J. Embryol. exp. Morph. Vol. 35, 2, pp. 355-367, 1976
Printed in Great Britain
355
Isoenzyme transitions of creatine
phosphokinase, aldolase and phosphoglycerate
mutase in differentiating mouse cells
By EILEEN D. ADAMSON 1
From the Department of Zoology, University of Oxford
SUMMARY
Extracts of embryonic mouse tissues (skeletal, cardiac and smooth muscle, and brain) were
analysed by Cellogel electrophoresis, for their isoenzymic distributions of three enzymes,
creatine phosphokinase, aldolase and phosphoglycerate mutase. Embryonic tissues from the
12th day to the end of gestation were examined for isoenzyme transitions, and it was found
that the adult forms of these enzymes appeared during gestation. Extracts from cloned
teratocarcinoma cells were similarly examined in order to determine their degree of biochemical differentiation. Undifferentiated embryonal carcinoma cells contained only the
early embryonic forms of all three enzymes, while differentiated cells formed in vivo, and in
some cases in vitro, started to express the adult types of creatine phosphokinase and aldolase.
Thus, biochemical parallels have been demonstrated between developing embryonic tissues
and teratocarcinoma cells differentiating in vitro.
INTRODUCTION
It is known that cells of embryoid bodies and derived cell lines can differentiate
both in vivo and in vitro (reviewed by Martin, 1975). In most cases the differentiation has been followed by histology and they form recognizable muscle, nerve,
cartilage, pigmented cells and keratinizing and glandular epithelium. Biochemical studies have shown that during the differentiation of embryoid bodies
the specific activity of alkaline phosphatase and protease declines; these are
enzymes characteristic of the embryonal carcinoma cells (Bernstine, Hooper,
Grandchamp & Ephrussi, 1973; Hall et al 1975). As the differentiation proceeds
so the specific activity of acetylcholinesterase and creatine phosphokinase
increases and one study has suggested the formation of nervous tissue (Levine,
Torosian, Sarokhan & Teresky, 1974) while another showed striated muscle
formation (Gearhart & Mintz, 1974). The extent to which these enzymatic
properties can be taken as markers of particular cell differentiation was not
established.
Here I have compared the appearance of three enzymes in developing mouse
1
Author's address: Department of Zoology, University of Oxford, South Parks Road,
Oxford 0X1 3PS, U.K.
23-2
356
E. D. ADAMSON
embryos and in teratocarcinomas. Particular patterns of the isoenzymic forms of
these enzymes are characteristic of muscle and brain tissues in adult mouse and
their appearance in developing embryonic tissues is recorded. The enzymes are
creatine phosphokinase (CPK, EC 2.7.3.2), fructosediphosphate aldolase
(EC 4.1.2.13), and phosphoglycerate mutase (PGM, EC 2.7.5.3). These
enzymes were chosen because studies on other species have shown that the
distribution of their isoenzymes changes during the development of skeletal
muscle and brain.
CPK is a dimeric enzyme composed of sub-units M and B in homo- or heteropolymers MM, BB and MB. In the brains of a wide variety of species, only BB is
present, while in skeletal muscle only the MM form is found. Since the type of
CPK found in the early embryos of several species is usually exclusively BB,
there must be a transition of isoenzymic forms during the development of
skeletal muscle (Cao, de Virigilis, Lippi & Coppa, 1971; Turner & Eppenberger, 1973). PGM is a dimeric enzyme similar to CPK in the form and
distribution of its isoenzymes in several mammalian species. In addition, its
isoenzymic transitions are similar during human muscle development (Omenn &
Hermodson, 1975).
Aldolase is a tetrameric enzyme composed of distinct sub-units A, B or C.
Adult skeletal muscle usually has only one isoenzyme of aldolase, A 4 ; both A
and B sub-units are found in liver and kidney; A and C sub-units occur in brain
(Rutter et al. 1968). In rat embryos the predominant aldolase isoenzyme is A4 and
there is no change of type during the development of cardiac and skeletal muscle.
There is, however, a change in developing brain tissue, resulting finally in
approximately equal amounts of five isoenzymes, A4, A3C, A2C2, AC 3 and C4
(Turner & Eppenberger, 1973).
The transitions of the isoenzymes of CPK and aldolase that occur in developing skeletal muscle in vivo also occur during the maturation and differentiation
of chick myoblasts in vitro (Morris, Cooke & Cole, 1972; Turner, Maier &
Eppenberger, 1974) and rat myoblasts in vitro (Yaffe & Dym, 1972). I have therefore investigated the possibility of using isoenzymic analyses to determine the
type and the degree of differentiation of cultured teratocarcinoma cells.
MATERIALS AND METHODS
(1) Biological materials
Several stocks of mice were used in this study and no differences were detected
between them. PO mice were obtained from the Pathology department of the
University of Oxford, U.K., CFLP mice from Carworth Europe, Alconbury,
Hunts., U.K. The day of detection of the copulation plug was designated the first
day of gestation. Pregnant female mice were killed by cervical dislocation and
the embryos were dissected in ice-cold solution A of Dulbecco & Vogt (1954)
(PBS). Embryonic tissues were collected into centrifuge tubes, drained free of
medium and either frozen at - 20 °C, or processed immediately.
Isoenzyme transitions in differentiating mouse cells
357
Cultured teratoma cells and solid teratomas were obtained from 129/J and
C3H derived growths (Papaioannou, McBurney, Gardner, & Evans, 1975).
This material was kindly provided by M. McBurney of this laboratory.
OC15 SI is a cloned line of cells derived from a transplantable teratocarcinoma
of strain 129/J. Three other cloned cell lines from transplantable teratocarcinoma of strain C3H mice were also examined. These lines were maintained as
homogeneous populations of embryonal carcinoma cells. All the cell lines could
differentiate under suitable conditions of culture (McBurney, in preparation).
(2) Tissue extractions
Adult tissues were homogenized with four volumes of either 0*25 M sucrose,
0-025 M Tris-HCl, pH 7-4, 2-5 DIM disodium salt of EDTA (for brain tissue and
also for tissue culture cells), or four volumes of 0*25 M sucrose, 0-025 M TrisHCl, pH 7-4, 0-025 M magnesium acetate, 0-2 % (v/v) 2-mercaptoethanol (for all
other tissues).
Embryonic tissues were suspended in a volume of the above solutions approximately equal to that of the tissue and were sonicated for two bursts of 10 sec
each or until disintegrated.
Homogenates were centrifuged at 100000 g for 30 min at 2-4 °C in an M.S.E.
High-Speed 65 centrifuge. Supernatants were frozen at - 20 °C in small aliquots.
(3) Electrophoretic analyses
Three to five microlitres of tissue extracts were analysed electrophoretically on
Cellogel strips (Reeve Angel Scientific Ltd, Whatman Labsales Ltd, Maidstone,
Kent, U.K.), using a micro-scale applicator. The electrophoretic buffer and strip
soaking solution was 0-06 M barbitone buffer, pH 8-6, containing 0-lmM
2-mercaptoethanol (and also 5 HIM EDTA for aldolase determinations).
Electrophoreses (at 4 °C) were run at 250 V (25 V/cm) for 60 min in the case of
CPK separations, 60-75 min for aldolase and 3 | h for PGM on a Shandon
Model U77 electrophoresis apparatus (Shandon Scientific Company Ltd, 65
Pound Lane, London NW10).
Creatine phosphokinase isoenzymes were stained using an agar overlay
essentially as described by Dawson & Eppenberger (1970). Also included in the
reaction mixture was 1 HIM adenosine-5'-monophosphate in order to inhibit the
enzyme myokinase which also stains with this reaction mixture. Controls were
performed by staining duplicate Cellogel strips in a reaction mixture which did
not contain the substrate creatine phosphate.
Aldolase isoenzymes were stained similarly by the reaction mixture, essentially
as described by Penhoet, Rajkumar & Rutter (1966). A modification introduced
by Lebherz & Rutter (1969) was used in order to reduce alcohol dehydrogenase
staining. Control strips were incubated with the same agar overlay except that
fructose diphosphate was omitted. Cellogel/agar strips were incubated at 37 °C
in the dark for a period of 10-30 min or until a good formazan colour had
358
E. D. ADAMSON
developed. The strips were processed and 'whitened' by the procedure recommended by the manufacturers before being photographed.
Phosphoglycerate mutase isoenzymes were made visible on Cellogel strips by
a fluorescent method essentially as described by Omenn & Hermodson (1975).
After incubating 1-3 h at 37°, black spots (NAD produced by the enzyme)
could be seen against a background of fluorescent NADH in ultra-violet light
(365 nm). These were photographed at /3-5 for 45 sec on FP 4 film (Ilford Ltd,
Ilford, Essex, U.K.) using a dark green filter.
(4) Chemical materials
General chemicals were analytical reagent grade from Fisons Scientific
Apparatus, Loughborough, Leics., U.K., or from British Drug Houses, Poole,
Dorset, U.K. Enzymes and substrates were obtained from Koch-Light Laboratories Ltd, Colnbrook, Bucks., U.K.: from Sigma (London) Chemical Company,
Kingston-upon-Thames, Surrey, U.K.; or from the Boehringer Corporation
(London) Ltd, Lewes, E. Sussex, U.K.
RESULTS
Table 1 shows the distribution of the isoenzymes of CPK and aldolase in
adult mouse tissues. It is similar to that of rat and other mammals (Masters,
1968; Turner & Eppenberger, 1973). CPK B sub-unit homopolymer is the only
isoenzyme present in adult brain tissue, and is the predominant form present in
very early embryonic cells (see Table 2). Thus the tissues which must transform
their CPK isoenzyme type at some stage in development are skeletal (striated
muscle), cardiac muscle, and the smooth muscle of the intestine and bladder.
On the other hand, for aldolase the transition must occur in brain, kidney and
liver tissues since the major embryonic form is A4 (see Table 3).
(1) CPK isoenzymes in ontogeny
Table 2 shows the results of isoenzymic analyses of four kinds of muscle at
several stages of development. By the 15th day of development a significant
amount of MM activity above control (see below) is detected for the first time in
skeletal muscle. It is probable that both the M and B sub-units of the enzyme are
being synthesized because the heteropolymer MB is visible (Fig. 1 a, track 4) and
this would not appear unless either there was simultaneous production of both
sub-units in the same cytoplasm or continuous dissociation and reassociation of
the sub-units of the enzyme. The embryonic heart is highly developed morphologically by the 12th day, and this correlates with the very early production of
MB (Table 2).
Notice that in Fig. 1 there is a significant amount of staining activity at the
position of MM in early embryonic extracts. This activity is found in extracts of
all early embryonic cells as well as in teratocarcinoma cells. However, since the
Skeletal muscle
Cardiac muscle
Smooth muscle
(a) Uterus
(6) Stomach
(c) Bladder
Brain
Liver
Kidney
Spleen
Tissue
(+ )
MM
BB
B4
B,A
B,A9 BAa
A4
Aldolase
The intensity of enzymatic reaction is indicated by the number of + signs.
(+) means a very faint reaction.
* Similar activity was found on the control or blank strip.
MB
CPK
Table 1. Distribution of CPK and aldolase isoenzymes in various adult tissues of mouse
AC 3
+ 4-
c4
5
I'
S'
»••.
a
I
as*
v
360
E. D. ADAMSON
Table 2. Changes of CPK isoenzyme distribution in developing
mouse muscle tissues
Day of development
MM
MB
A. Skeletal muscle (hind limbs)
9th day whole embryo
( + )*
.
12th day whole bodies
+*
.
12th day skeletal muscle
+*
.
13th day skeletal muscle
+*
.
14th day skeletal muscle
+*
.
15th day skeletal muscle
++
+
16th day skeletal muscle
++
++
17th day skeletal muscle
+++
+
19th day skeletal muscle
+++
New-born skeletal muscle
++++
13th
14th
15th
16th
17th
day tongue
day tongue
day tongue
day tongue
day tongue
12th day
13th day
14th day
15th day
16th day
17th day
19th day
B. Skeletal muscle (tongue)
+*
.
+*
++
+++
++++
BB
++
+++
++
++
+++
+++
+++
+
+++
C. Cardiac muscle
(+ )
(+)
+
+
+
++
++
+++
++
+++
+•
+++
+•
D. Smooth muscle (intestine)
12th and 13th day bodies
+*
13th day smooth muscle
+*
14th day smooth muscle
+*
15th day smooth muscle
(+)*
17th day smooth muscle
(+)
Adult intestine
+
++
See notes to Table 1 for explanation of signs.
+++
control gels (with no creatine phosphate in the agar overlay reaction mixture)
showed identical staining (Fig. 1 b) at this position as well as at the myokinase
position, it is likely that this activity is caused by an enzyme activity other than
CPK. In Table 2 therefore, this kind of stained band is denoted by an asterisk.
The necessity for control incubations made at the same time as reaction
incubations is demonstrated by Fig. l(b). The spurious stained band at the
position of MM in some of the CPK reaction gels was not identified. It might
have been MM which also stained in the control gel because of the presence of
creatine phosphate in the cell extract. This would have been possible only if
Isoenzyme transitions in differentiating mouse cells
361
CPK
I
2
Origin
- |
"i 4
+
* *
i l
4
4
4
3
4
5
•
|
Origin
- \
•
I
4
4
€
6
t ft
MM MB BB
1
Myokinase
lb) Control
(o)
Fig. 1. Cellogel electrophoretic analysis of CPK at various stages in the development
of skeletal muscle in the hind-limb of mouse, (a) Reaction strips incubated in the
presence of substrate, (b) control strips corresponding to (a). Sample 7 was run at a
different time to samples 1 to 6. 1, 12th day of the gestational period; 2, 13th day;
3, 14th day; 4,15th day; 5, 16th day; 6,17th day; 7, adult skeletal muscle. Note the
similar amount of staining at the position of MM in (a) samples 1 to 4, and in (b) the
control samples. This enzymatic activity was assumed not to be MM (see text for
details). For experimental details see the Materials and Methods section.
Table 3. Changes of aldolase isoenzyme distribution in
developing mouse brain tissue
Day of development
A4
A3C
AC 3
12th day bodies
12th day brain
13th day brain
14th day brain
15th day brain
16th day brain
17th day brain
18th day brain
Adult brain
See notes to Table 1 for explanation of signs.
cieatine phosphate co-electrophoresed with the MM form of the enzyme. This
was tested by adding increasing amounts of creatine phosphate to an adult
skeletal muscle extract, electrophoresing these mixtures as usual, and staining
as for a control gel. None of the samples stained at the MM position. Thus the
unknown band is unlikely to be MM. Myokinase, however, which has a mobility
intermediate between that of MM and MB, was present as a stained band whose
intensity increased with increasing concentrations of creatine phosphate in the
362
E. D. ADAMSON
(P)
(a) Origin
Origin
1
2
k
3
4
5
li
|
11
?
2 Control
/
Spurious bands
6
7
8
fit it
•
A
1 Adult brain
2 Adult cardiac muscle
Aldolase
tf
o<!
*f i
c4
A2 c
2
Fig. 2. Cellogel electrophoretic analysis of aldolase (a) in developing mouse brain at
the increasing times in the gestational period 1, 12th day; 2, 13th day; 3, 14th day;
4, 15th day; 5, 16th day; 6, 17th day; 7, 18th day; 8, adult brain tissue. Not all
samples were run at the same time, (b) Samples stained for aldolase sometimes
revealed a contaminating set of stained bands. The lower two tracks are the corresponding control strips run in parallel with 1, adult brain tissue; 2, adult cardiac
muscle. Note the control stips are similarly stained and that the mobilities of the
spurious bands are slightly different from true aldolase isoenzymes.
original sample. This suggested that creatine phosphate was co-electrophoresing
with myokinase rather than with the MM form of CPK.
(2) Aldolase isoenzymes in ontogeny
Table 3 depicts the aldolase isoenzyme transition in brain tissue. By the 14th
day of development well-defined heteropolymers of A and C sub-units are
present, though these are faintly visible also in the 12th and 13th day brain
(Fig. Id). About 95 % of all aldolase activity in early embyonic cells is that of
A4 with 5 % A3C. During the development of all types of muscle, this remains
about the same.
In a small proportion of electrophoretic separations of aldolase isoenzymes,
a set of five faint bands moving faster than aldolase was visible in both the reaction gel and the control gel (Fig. 2b). The slower-moving bands of this set could
easily be identified erroneously as A3C, A2C2, etc., and illustrates the necessity for
control gels run in parallel with the reaction strips.
(3) Ontogeny of PGM isoenzymes
The data for PGM isoenzyme patterns is shown in Table 4. In the developing
skeletal muscle of the hind limb, a transition from BB to MM occurs and
the first appearance of MM is observed at the 15th day of gestation as it is
with CPK. In contrast to the latter, the BB isoenzyme of PGM remains the
Isoenzyme transitions in differentiating mouse cells
363
Table 4. Changes of PGM isoenzyme distribution in developing
mouse muscle tissues
Day of
development
MM
MB
BB
A. Skeletal muscle (hind limbs)
12th day
13th day
14th day
15th day
16th day
17th day
Adult
B. Skeletal muscle (tongue)
13th day
14th day
15th day
16th day
17th day
Adult
C. Cardiac muscle
12th day
13th day
14th day
15th day
16th day
17th day
Adult
PGM
t
t t
BB MB MM
Fig. 3. Cellogel electrophoretic analysis of PGM in developing hind-limb muscle of
mouse. 1, Skeletal muscle at the 12th day of gestation; 2, 13th day; 3, 14th day; 4,
15th day; 5,16th day; 6,17th day; 7, adult skeletal muscle. The adult form (MM) is
just visible in 15th day muscle but is only clearly stained in the 17th day extracts.
See the Materials and Methods section for experimental details.
364
E. D. ADAMSON
+
Origin
Origin
1 myc
MM
MB
(") CPK
t
BB
t 1
q <
A 4<A 2c2
(b) Aldolase
Fig. 4. Isoenzyme analyses in teratocarcinoma cells, (a) CPK, (b) aldolase. Track 1,
embryonal carcinoma cells of clone OC15 SI; 2, differentiated OC15 SI; 3, solid
teratoma tissue. This was produced in vivo from the same teratocarcinoma line
(OTT 6050) which was cloned to give OC15 SI.
predominant form to a much later time in development and the Cellogel reaction
strips have to be rather over-incubated to detect the low proportion of the MM
form of very early embryonic muscle (Fig. 3). As in the case of CPK, the first
appearance of MM in tongue and heart muscle is earlier (14th and 13th day
respectively) than in the skeletal muscle of the hind-limb. MM does not become
the predominant form of PGM until after the 17th day of development in all
types of muscle.
(4) Are these isoenzymes and their transitions detectable in teratocarcinoma cells?
Solid teratomas consisting of several different types of tissues gave the patterns
of CPK and aldolase isoenzymes expected of differentiated tissues. Fig. 4 (a) and
(b), track 3, shows that both muscle and brain type CPK and aldolase were
present in one extract. A cell line (OC15 SI) derived from this teratoma (see
Materials and Methods) and cultured under conditions to maintain it in an
embryonal carcinoma (undifferentiated) form, gave only the embryonic forms
of CPK (BB) (together with the spurious band at MM) and aldolase (A^. When
this cell line was allowed to differentiate and both nervous- and epidermal-like
cells were visible in the culture dish, the aldolase pattern had changed to a 5banded type typical of well-differentiated brain tissue (Fig. 4b, track 2). On the
other hand no change was detected in the CPK pattern (Fig. 4a), or in the PGM
pattern.
;
This result has been confirmed for several cell lines which were derived from
different sources of teratocarcinoma. Two lines were from embryoid bodies of a
C3H-strain tumour and one was from a solid tumour of C3H origin. Embryoid
bodies, like embryonal carcinoma cells in culture, contained only the BB form of
CPK and A4 aldolase. In all cases, after partial differentiation to nerve-type cells
during in vitro culture, the aldolase pattern had transformed into a 3- to 5banded type typical of brain tissue. In addition, in one dish, a moderate amount
of smooth muscle tissue appeared (visible by phase-contrast optics), and when
Isoenzyme transitions in differentiating mouse cells
365
the CPK isoenzyme pattern was examined a very small amount of MB heteropolymer was observed. There was no detectable MB or MM form of PGM in
any of the differentiated cell line extracts.
DISCUSSION
(1) Isoenzyme transitions in development
The study of isoenzymic transitions in developing mouse tissues gave results
summarized in Tables 2, 3, and 4. These are intended to serve as normal tables of
some of the changing biochemical events during differentiation. The adult forms
of CPK and PGM appear at different times in different muscle tissues; they are
apparent in cardiac muscle at the 12th day, in tongue by the 14th day, in most
skeletal muscle by the 15th day, and in some kinds of smooth muscle at the
17th day. Developing brain tissue acquires the typical adult-type isomers of
aldolase on the 14th day of gestation. In summary, CPK and PGM would appear
to be markers of some types of differentiating muscle cells, and aldolase of brain
cells.
Although the proportion of the adult form of PGM in early embryonic muscle
tissues was lower than that of CPK, the MB and MM forms of PGM appeared
at the same time in development. This was equally true of hind-limb skeletal
muscle, tongue and cardiac muscle, but the adult forms appeared at a different
time in each tissue. The degree of morphological development of the tissues
could be roughly correlated with the time of appearance of adult isoenzymic
forms. Possibly the parallel appearance of the differentiated forms of CPK and
PGM denotes a linked control system such as that suggested by Turner &
Eppenberger (1973). These authors argue that a number of proteins such as
CPK, aldolase and myosin may be co-ordinately regulated during muscle
development. It is interesting that striations are not obvious in sections of 15th
day hind-limb examined in the light microscope and so the above biochemical
changes occur in advance of the more obvious histological signs of differentiation.
Although the appearance of adult isomers of CPK and PGM was simultaneous
in all types of developing muscle, the latter enzyme could prove to be a more
reliable marker of the biochemical differentiation of muscle. This is because
extracts of all tissues at all stages in development showed no staining on the
control gel. There is therefore no ambiguity in the interpretation of the results.
(2) Isoenzyme transitions in teratocarcinoma cells
This is the first report of isoenzyme analyses of cloned teratocarcinoma cell
lines. Figure 4 shows that undifferentiated embryonal carcinoma cells are very
like early embryonic cells in having only the early embryonic isoenzymes,
namely, the BB form of CPK, the BB form of PGM (not shown), and the A4
form of aldolase. The patterns for embryoid bodies formed in vivo were similar.
When undifferentiated embryonal carcinoma cells of four different cloned cell
366
E. D. ADAMSON
lines produced in this laboratory were cultured under conditions to promote
their differentiation, they formed morphologically recognisable nerve, epidermal and muscle cells. Extracts from a cloned line (OC15 SI) which produces
a large proportion of nerve-type cells gave a highly differentiated brain-type
pattern of aldolase isoenzymes. One cloned cell line which differentiated to give
some patches of smooth-muscle-like cells as well as nerve cells, produced the
corresponding isoenzyme patterns, that is, a band (rather faint) of the MB form
of CPK and a set of four bands of A and C aldolases.
For mouse teratocarcinomas to be useful as an alternative to embryos in the
study of cell differentiation, they have to be shown to have properties which
closely parallel those of embryos. The stem cells of the tumours (embryonal
carcinoma cells) have been shown to be pluripotent (Kleinsmith & Pierce,
1964), and similar to early embryonic cells, both ultrastructurally (Damjanov,
Solter & Skreb, 1971) and antigenically (Artzt et al. 1973). Histochemical studies
have shown that the distribution of alkaline phosphatase follows the pattern
in embryos (Bernstine et al. 1973; Solter, Damjanov & Skreb, 1973). Some
biochemical properties of in vivo embryoid bodies allowed to differentiate in
vitro have been determined. Gearhart & Mintz (1974) followed the increasing
acetylcholinesterase specific activities of such cultures and identified striated
muscle fibres which eventually formed in them. Levine et al. (1974) showed that
specific activities of both acetylcholinesterase and CPK increase during culture
of in vivo embryoid bodies, but they correlated these with the production of
nerve cells on the basis of histology and the identification of the BB type of
CPK. Since embryoid bodies were shown (above) to contain the BB form of
CPK before differentiation, there was no transition of isoenzyme type during
differentiation.
It is important to show that clonal teratocarcinoma cells differentiating under
defined conditions in vitro reflect the orderly processes which occur during the
development of the embryo. Martin & Evans (1975) showed that one such
clonal line produced embryoid bodies in vitro, and these contained the correct
distribution of alkaline phosphatase. Here I have described the distribution in
embryos of the isoenzymes of three enzymes, and have shown that differentiating
teratocarcinoma cell clones change their patterns similarly.
I wish to thank Miss S. E. Ayers for skilful technical assistance. I am grateful to Dr C. F.
Graham for helpful discussions and criticisms, and to Dr M. McBurney for cloned teratocarcinoma cell lines. This work was supported by the Cancer Research Campaign.
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(Received 24 October 1975)