HHKaCT IIOJj j[OHCTBIIeM pOIITTCHOBCKOI'O o6Jlytlell1a.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
FOLIA BIOLOGICA
DaroiIII3HC HjieT0 cTa4IYIJIoJoHJ a
M. P03EHBEPI'
Bnon10rBgecR141t Ir1HCTHTyT Me Bi UHCHOro 41axyJlbTeTa YHHBepclTeTa B Bpno
Hocmynu/ o e peaan ulo 3 I V 1956
B noc7Iej(Hiie roJjbi npo611eMe 6ahTepnol ara nOCBHI1jaeTCH nOBbIIIIeHHOe BHFI-
MalHe, - HecoMHeHHO, IIOTOMy, LTTO 6awTepno4)arFH TeCHO CBH3axa c npo6JleMaMH
BHpycoMornH, a KpOMe Toro HMeeT H 0611je6H0J10r11eCH0e 3HaLIeHHe. Oco6eHHO
HHTeHCIIBHO C 1IOMOHjbIO 3JICHTpOHHOTO MuHpocHOHa nCCJIejjOBarIaCb MOp1lOJIOP111I
6aHTepnoc ara u MexaHn3MbI ero o6pa3OBaHHH.
Ogellb nojjpo6HO 3Ty npo6iieMy H3yuaJi Pepqiu (1953), KHHra KOTOpOFO Kjjy Jjei1CTBHeM 6aKTepno4 ara Ha hyBCTBHTeJlbxble KJIeTHn
H JjeI1CTBHeM yJ1bTpa4IIIOJIeTOBb1X JlyileH Ha JIH3oreHHMO KJIeTHH BeJTIIKa. BecbMa
BepowTHO, LITO IIpoljeccbl, BeJjywne B o6011X cjiyuanx K o6pa3oBannlo 6aKTepno(ara,
owHb 6JI43Hn jjpyr H jjpyry. HeCOMHeHHO, rnaBHylo pOJlb B 3Tnx upoi eccax nrpaeT
6aRTepnaJlbHan KJTeTKa, Tax KaK JMHyJlbc, BbT3hIBaiomHI3 BOCHPOII3BejjeHne 6aHTe-
pHol ara, MOMeT 6bITb coBceM Hecneljn(fI41IecKHM.
1. C HOMOnlbIO I a3oBOFO M1HpoCKOna HCCJIejjOBaJICH J1n3HC JIH3oreHHOrO IHTaMMa
cTaf HJIOKoKKa LS 2 HoCJte yJlbTpa( Ho;1OTOBOFO o6JlyheHnfl. AJTH cpaBHeHIH Ha61llo-
jjaJicH pacna)j 'IyBCTBHTeJTbHOr0 IIITaMMa cTa(njToKoKKa S 3 nojj BJIlHHneM 6aKTepno-
4ara, n30JI11 OBaHHoro H3 JlH30FeHHOrO IHTaMMa LS 2.
2. 113 cepnn M1Kpo4)OTorpa(ni1 ogeB11 HO, 11TO TeheHHe 6aKTepnoJ II3a B o6o11X
CJIyianX BecbMa cxo2Ke.
3. O6JIytIaBInneeq HJIeTH1 6aHTepHii nepejj pacnaJloM He ZjeJIfITCH, HO Bcerjja
3Hai1HTeJTbHO yBeJI1h11B IOTCH B o6'beMe.
4. JIITWIeCHne npOIjecebl, OTMeh HHite B Jl13oreHHOI1 KyJlbType noc rie yJlbTpa-
(rnOJIeTOBOro o6JlytleHHf1, npOTeHa1OT MejjJleHHee, item npoiiecebi pacnajja hYBCTBn-
TeJlbHbIX KJIeTOK, 3apa5KOHHLIX 6aKTepno4IaroM.
(Ta6.x. XIX, XX)
JlxTepaTypa
r p a g e u H a fi, 3.: BJIHIIHHe yJ1bTpac no.IeTOBbIx .uyueu Ha 6aRTepun Esch. coli. 11ca. Bxo-
JlornH 1 : 348, 1952.
B a i 1, 0.: Der Kolistamm 88 von Gildemeister and Herzberg. Med. Min. Wschr. Munchen
21 : 1271, 1925.
B o r d e t, J.: Ann. Inst. Pasteur 39 : 711, 1925. Cit. Lwoff, A., Bact. Rev. 17 : 269, 1953.
Lysogeny.
H e r 6 i k, F.: U6inek ultrafialoveho sv6tla na tvorbu bakteriofaga. Biol. listy 30 : 169, 1949.
J a c o b, F.: Induction de la lyse et de la production de bacteriophages chez un Pseudomonas
pyocyanea lysogene. C. R. Acad. Sci. Paris 231 : 1585, 1950.
L a t a r j e t, R.: Induction par les rayons X de la production d'un bacteriophage chez B. mega-
therium lysogene. Ann. Inst. Pasteur 81 : 389, 1951.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
L w o f f, A., S i m i n o v i t c h, L., K j e I d g a a r d, N.: Induction de la production de
bacteriophages chez une bacterie lysogene. Ann. Ins. Pasteur 79 : 815, 1950.
L w o f f, A., S i m i n o v i t c h, L.: Induction du developement du prophage par its sub-
stances reductrices. Ann. Inst. Pasteur 82 : 676, 1952.
L w o f f, A.: Lysogenic Bacteria. Endeavour 11 : 72, 1952.
Rose n b e r g, M., S m a r d a, J., J a k u b i k, J.: Produkce nekolika typo bakteriofagu
lysogennim kmenem Staphylococcus aureus. Cs. biologic 4 : 457, 1955.
Rose n berg, M., S mar d a, J., J a k u b i k, J.: Srovnani nekterych vlastnosti
bakteriofagu, produkovan~ch lysogennimi bakterierni a bakteriofagu, odvozenych z nich
pasdli. Cs. biologic 4 : 449, 1955.
R o s e n b e r g, M., S m a r d a, J.: Staphylococcal Bacteriophages of Lysogenic Origin
and a Comparison of these with Bacteriophages Subjected to Passage. Fol. biol. (Praha)
1 : 339, 1955.
T o p 1 e y - W i 1 s o n, G. S.: Principles of Bacteriology and Imunity. London 1947.
The Dynamics of the Breaking-down of Lysogenic Cells
Irradiated by Ultra-Violet Light
Summary
Lysis of a lysogenic staphylococcal strain was observed in the phase microscope
after irradiation with ultra-violet light. For comparison, the breaking-down of the
sensitive staphylococcal strain S 3 was observed, following the action of bacterio-
phage released by the lysogenic strain LS 2. It is evident from the series of micro-
photographs that the course of bacterial lysis is similar in both cases. The bacterial
cells irradiated by ultra-violet light do not divide before being broken down, but
always considerably increase in size. The lytic processes which were observed in the
lysogenic culture following irradiation with ultra-violet light have a slower course
than following the infection of sensitive cells with bacteriophage.
(Plates XIX, XX)
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
The Character of Leucocyte Reaction Following X-Irradiation
Z. KARPFEL
Institute of Biophysics, Czechoslovak Academy of Science, Brno
. After initial leucocytosis in the reaction of the blood to ionizing irradiation has
receded, leucopenia develops as a result of the cytostatic action of the X-rays on the
tissues of the organism. As a result of regeneration processes, the leucocyte level,
after a certain period, returns to normal, the time required for this showing a rela-
tionship to the dose used and the manner of administration, to the functional
condition and individual resistance of the blood-forming organs, alongside other
factors which form a concrete potential for the general capacity to control the
consequences of irradiation sickness.
As far as the initial increase in leucocytes is concerned, most radiohaematologists
are agreed that its occurrence is a characteristic feature after the administration of
high doses of ionizing radiation. On the further course of the post-irradiation leuco-
cyte curve there are altogether three opinions, which are based on experimental
results.
One group of authors describes the regeneration phase following a sudden or gradual maximal
decrease, as being more or less irregular, with frequent fluctuations and a gradual increase in the white
blood components in the surviving animals (Yegorov 1955).
The second group described another phase, within this one-an abortive rise in the leucocytes-
which appears between the second and eleventh day after irradiation (Jacobson et al. 1949-in experi -
mental animals) or even later (Hempelmann et al. 1952-in human subjects affected by ionizing rays).
Only a few communications mention that the post-irradiation course of the reaction of the blood is
characterised by a periodic, wave-like form. The first to describe this wave-like characteristic were
Langendorff and Paperitz (1939), on the basis of their observations on the bone marrow and peripheral
blood of irradiated mice. In their series, the minimum and maximum values in the curves are reached
after about 40 days. The rhythm they describe is thus of relatively long duration. Regeneration waves in
haemopoietic tissue were also observed by Bloom and Murray and by Jacobson and Block. Derer (1953)
observed periodic decreases in leucocytes following X-irradiation of leukaemic patients, with an interval
of approximately six days. A periodic leucocyte reaction was also described by Uher (1952) in his
communication on the time factor in regulation of the blood and by Thalhammer and Janicek (1951)
in their experiments with vitamin D.
The aim of our experiments was to verify the periodic response of the white blo od
component following irradiation with X-rays and to ascertain the relationship b e-
tween this response and the dose used and fractionation of the dose and, finally, t o
study the development of the reaction after irradiation of experimental animals
during narcosis (these latter experiments were carried out by Dr Praslioka).
The experimental animals were rabbits, Vienna breed, blue-eyed, weighing from 2,000-3,000 g.
Blood was collected from the ear vein daily, at the same time. The blood picture was studied for two
months after irradiation. All the animals were subjected to total irradiation with a Super-Sanax lamp
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
at 180 kV, 15 mA, F = 50 cm., filter 1 mm. Al, 0.5 mm. Cu with a feed of 33 r/min. The single doses
were as follows: 200 r (6 animals), 350 r (8) and 800 r (11); on fractionation a total dose of 1100 r was
divided into 11 daily doses of 100 r (10 animals), while the narcotized experimental animals were irra-
diated with a single dose of 350 r (4 animals). The narcotic used was ether, in a concentration which
guaranteed deep narcosis during irradiation; narcosis was also maintained for a further 60 minutes after
the lamp had been switched off. In every group of experimental animals the blood picture of three
control animals was also studied; the initial values before irradiation were determined from eight
specimens.
In our case, the curve showing the leucocyte response to the action of ionizing
radiation results mainly from two antagonistic processes. On the one hand there
is the protracted destructive action of radiation on blood formation, and on the other
manifestations of regeneration. The third possible factor, i. e. changes in the
distribution of blood, does not participate to any appreciable extent in the prolonged
study of changes in the blood, particularly with the method of total irradiation used
by us. In studying blood changes we ascertained when the fluctuations in the
number of leucocytes took place in the time curve, both from the positive and the
negative aspect.
The following findings were made: The incidence of the initial rise in the number
of leucocytes, due to an increase in the number of heterophils, shows a relationship
to the dose used. It occurs mainly on using higher doses. In our series this first
positive deflection was not recorded after 24 hours on using a dose of 200 r. It was,
however, recorded in two cases (out of eight) with a dose of 350 r. and in eight cases
(out of eleven) with a dose of 800 r. On fractionation of the dose the deflection was
not found and in irradiation of narcotized animals with a dose of 350 r, it occurred
once (out of four cases). The peak of the first negative phase is related to the indivi-
duality of the reaction of the various animals and occurs between the second and
fifth day. The maximal decrease occurs most frequently on the third day (50%)
or on the fourth day (20%). The incidence of the subsequent positive phase varies
still more in the individual animals, its peak being reached between the second and
the twelfth day. It occurs most frequently between the fourth and the sixth day
(70%). The peak of the following descending phase occurs between the fifth and the
15th day, most frequently between the seventh and the eight day (50%) and then
from the sixth to the 12 day (15%). A later incidence of this descending phase is not
related to the time incidence of the minimum in the previous wave, but follows
retarded commencement of the preceding ascending phase. A further increase again
occurs in the number of leucocytes, which reaches its peak, according to the indi-
vidual cases, between the eighth and the 16th day. With a dose of 200 r, no further
deflections occur. Processes of regeneration evidently predominate over destructive
processes and the white blood picture tends to regain its normal level. With higher
doses (in our case 350 and 800 r) and with fractionation, further deflections often
occur.
It is not possible to speak of any regularity, of a chronologically identical, periodic
incidence of limit values in the number of leucocytes, either maximal or minimal.
The incidence of alternating maximal and minimal values and their time curve tend
rather to give a picture of an irregular sinusoid, in which the length of the individual
waves varies more or less from case to case with the individual animals. On the
basis of our experiments it is impossible to say whether, and how far, the length of
these waves depends on the size of the dose of ionizing radiation. With a dose
of 200 r it appears to be shorter, since the average length of the interval between the
negative deflections is three days and between the positive deflections 4.3 days.
With the other methods of irradiation the average length of the intervals was as
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
follows: with 350 r, six days between the minima and 7.1 between the maxima;
with 800 r, six days between the minima (figures were not obtained for the maxima,
since the intervals varied markedly). With fractionated irradiation the average
length of the intervals between the minima was 5.4 days and between the maxima
5.3 days.
As far as the absolute values of the decreases are concerned, it was observed that
the maximal decrease occurred with doses of 200 r and 350 r in the first negative
phase, i. e. between the second and fifth day. Following irradiation with a dose
of 800 r, the lowest negative amplitude was mostly found in one of the following
negative phases and in fractionation of radiation this was always the case.
The results of irradiation in narcosis did not differ basically from the results with
a single administration of the corresponding radiation dose without narcosis.
The incidence of the initial rise in the number of heterophils is in agreement with
the data of Jacobson et al., who state that a significant increase takes place in the
heterophils in the peripheral blood of rabbits after doses of 400 r and more. This
does not mean that this increase, which often results in a rise in the absolute number
of leucocytes, could not also be manifested in the case of somewhat lower doses
(in our case 350 r), as a result of the different individual reactivity of the experimental
animals.
With a dose of 800 r, an increase in the number of heterophils was found in all
cases. As already stated in the results, in eight cases this led to an increase in the
absolute number of leucocytes. In the remaining three cases, the initial decrease
in the number of lymphocytes was so sharp and occurred so soon that the increased
number of heterophils was unable to equalize the loss in absolute numbers. This is
a frequent result in those types of organisms in which the percentual representation
of the main white blood components (heterophils and lymphocytes) is mainly in
favour of the latter and contrasts with the results in affected human subjects"'
(Hempelmann), in whom, especially after high doses, the phase of initial leucycytosis
may be prolonged to as much as seven days. According to Yegorov, the duration of
this phase depends both on the dose and also on the type of nervous system of the
organism, which is responsible for the individual reaction of the blood-forming
tissue.
In the initial changes in the number of leucocytes, an important part is doubtless
played by the pituitary-suprarenal system. This factor does not, however, serve to
explain the further course of irradiation sickness.
The question of the further trend of the leucocyte curve is a complicated one.
The increase in the number of leucocytes described between the second and the
twelfth day, which follows a negative phase, is identical with what Jacobson et al. and
Hempelmann et al. call the "abortive rise". Bloom and Jacobson associate the
etiology of this rise with the incidence of degenerate, temporary forms, which develop
in the bone marrow, chiefly in the elements of the myeloid series, and are flooded
out into the periphery. Hempelmann tends to take the view that the initial and
abortive increases are a secondary, non-specific reaction to damage to the tissue.
This reaction appears, for example, after burns, in which the "abortive increase"
can be attributed to associated infection. It is certain that the factors of both these
hypotheses can be manifested, but it is problematic whether degenerate blood ele-
ments appear in such large numbers in the corresponding period; on the other hand,
an associated infection is not present in all cases in irradiated animals.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80TOO246AO02900500012-2
Langendorff and Paperitz, who described long duration of the periodic character
of post-irrradiation leucocyte reaction, analyse the morphology of changes in the
bone marrow and peripheral blood and come to the conclusion that every type of
blood element is characterised by a special wave-like curve of its own. According
to them, the effective factor is the length of the life of the individual elements. They
evaluate their results at intervals of four days and that is why their rhythm has
a prolonged character. Derer holds the view that the persistent action of cytostatic
substances is a result of irritation of the functional centres for blood regulation by
tissue changes, which in addition reflexly produce synchronisation of the rhythm by
means of which the central nervous system governs the formation and destruction
of the blood elements. The exact periodic reaction described after cytostatic action
in an organism in which the state of the blood formation is not normal, cannot be
verified in normal organisms. Even if one takes the view that the nervous system is
largely responsible for the wave-like character of the post-irradiation leucocyte
reaction, it cannot easily be conceived that impulses proceeding in a regular rhythm
from the central nervous system and possibly caused by a functional modification
of the process of internal inhibition due to radiation, should produce absolute
regularity of the decreases in the leucocytes in the peripheral blood in circumstances
as complex as those which develop following irradiation of the whole organism. This
regularity is no doubt disturbed particularly by the positive deflections in the leuco-
cyte curve, which are caused by adaptation phenomena in the organism, associated
infection and other factors, and also by the short life of several generations of leuco-
cytes following irradiation.
Summary
Following irradiation of rabbits with a single dose of 200, 350 and 800 r, and also
with fractionated irradiation with 1100 r in 11 daily doses of 100 r, alternating
positive and negative deflections occur in the leucocyte curve, which on a graph
take the form of an irregular sinusoid, in which the individual wave lengths range
within considerable limits, according to the individual reactivity of the various
animals. The length of the intervals between the maximal and minimum values in
the trend of the leucocyte curve varies within an average period of four days with
a dose of 200 r and within an average period of about six days with a dose of 350
and 800 r and fractionated radiation of 11 X 100 r.
B 1 o o in, W., M u r r a y, R.: Ilistopathological Effects of Total Body X-Irradiation.
NNES, Div. IV, Vol. 1 (cited by Jacobson, Marks and Lorenz).
D e r e r, L.: 0 periodickej u6innosti chl6ralkylaminu, rtg oiiarenia a ACTH pri leukemii.
Bratislavske lekarske listy 33/8 : 545, 1953.
Hempelmann, L. II., Lisco, Ii., Hoffinann, J. G.: The Radiation Syndrome:
A Study of Nine Cases and a Review of the Problem. Ann. Int. Med. 36 :279, 1952.
J a c o b s o n, L. 0., B 1 o c k, M. H.: Not published (cit. by Jacobson and Marks).
J a c o b s o n, L. 0., M a r k s, E. K., L o r e n z, E.: The Hematological Effects of
Ionizing Radiations. Radiology 52 : 371, 1949.
L a n g e n d o r f f, H., P a p e r i t z, W.: tlber die Wirkung einer einzeitig verabreichten
Rontgendosis auf das Knochenmark der weissen Maus. Strahlentherapie 65 : 624, 1939.
P r a s l i 6 k a, M.: Vplyv niektorych nark6z a anoxie na 66inok ziarenia. Diss. Brno 1954.
T h a l h a in in e r, 0., J a n i c e k, L.:. Die unspezifische Therapie (Chorea minor) im
Lichte einer neuen vegetativ-regulatorischen Leukozytenreaktion. Wiener Klin. Wchschr.
63 :198, 1951.
U h e r, V.: Casovy faktor v regulaci krevni. as. lek. ces. 91 : 744, 1952.
E r o p o n, A. II., B o ~i x a p e u, B. B.: EponeTnopenne it noxnsxpyioulan paAnai;nu.
Mocxsa 1955.
Approved For Release 2008/04/10: CIA-RDP80TOO246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
XapaKTep JieHxoJ HTapHoH peaxnnH riociie peHTreHoBcKOro o6JlytIeHI4H
3. HAPII(DEJtb
IIocJIe OAHOxpaTHOFO o6JlyMennH HPOa HHOB Ao3aMn B 200, 350 n 800 r, a TaHwe
ITpn c)paKunoHIlpOBaHHOM cnoco6e o6JIy~leHlf B Tegenne 11 AHeII 110 100 r B AeHb,
B JIeIIKOu4TapHoI{ Kp1IBOII Ha6JIloIaeTCH LIepeAOBaHHO HOJIOSKHTealbnbIX n OTpHIja-
TeJlbHblX BOaTH, KOTOpoe MO?KHO n306pa3HTb rpa411I eCKH B BHAe HenpaBHalbHOI4
CHHyCOHALI.,jJTHHa OTAerIbHbIX BOJIH Kp1BO1 3Hagl4TeJIbHO KOJIe6 eTCH B 3aBHCHMOCTn
OT HHAIBHAyaJIbHOII peaKTHBHOCTII MHBOTHNX. rIpOAOJI>KITeJIbHOCTb IHTepBaJIOB
MeeKAy OTAeJlbHbIM1 Ma1CHMyMaM1 H OTAeJIbHbIMH MHHHMyMaMH JIe1KO1j14TaPHOII
KpfBOn EoaIe6JIeTCH Hpn Ao3e B 200 r OKOJIO cpeAHerO BpeMeHH B 4 AHH, a IIpu A03e
B 350 14 800 r n npH cpaJIUIIOHnpOBaHHOM o6ny*IeHH11 11 pa.l Ilo 100 r - oxono
6 AHeII.
ABTOP oTCTaHBaeT B3rJIHA, MTO BOalxo06pa3HbII3 XapaKTep peaKunn Ha o6Jlywlllle
O6yCJIOBJI14BaeTCH B 3HaMIITeJTbllOH Mepe pOJIbIO HepBHO] CHCTeMbI. l43BeCTHaJ Ilpa-
BHJIbHOCTb p1TMa HMnyJIbCOB, 1OCpeACTBOM KOTOphIX ueHTpaJIbHaH HepBHa1 CHCTCMa
ynpaBJIneT 06pa3OBaHneM n pacnaAOM 3JTOMOHTOB HpOBH, 06ycJIOBJIHBaeTCH, B03-
M05KHO, (JYHKUIIOHa1bHbIMYI 143MeHeHllMII npouecca BHyTpeHHCT0 TOpMO?KeHnH n0A
AeIICTBHeM o6JlytJeHllg. IlpaBHJlbHOCTb KoJIe6aHHII KOJIHqeCTBa TIeIIKOu1TOB B HepH-
( epHLIeCKOII KPOB14 HapyIHaeTCH, rJIaBHMM o6pa30M, Ko1Ie6aHHHMH B IIOJIO?KHTeJIbxylO
CTOpOHy, - HOTOpble CBH3aHbI C HBaieH1HMn aAa1TaIHu opraHn3Ma, - IOnyTHbIM1
1HlT)eKuwIM14 H APYFHMH (aRTopaMII, a TaHxHe co pan eHneM npOAOJISKHTeJIbHOCTH
5Kn3Hn HeCKOJIbKHX reHepaunn alen1OulTOB n0CJIe 06JIy*TeHHH.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
FOLIA BIOLOGICA
Sporulation of Bacilli
Consumption of Calcium by the Cells and Decrease in the Proteolytic Activity
of the Medium during Sporulation of Bacillus megatherium
V. VINTER
Institute of Biology, Czechoslovak Academy of Science, Department of Microbiology, Praha
The transition of the vegetative cells of bacilli into the resting form, spores, is
accompanied by a series of biochemical changes, an analysis of which throws light
on the deeper physiological laws of this process. One of these changes is a sharp
decrease in the proteolytic activity of medium, which takes place under certain
conditions on sporulation of Bac. megatherium.
In the present work a study is made of the influence of calcium and other ions on
this process and on the transition of the calcium from the medium into the sporulat-
ing cells. An investigation was made also of the effect of adding cysteine to the cul-
ture, before sporulation, on the decrease in the proteolytic activity of the medium.
Culture: A strain of Bacillus megatherium from the collections of the Institute of Biology of the
Czechoslovak Academy of Science was used.
Nutrient medium: Composition - 0.3% casein hydrolysate (Amigen), 0.1% glucose and 0.34%
KH2PO4. One mililitre of a concentrated mixture of certain salts and important ions was added to the
medium, composed as follows: 17.4 g. K2S041 12.3 g. MgSO4 . 7 H2O, 0.22 g. MnSO4 . 7 H2O, 2.0 g.
FeSO4 . 7 H2O, 1.44 g. ZnSO4 . 7 H2O, 18.3 g. CaCI, . 6 H2O to one litre. The proportion of these
salts is similar to that originally suggested by Grelet (1951). Two media were used for culturing-
medium A, in which the precipitate of calcium sulphate and phosphate was filtered off, and medium B,
which was the same as medium A, but enriched by the addition of calcium (10 ml. 5. 10-3 M CaCl2
per litre medium). The pH of the medium was adjusted to 7.2.
Method of inoculation and culturing: The nutrient medium was inoculated with 3 ml. of a suspension of
spores freed after autolysis of the sporangia. The suspension was stored at 5? C. From 90 to 100 ml.
medium was cultured in 500 ml. flasks, on a shaker (96 deflections/min., length of swing 9.8 cm.) at 2 7 ? C.
The percentage of spores was determined by direct microscopic observation and counting (Vinter 1955).
Determination of degree of turbidity: The degree of turbidity of the culture was determined in the
undiluted solution on a Lumetron colorimeter with a filter of 370 mg. Clear supernatant fluid was used
as the control.
Determination of the proteolytic activity of the medium: The supernatant fluid was incubated with
a 1% solution of casein with a pH of 7.0 in a water bath at 37? C. Unless otherwise stated, the length of
incubation was 60 minutes. Two methods were used for determining the intensity of proteolysis-the
Anson method (1938), in the modification of Chaloupka (1955) and a modified biuret test (Slavik and
Smetana 1953). In the case of the first method, the intensity of proteolysis was expressed in amounts
of milliequivalents of freed tyrosine, in the case of the second method in amounts of milligrams of digested
casein.
Staining of the spores: The spores were stained with malachite green and the cells counterstained
with basic fuchsin (Schmidt 1950).
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
Determination of the calcium content of the cells: Calcium was determined in the cellular sediment
(300-400 mg. dry weight), washed with distilled water and 0.01 N HCl, dried and mineralized in a sand
bath with a mixture of HNO3and HC104. The actual determination was carried out using a flame photo-
meter (VEB Carl Zeiss, Jena, model III).
Decrease in the proteolytic activity of the medium during sporulation
of B. megatherium
Both on media A and B there ;s a rapid decrease in the proteolytic activity of the
medium on sporulation to very low values (fig 1). The curve of the decrease is
steepest at the period of mass formation of the spores (fig. 2).
The course of the decrease in the proteolytic activity of the medium differs in the
culture and in the supernatant fluid, whether shaking is continued or whether the
medium is left at rest. The following procedure was used: two thirds of the contents
were removed from the culturing flasks under sterile conditions; they were centri-
fuged and the remaining third was left in the flask on the shaker. Half the supernatant
fluid was placed in a sterile flask of the same size and put on the shaker, while the
other half was left at rest at a temperature of 270 C. Proteolytic activity was deter-
mined in all three parallel specimens, immediately and after three hours. Sporulation
was confirmed by calculating the percentage of spores in the culture on collection
(tab. 1). In a culture in which sporulation had not yet commenced (12 to 121/2 hours
culturing), proteolytic activity decreased in the culture after three hours to very low
values, as usual; in the supernatant fluid on the shaker it decreased only slightly or
even showed an increase, and in the supernatant fluid at rest it showed an evident
increase. In the specimen taken at the commencement of sporulation (up to 10%
spores), there was a normal decrease in the culture and usually a slight decrease in
the supernatant fluid, whether on the shaker or at rest. In the flasks in which mass
Fig. 1. Relative proteolytic activity in the course Fig. 2. Relative proteolytic activity of medium
of culturing B. megatherium. x - axis: age of during sporulation of culture. x - axis: age of
culture in hours, y - axis: mg. digested casein per culture in hours, y - axis: left - mg digested
unit turbidity. Commencement of sporulation casein per unit turbidity , right-%
denoted by arrow. of spores in culture - - - -.
sporulation had begun, the difference between inactivation of the supernatant fluid
left at rest and the supernatant fluid on the shaker gradually increased. Although
there were considerable differences between the values in the individual flasks, this
general tendency is quite evident.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
Table 1. Properties of supernatant fluid taken before and during sporulation. All specimens were incu-
bated for 30 minutes. Proteolytic activity is expressed in a.10-4 milliequivalents of tyrosine; the values
in the col. headed "After 3 hours" gives the increase or decrease in proteolytic activity as a percentage;
proteolytic activity in the supernatant fluid immediately after collection 100?/0
?/0 of ,pores in cul-
ture at moment of
collection
Proteolytic activity
immediately after
collection
After 3 hours=
Supernatant on
Supernatant at re t shaker at 27?C Culture
Before sporulation
(age of culture
12 hours)
92.00
+ 14.68
17.00
98.37
Before sporulation
(age of culture
121/2 hrs.)
94.50
+ 28.20
+ 10.10
98.42
3.5
97.00
-26.30
28.90
99.13
4.2
128.00
- 11.70
- 21.50
99.34
-
4.5
130.00
- 23.10
- 27.00
-
-99-39
5.0
115.00
--- 8.70
20.90
99.44
7.8
76.00
0
5.29
98.95
9.4
71.50
- 4.20
13.30
99.17
12.2
91.00
- 4.94
- 30.77
98.69
14.5
89.00
-- 16.86
- 47.75
98.32
29.2
22.65
+ 2.70
- 20.51
98.02
29.5
82.50
- 12.74
- 16.40
---99.04
48.7
5.75
0
47.83
98.61
48.9
31.35
+ 5.40
86.43
97.29
Table 2. Determination of the presence of an inhibitor in the supernatant fluid from a sporulating
culture. This supernatant fluid is denoted in the table as "Inhibitor". A supernatant fluid from a cul-
ture of different age is used as "Enzyme". Proteolytic activity is expressed in miliequivalents of freed
tyrosine. 10-4. Incubation: 30 mins. Values: above - activity immediately after mixing: below - after
standing for three hours at 27 ?C.
% of spores in
culture at mo-
Age of culture from
ment of collecting
"inhibitor"
which supernatant
fluid with "enzyme"
"Enzyme" diluted
"Inhibitor" diluted Mixture of same
supernatant
activity collected
with same vol.
with same vol. vols. "enzyme" and
filuid
of water
water 'inhibitor" .
44.00 1
35
47
00
62.8%
9 his.
40.00
.
2.05
.
42.25
29
75
2
20
34
60
65.9%
hrs.
711
.
.
.
2
27.10
2.05
29.00
40.75
1
65
43
60
70.6%
9 hrs.
36.25
.
1.70
.
39.40
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
In a further experiment, the possibility of the production of inhibitors of protease
during sporulation was tested. The supernatant fluid from a culture 6.5 to 9 hours
old, in which it had been demonstrated that proteolytic activity would not change
for three hours at rest at a temperature of 270 C, was diluted with supernatant fluid
taken in the course of sporulation. Both supernatant fluids were diluted with the
same volume of water and the sum of the proteolytic activity of these specimens
was compared with the activity of a mixture of the same volumes of both superna-
tant fluids. The culture fluid was collected in the course of sporulation at the moment
when proteolytic activity was very low. If an inhibitor were present in this super-
natant fluid in an effective form, then the reaction between this inhibitor and the
active enzyme would result in a decrease in the activity of the mixture. In actual
fact, no decrease in activity occurred; on the contrary, there was usually a slight
increase, which was equalized after three hours' interaction at 270 C (tab. 2).
Effect of the addition of certain ions on the decrease in the proteolytic
activity of the medium.
All the ions named below were added after 12 hours' culturing, i. e. about 1-2
hours before sporulation commenced. Culturing was carried out on an A medium.
The addition of calcium ions in a concentration of 5. 10-3M, which proved the
optimal concentration for stability of proteases of actinomybes (Chaloupka 1955)
prevents a decrease in the proteolytic activity of the medium, while the course of
sporulation remains normal. The calcium was added in the form of a solution of
CaC12. A ten times smaller concentration of Ca-- ions, 5.10-4M, also prevents a decrease
(fig. 3). A lower concentration of calcium in the medium (5. 10-5M) only delays
the commencement of the decrease in proteolytic activity, while a concentration
of 5. 10-6M has no influence on it (tab. 3).
Table 3. Influence of the addition of calcium in lower concentrations on the decrease in proteolytic
activity of the medium. Proteolytic activity expressed in mg. casein digested in 30 mins.
le
Sam
Age of culture
p
12 hrs 14 hrs. 16 hrs.
Control (with
Prot. act.
28.3
1.2
0.3
added H20)
Sporulation
0%
66.8%
81.3%
Ca-- ions added in
Prot. act.
26.2
26.8
0.6
concentration
Sporulation
0%
37.7%
85.2%
5.10 M
10 e M
5
Prot. act.
24.0
0.5
0.6
.
Sporulation
0%
67.9%
85.9%
NIg?? ions, which are known to have a mildly stabilizing effect on bacterial proteases
(Gorini 1950) when added to the culture in the form of MgSO4 (concentration
5.10-1M) had only a weak stabilizing influence. The addition of Mn-- ions, which,
according to the data in the literature, are necessary in the presporulation period
in Bac. subtilis (Weinberg 1955) and, according to other data, activate proteases
of Bac. megatherium (Levinson and Sevag 1954), had no influence, with media of
our composition, either on proteolytic activity or on sporulation.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
Influence of the addition of calcium ions on the stability of proteases in the
supernatant fluid.
The supernatant fluid was collected in the period of advanced sporulation and was
divided between two flasks. Calcium was added to one of these in a concentration
of 5.10-4M and the same amount of distilled water to the other; both flasks were
then shaken at 290 C. In the flask to which calcium had been added, the decrease
which took place in the proteolytic activity of the supernatant fluid during shaking
was considerably less than in the flask containing no calcium (fig 4). The calcium
therefore clearly increased the stability of the proteases in the supernatant fluid
against shaking.
The addition of Ca" ions to the specimens
of supernatant fluid collected in the course so
of sporulation does not lead to an increase in
proteolytic activity as compared with the
controls with H2O.
Fig. 3. Influence of addition of Ca-- ions to culture
before sporulation on further proteolytic activity. x -
axis: age of culture in hours, y - axis: left-proteolytic
activity in mg. casein digested in 30 mins., right -%
of spores in culture. 1 - - - - - - Proteolytic activity in
culture with added H2O. 2 % of spores in
culture with added H2O. 3 Proteolytic activity
in culture with added Ca-- ions. 4 - - - - % of spores
in culture with added Ca" ions.
Fig. 4. Influence of Ca" ions on stability
of enzymes in supernatant fluid taken in
period of advanced sporulation (29.601)
spores). x - axis: duration of shaking of
supernatant fluid on shaker at 29? C, in
hours; y - axis: proteolytic activity in mg.
casein digested in 30 mins. 1 - - - -
Supernatant fluid with added Ca" ions
2 Supernatant fluid with added H2O.
Uptake of calcium from the medium into the sporulating cells
The calcium content of the cells before and after sporulation was determined by
means of spectrochemical analysis. On the A medium, the calcium content of the
cells increased during sporulation from 0.011% to 0.041%. The addition of 2 mg.
calcium per 1 litre medium (B medium) resulted in a considerable increase in the
differences in the calcium content before and after sporulation, but was not sufficient
to maintain stability of the proteolytic enzymes in the medium during sporulation.
The association between the decrease in proteolytic activity and exhaustion of the
calcium from the medium was thus maintained.
Spectrochemical analysis showed that between the 12th and 15th hour of cultur-
ing, i. e. in the period when the spores are being formed, a considerable increase
took place in the calcium content of the cells (fig. 5). The spores formed in the cells at
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
this period have all the appearance of spores, but they are still immature and do not
yet possess the property of retaining a stain on decoloration. This property is ac-
quired only after further hours of culturing.
When calcium was added before sporulation in considerably higher concentrations,
no increase occurred in the calcium content of the cells which completed sporulation,
with concentrations higher than 5.10-4M (fig. 6).
Fig. 5. Consumption of calcium by sporulating
cells and influence of this process on proteolytic
activity of medium. x - axis: age of culture in
hours, y - axis: calcium content of cells in %
of dry weight. 1 Calcium content of
cells in % of dry weight. 2 - - - - - Prot.
activity in mg. casein digested in 30, mins.
3 -.-.-.-.- % of spores.
Fig. 6. Calcium content of cells completing
sporulation in presence of varying concentra-
tions of calcium. x - axis: concentration of Ca
added after 12 hours culturing (10 ml. to 90 ml.
culture). y - axis: calcium content of fully
sporulated cells (17 hours culturing) in% of
dry weight.
Influence of the addition of cysteine to the culture before sporulation on the proteo-
lytic activity of the medium and on sporulation
After 12 hours' culturing, 10 ml. 0.01 M 1-cysteine solution (pH 7.0) were added
to flasks containing 90 ml. A medium, so that the final concentration of the cysteine
in the culture was 0.001 M. Proteolytic activity was determined immediately after
mixing and further after two and four hours' shaking. In the control flasks, 10 ml.
distilled water was added and the determination carried out in the same way as for
the actual specimens. In the intervals, the state of the cells in the culture was also
controlled microscopically. Whereas in the control flasks, to which water had been
Table 4. Influence of the addition of cysteine to the culture before sporulation on the proteolytic activity
of the medium and on sporulation of B. megatherium. Concentration of cysteine in the culture 1.10-3M.
Proteolytic activity expressed in mg. digested casein.
12 hrs. (immed. after
mixing)
14 hrs.
16 hrs.
Prot. act.
Sporulation
Prot. act.
Sporulation
Prot. act.
Sporulation
Control (with 10 ml.
water added)
20.40
0
2.60
53.1%
2.50
89.3 %
Specimen (with cysteine
solution added)
19.30
0
17.00
0
16.50
0
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
added, the usual decrease in proteolytic activity and normal sporulation took place,
in the flasks containing cysteine, no decrease occurred in proteolytic activity and
no spores were formed during the four hours (tab. 4). Similar results were obtained
adding cysteine in a 10 times weaker solution, when the final concentration in
the medium after mixing was 0.0001 M
(fig. 7). The only difference was that
during the first two hours about 2%
roundish, less shiny spores were formed.
By the end of the fourth hour they had
not increased in number.
Fig. 7. Influnece of the addition of cysteine to
culture before sporulation on proteolytic activity of
the medium and on sporulation of B. megatherium
(concentration of cysteine 1.10-4M). x - axis: age of
culture in hours: y - axis: left-mg. digested casein
(flasks with water added - - - - - ,flasks with
cysteine ), right % ofspores in culture(flasks
with water-. . , flasks with cysteine .......... ).
We also influenced the supernatant
fluid from cultures of varying ages by
1-cysteine solution. Tab. 5 shows the
values of proteolytic activity of super-
natant fluids from 7, 13 and 14.5-hour-
old cultures, immediately after adding
water or a solution of cysteine in
varying concentrations. The table shows
that in a supernatant fluid taken during
vegetative growth of the culture, cy-
steine brought about only a slight
decrease in activity; in a supernatant
fluid taken after a decrease in proteo-
lytic activity, the values remained too
low for accurate estimation.
Cysteine was also added to supernatant fluid taken in the course of sporulation and
its influence determined after further shaking. The whole supernatant fluid was
equally divided between two 500 ml. flasks, to one of which cysteine was added and
to the other distilled water. Proteolytic activity was determined immediately and
after a certain period of shaking. The supernatant fluids were diluted so that the
final concentration of the cysteine was 1.10-4M (tab. 6). The results show that the
decrease in the proteolytic activity of the medium during shaking is almost the
same in the mixture containing the cysteine and in the control containing water and
that in this case the cysteine exerted no "protective influence".
Table 5. Influence of cysteine on proteolytic activity of supernatant fluid from 7,13 and 14.5 hours of
culturing. Proteolytic activity expressed in mg. casein digested in one hour. Values in brackets represent,
proteolytic activity of supernatant fluid after being diluted with water.
Age of culture from which super-
t
t fl
id
t
k
Proteolytic activity of supernatant fluid with varying
concentrations of cysteine
na
an
u
was
en
a
5 x 10-3 3 x 10-3 1 X 10-3
7 hrs.
13.70
14.70
15.00
(14.90)
(15.00)
(15.20)
13 hrs.
8.90
10.00
10.36
(10.50)
(11.10)
(10.50)
14 1/2 hrs.
1.20
-
1.20
(65?/a spores)
(0.90)
(0.80)
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
Table 6. Influence of cysteine (1.10-4 M) on activity of supernatant fluid containing proteases labilized
on sporulation, shaken at 270 C.
Period of
Proteolytic activity in mg. casein digested in 1 hr.
of sporu-
0/
0
shaking
lation
in hrs.
Immed. after mixing
After shaking
Water
Cysteine
Water
Cysteine
11.5
6
37.00
36.80
28.00
29.50
13.8
6
52.00
51.00
34.80
34.00
42.0
3
16.70
17.70
3.00
5.60
From the data in the literature we know that certain ions and salts are necessary
for the sporulation process in bacilli and clostridia (Curran and Evans 1954, Fabian
and Bryan 1933, Foster and Heiligman 1949, Charney, Fisher and Hegarty 1951,
Grelet 1952a, 1952b, Knaysi 1945, Leifson 1931, Weinberg 1955) and also for the
activity and stability of bacterial proteases (Gorini, Fromageot 1949, Gorini 1950,
Gorini and Crevier 1951, Levinson and Sevag 1954). The present work aimed at
ascertaining whether, in Bac. megatherium, certain ions necessary for the stability
or activity of proteolytic enzymes were not exhausted from the medium before or
during sporulation.
The results of the experiments show that in the presence of a small amount of
calcium in the medium an association is displayed between sporulation and the
state of the proteolytic enzymes in the medium. If the concentration of calcium in
the nutrient medium is higher than that required by the sporulating cells, this
association is not seen. The question arises as to whether the small calcium content
in the A and B medium used by us was not the cause of the lability of the proteolytic
enzymes throughout the whole period of culturing and not only during sporulation
and whether the decrease in proteolytic activity during sporulation was not only
the result of the termination of the production of proteases. In that case the high
proteolytic activity in the culture before sporulation would have been due only to the
continuous renewal of the level of the enzyme in the medium, in replacement of the
denatured molecules of the enzyme. This possibility is suppressed by the following
findings :
1) Sensitivity of the proteases to shaking is not noticeably increased until during
the actual sporulation process.
2) The decrease in proteolytic activity is far more rapid in the presence of sporu-
lating cells than in the shaken supernatant fluid.
3) The considerable increase in calcium in the cells during sporulation on the
B medium is evidence that up to the time of sporulation, this amount of calcium
was "available" for the stability of the proteolytic enzymes in the medium.
4) The interruption of the sporulation or presporulation mechanism by cysteine
results in high proteolytic activity being maintained in the medium, even the A me-
dium.
It is also known from the literature that the spores of bacilli contain more calcium
than vegetative cells and the ability to accumulate calcium is mentioned in asso-
ciation with the thermoresistance of spores which have developed (Curran, Brun-
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
stetter and Myers 1943, Sugiyama 1951, Grelet 1950, 1952, Powell 1953, Tinelli 1955a,
1955b). Our work shows that the uptake of calcium occurs during formation of the
spores and that this exhaustion of calcium may lead to significant changes in the
medium.
From the data in the literature it is also known that a number of proteases require
the SH group for functioning. The results of our experiments show that in this case
the cysteine does not act on the proteases but on the actual sporulation process.
Summary
1) In the course of the sporulation of Bacillus megatherium a sharp decrease can
take place in the proteolytic activity of the medium to negligible values.
2) The decrease in the proteolytic activity of the medium on sporulation is not
due to the production of an inhibitor by the cells into the culture medium.
3) In a medium deficient in calcium, an increase occurs during sporulation in the
sensitivity of the proteolytic enzymes in the medium to shaking.
4) The proteolytic enzymes produced during vegetative growth and after it has
ended, require calcium to maintain their stability. A decrease in proteolytic activity
in a calcium-deficient medium can be prevented by the addition of an excess of Ca"
ions to the culture before sporulation.
5) Spectrochemical analysis showed that after the formation of young spores, the
cells of Bac. megatherium contained several times more calcium than the vegetative
cells before the commencement of sporulation.
6) A decrease in proteolytic activity takes place during sporulation of Bac.
megatheiium in a nutrient medium which does not contain more calcium than is
required by the cells for the formation of the spores.
7. The addition of cysteine to the culture before sporulation (concentration
1.10-4M) destroys the ability of the cells to form spores and prevents a decrease
in the proteolytic activity of the medium.
8. A concentration of cysteine of 1.10-;M does not prevent a decrease in the
proteolytic activity of supernatant fluid taken in the period of advanced sporulation
and shaken on a shaker at 27~ C. Nor does cysteine increase the proteolytic activity
of the supernatant fluid at rest.
A n s o n, M. L.: Estimation of Pepsin, Trypsin, Papain and Cathepsin with Haemoglobin.
J. Gen. Physiol. 22 : 79, 1938.
C u r r a n, H. R., B r u n s t e t t e r, B. C., M y e r s, A. T.: Spectrochemical Analysis of
Vegetative Cells and Spores of Bacteria. J. Bact. 45 : 485, 1943.
C u r r a n, H. R., E v a n s, F. R.: The Influence of Iron or Manganese upon the Formation
of Spores by Mesophilic Aerobes in Fluid Organic Media. J. Bact. 67 : 489, 1954.
F a b i a n, F. W., B r y a n, C. S.: The Influence of Cations on Aerobic Sporogenesis in
a Liquid Medium. J. Bact. 26 : 543, 1933.
F o s t e r, J. W., H e i l i g m a n, F.: Mineral Deficiencies in Complex Organic Media
as a Limiting Factors in Sporulation of Aerobic Bacilli. J. Bact. 57 : 613, 1949.
G o r i n i, L., F r o m a g e o t, C.: Une proteinase bacterienne (Micrococcus lysodeicticus)
necessitant Pion calcium pour son fonctionnement. Compt. rend. 229 : 559, 1949.
G o r i n i, L.: Le role du calcium dans l'activite et la stabilite de quelques proteinases bacte-
riennes. Biochim. Biophys. Acta 6 : 237, 1950.
G o r i n i, L., C r e v i e r, M.: Le comportement de la proteinase endocellulaire de Micro-
coccus lysodeicticus au cours de la lyse de cet organisme par lysozyme. Biochim. Biophys.
Acta 7 :291, 1951.
G r e 1 e t, N.: Le determinisme de la sporulation de Bacillus megatherium I. L'effet de 1'epuise-
ment de l'aliment carbone en milieu synthetique. Ann. Inst. Pasteur 81 : 430, 1951.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
G r e 1 e t, N.: II. L'effet de la penurie des constituants mineraux du milieu synthetique. Ann.
Inst. Pasteur 82 : 66, 1952a.
G r e 1 e t, N.: IV. Constituants mineraux du milieu synthetique necessaires h la sporulation.
Ann. Inst. Pasteur 83 : 71, 1952b.
C h a 1 o u p k a, J.: Proteolyticke enzymy aktinomycety Streptomyces griseus. (Js. biologie
4 : 206, 1955.
C h a r n e y, J., F i s h e r, W. P., H e g a r t y, C. P.: Manganese as an Essential Ele-
ment for Sporulation in the Genus Bacillus. J. Bact. 62 : 145, 1951.
K n a y s i, G.: A Study of Some Environmental Factors which Control Endospore Formation
by a Strain of Bac. mycoides. J. Bact. 49 : 473, 1945.
L e i f s o n, E.: Bacterial Spores. J. Bact. 21 : 331, 1931.
L e v i n s o n, H. S., S e v a g, M. G.: Manganese and the Proteolytic Activity of Spore
Extracts of Bac. megatherium in Relation to Germination. J. Bact. 67 : 615, 1954.
P o w e 11, J.: Biochemical Changes Occurring during the Germination of Bacterial Spores.
Biochem. J. 54 : 210, 1953.
Schmidt, C. F.: Spore Formation by Thermophilic Flat Sour Organisms I. The Effect
of Nutrient Concentration and the Presence of Salts. J. Bact. 60 : 205, 1950.
S I a v i k, K., S m e t a n a, R.: Stanoveni aktivity proteolytickych enzyme biuretovou
reakci. Chem. listy 46 : 649, 1952.
S u g i y a m a, H.: Studies on Factors Affecting the Heat Resistance of Spores of Clostridium
botulinum. J. Bact. 62 : 81, 1951.
T i n e 1 1 i, R.: Etude de la biochimie de la sporulation chez Bacillus megatherium. I. Compo-
sition des spores obtenues par carence des differents substrats carbones. Ann. Inst. Pasteur.
88 : 212, 1955a.
T i n e 1 1 i, R.: II. Modifications biochimiques et echanges gazeux accompagnant la sporula-
tion provoquee par carence de glucose. Ann. Inst. Pasteur 88 : 364, 1955b.
V i n t e r, V.: Nerovnocennost bunek Bacillus megatherium v prubehu sporulace. Cs. biologie
4 : 294, 1955.
W e i n b e r g, E. D.: The Effect of Mn" and Antimicrobial Drugs on Sporulation of Bacillus
subtilis in Nutrient Broth. J. Bact. 70 : 289, 1955.
B H H T e p, B.: HepaBHoI eaaocTb H.neTOH Bacillus megatherium B Teueuae cnopoo6pa3oBaHHH.
Fol. biol. (Praha) 1 :188, 1955.
X a JT o y n H a, I0.:. ITpoTeoJ1nTT1YecKae 3H3IIMbI aHTBHOMHIIeTa Streptomyces griseus. Fol.
biol. (Praha) 1 : 144, 1955.
Cnopoo6pa30BaHHe 6aWiIJLJI
rlepexojj HaJIbiiua B HJIOTHH H HajjeHHe HpoTeoJIHTHuecHoH aKTIBHOCTH Cpejjbl hpH
cilopoo6paaoBaHHH Bacillus megatherium
Mbl HyJTLTHBHPOBaJTH BITaMM Bacillus megatherium Ha HagariHe B IIHTaTOJImHOH
cpejje C r11)jp0JIH3aT0M Ha3eHHa H C He6oJ1bmuM cogep?KaHHeM HaJibI[HH. 3TOT WTaMM
BbljjeT1HJl B cpejjy 3HagHTeJibHb1e HOJIHIIeCTBa HpoTea3. B HpOIjecce cnopoo6pa3oBaH11H
Ha6JITOj;aeTCH pe3Hoe CHHSHeHHO Hp0TeoJTHTHueCHOH aKTHBHOCTH cpejbI - BHJIOTb
jjo caMblx He3Ha'1HTeJ1blbIX BeJIHLIHH. 8TO CHHmeHIle He 06yCJIOBJIexo BbijjeJieHHeM
HJTeTHaMH B HyJ1bTHBaIJHOHHyIO epejjy I3HrII6HTOpa.
rlpoTeoJTHTHuecHHe 3H314Mb1, BbljjemTHeMble B cpejjy B TeueHHe BereTaTHBHOro poCTa
H noc ie ero oK0HL1aH11H, jjJIH CB0eI3 yCTOHLTHBOCTH Hy?HjjaIOTCH B HaJlbuHH. C HOMOWbMO
crieHTpaJIbxoro aHarIH3a MbM yCTaHOBHJlkl, qTO IocJle o6pa3OBaH14H chop HJTeTHH Bac.
megatherium cojjepmaT B HeCROJIbHO pa3 6oJIbme Ha2muI H, ileM BereTaTHBHble
KJ1eTHH Hepejj HailaJIOM chopoo6pa3oBaH14H. 3TOT Hepexojj KaJibIUHH B KJTeTHH
B TeuueHHe Hpoijecca o6pa3oBaHhH CHOP B 6ejjHOJI HaJlbijHeM cpejje Bb13LIBaeT JIa6HJIb-
HOCTb HpOTeOJIHTHueCKHX 3H3HMOB epejjbi, HPOHBJIHTOHMYIOCH B TeueHHe CHopoo6pa3O-
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246A002900500012-2
BaHHH HoCTeneHrlbIM lOBbImeHHeM IIVBCTBIITCJIbIfocTH npoTeaa DO OTIIOIBCHBIO
H BCTpnxHBaHffio Ha KataT1Ke npH 27 `C. HpeJjyIIpeJjIITb cHn?KCIIHe npoTeWJIHTH4CCKOii
aKTHBHOCTU B 6e;ji4oA KaJlbJ HCM epee MO}KHO 11yTCM np16aBJICHIIfI B Ky:lbTypy IIepe
HaiIaJIOM cIIOpO06pa3OBaHII1I II36bITHa I40HOB Ca". IIoJ[O6HLIM 06pa3oM, IIpH6aBJICCI
JjOIIOJIHIITCJIbHO H36bIT0K KaJ1hI[HH, MO?EHO CTa614aII3upOBaTb TTPOTea3bI Cy'HCpHaTaIIT-
HOII 7HHj[K0CTI4, B3IITOII B nepHOJL pa3BepuyTOii cuopyJI n i1ii u gaJTCKO aaIIIe llIeir
JIa6HJIbH0CTII IIpOTea3. TaKHM 06pa30M, naJjeHIIe IIpOTC0JIIITH9eCKOII aI:THBHOCTH
upH cHopoo6pa30BaHIIH Ha6JIIOJ1,aeTCH B TaKOii nHTaTeJibHOii CpCJ1,C, KOTOpan coJjep-
?KIIT He 60JIbme KaJIbItlf, 9CM CKOJIbHO Tpe6yeTCH JLJIfI o6pa3oBaHHA chop.
Hepexo KaabuHFI B chIOp006pa3yIOIIjhe KJICTKII IIpoTel:aeT B nepi0Jj, KOrJja
B03HIIKaI0T MoJIOJjhIe enopTI, 061TajjaxhI le y?Ke OHTII9ecHIIMH CBOIiRTBaM41 CHOp,.
Ho OTJIII9aIOIUl4ec} OT 3peJIbIX chop 110 CBOeHi oKpamHBaCMOCTH.
1Ianee B pa6oTe oIIIICbIBaeTCH BJItIHIIIIe Ilph6aBJIeHIIH B KyJIbTypy IlepeJj CIIOpO-
o6pa3oBau1IeM W1CTeuHa Ha cIIH?KeHEIe I1poTeoJI14T149CCKOII aHTIIBHOCTH CpCJjbl H Ila
cnopoo6paaoBanne Bac. megatherium. B KOHI eHTpaJUIIH 1 . 10-3 H 1 . 10-4 M
unCTeuH HapymaeT cnoco6HOCTb 11 teTO1 K o6pa3OBamno chop H IlOJIHOCTbIO HOJjaB-
JIHCT cBII?Kerrie npoTeoJI14TII9ecRCOII aIITHBHOCTII CpejjhT. (14aKT, 'ITO 1 h1cTCNH JjCHCTIIyOT
Ha CIIOCO6HOCTb IUIICTOK K enOpOO6pa30BaHHIO, HOL TBep?KjjaeTCH Ti TOM, 9TO IUIICTCIIII
He IIpCTIHTCTByeT CHH?KeHIIIO IIpOTC0JTHTTT9ec1OH aKTHBHOCTJT cyHCpllaTaHTHOH ?Rhi -
KOCTIT, B3fIT0H B HCpno pa313epHyT0h CTIOpyJIHLjIIH H BCTpHXT1BaeM0II Ba Ka9aJhcc
npu 27 IC, II He BJIBBCT Ha I1pOTOOJIIITH9ecuyTO aKTIIBHOCTb Cyhep]IaTaBTHOIL ?1CBJI-
KOCTII H upl ee xpaHCHIIII B IIOKO0.
226
Approved For Release 2008/04/10: CIA-RDP80T00246A002900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
FOLIA BIOLOGICA
On the Transglucosidatory Activity of Enzymatic Preparations
of the Fungus, Aspergillus niger
M. BURGER and K. BERAN
Institute of Biology, Czechoslovak Academy of Science, Department of Microbiology, Praha
If a fluid culture or extract of a culture of Aspergillus niger is incubated with
maltose, certain oligosaccharides are formed in addition to the hydrolysis of maltose
(Pan, Andreasen and Kolachov 1950, 1951). The containing of a 1.6-glucopyranose
bond is characteristic for these substances (panose, isomaltose). In addition, there
are usually also traces of maltotriose (Burger and Beran 1956, a ,b).
There are still too few facts available to be able to say by what mechanism these
products develop. The solution of this problem is important for several basic questions
connected with the interaction of the substrate with the enzyme in the hydrolysis
of poly- and oligosaccharides.
It is now known that the formation of these oligosaccharides takes place through
transglucosidation, i. e. by transferring the glucose residue from maltose or starch
to the corresponding acceptor (maltose, glucose etc.) (Pan, Nicholson and Kolachov
1952). It is interesting that in the experiments of Pan et al., the series of oligo-
saccharides, apart from maltose, did not act as substrate for the formation of iso-
maltose or the higher oligosaccharides with a 1.6-glucopyranose bond (Pan, Nichol-
son, Kolachov 1952).
The aim of the present work was to ascertain whether reducing oligosaccharides
with a 1.6-glucopyranose link are formed from certain substances after incubation
of short and long duration with a preparation of Aspergillus niger.
Enzymatic preparations used. An enzymatic preparation from a culture of the fungus Asperg:llus niger
was used for the experiments. The preparation of the cultures on bran and the extraction of the enzymes
from these cultures has already been described in a previous communication (Burger and Beran 1956a).
The characteristic property of this preparation is that it has a high maltose activity.
Working method. Incubation of the individual substrates by the enzymatic preparation was carried
out for 75 minutes and 18 hours at 30? C. The total volume of the incubated mixture was 1 ml. and con-
tained 3% enzyme solution, an acetate buffer in a final concentration of 6.5. 10-2 M and with a pH
of 4.5 and in some cases glucose in a final concentration of 0.3%. The glucose was added to the substrate
as a possible acceptor. In order to prevent growth taking place during incubation, a few drops of toluene
were added to the mixture. Enzymatic activity was arrested by placir g the test-tube in a bath of boiling
water for five minutes. Of the specimens, 20 pl. was dripped on to What man 1 chromatographic paper.
Analytical Methods. The incubation products were ascertained by the chromatographic method of
Greene and Stone (Green and Stone 1952) with our own modification (Burger and Beran). A sample
of maltose from incubation with an enzymatic preparation of A. niger was taken as standard. The com-
position of the sugars of the sample is known (Burger and Beran 1956 b).
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246A002900500012-2
Action of the Enzymatic Preparation on Various Oligosaccharides
Fig. 1. gives a chromatographic analysis of the products produced after 75 min-
utes' incubation of the enzymatic preparation with maltose, saccharose, lactose,
raffinose and cellobiose. During this period hydrolysis of these substrates took place
to varying degrees. In the case of raffinose, it could be seen that the breaking-down
of fructose takes place with greater intensity than hydrolysis of melibiose. A spot
of raffinose and saccharose appeared on the paper obviously after hydrolysis of these
sugars, during development of the chromatogram. In the case of maltose, the form-
ation of panose and isomaltose is evident, in the case of cellobiose the formation
of isomaltose could be seen and also of another product, which was situated on the
paper between maltotriose and panose. In the case of the other oligosaccharides no
reducing sugar was produced. Since, with the method used for developing the chro-
matogram, lactose is situated in the same place as isomaltose, it can be stated that
this oligosaccharide does not form higher reducing oligosaccharides.
Fig. 2 shows the situation after 18 hours' incubation. Maltose and cellobiose were
by now virtually completely hydrolysed. Panose and an unidentified oligosaccharide
which developed from cellobiose had also been broken down and only isomaltose
remained. Not even after this period did raffinose (or melibiose), lactose and saccha-
rose produce reducing oligosaccharides with a 1.6-glucopyranose link similar to those
which developed from maltose.
The chromatogram further shows that from maltose and cellobiose traces of oligo-
saccharides developed, which were situated on the paper between maltose (or cello-
biose) and isomaltose. These are probably oligosaccharides with a bond other than
1.4- or 1.6-glucopyranose and they still remain to be identified.
As far as raffinose, lactose and saccharose are concerned, our findings confirm
those of Pan et al. (Pan, Nicholson and Kolachov 1952), who also found no reducing
oligosaccharides after incubation of a fluid culture of A. niger with the sugars named
above. As regards cellobiose, our findings are at variance with those of the authors
mentioned, who found no oligosaccharides after the incubation of cellobiose with
a fluid culture of A. niger (Pan, Nicholson and Kolachov 1952).
Fig. 3 gives results following the incubation of trehalose (x-D-glucopyranosyl-,
u-D-glucopyranose) with an enzymatic preparation of A. niger.
With this oligosaccharide also there was no formation of a new product apart
from glucose. As is seen from the illustration, trehalose gives a spot between maltose
and isomaltose. As a non-reducing oligosaccharide it completely reduced silver
following preliminary hydrolysis on paper during development of the chromatogram.
Fig. 3 shows that even after 18 hours trehalose was hydrolysed only to a small
degree. The traces of fructose and pentoses on the chromatogram are from the
enzymatic preparation. .
Action of the Enzymatic Preparation on Some Heterosaccharides
Further experiments dealt with the action of an enzymatic preparation of A. niger
on methyl-x-D-glucose, methyl-x-D-mannose and glucose-l-phosphate. The results
are given in figs. 4 and 5.
As shown in fig. 4, the results are similar to those in the previous experiments.
Hydrolysis of methyl-glucose occurred within 18 hours, but even after this period
oligosaccharides were not formed. Fig. 4 also shows that only traces of methyl-
mannose were hydrolysed.
Approved For Release 2008/04/10: CIA-RDP80T00246A002900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
T
IM
S2 a b C d S,
S2 MM MI'1 S3 MG MG S,
G G
Fig. 3. The Action of an Enzymatic Preparation of A. niger on Trehalose. S2 = specimen of trehalose,
a = trehalose with glucose after 75 minutes' incubation, b = trehalose after 75 minutes' incubation,
c = trehalose with glusoce after 18 Fours' incubation, d = trehalose after 18 hours' incubation, G =
glucose, M = maltose, T = trehalose, IM = isomaltose, P = panose.
Fig. 4. The action of an Enzymatic Preparation of A. niger on Methylmannose and Methylglucose.
S2= specimen of methylmannose, MM = methylmannose, MM + G = methylmannose with glucose,
S2 = specimen of methylglucose, MG = methylglucose, MG + G = methylglucose with glucose, Ma =
mannose, G = glucose, M = maltose, IM = isomaltose, P = panose.
Similar results were also obtained on the incubation of glucose-l-phosphate with
an enzymatic preparation (Fig. 5). Not even after 18 hours was any new product
formed, apart from glucose. Even hydrolysis of glucose-l-phosphate was not complete
under our experimental conditions.
Action of the Enzymatic Preparation on Glucose
In the previous experiments a study was made of the transfer of the glucose radical
from holo- or heterosides to a carbohydrate acceptor.
Further experiments aimed at ascertaining whether direct synthesis of the oligo-
saccharides mentioned above takes place. Since it was assumed that possible synthesis
would take place as a reversal of the hydrolytic process, the experimental condi-
tions were adapted in such a way that the synthetic reaction could take place as far as
possible. For this purpose the active concentration of water was reduced by adding
an excess amount of glucose or by adding glycerin to the mixture. The final con-
centration of glucose was 5, 10, 30, 40, 50 and 60%. The glucose was therefore both
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
substrate and acceptor ir. the mixture and, in excess concentrations, it also absorbed
water from the mixture. Otherwise the conditions of incubation were the same as
in the previous experiments. The results after 18 hours are given in fig. 6 and 7.
a b C S2 d e
Fig. 5. The Action of an Enzymatic Preparation of A. niger on Glucose-1-Phosphate a = glucose-l-
phosphate after 75 minutes' incubation, b = glucose-l-phosphate with glucose after 75 minutes' incu-
bation, c = enzymatic preparation after 75 minutes' incubation, S2 - specimen of glucose-I-phosphate,
d = glucose -l-phosphate after 18 hours' incubation, e = glucose-l-phosphate with glucose after 18 hours'
incubation, f = enzymatic preparation after 18 hours' incubation.
Fig. 6 shows that under our experimental conditions, synthesis of two oligo-
saccharides from glucose occurred, viz. maltose and isomaltose. It should be noted
that whereas maltose was formed only in the presence of higher concentrations of
glucose (from 30%), isomaltose was already formed when only 5 and 10% glucose
was present in the mixture.
After 150 minutes' incubation, the chromatogram presented a similar picture to
that after 18 hours, the only difference being that the amount of products formed
was smaller.
Fig. 7 shows, in addition to the incubated mixtures, control glucose solutions
in the same concentrations as in the mixture. Fig. 7 therefore shows that the oligo-
saccharides which developed were formed by enzymatic synthesis and were not
present in the specimens of glucose in the form of impurities.
In the previous work a number of facts were given, demonstrating that the forma-
tion of isomaltose and panose on the incubation of maltose with an enzymatic pre-
paration of A. niger, is not the result of the activity of a special transglucosidase, but
that the development of the oligosaccharides named from maltose is due to the
catalytic action of maltase (Burger and Beran 1954, 1956b). It is known, however,
that maltase also hydrolyses a number of substrates, in which the formation of oligo-
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80TOO246AO02900500012-2
saccharides was not found (Summer and Myrback 1950) (e. g. methylglucose and
trehalose). The question arises as to why the formation of the oligosaccharides men-
tioned did not take place with these substrates under our experimental conditions.
It is known, and was also clearly demonstrated on our chromatograms, that these
substrates are hydrolysed by maltase much more slowly than maltose.
It follows therefore that the affinity of maltase for trehalose and methyl glucose is
much less than its affinity for maltose. It is probable that the same will apply if a com-
parison is made of the affinity of maltase for the substrates concerned and of its
affinity for water. The transglucosidation products catalysed by maltase can
develop, however, only if an acceptor with a sufficiently greater affinity for
maltase than water is present in the system. It is clear that trehalose and
methylglucose do not possess this affinity. Nor could glucose be the acceptor
in these experiments, since there was a low concentration of glucose in the solution.
The formation of isomaltose and cellobiose is interesting, since from these facts
it can be assumed that in this case isomaltose is formed by a similar, or identical
mechanism, as from maltose. No further experiments are, however, available for
confirming this assumption.
As far as saccharose is concerned, it is evident that hydrolysis took place by means
of invertase (19-fructosidase) and that development of the reducing oligosaccharides
which develop on the hydrolysis of maltose was therefore not possible (Bealing and
Bacon 1953).
It must be emphasized that the method employed by us (4-fold development of
the chromatogram) does not reveal oligosaccharides with more than four or five
glucose fractions in the molecule. The formation of higher saccharides in our experi-
ments is therefore not excluded.
The experiments in which varying amounts of glucose were incubated demonstrated
that the enzymatic preparations studied by us are capable of resynthesizing isomal-
tose from glucose, and that if the concentration of water is reduced to a sufficient
limit, resynthesis of maltose also occurs. The formation of isomaltose from maltose
did not take place as a secondary process, as is demonstrated by the fact that with
a lower concentration of glucose (from 5 %), only isomaltose was formed and no
maltose. It follows, therefore, that an enzymatic preparation of A. niger catalyses
the synthesis of isomaltose not only by the route of transglucosidation:
maltose + glucose ? isomaltose + glucose,
but also on the basis of the following equation:
glucose + glucose ? isomaltose.
The calculations show that these reactions are thermodanymically possible, if it is
borne in mind that the isomaltose or maltose were formed in amounts over 1,000
times less than the concentration of glucose.
(Plates XXIV, XXV)
B e a 1 i n g, F. Y., B a c o n, I. S. D.: The Action of Mould Enzymes on Sucrose. Biochem.
J. 53 :277, 1953.
B u r g e r, M., B e r a n, K.: K otazce mechanismu hydrolysy dextrinii plisi ovymi enzyma-
tickymi preparaty. Chem. listy 48 : 1395, 1954.
B u r g e r, M., B e r a n, K.: 0 mechanismu ueinku maltasy plisne Aspergillus niger I. Vliv
teploty na aktivaci hydrolysy skrobu plisnov~mi enzymatickymi preparaty. Chem. listy
50 : 133, 1956a.
Approved For Release 2008/04/10: CIA-RDP80TOO246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
B u r g e r , M., B e r a n, K.: 0 mechanismu 1ieinku maltasy Aspergillus niger II. Trans-
glukosidaLni reakce. Chem. listy 50 : 821, 1956b.
G r e e n, S. R., S t o n e, I.: Fermentability of Wort Trisaccharide a Factor in Variable
Attenuations. Wallerstein Lab. Comm. 51 : 347, 1952.
Pan, S. C., A n d r e a s e n, A. A., K o l a c h o v, P.: Enzymic Conversion of Maltose
into Unfermentable Carbohydrate, Science 112 : 115, 1950.
P a n , S. C., N i c h o l s o n , L. W., K o 1 a c h o v, P.: Isolation of a Crystalline Tri-
saccharide from Unfermentable Carbohydrate Produced. J. Am. Chem. Soc. 73 : 2547, 1951.
P a n , S. C., N i c h o l s o n , L. W., K o l a c h o v, P.: Enzymic Synthesis of Oligo-
saccharides - a Transglycosidation. Arch. Biochem. Biophys. 42 : 406, 1952.
Summer, J. B., M y r b a c k, K.: The Enzymes. New York 1950.
BOHpocy TpaHerjnoU03HAHpy1OMeIi geHTeHbHocTH 3H3HMaTI4gecRi4x
HpeHapaTOB Aspergillus niger
BccJIeAOBaiioCb o6pa30BaHHe BoccTaHaBJIHBaIou HX o.IIIrocaxapHJ[OB, o6JIaJ1aIOIL iix
1,6-rJIIOHOHIIpaHO3HOpi CBH3bIO (143oMa.IbTO3a H Haxo3a) npll 14Hny5aL[HH 3H3IIMaTH-
ileCHoro 3icTpaI{Ta HJieceHH A. niger (nyJIbTHBHpOBaBmeikcn Ha oTpy6nx) c pacTBO-
paMLI pa3JiHMHbIx OJIHrocaxapIs oB H rJIIOH03bl B pa3JIIItIHOIi xoHlleHTpa41I14. Xpo-
MaTorpa(mn1ecnlIfi aHaJIH3 HceiieAyeMblx OJIHrocaxapHAOB H OI13BO;{HJICn nOCJTe
HpaTnOCpOgHOII II JAJIHTeJIbHOfi HHI{y6agYIH.
B COOTi1eTCTBI41I C npOII3Be;AeHHLIMH paHee HCCJIejOBaHIIHMH Pan-a C CoTpyJJIIH-
HaMH MbI yCTaHOBHJIH, TO 113 MaJlbT03bI 06pa3yeTCH nan H30MaJIbT03a, Tan H 311aw-
TeJlbHble no3IIITIeCTBa naH03bI. HpF J[JIIITeJIbHO1%I 1Hny6agHH HaHOHJIneTCn TOJIbHO
U3oMaJIbTo3a. 143 pa(J)HH03bl, JIaHT03bI, caxapo3bI H rJIiono3LI-1-(fioc())aTa BbIHIeHa-
3BaHHbie o iHroeaxap11 bi He o6pa30BajIHCb, XOTH HX ru;gOJIH3 OCyIL[ec BJIHJ1cH.
B OTJIHLIHe OT / aHHbIX Pan-a ii g p. Mbl y6e//HJIIICb, LITO 143 LLeJIJI06II03bI O pa3yeTCH
HBoMaJIbT03a H TogHee He onpeqeJIeHHblf'i HaMII w 14roeaXapIJS, hOTOpbifi Ha x oMaTO-
rpaMMe noMelIjaeTCH Me?Hgy MaJIbTO30IT H naHO30fi.
B3 gpyrlix H3ymaBHIHXCH HaMII BeU1eCTB (Tperaj103a, McTHJI-rJIIOno3a, McTHJI-
MaHHo3a) BocCTaHaBJIHBaIou He OJTHrocaxapHgbi e 1,6-rT1IOHOnHpalI03HOfi CBII3bIO
TaHme He o6pa3OBaJIHCb, XOTH Y HHX Ocyn1ecTBJIHJICH no npatiiiefi Mepe gaCTHtIHbIi4
riigpo.li13.
BpH IIHny6agHi4 6o.nee iIJIII Mellee Ko1neHTpnpfBaHHb1x paCTBOpOB r.la)no3bl pe-
CHHTeTHanpyeTen 143oMaJIbTO3a (Ha' HHaH c 5% r.TIOH03EI H 600bn1e) H MaTIbTO3a
(HaiIHHaji e 30 H 6onIee HpOgeHTOB ralOn03bI). O6pa3oBaHHe 1430MaJIbTO3bI 143 rJIIO-
Ho3bI ocymeCTBJIHJIOCb He tiepe3 MajIbT03y, a HpFIMO H3 rJI1on03bl. 3TO J~Ona3blBaeT,
MTO o6pa3OBaHHe H3OMaJIbTO3bI MO?neT - npoMe peaniiiIH TpaHcr.niono34JraIITH 113
MaJIbT03bI - OcyigeCTBJIFITbCH Tan9ne nyTeM peCHHTe3a 143 rJIIono3bI.
(Ta6z. XXIV, XXV)
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
Das Auftreten von Olmohnbakteriosen in der Tschechoslowakei
J. ROZSYPAL and F. MRAZ
Biologisches Institut der Tschsl. Akademie der Wissenschaften, parasitologische Abt., Praha and
Institut fur Getreideforschung, phytopathologische Abt., Kromeii'c
Der Olmohn (Papaver somni f erum L.) gehort unter unseren Zuchtungsbedingungen
zu den landwirtschaftlichen Produkten mit sehr schwankenden Ernteertragen,
deren Hohe unter anderem vom Gesundheitszustand der Pflanzen, also von der
Summe verschiedener Krankheits- and Schadlingseinflusse abhangt.
Bei Untersuchungen von Zuchtkulturen des Mohns (1953) erweckte unser Interesse
das Aufreten and die Verbreitung zweier unterschiedlicher Bakteriosen, deren
Merkmale uns zwar bereits aus fruheren Jahren bekannt waren, bisher aber nicht
gebuhrend betrachtet wurden. In der Praxis wurden diese Bakteriosen meistens
ubersehen and die Erkrankung der Pflanzen anderen Ursachen zugeschrieben. Wir
sahen uns daher zu ihrem eingehenden Studium veranlasst.
Nach ihren typischen Erscheinungsformen bezeichnen wir diese Erkrankungen
im weiteren als "Blattfleckenbakteriose des Mohns" and als ,Stengelbakteriose des
Mohns".
Die Blatt fleckenbakteriose des Mohns
Die in der Literatur angefuhrten Blattbakteriosen betreffen vorwiegend wild-
wachsende Mohnarten and ihre Varietaten, die in den USA and in England als
Zierpflanzen gezuchtet werden (Clinton 1909, Bryan and Mc Whorter 1930, Dowson
1949, Nance 1951). Der Krankheitserreger wurde von Bryan and Mc Whorter als
Bacterium papavericola n. sp. beschrieben and nach der spateren Taxonomie als
Xanthomonas papavericola eingeordnet. Die Blattfleckenbakteriose kommt nach
Berichten auch beim Olmohn in der UdSSR vor (Beloselskaja and Silvestrov,
1953, Gorlenko 1953).
Die Pflanzen werden hauptsachlich in den Sommermonaten befallen, also zu einer
Zeit, in der unter dem Einfluss abwechselnder Regenfalle and starkerer Besonnung
im Boden and in den Kulturen ein dunstiges Mikroklima herrscht. Der Mikro-
organismus gelangt zunachst aus dem Boden durch abprallende Regenspritzer oder
vom Wind angewehte Teilchen der Ackerkrume auf die untersten Blatter. Die
weitere Verbreitung erfolgt durch gegenseitige Beruhrung der Pflanzen, durch
Regenspritzer oder auch durch Insekten. Die feuchten Blattflachen ermoglichen das
Vordringen des Mikroorganismus durch die Spaltoffnungen in das Gewebe. An der
Infektionsstelle entstehen zunachst helle, wasserige, sich vergrossernde Flecke, die,
von der Nervatur begrenzt, eine unregelmassig eckige Form einhalten. Spater
nehmen sie eine gelbliche Farbung an, trocknen ein and bleiben, zum Unterschied von
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
den sehr ahnlichen, durch Helminthosporiose hervorgerufenen Flecken, transpa
rent. Die schiitter verstreuten Flecke sind gewohnlich grosser (5-10 mm), rundli-
cher and lassen manchmal eine Zonenbildung erkennen. Bei dichtem Besatz gehen
sie ineinander fiber and das Blatt vertrocknet schnell. Eine Verbreitung durch die
Gefassbundel auf grossere Entfernung wurde nicht beobachtet. Unter giinstigen
Bedingungen, hauptsachlich bei genugender Feuchtigkeit and Warme, kommt es zu
einer epidemischen Ausbreitung der Krankheit auf die ganze Kultur. Der Verlust
der Assimilationsflache iibt bei starkerem Befall einen bedeutenden Einfluss auf die
Entwicklung der Mohnkapseln and Qualitat des Samens aus. Beim Klatschmohn
werden vom Mikroorganismus nicht nur die Blatter, sondern auch Stengel, Knospen
and Kapseln befallen, was bei Olmohnkulturen nicht beobachtet wurde.
Eine Verbreitung der Blattfleckenbakteriose wurde bisher nur gelegentlich in
einigen Gebieten der Republik festgestellt.
Der Mikroorganismus konnte im allgemeinen leicht aus frischen, Hoch nicht
eingetrockneten Flecken isoliert werden, wo er in einer fast einheitlichen Assoziation
vorzufinden ist. Das Ausgangsmaterial fur die reinen Schalenkulturen bildeten auf
dem Nahragar die an der Randzone der ausgestanzten Blattfleckenstiicke oder
in den Ausstrichen des im Wassertropfen zerriebenen Blattfleckenmaterials entste-
henden Kolonien. Mit den Subkulturen wurden die Blatter junger, im Beet geziichteter
Pflanzen durch Bestreichen mit verdiinnter Bakterienemulsion infiziert. Die Pflanzen
wurden durch Glaszylinder mit Kaliko-Verschluss durch zwei Tage hindurch verdeckt.
Nach 4-5 Tagen machte sich die Infektion in dichten kleinen Flecken bemerkbar,
die stellenweise in zusammenhangende Flachen ubergingen. Die Identitat des
Mikroorganismus wurde durch Ruckisolierung nachgewiesen.
Die Infektion des Stengels wurde snit einem sehr dunnen Kapillarrohrchen durch-
gefuhrt, das 1-2 mm3 wasserige Bakteriensuspension enthielt and his zum Bereich
der Gefassbundel eingestochen wurde. Nach 7-9 Tagen trat nur eine lokale Ober-
flachennekrose unmittelbar urn. den Einstich herum and eine Nekrose des inneren
Gewebes his zu 2 cm ober- and unterhalb des Einstichs in Erscheinung. Nach Ablauf
von 2-3 Wochen breitete sich die Nekrose in schmalem Streifen nach oben and
unten auf die Entfernung von einigen cm aus. Diese Pflanzen konnen zwar in ihrer
Entwicklung geschwacht sein, doch tritt keine Faule ein wie bei der Stengelbakteriose
des Mohns.
Die Bakterien haben die Form eines kurzen, ovoiden, 0,9 x 0,6 p. grossen Stabehens; sie sind beweg-
lich, gramnegativ, besitzen eine einzige, polstandige Geissel and bilden bei dlteren Kulturen Hullen, aber
keine Sporen.
Auf Fleischextrakt-Peptonagar wachsen im Verlauf von 24 Stunden 1 bis 1,5 mm grosse, runde and
gewolbte Kolonien von senfgelber glanzender Farbung and mit geradem Rand; im durchscheinenden
Licht sind sie braungelb and gegen das Zentrum zu dichter granuliert. Auf Schragagar bilden sie einen
dunnen, sattgelben Belag von schleimiger Konsistenz. Auf Kartoffelagar verlauft ihr Wachstum uppiger
and die Farbung ist sattgelb; Starke wird hydrolysiert. Bei Milch erfolgt am zweiten Tag Koagulation,
am sechsten Tag Peptonisation. Der Mikroorganismus bewirkt Garung ohne Gasbildung bei Dextrose,
Galaktose, Saccharose, Fruktose, Laktose, Maltose, Arabinose, Mannit, Mannose and Glyzerin, reduziert
Nitrate, verfliissigt Gelatine langsam (in 6 Wochen his zur Halfte der Normalsaule) and produziert
Ammoniak sowie Schwefelwasserstoff, nicht aber Indol. Der Mikroorganismus ist oxybiotisch; im
Eintrocknungszustand betragt seine Lebensbestandigkeit 7-9 Monate. Temperaturoptimum 25- 30? C;
pH Wert 6,5-7,5.
Nach diesen Eigenschaften wird als Erreger der bei uns verbreiteten Blattfleckenbakteriose des
Olmohns bestimmt:
Xanthomonas papavericola (Bryan et Me Whorter) Dowson.
Synonymum: Bacterium papavericola Bryan et Whorter.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
Die Stengelbakteriose des Mohns
Diese durch Schwarzwerden and Nassfaule des Stengels in Erscheinung tretende
Krankheit wurde ursprunglich beim Opium- and Klatschmohn in Indien festgestellt
(Hutchinson 1916, Ram Ayyar 1917); ihr Erreger wurde erst spater unter der
Bezeichnung Bacillus papaveris n. sp. beschrieben (Ram Ayyar 1927).
In unseren Kulturen beobachteten wir Pflanzen mit Merkmalen, die jenen glichen,
die von diesen Autoren angefuhrt wurden. Die ersten Krankheitssymptome machten
rich bei den Pflanzen im Stadium ihrer starksten Entwicklung zu Beginn der
Knospenbildung bemerkbar. Die Erkrankung beginnt mit dem Welken der Gipfel
and breitet sich binnen 2-3 Tagen fiber die gauze Pflanze aus. Der Infektionsbeginn
verrat sich durch eine dunkle, weiche Stelle, am haufigsten in der oberen Stengel-
halfte, an der gewohnlich der Stengel bruchig wird. Im Querschnitt des Stengels
erscheint das Mark gebraunt bis schwarzlich and ist zum grossten Teil zerstort; die
Innenwand ist von weisslichem bakteriellem Schleim bedeckt, der aus der Bruchstelle
fliesst. Es folgt dann der Zerfall aller ubrigen, bisher nicht betroffenen Pflanzenteile.
Jiingere Pflanzen unterliegen dem Zerfall bedeutend schneller, sodass oft nur auf der
Erde liegende vertrocknete Blattreste iibrigbleiben. Die in ihrer fortgeschrittenen
Entwicklung befallenen Pflanzen werden zwar nicht bruchig, sterben aber ebenfalls
schnell ab. Beim Klatschmohn sind die Krankheitssymptome ahnlich. In Kulturen
werden zunachst einzelne Pflanzen befallen, von denen aus dann die Krankheit
nestartig um sich greift, wobei besonders feuchte Boden ein Reservoir dieses Mikro-
organismus bilden. Bei der Infektion scheinen eine grosse Rolle beissende and
saugende Insekten zu spielen, die in der weiten Umgebung des Standortes einen
Bestandteil der Biozonosen der Mohnkulturen and der. Phytozonosen bilden. Die
betrachtliche Menge des bakteriellen Schleims, mit dem die Pflanzen im vorge-
schrittenen Faulezustand bedeckt sind, ermoglicht eine Vbertragung der Infektion,
die, wie experimental nachgewiesen wurde, auch durch die Weichwanze Calocoris
norvegicus Gmel. erfolgen kann.
Die Stengelbakteriose des Mohns wurde bisher bei einer grosseren Zahl von Lokali-
taten in Mahren and in der Slowakei beobachtet.
Zur Isolierung des Mikroorganismus wurde das Material der Schleimsubstanz
verwendet, die das Innere der faulenden Stengel bedeckt. Die kiinstliche Infektion
zahlreicher Pflanzen verlief durchwegs positiv and rief alle Begleitsymptome
natiirlicher Infektionen hervor. Nach 48 Stunden begann das Welken der Pflanzen,
am dritten Tag erschienen am Stengel die ersten Bruchstellen and im Verlaufe einer
Woche waren 90 % der Stengel von Versuchspflanzen bruchig, was auf eine grosse
Pathogenitat des Mikroorganismus hinweist.
Nach Infektion durch Auflegen eines befallenen Gewebeteils in die Blattachsel
kam es unter typischen Erkrankungserscheinungen zum Absterben der Pflanze
bereits nach 12 Tagen. Ebenso wurden gauze Pflanzen nach Infektion des Haupt-
nerven eines Blattes befallen, die ungefahr 5 cm von der Insertion entfernt gesetzt
wurde; dabei konnte eine Verbreitung der Bakterien durch die Gefassbundel beob-
achtet werden. Das Aufstreichen der Suspension auf Blatter rief keine Flecken-
bildung hervor, die bei der vorherbeschriebenen Blattfleckenbakteriose erzielt
werden konnte. Die infizierten Knospen trieben entweder noch aus and entwickelten
verschiedentlich deformierte Mohnkapseln, oder wurden Schwarz and vertrockneten,
wobei die Infektion oft durch die Gefassbundel auch auf den Stengel ubergriff and
manchmal bis in die Wurzel vordrang. Ahnlich verlief die Infektion in den verschie-
denen Organen des Klatschmohns and unter den verwandten Pflanzen beim gemeinen
Erdrauch (Fumaria oflicinalis L.), wahrend sie beim Scholkraut (Chelidonium
mains L.) and beim Jungfernherz (Dicentra spectabilis L.) negativ blieb.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
Beschreibung des Mikroorganismus
Die Bakterien haben die Form kurzer, 0,4 X 0,5 bis 2,7 r grosser Stabchen mit abgerundeten Enden.
Sie rind beweglich, gramnegativ, seitenstandig begeisselt and bilden weder Mullen noch Sporen. Die
Kolonien erreichen auf Fleischextrakt-Peptonagar eine Grosse von 0,5 bis 1,5 mm and rind schwach
kuppelformig, rundlich mit geradem hellem Rand, graulich weiss, glanzend, schwach blau opalisierend,
im durchscheinenden Licht gelbbraun and feinkornig. Altere Kolonien haben ein annahernd stiirzenartigeIg
Aussehen mit erhohtem gelblichem Zentrum and unregelmassig gewelltem, radial gefaltetem Rand.
Auf Schragagar bilden die Kolonien einen iippigen, grauweiss glanzenden and im durchscheinenden
Licht gelbbraunen Belag mit fein gewolbtem Rand, auf Kartoffelagar einen schwach erhohten, grau-
weissen, stark glanzenden Belag mit gelapptem Rand. Bouillon wird nach 24 Stunden in der ganzen Saule
getrubt, Milch am dritten Tag ebenfalls in der ganzen Saule gefallt. Der Mikroorganismus vergart
Dextrose, Fruktose, Saccharose, Laktose, Mannose, Glyzerin, Xylose, Mannit and Arabinose unter
schwacher Gasbildung, die am dritten Tag ausgepragter ist. Maltose gart nicht oder nur sehr schwach
and ohne Gasbildung. Positive Reduktion von Nitraten. Gelatine wird anfanglich schusselartig, nach
zwei Tagen sackartig and am sechsten Tag in der ganzen Saule verfliissigt. Ammoniak and Schwefel-
wasserstoff wird nur sehr schwach, Indol dberhaupt nicht produziert. Sehr schwache Hydrolyse der
Starke. Milch mit Lakmus and Methylenblau farbt ab. Der Mikroorganismus ist fakultativ anaerob,
seine Lebensfahigkeit betragt im Eintrocknungszustand ungefahr 4-5 Monate mit einem Wachstums-
optimum bei 26-30? C and pH 7-8.
Im Hinblick auf these Eigenschaften gehort der Erreger der bei uns beobachteten Stengelbakteriose
des Olmohns zur Gruppe der pathogenen Stamme, die taxonomisch reprasentiert wird durch die Art:
Bacterium carotovorum (L. R. Jones) Lehmann, Neumann.
Synonyma: Bacillus papaveris Ayyar,
Erwinia papaveris (Ayyar) Magrou,
Erwinia aroideae (Towsend) Bergey.
Die erkannte Tatsache, dass beide Krankheiten auch wildwachsende Mohnarten
befallen and durch Insekten iibertragen werden konnen, erhoht die Bedeutung dieser
Bakteriosen unter unseren Ziichtungsbedingungen. Vom Standpunkt des Pflanzen-
schutzes sind vor allem Praventivmassnahmen notwendig, also die Auswahl nicht
allzu feuchter Boden, Einhaltung einer richtigen Fruchtfolge and Verwendung von
gut geerntetem and gereinigtem Saatgut aus nur gesunden Kulturen. Durch
rechtzeitige Bekampfung der Schadlinge mit Insektiziden kann auch die Ausbrei-
tung dieser Bakteriosen eingeschrankt werden.
(Bildtafeln XXVI, XXVII, XXVIII, XXIX)
A y y a r, C. S.: A Bacterial Soft Rot of Garden Poppy. Memoirs of the Department of Agri-
culture in India. Calcutta 2 (2) : 29, 1927.
B a 1 1 a r i n, C.: Untersuchungen iffier Helminthosporium papaveris. Phytopathol. Zschr.
6 : 339, 1950.
B e r g e y, D.: Manual of Determinative Bacteriology. London 1948.
B r y a n, K. M., Mc W h o r t e r, P.: Bacterial Blight of Poppy Caused by Bacterium papa-
vericola, sp. nov., J. Agr. Res. Washington 40 : 1, 1930.
D o w s o n, W. J.: On the Systematic Position and Generic Names of the Gram-Negative
Bacterial Plant Pathogens. Zbl. Bakter. II. Abt. 100 : 177, 1939.
D o w s o n, W. J.: Manual of Bacterial Plant Diseases. London 1949.
H e i n z e, K.: Saugschaden durch Weich- oder Blindwanzen Capsidae an Kartoffeln and
Ruben. Nachrbl. dtsch. Pflanzenschutzdienst (9), 1950.
M a h I e, E., K u h f u s s, K. H.: Neuartige and ernste Schaden durch die Mohnstengel-
gallwespe Timaspis papaveris Kieff. Nachrbl. dtsch. Pflanzenschutzdienst (12) 229, 1953.
N a n c e, N. W.: Some New or Noteworthy Plant Disease Records and Outstanding Develop-
ments in the United States in 1949. Plant Dis. Reptr. Suppl. 194 : 364, 1951.
N o 1 t e, H. W.: Krankheiten and Schadlinge der blfruchte. Berlin 1950.
N o 1 t e, H. W.: Die Kapselvergilbung des Mohns. Eine Gallwespe als newer deutscher Mohn-
schadling. Zschr. Pflanzenkrankh. 58 : 89, 1951.
R o z s y p a 1, J.: Sledujeme rozsireni dosud malo znamych s"kudcu maku. Za vysokou urodu
2 : 62, 1954.
S t a r y, B.: Novy skudce maku. Ochrana rostlin 17 : 88, 1941.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
W e r n h a m, C. C.: The Species Value of Pathogenity in the Genus Xanthomonas. Phyto-
pathol. 38 : 283, 1948.
B e JI o c e JI c K a H, E. P., C H JI B e C T p o B, A. J.1,.: BpeAHTe3IH H 6oJIe3HH IjBeT04HBIx
H OpaHHiepefIHbIX pacTeHHtl. MOCKBa 1953.
I' 0 p JI e H H o, M. B.: BaHTepHa3lbable 6o31e3HH pacTeHHit. COBeTCKaH Hayxa 1953.
X (H C T o (1, A.: EAHa HOBa 6aKTepHIkHa 6o3IecT HO Maxa as oUHyM IIpII 'IHHOBHBaHa OT Bacillus
Erwinia) papaveris H. cH. CHHcaHHeTO Ha 3eMeg. OHHTHH HHCTHTYTH 5, 9-10. Co~Ha 1953.
BaHTepu03M ceflHHoro MaJa, HariIIOAaBIUHecfi B 4exocJIOBaKI4II
II. P03CbIIIAJI H (D. MPA3
Pea o e
HPHBOAHTCH CBeJeHHH 0 Haxo?KAeHHH H paCHPOCTpaxeHHOCTH B LICP ABYX pa3-
JIHiIHbIX 6a1TepHaJlbHbIX 3a60JIeBaHHI3 COHHHOFO Maxa, KOTOpble Heo6XOJ(nMo npu-
COOAHHHTb K H3BeCTHbIM AO CHX HOP X03HHCTBeHHO Ba?KHbIM 3a6oJIeBaHHHM MOM
HIHpoKO pacHPOCTpaHeHHoro y Hac MaCJIHLIHOro.
HO HpH3HalaM MM O603HatIaeM SIX H H e6aITepHaJlbHyIO HHTHHCTOCTb JIHCTbeB
Maxa> n e6aMepHo3 CTe6JIH Maxae.
Hepnoe 3a6orIeBaHne HOHBJIHeTCH B JIeTHHe MecnIjbI, Korga Hpli uepeAoBaHHH
AO?KAeH H HHTeHCJJBHOFO CoJIHeliHoro o6JIy'IeHHH IIO'IBa u noceBbI 3anpenaIOT.
MHKpoopraHH3MbI IIonaAaioT Ha paCTeHHH H3 HOIqBbI BMeCTe C OTCKaKHBalolljHMI
AO?H ( BbIMH Ka1JIHMI H HaBeHHHbIMH BeTPOM tiaCTHIjaMH 3eMJIn. f1BJIHHCb HOABHSK-
HbIMH, OHH Jlerlo HpOHHKaIOT 110 BJia?KHOI nOBePXHOCTH JIHCTa B yCTbHlja H B Me?K-
KJIeTOgHOe npOCTpaHCTBO. 3AeCb B03HHKaIOT H(eJITOBaTble, no3AHee 6ypeioliwwe
H 3aCbIXaIOIIIHe, orpaHHLIOHHbie HO KOHTypaM id1JIKaMH IIHTHa HenpaBHJIbHOH 4IOpMbl
H pa3JIHLIHbIX pa3MepoB. Hp1 COOTBeTCTBeHHbIX yCJIOBHHX 60JIe3Hb 3nhAeMitieCKH
paenpOCTpaHHeTCH 110 BceH KyJlbType. YTpaTa 60JIbmeI3 T1aCTH accHMHJ1IipylolljefI
IIO XHOCTH pe3KO BJIHHeT Ha pa3BHTne MaKOBOK H KagecTBO 3epua. BaHTepH03
Ha6JInoAa3Icn n0Ka B HecROJIbRHX 06JIaCTHX i1CP. BaKTepHH He TpyAHO H3OJIHpoBaTb
H3 MOJIOAbIX, elije He 3acoxmiix 1HTeH, rAe OHM 06pa3yIOT IIOLITH OAHOPOAHOe co-
o611jeCTBO. PacTHpaa 3MyJIbCHIO LIHCTOH KyJlbTypbl Ha JIHCTbH, He TpyAHO Bb13BaTb
HCRYCCTBeHHOC 3apa?HeHHe.
BaKTepHH HMeIOT I IOPMY KOpOTKOI1 HI3IjeBHAHOYi naJIOiIKH pa3MepaM11 B 0,9 : 0,6 p.
Ha MHCOIOHTOHHOM arape OHH uepe 48 giac. o6pa3yIOT BbIHyHJIbIC Kp}'FJlble ROJIOHIIH
C rJIaAKIMH KpalMH, pa3MepaMH B 1-1,5 MM, rop iwiHO-?KeJITble, 6JIOCTHIIjne, Ha
CBOT KopHilHeBO-SKeJITble, K IjOHTpy 6o11ee rycTO3epHHCTble. Ha KOCOM arape OHIL
06pa3yIOT HpKO?KeJITbIH CJIHBHCTbIH Ha11eT. BaRTeplH MOAJIeHHO pa3?KH?KaIOT ?Ke-
JIaTHH; BbI3bIBaIOT CBepTbIBaHHe, a n03AHee neHTOHH3al[HIO MOJ1o}a; C6pa?KHBaIOT
AeKCTp03y, raJIaHT03y, (pyKT03y, caxap03y, JIaHT03y, MaJIbTO3y, apa6HHO3y,
MBHHT, MaH03y H rTT11IOPHH 6e3 o6pa3OBaHHH ra3a; BOCCTaHaBJIHBaIOT HnTpaTbl;
BbIAeJIHIOT aMMnaK H cepOBoAOpOA, HO He HHAOJI. PaKTepHH KHCJIOpOAOJII061BbI.
B 3aCymeHHOM COCTOHHHH OHH COXpaHHIOT ?KH3HecnoCo6HOCTb B TeueHHe 7-9 Me-
CHIjeB. OHTHMyM pocTa IIpH 25-30 ?C, pH 6,5-7,5.
Ho 3THM npl3HaKaM B036yAHTeJIeM Ha6JIIOAaBmeiicH HaMH 6aHTepHaJlbHOIl nHTHI'5-
CTOCTI JIHCTbeB MaHa HBJIHeTCH Xanthomonas papavericola (Bryan & Whorter)
Dowson.
CIHOHHM: Bacterium papavericola Bryan & Whorter.
BTOpoii 6aHTeplO3 MaKa npoHBJIHeTCH IIOiIOpHeHHeM H MOHp0I rHHJIOCTbIO
CTe6JIeil, - iIanje BCero B HeplOA HaH6owlee 6ypHOrO pocTa, B HaiIaJle nepHOAa
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
o6pa3oBaHll 6yTOHOB. BoJIe3Hb TIpOHBJIHeTCH 3aBnjjaHneM :30TICHb1X paC'IeH1111,
IIa=IHHan e BepxylueIC. 0 Ha-iajie 3a6oJIeBaHnn 1'OBOp11T 60JIee TCMHOC 14 MH1'HOC MCCTO
B BepXHeli 'IaCTH CTe6JTH, rAe OH o6bI iio HajjJlaMb7BaeTCH. IIa pa3pe3e BHjjHO, 'ITO
MH1OTb cTe6JIH nozleplleiiia, 6oJTbmeli'JaCTbI0 pa3pymena, a GTeHTCa C're6JIH C BllyTpeH-
HeH CTOpCHbI noHpbl7'a 6CJIOBaTO11 CJ1143bI0 6aFTep111. B HOCCBaX 6OJIe3I1b IIOHBJIHCTCH
CHa'a.Tla Ha OT'TjeJIbHbIX paCTeHHHX, BOHpyr HOTOpbIX pacllpOCTpaHHCTCII riio i oo6pa3-
no. Po3epiiyapoM Mn1SpoopraHn3Ma HBJIHCTCH, 1' TaBHhIM 06pa3oM, BJIa?i iax IIO'(Ba.
'3 IIa911Te,IbnvIO pOJIb Ilp11 11H(4)eKljn11 HrpaloT, ICaK Ka?KOTCH, H(JI10III11e It COCyIIjBC
HaceHoMblo. I I a O11b1",'e IO, Ia 110;jTBep?I:jjeHa BO3MO?IiHOCTb 11epe iaqu 11H4)C1{U1l i
KJTOIIOM Calocoris norvegicus Gmel. BCHyCCTBCHHOC 3apaiiwmjo MHOI'Oilneaem ix
paCTe7111n B3B0CbIO ~111CTOIT HyabTypbl (B GTe6eJIb, 6}'TOIIb1 it Ma1OBK11) BCCI'jja jjaBajio
IIOJTO?K11TeJIbnbiC I)C:3yJIbTaTbI. B1CJIajjblBan ICyc0IHn 3apa?ICCHH07T THaHH B lia:iyxy
JIHCTa TO?hC Mo MIo BbI3BaTb 3a60JIeBaune. PacTnpaHlle B3BCCH 110 JIICTI,BM 110 Bbl3bl-
BaJio HHTHHCTOCTI. 3TO CBi1TjCTeJIbCTByCT 0 TOM, 'ITO B yCTbBTja JI11CTbCB 6a1Tepn11
He nponnHax)T.
Bo36yRHTCJIb - 3TO BecbMa 110jjBn?HHMle llaJtol1Hir, pa3MepaMl1 B 0,4 : 0,5-2,7 C 3ai pyrJTCHHbIM11 HoituaMH H HCCKOJ1bHUM11 11Cp1ITpnxaJlbHbIMn ?Kl'yTIIHaMIT, rpaM-
OTpFIjaTCJlbHble 11 Tie o6pa3y101jne 1111 Cnop, II6I iCancyji. Ila MHCOIICIITOnHOM arape
Iope3 24 'laca Bb1paCTaIOT Hp}'rJIble, CJICrxa i ynoJloo6paauble HOJIOIR(11 c 6oJICe
CBCTJIb1MU rJTaRH11MH HpanMu, pa3MepaM11 B 0,5-1,5 MM B jjnaMCTpe, CepOBaT0-
6eJTble, 6JICCTHIIj11e, OTJI11BaIonjlle C11HCBaTbIM, Ha CBOT ?IieJITO-1Opn11HCB61e, MICJIKO-
3CpHlIcTLle. CTapble I{OJIOnnn IMCIOT HCC1-OJ1bIC0 BhlIi Ic rylo (I)OpMy, C HC11paBl1JIb110
BOJIHOO6pa311bIM1'1, JTyileo6pa3lIO CMOp11jeHHbJM11 KpaHMu. Pa; -I ukI eHno ?ienaTnua
TTPOX0U14r 6bICTpO, - Ha 6-oft jjenb no BCCMy cTOJ16ut y. haRTeplln ('.6pa?hIIBaIOT
jjei{GTpO3y, OpyHTO3y, Caxap03y, JIaHT03y, Mano3y, I'J1n1jeplln, HCino3y, MaunT,
apa61ni03y 711)11 He3Ha'IHTCJTbHOM o6pa30BaHHn ra3a, 110 HC c61)a?Kn13aJ01' MaJIb'1'O3bI;
IIOCCTanaBJIHBa1OT HHTpaTbT. Bh1jeJIeT1J1e aMMHaHa it CCl)oiiw opOja 13CCI,IV1a iie;Ola`In-
TCJ11,110, 11HTLOJI 11e BITpeJlBeTCH. BaK7'epuIT MOT}'T 1K11'rb aiia3pO6710, 13 :3a(- 1nenn11OM
('OCTOHnnn COXpaHHH)T ?KH3IICCII0006HOCTb B T011CHIIe 4-5 Me('HIICB. O1PlnMVM pocTa
11pH 26-30 ?C 11 pIl 7-8.
Ho 3TIM np113naKaM Bo36yjj1,1TeJ1b Ha6J11ojjaBHIeroCH IIaMii 6aRTep iwa MaHOH6Ix
CTC6JICH 11p11HajJTC?KHT H 1'py11HC TTaTOrCHHbTX H]TaMMOB, raiCO1ToMnwieciH Hpejj-
cTaBJICHHbIX B11joM Bacterium (arotovorunl (L. B. Jones) Lehmann, Neumann.
CnHOHHMII: Bacillus papaveris Axyar.
Erwinia papaveris (Ayyar) Magrou
Erwinia aroideae (Towsend) Bergey.
IIa6JIIop;eH11e, 'ITO o6a 3a6oJ1e13aIIIIH nopaiRaJOT it jjuHopacTynjuII copnblii MaH
11 MOlYT nepepaBaTbCH it ITaceHOMLTMI, rlOBbnnaeT ux ,ma'Ien11o TkJIH iialI]nx JIOCOBOB.
C TO'IHn 3PCHHH :3anjl1Thl HCO6XOjjllMO 3a60TIiTbCH npeHjC BCCTO 0 11pO(j)nJ1a7iTH-
'ICCRHX MepOllpIHTITHX, iiop;6ope tie CJTHImCOM BJIa?i ioro y'lacTR , C06J1IOjjelIHn
IIpaBHJIhHbIX CPOHOB H CHOCO6OB HOCOBa H o6 IICIIOJIb30Ba1Inn upaBFTTIbIIO co6paHllbTX
it otmitjeHHbIX CCMHH TOJIbH0 113 3TkOpOBbIX HVJIbTyp. C130enpeMC11J10e llpnMCHennO
n1CCHTi1jn;_j}}Ix CpejjCTB MOIKeT OrpaHn9nTb paCfPOCTpaHCHne 3TIX 6aKTepn03011
Bpej wreJIHMn.
(Ta6,,i. XXVI, XXVII, XXVIII, XXIX)
238
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
FOLIA BIOLOGICA
I~ Bonpocy ytIacTHSI HeonJloAoTBOpsIIOIuHx M14BBH1 oB
B HOJIOBOM npouecce
M. BOIITHIHHOBA
BnoJIorwiecxu i 1HCTIITyT LICAH, 3xcnepHMeHTaJlbxaa 6no.lornn a reneTaHa, Hpara
Ilocmynuno e pe8axifu o 12 V 1956
OTKpbITne, LITO )ICIIBgKHII MwICHO HaRTH B napa(JnHOBbIX cpe3ax TKanel-i IIOJIOBOrO
TpaKTa CaMOIC (Herwerden 1905, Kohlbrugge 1910, 1911, FeHnH 1950, 1951, 1953
n 1955, XypbiH H Jjp. 1954, BOHTHIIIKOna 1955) Beim is 3aiuiiouean1o o HaJinunH
IlplMfHHOII 3aBHCHMOCTn Me?KJjy npOHluHOBeHneM ?HFBgHKOB B coMaTHgecKne TKaHI
IOJIOBOFO TpaKTa caMKn n HBJIOHIHMH TeJIerOHln H KCeHnn, a OTzlacTII 3((eKTnB-
HOCTbIO reTepocnepMnn (CapuncHH 1952, Hylunep 1954).
MbI CKOHTpOJInpoBaJIn no jo6Hble cJiyuan H03HTIIBHOTO Jjuaruo3a C HOMOIUbIo
BHJjOH3MeHeHHOrO McTOJja H pnga MOJ[CJien OHbITOB, pe3yJlbTaTbI KOTOpbIX CBnjjeTeJib-
CTByIOT 0 TOM, i1TO CJIygan Haxo?KJjeHnn ?KHBLIIHOB
B TKaHHX - H HaMH, It J(pVPHMn IjnTnpyeMblMn
aBTOpaMH - HBJISIIOTCH apTecauTaMH n o6ycJio-
BJIeHbI !cnoco6amii rncTOJIOrngecuol o6pa6OTHn.
3xcnepumenmarcbna.g 2lacmb
I. IIoJlosofi TpaHT Hypnubi
A. Ilapag5unoebae u lfe/i.2ouauH0ebae epe3bl. Rypbi, 1430-
J1nponannbie 3a 45 gne# go onblTa OT HeTyxa, ncHyccT-
BeHHO oceMeHnJIncb (Seiden 1947, OpeJI 1949) Ro3aMn
B 0,4-0,8 MJI afHyJIHTa 3a 26 nac., 6 `l. 30 M., 4 q. 30 M.,
2 'iaca n, HaHoxell, 3a 10 MnH. go yMepnnBJleHHn. ITOCJie
6bICTpO npenapoBHn Becb nfil;enoA Hypnubl norpyxca.ncn
B 10% (1opMoni, 9epe3 2 9aca BbIHIMaTIcH, pa3J{eJIxJIcH
Ha 6JIOHn n noCJle OHOH`iaTeJIbHOrl (JnHcal[Hn nOJ;BepraJICH
Jca2IbHefInef'i o6pa6OTHe. LlacTb Ha%Hgoro OTpe3Ha BilijeBOga
Mbi 3aJiHBaJin B napa(jnH, ailaCTb-B IIeJIJIouJ;nn. TOJIWIIHa
iiapaq)MHOBbIx cpe3OB 6binaiia 6-8 ?, ueJiIionl;anOBbix
-10 ~t. OHpacHa npon3BOJ;uJlacb 6omlbiuea giaCTblO reMa-
TOHCnJInH-3o3HHoM no MOAH(4nHal(nn Harris-a.
PMC. 2. CxeMa nol{Bx3binannn neTeJlb
nftienoga. 1 - MecTO HanoaleHaa
nepBO# JinraTypbl; 2, 3 - MecTa
HaJioHHeHnn BTOpof1, o6iueil Jinra-
Typbl. CTpeJino1l O6o3Ha*leH xoJ;
n]tueBOga no HanpaBJieHino H
HnilHFHy.
B. Hecr(e8oeanus nepumoneaAbuou nosepxHocmu u eneuiHux cAoee scuifeeo8a xypurfbi Home oce-
.hGeHeHua. Tpu HypnIIbl 6b1JIn Ha 40-75 J;Heu H30JIHpOBaHbI OT neTyxa. TITO6LI HCHJIIOYHTb BO3MO?H-
HOCTb npOHnHHOBeHnn H HB'IHKOB Ha nepHTOHeaJIbHyIO nOBepXHOCTb nfli;esol{a 143 ero npocneTa
4epe3 IIOJIOCTb 6pI0IIIHHnI, npOMBOJjHJlacb c2legyioHlaH onepagwi: Bee Hypbl onepHpoBaJlncb nog
ypeTaH-6ap6nTaJIOBbIM Hapno30M (10 r ypeTaHa, 0,75 r 6ap6MTyponot HHCJIOTbI, 100 MJI H2O
B nOJIOCTb 6pIOInnHbl 3a 2-3 iaca J;o onepar nn). Hocue JlanapOTOMnn npOH3BOgMJlocb gBO#Hoe
nOJ;BH3blBanne neTJIM nllIAeBOga B ero cexpeTOpHO# LiaCTM (gBO#HO# IIIeJIHOBOt HHTbIO nOBepx
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
ToJICTOro clIon MapJIn - pHe. 2). B nyHKTe I 6blJia Ha.Io}Kexa nepBaa JInraTypa, B nyHKTax
2 H 3 - BTopan, o6ii;an. Ha BTOpOHI Aenb nocile onepai;Hn y BCeX 3 Kyp 6bLJIO npoH3BeAeH0 HCKyc-
CTBeHH0e oceMeaenne. HOTOM y 1-oil KypHlubI HilueBOA 6bIJl oTnpenapfpOBax H BbIHyT 143 6plomnoik
UOJIOCTH, a y 2 Apyr lx - TOJIbKO pa3BH3aH, HO OCTaBJIeH B 6plomHOH nOJIOCTH. C IOBepXHOCTII
H139eBOAa, MaTKH H isthmus tubae TOHKHMH HOHSHHI;aMH HJIH TOHKHM IipiO*IKOBbIM HHHneTOM OTpe-
3aiIHCb MaJIeHbKHe `IaCTHI[bI TKaHH, KOTOpble paCTHpaJIHCb H HaHOCIJIHCb B (opMe Ma3KOB Ha npeA-
McTHOe CTeKJIbIIHKO. IrOCJIe npnrOTOBJIeHHH Kamcoro npenapaTa HHCTpyMeHTbI 3aMeHHJIHCb
gHCTbIMH. HpeAMeTHble CTehJIbImKII HyMepOBaJIHCb B 3aBHCHMOCTn OT McCTa B3HTHH o6pa31;a
H KOJIHUeCTBa O6pa3gOB, B3HTbIX C oAHOFO H Toro HSe McCTa HIlI;eBOAa. Hoene 3TOr0 BCKpbIBaIICH
HHI;eBOA H AeJIaiIHCb Ma3KH-oTneuaTiH CJIH3HCTOH, K KOTOpOH Hp HKJIaSbIBaJIHCb npeAMeTHLle
CTeKJIMIHKH. fILII;eBOA BCHpbIBa.ICH B CJIeAyIOn;eM nOp1l Ke: MbI BeJIH HpOAOibHain pa3pe3 CHauaJla
Me1HAy TiuraTypaMH B nyHKTax I H 3, noTOM MeiKAy JIHraTypaMH 1 H 2 H, HaKOHeJ;, nepeA HyHKTOM 2.
MbI CJIyuaJIH Ha 10 nac. (Ha Houb) 50 6eJIbIX Mbime6 (no I caHi y Ha 2 caMoi) n 40 Kpblc (no
2 caMga Ha 3 caMoi), nocJle uero y Bcex cantor AelallHCb BTaraJIHI1;Hbie Ma3KH, rsoTopbie orspamH-
Bajincb no Giemsa. Hajinune HSHBUHKOB 6biJlo ycTaHOBJIeno TOJIbKO Y 1 MLIHH ii 8 Kpbic. DT14X
9 caMoK 6biiIo yMepiuBJieHO. hIHTepBaii MeinAy oceMeaeHHeM H yMep1;BJIenneM 6LiJi He 6oJlbme
12 uac. HoJloBble TpaKTbl 3THX caMOK (BJiarailHu;e c 06OHMH yrllaMi MaTKH n HNIZeBOAbl) 6birni
oTnpenapnpoBanbl m 06pa6oTanbl (nyTeM 3a7I4BKn B I.jeJlJloIIAHH).
A. Peanifun mnaneu na enpblcnueanue cnepMuee. Mbi HCXOAnJ1H 143 npeAnoJIOHSOHHH, 'ITO TaKHe
6oJibmlie KolIHUecTBa HSHBUHKOB, KaKne MLI HaXOAHJIH Ha napa4)HHOBLIX 14 1 eJIJIOHAHHOBbIX cpe3ax
nOJIOBbIX opraHOB KypHI(bI, AOJIHiHbI 6bIJIH 6bI BbI3bIBaTb 6ypHylo peaNgHIO (barOIUHTO3a, eCJIH 6m
}KHBYIIKH aKTHBHO npOHHKaJIH B TKaHH 7E1BOr0, CHOCo6HOP0 pearllpOBaTb opraH113Ma caMKH.
Tars Kai Hal He Ha6JIIOAaJIIi nO9o6HOl peaHJ I4H, MbI nOCTaBHin CJIeAyIou;14H OBLIT: 'IeTblpeM I;eeap-
HaM 6blJIO BBeAeHO nOA rso}Ky, B ABOHHy1O KOi1HyIO CKiaAHy McHiAy naegoM it IOCTLMH npeAfJIe'lba
HO 0,5 MJI B3BeCH npOMLITbIX iiliBYHKOB neTyxa nopoAbi JIerropH B ())H3noJIorIVIecroM pacTBOpe.
Cnoco6 npOMbIBKH: noiIyueHHLi6 3HHyJIHT TpHHc LI npOMbIBa.ICH B 5-7 pa3 6oJIbnIHM KOJIn-
ueCTBOM ()) H3HoJIorngecloro paCTBOpa. GCaAoK pa3BOAHJICH B COOTHOmeHIIH rip 116JIH3IITOJIbHO
2 uaCTi HSIIBUHKOB Ha 3 uacTii (bn3nOJIOPHYecioro paCTBOpa. IjecapHH y6HBaJIHCb uepe3 pa3JIli4Hble
npoMel+SYTKH BpeMeHH nOCJIe BnpaICKHBaHBH iSHBUHKOB: uepe3 1, 6, 24 if 48 uac. Y BCOX 4 I;ecapois
McCTO ylolla 6bIJI0 3aMeTHO MaKpoeKonHUeCKH. Y nepn06 H BTOpOH I;ecapoK Ha KpbIJIbHX npollSynLl-
BaJIHCb (D JIIOKTHpyIOII;He yTOJIII;eHHH BeJIHUHHO6 C He6oJlbmyio rOpOmHHy. TaK 6bIJI0 H y I;ecapim,
y6HTOil uepe3 24 uaca, a y y6HTOH uepe3 48 uac. Ha KpaIJlbrX npOiuynblBaJIHOb nJIOTHble, He(hJIIOKTII-
pylo1;He yTOJI111eHif BeJIH`IHHOB C rOpOIHHHy. HorspaCHeHiIH He Ha6JIloAaJlocb. CKJiaAKII ISOaaSH
BbIpe3aJIncb BCerAa AaJIeKO OT MecTa yKOJIa H Ha 2-3 data norpy}KaJIHCb B 10% ()opsoJI, ToJIbISo
nOCJIe Toro, Karl 6biBaiia o6ecneuena no l{patiHei Mepe nepBHUaaH HoaryJlnuun u He rpo3IIJIa yaKe
onacnocTb, 'TO HSHBUHHH 6yAyT 3anecenbI B OHpyHSai0IIIne TxaHH, c1JIaAKH KOHS1i pa3pe3aJIHCb AO
Tpe6yeMbIX pa3MepoB H nOCJIe orsonuaTeJlbno6 ()HKCai;HH 3aJIIIBalT4cb B napa(J)HH.
B. HCue'iunu e cneilemnoL ,1tbiuife. CKeJieTHan MMml;a (6eApeHHan MbIr1ua Kypnwi 14JI14 1-AneB-
Horo IjaInJIeHlia) norpyHSaJIaCb AO 1/3 AJiBHbi Ha 2 uaca npr KOMHaTHOlI TennepaType B 3HKyJIHT.
HeROTOpble MaimlmbI norpynsaJIHCb B HopMaJlbHbili 3HKyJIHT c asunaiMu nKHBUHKaM1i, IOTOI)LIO uepe3
2 uaca OCTaBaiincb eiue HOpMaJIbHO BOABHHSHbIMH. /pyrne Mbiml;bl noMe11;aJIHCb B 3HrsyJIHT, H KOTO-
poMy 6riiia npH6aBlTeHa 1 KanLIn 8% c opMoJla, HeMOAJIOHHO y6HBa,IB,maH HSHB'IHKOB. Llepe3 2 uaca
MbIm1[bI o6pa6aTbIBaJIIICb rHCTOJIorHYecrsH, nyTeM 3aJIHBKn B napay~HH n n,eJIJI01IA11H.
B. Ilona3 ucnycemeennozo npuenecenuJr 'iyalcepo(nbrx n/cemon e mnanu.
a) 3pHTpOI[HTbI KpOJIHKa B rlypIIHO ( MblmUe. HypHHaH Mbi1I11 1 norpyiiiaiiacb Ha 2 uaca upll
KOMHaTHO14 TeM lrepaType B uHTpaTHYIO KpOBb KpoJI11Ka. RpOJInubH rsppOBb B03BOJIHJIa OT.IIHU11Tb
C06CTBeHHble (HAepHble) ipaCHbie KpOBHHLIe Teilblua 1 aInJIeHKa OT MOry1I;11X npOHHKHyTb cioAa
6e3'bHAepHbIX 3pHTpo1 HTOB KpoJIHKa. L4epe3 2 uaca Mblml;a BbIHHMaJIaCb, CnOJIaCKHBaJiaCb (f1113110-
iIOrHUeCKHM paCTBOpOM 11 3aJIHBaJIacb B L eJIJIOnAHH.
6) }RHB'iHKH H3 (1HKcamIOHHOII HMAKOCTH Ha I1eJIJIO14AHHOBbIX Cpe3aX Mbilni;ai. Mbiuiga I;binJieHKa
(HrsCHpoBaJIaCb HOpMaJbHLiM CH00060M B 10% (DopMOJie, K KOTOpOMY MLI npH6aBJInJIH CBeisHti
3HKyJIHT (0,5 MJI 3HKyJInTa Ha 100 Mil 10% ( OpMOJIa) H nepeMemlBaJIH BCTpHXHBaHHeM. HOCJie
12-ilacOBOt (HKCaI;HH Mblml;a B Teuenne 24 uac. npoMblBallaeb B TeKyn eI1 BOAe 11 3aJIHBalIaCb
B ILeJIJIoHAHH.
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
Peayrlbmamu u Oucxyccua
UJIaBHai pa3HiH a McHHjjy KapTIIHaMYI napa( IIHOBblx H IjOJIJIOII IIHOBbIX cpe3oB
TKaxeI3 IOJIOBOI'O TpawTa oeeMeneHHoH KypHljbi 3aKJIIOIaeTCH B OTCyTCTBHH SKHBgIHOB
B 3nHTOJIHH H CJIH3HCTOI3 o6OJ1O'1Ke Ha UOJIJIOHj1iHOBbIX cpe3ax. B oTJIHLIHe OT Hapa4nl-
HOBbIX, Ha ijeJIJIOHJjHHOBbix cpe3ax He 6bLJIH Haflgexbi npoHHKalo1uKe SKHBLIHKH,
a TOJibKO 91inB'IHKn, HpiCTaBHine H pecHHiIKaM. IiapTHxa cnepMHeB, HaxojjHMbix
B Mblfegixofi H CoegHHHTeJlbxoll TKa1H, To}KJjeCTBexna c KapTHHOII napa(HHOBNIx
cpe30B (pile. 1). HaJin'1ne ?KHBLIIIKOB B 3niTeJIHH n CJIH3HCT0II o6oJIo'IKe H npnCyT-
CTBHe pOBHLIX, Kau 6bI BbITHHyThIX ?KHB'1HKOB B npOCBeTe, 6JI143 nOBOpXHOCTII
3n1ITeJIHH Ha Hapa(JlnHOBMX cpe3ax MO RHO o6'J 1CHHTb MexaHugecKYIM nX npOTacxn-
BaHHeM HO11 OM MHKpOTOMa npn pa3pe3e. OjjxaKo Mbi no rIaraeM, LITO H jlpyriie }KHB-
'InKH MoryT IIO11 ) aTb Ha Cp03 B pe3yJibTaTe HaMbiBaHHH H3 npocBeTa HIIueBOjja
BO BpeMH yJjaJIeHHa napar HHa nepejl oHpacKoii, TaK KaK Ha cBexce3aJlnTbIX B 6aJIb3aM
npenapaTax LIacTO BcTpeLIaioTCH cBo6ojjno JjBH?KyIIjHeCH CnepMnn, a MaCCIIBHble
HX CKOHJIeHHH BcTpe'IaIOTCH H cpaBHHTeJIbHO BbICOKO Hag IIJIOCHOCTbIO pa3pe3a.
RpoMe Toro MM noKa3a1IH, 'ITO B napar2HHoBMX cpe3ax HaXOAHTCH H MepTBbie,
H 1KHBLIe cnepMH}, - game B TeX CJiy'Iasx, Horjja 6J1OKH pa3pe3aiOTCH 110 HanpaB-
JIOHHIO OT He norpy}KaBHIei1CH B 3HKyJIHT 1OBCPXHOCTH MbIIHIjbI, OTKyjja B03MO RHOCTb
MexaHH'1OcKoro nepeHeCOHJH mHB'IIIKOB Ha Cpe3 XOTH n He HCKJIIO'1eHa, HO n0
KpaiHen Mepe orpaHHileHa. 3TOT B3rJIHjj nOjjTBepxijlaeTCH TeM, '1T0 Ha ljeJ1J101Ijjli-
HOBIiIX Cpe3aX mHBiiHHH He BCTpe'IaIOTCH HH B 3nHTeJInH, HK B CJIH3HCTOn, HH B B14j1e
CKO1JIeH1iI Hag yJOBHeM cpe3a.
I'13 HaJIn'1HH 7HHB'1HHOB B MbMme'IHOii 11 COej11HHTeJIbHOII TKaHHI Ha napaC HHOBbIx
H IjeJIJIOHjjHHOBbIX Cpe3aX BbITe1aCT ewe OjLHa BO3MOWHOCTb HCKyCCTBeHHOro, naCCHB-
Horo nonajjannn ?KHB'IHKOB B TKaHH: H McXaHH'ieCKOrO, npn pa3pe3ax CBemen
TKaHH nepejj (nlccauneli, H BMOCTC CO BHHTbIBaIO11 eicn B TKaHb (1KCau110nHOii
SKIIjjKOCTbIO. HO3TOMy jjJIH CJle)jyIOnjerO OHLITa MbI n36par114 Taion npHeM, KOTOpMII
H03BOJIHJI 6b1 HCHJIHO'IHTb 3TO no60'IHoe jjeHCTBHe r1CT0JIOri3'IeCK0I3 o6pa60TKn.
MbI HCXOjjHJIH H3 cJiejjyiowero npejjnWJI05KeHHn: eCJIn mHB'IHKlI llpoHHKaIOT B 31111-
TeJIHIi H B rJiy6me paCn0J103KeHHble MbMHIe'1Hblii H CePO3HbIH CJIOH HIIIj0Bojja B pe3yJIb-
TaTe aKTHBHOPO j1Bn}1ieHHH, MbI MOmOM rOnbITaTbCH HalTH nX TaM, He ilpHMeHHH
nepejj B3HTIIeM o6pa3uoB KalcHx 6M TO Hl3 6bIJI0 BMemaTeJIbCTB HJIH peareHTOB.
Pe3yJIbTaTbI 3TOro OHbITa: T. C. yCT3HOBJIeHHOe HaMH OTCyTCTBHe mIIB'IHKOB B 3THX
CJIOHX HIIIjeBoga y BCex 3 Kyp, Rai- n nx OTCyTCTBIIe B IIPOCBeTe j1HCTaJIbnofi TpeTli
cexpeTopnolI 'IaCTH HIIIjeBOjla Kypnubi, oceMeHeHHoII 3a 20 win 10 MLH. go yMeplU-
BJIeHIIH, - jjoHa3bMBaIOT HCKyCCTBeHHbili xapaxTep, Bo-IlepBbMX, noua TaHHH cnep-
MHeB B MbI ue'1Hy10 H COejjFHHTe2IbHyIO TuaHb Ha uapa(~lHOBIIX n 1jeJIJIOIIAHHOBbIX
cpe3ax, a BO-BTOpbIX, 11 HaJIl'1HH CHepMHOB B napa() HHOBMX Cpe3aX npOHCHMaJIbHbIx
y'IaCTKOB 1T "eBoga H CTpOMbI HH'IHHEa KypHubi, y6HTOII 'Iepe3 10 MHH. JOCJIO
OCeMeHeHmi, - TaK Iiaii 3a TaKOi IcOPOTI{HI CpOK m14B'1HKH He CH0006HEI npOHHKHyTb
Talc BbICOKO Bj10Jlb IOJIOBOrO TpaiTa uypuubl. BepOHTHO, 3j1eCb Cbn'p3JTH pOJlb Te 5Ke
()aiTOpbI, KaK H npH Mojjejln OnbITa, Korjja Mbi (HHCHPOBaJIH MbIIHIJY B (J)OPMOJIO
C np16aBJleHneM 3aHyJIHTa n KOrjLa B ueJIJI0nj1uHOBOM epe3e Tome 6bIJln HanjjeHbl
)I HWHIIH (plIC. 8). OTpJIuaTeJlbHble pe3yJlbT3TbI jjana, HauoHelj, n o6pa6oTKa in toto
HIIu0BOj10B MbHHel H KPbIC (pnc. 3), npn KOTOpoli IICKJIIO'IaeTCH H BjjaBJIHBaHIIC
ClrepMHeB B Cpe3bi HO}ROM, H IIX BCacbIBaHue B MemTKaHOBbie IljeJIH.
4To6b1 np0BePHTb McXaHn3M naCCHBHorO BHeCOHHH mHB'IHKOB B TKaHi, MbI IOCT3-
BHJI14 ewe j1Ba MOjjeJlbHbIX OnbITa (KpOMe y1OMBHyTOPO y5lie OnblTa (fnKCaUHH B (Op-
MOJle c npn6aBJIeHHeM 3HKyJIHTa). Hpi nePBOM OnbiTe MbI yCTaHOBilJIII, 'ITO HcFIB'11IKIi
npoHHKaIOT B TKaxn TaHme in vitro (pnc. 5), npH BTOpOM Hie y6ejLWJIHCb, 'ITO HpaKTII-
'IecHH Tau me BOj1YT ce6H H MepTBble cnepMnn H game HpOJIH'IbJ 3PHTpOIIHTbi
Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
 Approved For Release 2008/04/10: CIA-RDP80T00246AO02900500012-2
(pnc. 6, 7). Pa3Hnua 3JjOCb TOJIbKO KOJIJLIeCTBeHHaII: McPTBbIX CHe MHeB B Cpe3aX
HaxOJjHTCH ropa3Jjo McHbIIIe, LIeM ?KHBBIX. 3Ta pa3Hnua 06'bHCHHeTCH TeM, qTO
?KIIBLIBKH, y6HTble ( )OpMOJIOM H BHHTaBHIHOCH B Me1KTKaHeBble npOCTpaHCTBa npn
nOCJIeJjylOHjeH o6pa6oTKe (rJIaBHbIM o6pa3oM, npOMbIBKe), 6onbfHeI3 gaCTbIO CHOBa
BbIMbIBaIOTCH, Toria KaK HHBble CnepMnH, nOJjBepraioiuueCH MaCCOBO1 KoaryJlnunu
TOJIbKO IIOj[ Jje1CTBHeM 4 HKCnpy1OIHeii ?KHJjKOCTH, He BbIMbIBaIOTCH. HOJ[TBep?KJjeHHeM
3TOrO Mor 6bi CJIy?KnTb H TOT ()aKT, TITO H OPITPO111TOB 143 UHTpaTHOII KPOB1 HaXO-
Jj1TCH B TKaHHx ropa3Jjo 6oJIbHIe, gem y6nTbIX ?KHBLIHKOB. Mbi nonIaraeM HOaTOMy,
9TO BO BCex CJIyLIanx ueJio HAeT JI1IIIb 0 II CCHBHOM JjB1?KCHHII ?KHBLIIIKOB, - 6e3-
pa3JIHtrHO, ?KHBbIX 14JI1 y6nTbIX. HaKoHeu, KOCBeHHbIM JjOKa3aTeJIbCTBOM He B KOJIb3y
aITHBHOFO npOHHKHOBOHHH ?KIBMHKOB B TKaHH IIOJIOBOrO TpaKTa caMKH in vivo
BBJIHeTCH, 6bTTb MO?KeT, n TO, MTO B 3THX TKaHHX He 6bIBaOT rjeJIJHOJ1HpHOI peaiunn.
TaK Ka1 npn IIOJjKO?KHOM BBOJjeHHII CIIepMneB TaKaH peaKUHH n (aFOIUHTO3 ?KIBgIKOB,
JjeHCTBHTeJIbHO, Ha6JllojalOTCH (pHc. 4), MO?KHO 6bIJIO 6bI 0?KHJjaTb, iiTO ariajioril9TIaa
peaHun i HaCTynnT H B TKaxHx HOJIOBOrO TpaKTa.
;ii HHbie Kohlbrugge (1910, 1911), HaxOR1BIHeFO CnepMnn B 3ITHTeaIHn MaTKII
Y pa3JIHiiHbIX ?KHBOTHLIX, KPHTHKOBa1I yxie Sobotta (1911, 1920). HCTo IHHK 01111400K
Kohlbrugge OH BIIJjeJi B aleHKOUHTax, nHKHOTU'Ieclne Hupa KOTOpbIx Kohlbrugge
HpHHHMaJT 3a yBeJlntleHHble rOaOBHH CHepMHeB. OJjHaKo rJIaBHbIM HeJjOCTaTKOM
pa6oT Kohlbrugge 11 JjpyrnX TUHTnpyeMbIX HaMH aBTOpOB 6bIJIa, Kai; K3IKOTCH, IIpn-
McHHBIHaHCH lMn McTOJjHEa 3aJI4BKH B IlapadJHH. TaKHM o6pa3oM, y Kohlbrugge,
6ecCHOpHO, peub nJZeT 0 ?KHBiInHax, HCKyCCTBOHHO HpHBHeCeHHbIX B TK3HH.
HaiuH nepBbie OIIbITUI c Kyp3Mn (BOnTnIIIKOBa 1955) C IIp1MCHOHHOM 33JIHBKH
B napac)HH IIpIIBCJII H3C K TaKIIM xce BbIBOJjaM, K3Kne cJjenlajin H OCTaJIbHble unTn-
pyeMbie aBTOpbI. H pry HeCOOTBeTCTBHII, KOTOpbIX HIIHTO 113 HHX He o6'bRCHnai,
TIpHHaJ[Jie?KHT, Hanp., n TO, 1ITO HeBO3MO?KHO, LIT06bI CHepMnn Tau 6bICTpO npOJjB0-
raJlHcb no HllljeBOJjy n y?Ke LIepe3 10 MHH. noc.le CapniI HonaP3JIn B CTeHK1 nOJIO-
BOro TpaKTa: JjB1?KOH4e ?KHBM1KOB npoJjoJJ?KaeTCH o6bIKH0BeHH0 HeCKOJIbKO gaCOB,
npexsJje MeM OHn JjOXOJ1HT JO Hlilja (2-4-8 'JaCOB y OBeu - Dauzier, Winterl-
berger 1952, Buisson, Dauzier 1955; 4 jaca y Kpo2lnxa - Chang 1951). Kohl-
brugge (1910, 1911) H FeHHH (1950, 1951, 1953, 1955) Ha61liolanH, Ka1 11 Mbl, B napa-
( )TIHOBLIX cpe3ax cnepMnn, HanpaBJIHIOIuneCH K HOBePXHOCTII 3nfTeJ1I4H 1JIH
np0HHKalOnjne B 3HHTeJf I i. no HOBOJjy CJIOB reHHHa,'ITO B 3IHTe2IHII ?KIBLIHK1 BCTpe-
qaloTCH peJj1O, Ho 3aTO B coeIjHH1TeWTbHOli n MbIIIlOTTHOI TKaHII - B 60JIbIHHX 140JIH-
IIeCTBaX, MO?KHO CK333Tb TOJIbIO TO,'iTO pa3JIlLIIH B KOJI1MeCTBe ?KHBLIHKOB, 3aHeCeH-
HbIX Ha Cpe3 143 npoCBeTa HO?KOM, n ?K1BLIIKOB, BHHTaBHIIIXCH B Me?1TKaHOBble IueJln,
BIIOJIHe IOHHTHbI, KaK MbI n0K33aaH Ha MOJjeJT1 OIIbITa. 'ITO KacaeTCH yTp3TbI ?KI'y-
TnKOB, HKo6bi HacTynaioujell npn npOHHKHOBeHH1 H 1BtiHKOB B TKaHH, CJIejjyeT
3aMeTHTb, iiTO CnepMHH co ?KryTHKaM1 II 6e3 HHX MO?KHO HalTH B II OCBOTO nOJIOBOTO
TpaKTa CaMOK oileHb CKOPO nocae CJIy'Kn.
HpntrnHy Ha6JIIOJjaBIHeITCH FeHHHbIM (JparMeHTaunn ?KlBLIHKOB B TKaHHX (no ero
MHeHHIO, OHH HKO6bi 331OHOMePHO TepHIOT Ty LIaCTb rOJIOBKII, KOTOp3H B MOMeHT
II OHIIKHOBeHHH HanpaBJreHa K 3HHTeJIHIO) JIerKO OTKpbITb B TOM, 'iTO 4)parMeHTaljIH
TOJIOBOK HacTynaeT y?Ke B IIpoCBeTe, Tau '-ITO cpeJjll ?K1BIHIKOB, 3aHeceHHbIX B TK3HII
H3 npoCBeTa, JjOJI?KHbI BCTpe'aTbCH CaMble pa3HOO6pa3Hbie o6JIOMKH.
Pe3yJIbTaTbI OnbITOB FeHHHa C npOHHKHOBeHIIeM ?KIBWKOB B pa3JIIILIHbie TKaHIT
in Vitro 3T0 TOH1e apTelaKTbI, Kau MbI y?Ke nOKa3amrn.