J. Embryol. exp. Morph. Vol. 52, pp. 127-139, 1979
Printed in Great Britain © Company of Biologists Limited 1979
\ 27
Enzyme analysis of mouse extra-embryonic tissues
By MICHAEL I. SHERMAN 1 AND
SUI Bl ATIENZA-SAMOLS 1
From the Roche Institute of Molecular Biology, New Jersey
SUMMARY
We have separated for enzyme analysis the following layers that surround the conceptus at
midgestation: decidua, trophoblast, parietal endoderm (including Reichert's membrane),
visceral endoderm, yolk-sac mesoderm and amnion. Measurement of several catabolic
enzyme activities (N-acetyl-/?,D-hexosaminidase, /?-glucuronidase, alkaline and acid phosphatases and non-specific esterases) in these tissues indicates that they are biochemically
distinct, perhaps reflecting the different functions that they perform in providing the embryo
proper with a desirable environment for differentiation and development. Our studies also
provide an example of how visceral endoderm cells can effectively block passage of maternal
macromolecules (in this case a serum esterase) in the fetal circulation. Finally, since there is
often difficulty in distinguishing among early embryonic and extra-embryonic cell types
produced in teratocarcinoma cultures, we have considered how our observations might be
of use in this respect, particularly in discriminating between visceral and parietal endoderm.
INTRODUCTION
A number of structures intervene between the midgestation embryo and the
decidua, which is the maternal moiety of the placenta. Cells deriving from all
three germ layers are represented in these structures (see Snell & Stevens, 1966)
which include trophoblast, parietal endoderm, yolk sac and amnion. Together,
these cell layers provide the embryo with a desirable and well regulated environment for growth and development. This they do in part by safeguarding the
embryo from mechanical injury, by chanelling nutrients to the embryo, by
preventing passage of undesirable elements from mother to fetus, and by
protecting the embryo, which is expressing foreign (i.e. paternal) surface
antigens, from immunologic rejection.
Structural and ultrastructural studies have provided some information concerning the functions of the various cell layers (see, Amoroso, 1952; Brambell,
1969). For example, the permeation of the mouse trophoblast layer with
maternal blood and the active phagocytic activity of trophoblast cells indicate
a role in acquiring maternal nutrients for consumption by the embryo (Fawcett,
Wislocki & Waldo, 1947; Bridgman, 1948; Jollie, 1965). Ultrastructural and
histochemical studies suggest extensive pinocytosis and lysosomal activity in the
1
Authors' address: Roche Institute of Molecular Biology, Nutley, New Jersey, 07110,
U.S.A.
9-2
128
M. I. SHERMAN AND SUI BI ATIENZA-SAMOLS
visceral endoderm layer of the yolk sac (e.g. Beck, Lloyd & Griffiths, 1967 a, b);
it is likely that these cells are involved in absorbing and subsequently
degrading maternal macromolecules, including potentially harmful ones, to
elementary substrates useful in metabolic processes in the embryo proper. The
establishment of a circulatory system by the mesodermal layer of the yolk sac
provides a convenient route by which these nutrients can reach the embryo
(e.g. Payne & Deuchar, 1972). 7'he thick extracellular matrix secreted by parietal
endoderm cells (Pierce, Midgely, Sri Ram & Feldman, 1962) might serve as a
physical barrier to undesirable elements of maternal origin (Jollie, 1968), and
that produced by trophoblast cells might act to decrease antigenicity of these
cells, thereby reducing the risk of immunological rejection (Kirby, 1968; but
see Edidin, 1976).
In order to better understand how these different extra-embryonic cell types
develop, function and interact to serve as a support system for the embryo, we
have been analysing these cells biochemically. We have, for example, found
biochemical and functional markers for trophoblast (see Sherman, 1975a, for
a review; Strickland, Reich & Sherman, 1976; Sherman, Atienza, Salomon &
Wudl, 1977), parietal endoderm (Strickland et al. 1976) and yolk-sac (Sherman,
1972a; Bell & Sherman, 1973; Shapiro & Sherman, 1974; Sherman, 19756)
cells. However, when determining activities in trophoblast cells, we had not
separated them from the parietal endoderm layer, and we had not assayed
either the latter cell type or amnion cells for a number of trophoblast and yolksac markers. Furthermore, we were unable in our previous studies to ascribe
yolk-sac markers to the endodermal or mesodermal component. In this report,
we have separated the various cell layers and analyzed them individually in order
to investigate similarities and differences among cells which have derived
directly from the same progenitors (parietal and visceral endoderm) or which
have become intimately apposed to each other (parietal endoderm and
trophoblast, visceral endoderm and yolk-sac mesoderm).
METHODS
Preparation of tissues
Tissues were obtained from the uteri of SWR/J mice that had been mated
with SJL/J males. On the 11th and 13th days of gestation (the day of observation of the sperm plug is considered the first day) the pregnant mice were killed
by cervical dislocation. Uteri were removed and placed in phosphate-buffered
saline (PBS; solution A of Dulbecco). The trophoblast layer was separated
from the decidua as described previously (Sherman, 19726); yolk sac, amnion
and embryo proper were also separated with watchmaker's forceps. The
parietal endoderm layer was separated from trophoblast tissue by pinching the
latter with watchmaker's forceps on the inner surface of the point of juncture of
giant cells with the placental disc. The thick Reichert's membrane, studded with
Mouse extra-embryonic tissue enzymes
129
parietal endoderm cells, is most easily pulled apart from adherent trophoblast
cells at this location, and the entire structure could be removed cleanly as one
or a few pure and continuous sheets. This is essentially the same dissection
procedure used by Minor et al. (1976).
The procedure for separating the endodermal and mesodermal moieties of
the yolk sac is the same as that used by West, Frels, Chapman & Papaioannou
(1977), and virtually identical to the procedure described by Levak-Svajger,
Svajger & Skreb (1969) for separation of germ layers of the rat embryo. Briefly,
yolk sacs were washed with PBS and placed in a mixture of trypsin (0-5%;
Sigma Chemical Co., St Louis, MO) and pancreatin (2-5%; Microbiological
Associates, Bethesda, MD) in Hanks basic salt solution. After 2 h incubation at
4 °C the tissues were again washed with PBS and placed in a culture dish containing PBS. Under a dissecting microscope the endodermal and mesodermal
cells were separated by catching an edge of the tissue with watchmaker's forceps
and stripping the two layers apart as intact sheets. The mesodermal layer could
be distinguished from the endodermal one since it is vascularized and thinner
than the latter. The pooled endodermal and mesodermal layers were then freed of
residual proteolytic enzymes by low speed centrifugation in PBS. Previous studies
(West, Frels, Chapman & Papaionnou, 1977; Jetten, Jetten & Sherman, 1979)
have attested to the effectiveness with which the endodermal and mesodermal
layers of the yolk sac can be separated.
Enzyme analyses
After dissection as described above, the extra-embryonic tissue layers were
cleared of blood by two or three low-speed centrifugations in PBS. The pelleted
tissues were resuspended in distilled water (1 ml/100 jug wet weight) and stored
at —70 °C. Extracts were prepared by thawing the tissues and homogenizing
them, in the volumes of water indicated above, with teflon pestle homogenizers
(Thomas Inc., Philadelphia, PA). Protein assays (Lowry, Rosebrough, Farr &
Randall, 1951) were carried out on the homogenates. Protein contents were
adjusted to 1 mg/ml by the addition of water. The homogenates were then
separated into small aliquots and stored at - 70 °C until they were assayed for
enzyme activity.
To determine the effects of the protease treatment upon enzyme activities in
the visceral endoderm and yolk-sac mesoderm layers, yolk sacs were incubated
with trypsin-pancreatin but were not separated into the two components. These
yolk sacs were then washed by centrifugation and treated in the same way as
the other tissues.
Phosphatase assays were carried out at pH 5-0 (acid) and pH 10-0 (alkaline)
using jp-nitrophenyl phosphate as substrate (Sherman, 1975 c). iV-acetyl-/?,
D-hexosaminidase (NAH) activity was assayed with /?-nitrophenyl-iV-acetyl-/?D-glucosaminide (Sherman, 1975 c). /?-Glucuronidase (/?-G) was assayed as
described earlier (Bell & Sherman, 1973), except that /?-nitrophenyl-/?-D-gluc-
130
M. I. SHERMAN AND SUI BI AT1ENZA-SAMOLS
uronide (10 ITIM) was used as substrate, and the buffer was 0-1 M citrate, pH 4-5.
The procedure for polyacrylamide slab gel electrophoretic analysis of nonspecific esterase activities has been detailed previously (Sherman, 1975b);
a-naphthyl acetate and a-naphthyl butyrate were used together as substrates.
RESULTS
N-Acetyl-fi-D-hexosaminidase and fi-glucuronidase activities
The data in Table 1 demonstrate that 11 th-day yolk-sac NAH levels are much
greater than those of other extra-embryonic tissues, embryo proper and decidua
of the same gestation age. Since incubation of yolk sacs with trypsin and pancreatin reduced NAH and fi-G levels only slightly (Table 1), we used this
proteolytic treatment to separate the mesoderm and endoderm layers of the
yolk sac and then measured enzyme activities in homogenates of these cell
layers. As Table 1 illustrates, the visceral endoderm layer has a specific NAH
activity almost three times that of the mesoderm layer. By contrast, /?-G
activity levels in llth-day visceral endoderm homogenates are lower than those
of any of the other tissues studied. Consequently, when ratios of NAH//?-G are
determined, visceral endoderm ratios are 6-30 times higher than those of other
tissues. It was observed that parietal endoderm cells, which, along with visceral
endoderm cells, derive from the primitive endoderm layer of the blastocyst,
possess one of the lowest ratios of NAH//?-G activities among the llth-day
tissues analysed.
Whereas embryo, amnion and yolk-sac mesoderm cells all shown approximately a 50% increase in NAH activity between the 11th and 13th gestation
days, both visceral and parietal endoderm levels increase about fivefold
(Table 1). Once again, even though /?-G activity appears to have risen sharply
in visceral endoderm cells between the 11th and 13th gestation days, the NAH/
/?-G ratio in 13th-day visceral endoderm homogenates is 10-30 times higher
than that of embryo, amnion, yolk-sac mesoderm and parietal endoderm
homogenates.
Phosphatase activities
Table 2 confirms and extends earlier observations (Sherman, 1972 6) that
llth-day trophoblast homogenates possess very large amounts of alkaline
phosphatase activity compared to levels present in homogenates of other
embryonic and extra-embryonic cell types. In most of the other tissues, levels
of alkaline phosphatase are similar to, or less than, those of acid phosphatase.
Eleventh-day visceral endoderm and yolk-sac mesoderm possess similar levels
of both acid and alkaline phosphatase activity. However, homogenates prepared from the same tissues on the thirteenth day have quite different acid/
alkaline phosphatase ratios since, in the intervening period, alkaline phosphatase levels increase in visceral endoderm cells with no change in acid
67-5
81-8
125-9
19-6
ll±0-2
0-8 ± 0 1
0-7±0-l
l-6±0-2
3O±O-3
l-7±0-2
20±01
74-2 ±1-4
65-5 ± 6 1
88-1 ±4-2
31-4±0-3
13-5±2-0
12-6±O-5
220 ±2-9
4-5
7-4
110
4-2
50
NAH//?-G
l-3±0-2
1-9 ±0-2
p-G
Sp.act±s.D.
5-5±O-3
9-5 ±5-3
NAH
Sp.act.±s. D.
61
6-7
87-8
148-2
14-6
12 5
1-2 ±0-2
2-2±01
2-4±0-2
2-6±0-2
3-2±01
5-2 ±0-2
—
—
7-3 ±0-4
14-8 ±0-3
210-6±50
385-3 ±8-0
46-6 ±1-3
650 ±6-2
—
—
—
—
NAH//?-G
fi-G
Sp.act. ± S.D.
NAH
Sp.act.±s.D.
13th gestation day
Hi
s:
s
Hi
<TN
ibryonic tL
Enzyme activities are averages of quadruplicate determinations. N-Acetyl-/?,D-hexosaminidase (NAH) and /?-glucuronidase (fi-G) specific
activities are expressed in terms of nmole product formed/min/mg homogenate protein ± standard deviation. In the 'yolk sac Total, treated'
sample, yolk sacs were incubated with trypsin-pancreatin, but the two cell layers were not separated (see Methods). Data were not obtained
for 13th-day trophoblast and decidua because of the difficulty in separating these tissues cleanly at that stage.
Embryo
Amnion
Yolk sac
Total
Total, treated
Visceral endoderm
Mesoderm
Parietal endoderm
Trophoblast
Decidua
Tissue
11th gestation day
Table 1. N-Acetyl-ft, D-hexosaminidase and figlucuronidase specific activities in midgestation embryonic
and extra-embryonic tissues
se ext
ymes
90±3-9
3-5±2-l
2-2±0-3
2-3 ±0-5
2-2 ±0-6
33-5±3-4
235-2±33-6
27-0±4-9
19-8± 11
ll-6±2-4
20-5 ±1-7
16-3 ±0-8
18-8±2-0
15-3±l-5
29-9 ±7-4
32-8 ±5-2
Alkaline,
Sp.act. ± S.D.
9-3
71
8-5
0-5
01
1-2
2-2
3-3
Acid
Alkaline
16-3 ±3-2
16-3 ±5-6
25-7 ±7-6
21-7±10
—
—
14-9 ± 7 0
13-4±l-4
Acid,
Sp.act. ± S.D.
1-6
—
—
4-5
3-8
12-2
3-6±0-6
4-3 ±0-9
2-1 ±0-7
13-6+1-7
—
—
1-5
7-4
Acid
Alkaline
100±l-9
l-8±0-3
Alkaline,
Sp.act. ± S.D.
13th gestation day
Enzyme activities are averages of quadruplicate determinations, Specific enzyme activities are expressed in terms of nmole
Droduct formed/min/mg homogenate protein ± standard deviation.
Embryo
Amnion
Yolk sac
Total
Visceral endoderm
Mesoderm
Parietal endoderm
Trophoblast
Decidua
Tissue
Acid,
Sp.act. ± S.D.
A
11th gestation day
0
>
70
a
m
M
00
O
vs-v:
Table 2. Phosphatase specific activities in midgestation embryonic and extra-embryonic tissues
to
SUI BI
Mouse extra-embryonic tissue enzymes
1
2
133
3
Fig. 1. Electrophoretic esterase profiles of 11th and 13th gestation day yolk-sac
homogenates. Polyacrylamide gel profiles of non-specific esterases (acetyl and butyryl) were obtained from llth-day whole yolk-sac (lane 1) and 13th-day whole yolksac (lane 3) homogenates as described in Methods. The profile in lane 2 was obtained
after llth-day yolk sacs were treated with a trypsin-pancreatin combination but
were not separated into endoderm and mesoderm moieties; instead, the tissue
was washed by centrifugation to remove the proteases and then processed as were
the other samples. The band regions are lettered in accordance with our previous
designations (Sherman, 1972a, 1975a); however, with improved resolution of the
bands we can now detect sub-bands in most of the regions, and these are numbered.
E-region esterase activity is not present in yolk-sac extracts and is apparently
restricted to decidua (Sherman, 1972a). The direction of migration was from the
cathode (top) to the anode (bottom).
phosphatase levels, whereas the opposite is true for yolk-sac mesoderm cells.
Eleventh- and thirteenth-day parietal endoderm cells have higher alkaline
phosphatase activities than visceral endoderm or any of the other tissues studied
with the exception of trophoblast. For reasons that we do not understand, acid
phosphatase specific activities are tightly clustered within a threefold range
(10—30 nmole/min/mg protein) in the tissues under analysis, whereas alkaline
phosphatase levels vary by a factor in excess of 100 (1-8 nmole/min/mg protein
for 13th-day amnion homogenates to 235-6 nmole/min/mg protein for llthday trophoblast).
134
M. I. SHERMAN AND SUI BI ATIENZA-SAMOLS
10
25
Total
50 75 100
5
Endoderm
10 25 50 75
10
Mesoderm
25 50 75 100
Fig. 2. Comparison of electrophoretic esterase profiles of 13th gestation day yolksac endoderm and mesoderm. Extracts were prepared as described in Methods,
from total yolk sac and from the separated mesodermal and endodermal moieties.
From 5 to 100/tg of total homogenate protein (the amounts are indicated above
each lane) from the three preparations were subjected to analysis for esterase
activity as described in Methods. Sub-bands are not numbered in this figure but
scoring for presence and absence of sub-bands appears in Table 3. Also, some
faint bands of activity in the original gel are not clearly reproduced in the photograph,
but their presence is indicated in Table 3.
Non-specific esterase electrophoretic profiles
Eleventh-day yolk-sac homogenates possess a number of different esterase
activities as indicated by polyacrylamide gel electrophoretic analyses (Fig. 1,
lane 1). The esterase activity in the G region is not observed in embryo proper,
trophoblast or decidua cells (Sherman, 1972a, b). As Fig. 1 demonstrates, some
homogenate preparations show multiple G esterase band patterns suggestive of
polymorphism. The trypsin-pancreatin procedure which is used to separate
yolk-sac mesoderm and endoderm layers does not diminish detectably any of
the esterase activities (compare Fig. 1, lanes 1 and 2). Thirteenth-day yolk-sac
preparations (lane 3) possess substantially more activity in the A2, Cl, C2, D4
and F regions than do llth-day preparations.
To determine which cell layer(s) in the yolk sac contains G esterase activity,
endoderm and mesoderm were analyzed separately after trypsin-pancreatin
treatment. Fig. 2 and Table 3 illustrate that the bulk of the activity resides in the
visceral endoderm layer: whereas traces of G esterase can be detected in as
little as 5 /tg homogenate protein from that tissue, G region activity is detectable only in 50 /tg or more of homogenate protein from yolk-sac mesoderm. The
small amount of G esterase activity in mesoderm homogenates might be a
reflexion of minimal contamination of that tissue preparation with endoderm.
Further comparison of the yolk-sac endoderm and mesoderm esterase profiles reveals other differences between yolk-sac endoderm and mesoderm
homogenates. There is an absence of F esterase in the latter tissue, and meso-
_
_
D l
_
+
+
++
+
+
25
_
+
+
++
+
+
50
Yolk sac,
total
*
„
+
+
+
++
+
+
+
+
+
++
+
+
75 100
_
_
—
+
+
-
5
+
+
—
++
+
±
10
+
+
—
++
+
+
25
Yolk sac,
endoderm
*
+
+
+
++
+
+
50
„
—
+
-
-
10
+
+
-
-
25
w
-
-
-
+
+
_
+
+
—
+
-
-
A
^
-
++
+
+
• — —
+
-
5 0 100
Parietal
endoderm
75 100 25
++ ++ ++
±
-
-
50
- ) - _ _ _ _ _ _ - _
+
_
_
_
_
+
++
+
+
75
Yolk sac,
mesoderm
*
§
B '
>^
<{,
3
*
f§
^,
^
S
Data are taken from Figs. 2 and 3. Band identification is described in Fig. 1. Symbols are as follows: — = no activity observed; ± =trace V
of activity; + =band clearly observed; + + =strong band of activity. In some cases, bands are not clearly visible in the reproductions, but 3>
are apparent on the original gels. Yolk-sac mesoderm cells possess a band of esterase between the A and B regions that would, if present in o>
the other tissues, be obliterated by the large amounts of activity in the B region.
2
—
1^1
+
Bl
2
10
±
+
,
A 1
Band:
fig
protein
Table 3. Electrophoretic esterase profiles from 13th gestation day yolk-sac and parietal endoderm tissues
136
M. I. SHERMAN AND SUI BI ATIENZA-SAMOLS
5
10
Visceral
25
50
75
25
Parietal
50
100
Fig. 3. Comparison of electrophoretic esterase profiles from 13th gestation day
visceral and parietal endoderm. Tissue preparation and electrophoresis were
carried out as described in Methods. The numbers above each lane refer to the
number of/tg of total homogenate protein analyzed.
derm homogenates do not seem to possess some of the multiple endoderm bands
in the A, B, C and D regions. Overall, yolk-sac mesoderm appears to possess
substantially less esterase activity than visceral endoderm (Fig. 2; Table 3).
In Fig. 3 and Table 3, esterase profiles from visceral endoderm and parietal
endoderm preparations are compared. Once again, there is substantially more
esterase activity overall in the visceral endoderm homogenates. As is the case
with yolk-sac mesoderm, only traces of G esterase are detectable in 50/^g
parietal endoderm homogenate protein, and some of the C and D region bands
present in visceral endoderm profiles are not observed in parietal endoderm
patterns. Unlike yolk-sac mesoderm, however, parietal endoderm has a prominent band of F esterase activity.
Although not shown here, we have observed that 13th-gestation-day embryo
(Sherman, 1972a) and amnion (J. Flick & M. I. Sherman, unpublished observations) homogenates generate bands of esterase activity only in the A2, B and
D regions; neither tissue appears to possess F or G esterase.
Mouse extra-embryonic tissue enzymes
137
DISCUSSION
The results of the experiments described here indicate that extra-embryonic
tissues differ markedly with respect to the enzymic properties under study.
Tissues such as parietal endoderm and visceral endoderm, which derive from a
common primitive endoderm progenitor, show extensive biochemical divergence
within the 5- to 6-day periods between their initial separation and the time of
analysis in this study. Cell surface analyses of primary parietal and visceral
endoderm cell cultures (Jetten, Jetten & Sherman, 1979) provide further evidence
of this divergence. With few exceptions, tissues which are in close apposition
to each other, such as visceral endoderm and yolk-sac mesoderm, or parietal
endoderm and trophoblast, differ markedly in levels of the enzymes tested.
These observations may be taken to support the idea that the different extraembryonic membranes, regardless of their origin or ultimate location, act in
different ways to help support the embryo.
Previously, we proposed that yolk-sac cells, with their high levels of NAH
activity, were involved in breakdown of the sugar moieties of glycoproteins,
thereby slowing or preventing their further penetration into the embryo proper
(Sherman & Chew, 1972; Bell & Sherman, 1973). The present study strengthens
this idea. Upon separation of the layers of the yolk sac, the bulk of NAH
activity is associated with visceral endoderm cells. Furthermore, as Fig. 2 and
Table 3 illustrate, esterase activity is detected in the F region, only in the visceral
endoderm layer, not in the yolk-sac mesoderm. We have noted previously that
F esterase, probably the enzyme identified genetically as esterase-1, is synthesized by the mother and is taken up from the serum by the conceptus (Sherman & Chew, 1972). At the time we made this discovery, we were impressed by
the inability of the enzyme to penetrate beyond the yolk sac into the embryo
proper. The reason for this failure is now clear, since the enzyme appears not to
pass the visceral endoderm layer intact and, thus, cannot enter the circulation in
the mesodermally derived portion of the yolk sac. This is very likely a generalized phenomenon that is related to the selective transfer of proteins such as
immunoglobulins from mother to fetus (see Brambell, 1969).
The enzyme patterns described here, aside from providing insight into
differences among extra-embryonic cell types, might be pertinent to the identification of cells produced by teratocarcinomas. A number of embryonal
carcinoma cell lines differentiate in vitro into cell types which resemble endoderm
morphologically (e.g. Martin & Evans, 1975*3, b). A dramatic reduction in
cellular alkaline phosphatase activity has been a commonly used indicator of
embryonal carcinoma cell differentiation (Bernstine, Hooper, Grandchamp &
Ephrussi, 1972; Martin & Evans, 1975a, b; Linney &Levinson, 1977; Sherman
& Miller, 1978). However, as the data in Table 2 demonstrate, with the exception
of trophoblast, the embryo proper and the extra-embryonic tissues all have low
levels of alkaline phosphatase. Consequently, as we have pointed out elsewhere
138
M. I. SHERMAN AND SUI BI ATIENZA-SAMOLS
(Sherman & Miller, 1978), reduction in alkaline phosphatase activity should
only be used in conjunction with other more specific markers in the identification
of cell types differentiating from embryonal carcinoma cells.
One marker that might be useful for indicating the presence of visceral
endoderm cells in mixed teratocarcinoma cultures is G esterase, since this
enzyme is not produced in substantial levels by other embryonic or extraembryonic cell types. Furthermore, of several adult tissues tested, only kidney
cells produce an esterase with a similar electrophoretic migration rate (Sherman,
1972 b), and Stevens (1967) has reported that kidney cells are rarely, if ever,
encountered in teratocarcinomas. Also, the observation that G esterase appears in primary cultures of visceral endoderm cells (Jetten, et al. 1979), postblastocyst cultures (Sherman, 19726, 19756) and in certain blastocyst-derived
cell lines (Sherman, 1975 c; A. M. Jetten, S. B. Atienza-Samols & M. I. Sherman,
in preparation) suggests that the appropriate cells will express this enzyme
in vitro. We are presently attempting to obtain a G esterase antiserum; with
such an antiserum, we should be able to pinpoint which cells, if any, in mixed
teratocarcinoma cultures contain the enzyme.
We wish to thank Mr Jon Flick (Columbia College of Physicians and Surgeons) for
participation in some of the initial experiments.
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JOLLIE,
{Received 27 November 1978, revised 23 January 1979)