Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IN NORMAL ANDCYSTIC FIBROSIS FIBROBLASTS
MEMBRANE FLUIDITY S.T.
Christian and John A. Monti The Neurosciences Program, Department of Psychiatry and
Wayne H. Finley Laboratory of Medical Genetics Department of Medicine The University
of Alabama in Birmingham University Station Birmingham, Alabama 35294
Received
October
24,
1977
SUMMARY We have observed distinct differences in the polarization of fluorescence and temperature dependent emission intensity of the highly fluorescent phospholipid derivative (l-acyl-2-(N-4-nitrobenzo-2-oxa-l,3-diazole)--aminocaproyl phosphatidylcholine (NBD-PC), when incorporated in the plasma membranes of normal and cystic fibrosis fibroblasts. Fluorescence polarization measurements indicate that the fluorochrome has a much higher degree of rotational mobility in cystic fibrosis fibroblasts as compared with normal cells. Temperature dependent transitions in the emission intensity of NBD-PC incorporated in normal fibroblasts are indicated at 17.7 and 21.2O C while the abnormal cell membranes apparently undergo transitions at 8.7 and 13.5O C. These differences might be due to changes in plasma membrane composition and/or organization, in the case of the cystic fibrosis cells. The thermal
motion
to as "membrane
fluidity,"
major
role
in
mobility
various
a mechanism
to the
interior
lipid
layer
overall embedded trolled
for
its
conditions
now thought
sites
induction Therefore,
a direct of the
surface should
control
membrane
(2). (and
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
(1).
surface
fluid
state
on signals
that
exhibit
a given specific
protein
membranes signals and/or
of surface are
related
mobility cell
a
The overall
biochemical
ordered
and the thermal
referred to play
of plasma
of specific
of highly the
sometimes
by many investigators
on the
Consequently, does)
components,
mechanisms
the conduction
by the
may exert
membrane
regulatory
pertubations.
flexibility in
is
of receptor
may provide
conformation
various
cellular
of a rrlyriad
cell
of
lipid
membrane to the
of receptors
type
temperature
under
condependent
966 ISSN
0006-291
X
Vol.79,No.3,
BIOCHEMICAL
1977
transitions
that
Any change,
therefore,
whether tion
experimentally,
type
character
or resulting
process,
dependent
utilized
these
of the cell
in the physical
disease
temperature tion
are characteristic
induced and/or
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the
of the
in such measurements
is
investigation.
cell
from either
may be reflected
transitions
under
directly
metirane,
membrane,
a genetic
altera-
as a change
in the
providing
of sufficient
the
sensitivity
instrumentato detect
changes. In general,
degree
an efficient
of mobility
tool
of receptor
for
sites
characteristics
of melllbrane
tion.
most of the extrinsic
However,
are structurally
unrelated
Recently,
et al --*
Monti,
fluorescent
metirane
between
normal
cystic
fibrosis.
quantitative
evaluation
and of phase
transitions
(5,6)
fluorescent
to naturally
probes
occurring
transition fibroblasts
Utilizing
some striking
temperatures
constituents.
and properties
(7).
from
of a highly this
differences
compound
with
and the polarization
and fibroblasts
polariza-
used in such studies
membrane
on the synthesis
we have observed
of the
has been fluorescence
of phosphatidylcholine
probe,
to both
(3,4) layers
reported
derivative
as a metirane
lipid
the
respect
of fluorescence
individuals
afflicted
with
METHODS GM #142 CF cells Repository, studies
Camden, were
biopsy
Both
20 per
The cells
cent
were
centrifugation in isotonic again washing
buffer,
Control from
males
(N=3),
and were
types
of fibroblasts
fetal
calf
harvested
saline
from the
either
at 2,000
collected
obtained
New Jersey.
obtained
of adult
disorder. with
were
amniotic
which
without
they
five
was 0.05
0.5 ug/ml
(N=5)
The cells
genetic
in McKoys 5A media
had reached flask, washed M in Tris were
confluence. collected
twice buffer, then
by
by resuspension pH 7.4,
suspended
of NBD-PC, and centrifuged
967
Cell
or from skin
any identifiable
culture
minutes,
Mutant
used in these
cultured
the
by centrifugation. containing
fluid
were
by scraping
solution
fibroblasts
serum until
x g for
Human Genetic
and
in the as before.
Vol. 79, No. 3, 1977
The final saline
BIOCHEMICAL
pellet
of each cell
and 0.5
subsequent
ml aliquots
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
type
were
was then
taken
and diluted
phospholipid
derivative
1,3-diazole)-aminocaproyl
phosphatidylcholine
Shaw,
Inc.,
Avanti
intensity
Biochemicals
as a function
to equilibrate C/minute.
water
at +5'
cuvette
Forma Scientific
to insure using
that
gently the
a Farrand
were
with
a 3.0
a reference
system
light
for
suspensions.
to the
consisting
concentrations
of scattered
to the
cuvette
and the
with
with
1 indicates
the
a
in the Company
contents
in
each determination was measured
a 150 W xenon
source,
grating
correction
using
quartz
of cell
measurements,
fluorescence
signals
fluorescence
intensities
factors, polacoat
the
was measured
unlabeled
experiments,
temperature
dependence
968
cm.
suspensions
polarization
of an identical
of 1.0
the
cell
suspension.
contribution was s 3 per cent
RESULTS AND DISCUSSION Figure
of
to a
a pathlength
the turbidity
used in these measured
it
Fluorescence
, including
Since
light
at a rate
Instrument
rod before
of Chen and Bowman (8),
in fluorescence
of scattered
cell
ml quartz
filters.
were
and a W + W - 1100 recorder.
spectra
method
errors
slits,
Cells
The temperature
probe,
equipped
fluorescent
by installing
Springs
in suspension.
Walter
fibroblasts
C.
heating
bath.
a Yellow
a teflon
Dr.
and connecting
a thermistor
with
polarization
introduce
all
use in
fibrosis
was achieved
water
spectrofluorometer
and emission
At the protein
with
remained
done in
by the
contribution
control
from
to +40°
before
the fluorometer
and emission
obtained
excitation
from +5'
with
stirred
cells
Mark-I
Fluorescence
might
buffered
The relative
and cystic
15 minutes
continuously
adjustableetcitation
were
for
in
equipped
were
Experiments
in control
temperature holder
was monitored
cuvette
Alabama.
Company 2095 circulating
425C telethermometer the
ml for
was a gift
of temperature
Efficient
jacketed
cuvette
to 3.0
free
1-acyl-2-(N-4-nitrobenzo-2-oxa-
Birmingham,
of NBD-PC incorporated
was measured
0.2'
in probe
experiements.
The fluorescent
allowed
suspended
of NBD-PC emission
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
LOG
3.30
3.40
3.50
3.60
3 (OK )-'
4’0
T
9!F:
Plot of the fluorescence intensity of the NBD-PC phospholipid pro e incorporated into the plasma membranes of control (closed circles) and cystic fibrosis (open circles) fibroblasts as a function of the reciprocal of the absolute temperature. The exciting wavelength was 465 nm and emission was observed at 525 nm. Excitation and emission slits were 10 and 5 nm respectively. Experimental conditions are the same as those descri ed in the text. The cell concentration in each case was approximately 5 x 10 1 cells/ml in a 3 ml cuvette. The phase transition diagrams of the cystic fibrosis fibroblasts indicate both a different slope and widely different phase transition points from the normal cells. Phase transitions occur in these cells at 13.5' and 8.7' C respectively. Normal fibroblasts under identical conditions show phase transitions at 21.2 and 17.7 C respectively. All values are the average of triplicate determinations.
intensity types.
when the This
figure
shows
and cystic
fibroblasts
The normal
cells
abnormal previously
cells
with
in incorporated
a distinct respect
show discontinuities apparently
stated,
in the overall
fluorochrome
undergo
such differences
composition
of their
difference to their at 21.2' transitions could
between thermal
normal
of both
C and 17.7'
be a reflection cytoplasmic
cell
fibroblasts
transition
at 13.5'
respective
969
in the membrane
characteristics. C while
C and 8.7' of the
the C.
As
changes
membranes,
or
VoL79,No.
BIOCHEMICAL
3,1977
250
300
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
400
350
450
5w
X(nm) of fluorescence of the NBD-Cl phospholipid probe in Figure 2: Polarization the membrane of both normal and abnormal cell types measured as a function of the excitation wavelength. Emission was observed at 525 nm and excitation and emission slits were 10 and 5 nm respectively. The normal fibroblasts (closed circles) show a consistently higher degree of polarization than do Each data point represents the the cystic fibrosis cells (triangles). average of eight separate determinations in the case of normal fibroblasts and three separate determinations in the case of the cystic fibrosis cell.
alterations at this
in the time,
CF fibroblast Figure between
these
organization
of the membrane's
we do not
know what
membranes
might
two cell
types.
spectra
polarization
of the
be explained
by more restricted
in
the normal
a much lower
account
2 shows the differences
polarization
probe
specific
degree
different normal
cells
fibroblasts. of polarization.
for in the
These
data
lipid
difference(s) our
show that
is
than
970
the
mobility
The abnormal These
between
polarization
the two cell
rotational
(9).
However, normal
observation.
between
higher
layer
of fluorescence
types
but that
CF cells. of the
cells, data
are the
not only
appear
This
the may
fluorescent
however,
indicate
to be in agreement
and
BIOCHEMICAL
Vol. 79, No. 3, 1977
with
the
usually
transition indicate
a higher
temperatures.
transition
The data
presented
patients
higher
between
with
cystic
proteins
expect in these
chromosomal
markers
is
cystic
basis
sufficiently
sensitive
by utilizing
the
the
with
fibrosis
higher
indicate
and fibroblasts
the
muscular
obtained using
mobility
dystrophy,
would
contain
physical-
(lo-14),
surface
Our cwn data
a recessively
are present
it
inherited
which
themselves
is in the early that
to clearly
fibroblasts
(14).
is,
in the
of polarization.
appear
from patients
to be made, or the carriers
suggest
That
of
and more
suggest
regions
of
that
cellular
of greater
in normal cells.
membranes fibrosis
to change
lower
with
Cystic
membranes
finding
from than
report
transition polarization
temperature. the degree
temperatures
the
have reported
in cells
persons
to find
investigators
membranes
on a prenatal
do lower
Other
disease
findings
than
fibrosis.
Huntingdon's
area
viscosity
normal
recently,
in this
transition
probe
in this
techniques,
fluidity
higher
one might
the
differences
spin-label
since
as does the transition temperature,
membrane
membrane
of the phospholipid
same direction
chemical
data,
Consequently,
fluorescence
from
temperature
AND BIOPHYSICAL RESEARCH COMMUNlCATlONS
stage
cells
instrumentation,
phospholipid
allow
and no known
either
an in utero
to be identified.
of development,
may eventually
by obtaining
would
disease
Although
we believe
be possible from amniotic identify
fluorescent
fluid,
the carriers
probe--fibroblast
our work
that
to diagnose
diagnosis
our
the
disease
and using of the disease membrane
technique. ACKNOWLEDGEMENTS We thank Ms. Maureen Lynch Caste and Mr. Jerome technical assistance, and to W. 0. Romine for typing work was supported by NIMH grant 5ROl DA 01235-02.
McCombs for expert the manuscript. This
REFERENCES :: i:
Singer, S.J. and Nicolson, G.L. Science, Gitler, C. Ann. Rev. Biophys. Biom, Shinitzky, M I b M and Sachs, L. M:: and-Sachs, Inbar, M., Sh;niEzzz: L.
971
175, 720 (1972). 51 (1972). FEBS Letters, 24, 247 (1973). J. Mol. BioT.,X, -. 245 (1973).
Vol. 79, No. 3, 1977
i: 7. 9": 10. 11. 12. 13. 14.
BIOCHEMICAL
AND BIOPHYSICALRESEARCH
COMMUNICATIONS
Rudy, B. and Gitler, C. Biochim. Biophys. Acta, 288, 231 (1972). Cogan, U., Shinitzky, M., Wever, G., and Nishida, T. Biochemistry, 12, 521 (1973). Month, J.A., Christian, S.T., Shaw, W.A., and Finley, W.A. Proc. Am. Sot. Neurochem., 8, 75 (1977). R t- and Bowman R.L Science, 147, 729 (1965). Sk?:; A and Hudso; B 4 35 4; 449 ‘1. Buttekfilld, D.A., Che;nut,'D.B., Proc. Nat. Gael , Acad. Sci. U.S.A.; 71, 909.(19741. Butterfield, D.A., Roses, A.D., Cooper, M.L., Appe 1, S.H. , and Chesnut, D.B. Biochemistr 13, 5078 (1974). Butterd "C&nut, D.B., Appel, S.H., and Roses, A.D. Nature, 263. 159 11976). Butterfield, DIA., Roses, A.D., Appel, S.H., and Chesnut, D.B. Arch. Biochem. Biophys., 177, 226 (1976). Butterfield, D.A., Oesivein, J.Q., and Markesbery, W.R. Nature, 267, 453 (1977).
i
K76 ..
972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IN NORMAL ANDCYSTIC FIBROSIS FIBROBLASTS
MEMBRANE FLUIDITY S.T.
Christian and John A. Monti The Neurosciences Program, Department of Psychiatry and
Wayne H. Finley Laboratory of Medical Genetics Department of Medicine The University
of Alabama in Birmingham University Station Birmingham, Alabama 35294
Received
October
24,
1977
SUMMARY We have observed distinct differences in the polarization of fluorescence and temperature dependent emission intensity of the highly fluorescent phospholipid derivative (l-acyl-2-(N-4-nitrobenzo-2-oxa-l,3-diazole)--aminocaproyl phosphatidylcholine (NBD-PC), when incorporated in the plasma membranes of normal and cystic fibrosis fibroblasts. Fluorescence polarization measurements indicate that the fluorochrome has a much higher degree of rotational mobility in cystic fibrosis fibroblasts as compared with normal cells. Temperature dependent transitions in the emission intensity of NBD-PC incorporated in normal fibroblasts are indicated at 17.7 and 21.2O C while the abnormal cell membranes apparently undergo transitions at 8.7 and 13.5O C. These differences might be due to changes in plasma membrane composition and/or organization, in the case of the cystic fibrosis cells. The thermal
motion
to as "membrane
fluidity,"
major
role
in
mobility
various
a mechanism
to the
interior
lipid
layer
overall embedded trolled
for
its
conditions
now thought
sites
induction Therefore,
a direct of the
surface should
control
membrane
(2). (and
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
(1).
surface
fluid
state
on signals
that
exhibit
a given specific
protein
membranes signals and/or
of surface are
related
mobility cell
a
The overall
biochemical
ordered
and the thermal
referred to play
of plasma
of specific
of highly the
sometimes
by many investigators
on the
Consequently, does)
components,
mechanisms
the conduction
by the
may exert
membrane
regulatory
pertubations.
flexibility in
is
of receptor
may provide
conformation
various
cellular
of a rrlyriad
cell
of
lipid
membrane to the
of receptors
type
temperature
under
condependent
966 ISSN
0006-291
X
Vol.79,No.3,
BIOCHEMICAL
1977
transitions
that
Any change,
therefore,
whether tion
experimentally,
type
character
or resulting
process,
dependent
utilized
these
of the cell
in the physical
disease
temperature tion
are characteristic
induced and/or
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the
of the
in such measurements
is
investigation.
cell
from either
may be reflected
transitions
under
directly
metirane,
membrane,
a genetic
altera-
as a change
in the
providing
of sufficient
the
sensitivity
instrumentato detect
changes. In general,
degree
an efficient
of mobility
tool
of receptor
for
sites
characteristics
of melllbrane
tion.
most of the extrinsic
However,
are structurally
unrelated
Recently,
et al --*
Monti,
fluorescent
metirane
between
normal
cystic
fibrosis.
quantitative
evaluation
and of phase
transitions
(5,6)
fluorescent
to naturally
probes
occurring
transition fibroblasts
Utilizing
some striking
temperatures
constituents.
and properties
(7).
from
of a highly this
differences
compound
with
and the polarization
and fibroblasts
polariza-
used in such studies
membrane
on the synthesis
we have observed
of the
has been fluorescence
of phosphatidylcholine
probe,
to both
(3,4) layers
reported
derivative
as a metirane
lipid
the
respect
of fluorescence
individuals
afflicted
with
METHODS GM #142 CF cells Repository, studies
Camden, were
biopsy
Both
20 per
The cells
cent
were
centrifugation in isotonic again washing
buffer,
Control from
males
(N=3),
and were
types
of fibroblasts
fetal
calf
harvested
saline
from the
either
at 2,000
collected
obtained
New Jersey.
obtained
of adult
disorder. with
were
amniotic
which
without
they
five
was 0.05
0.5 ug/ml
(N=5)
The cells
genetic
in McKoys 5A media
had reached flask, washed M in Tris were
confluence. collected
twice buffer, then
by
by resuspension pH 7.4,
suspended
of NBD-PC, and centrifuged
967
Cell
or from skin
any identifiable
culture
minutes,
Mutant
used in these
cultured
the
by centrifugation. containing
fluid
were
by scraping
solution
fibroblasts
serum until
x g for
Human Genetic
and
in the as before.
Vol. 79, No. 3, 1977
The final saline
BIOCHEMICAL
pellet
of each cell
and 0.5
subsequent
ml aliquots
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
type
were
was then
taken
and diluted
phospholipid
derivative
1,3-diazole)-aminocaproyl
phosphatidylcholine
Shaw,
Inc.,
Avanti
intensity
Biochemicals
as a function
to equilibrate C/minute.
water
at +5'
cuvette
Forma Scientific
to insure using
that
gently the
a Farrand
were
with
a 3.0
a reference
system
light
for
suspensions.
to the
consisting
concentrations
of scattered
to the
cuvette
and the
with
with
1 indicates
the
a
in the Company
contents
in
each determination was measured
a 150 W xenon
source,
grating
correction
using
quartz
of cell
measurements,
fluorescence
signals
fluorescence
intensities
factors, polacoat
the
was measured
unlabeled
experiments,
temperature
dependence
968
cm.
suspensions
polarization
of an identical
of 1.0
the
cell
suspension.
contribution was s 3 per cent
RESULTS AND DISCUSSION Figure
of
to a
a pathlength
the turbidity
used in these measured
it
Fluorescence
, including
Since
light
at a rate
Instrument
rod before
of Chen and Bowman (8),
in fluorescence
of scattered
cell
ml quartz
filters.
were
and a W + W - 1100 recorder.
spectra
method
errors
slits,
Cells
The temperature
probe,
equipped
fluorescent
by installing
Springs
in suspension.
Walter
fibroblasts
C.
heating
bath.
a Yellow
a teflon
Dr.
and connecting
a thermistor
with
polarization
introduce
all
use in
fibrosis
was achieved
water
spectrofluorometer
and emission
At the protein
with
remained
done in
by the
contribution
control
from
to +40°
before
the fluorometer
and emission
obtained
excitation
from +5'
with
stirred
cells
Mark-I
Fluorescence
might
buffered
The relative
and cystic
15 minutes
continuously
adjustableetcitation
were
for
in
equipped
were
Experiments
in control
temperature holder
was monitored
cuvette
Alabama.
Company 2095 circulating
425C telethermometer the
ml for
was a gift
of temperature
Efficient
jacketed
cuvette
to 3.0
free
1-acyl-2-(N-4-nitrobenzo-2-oxa-
Birmingham,
of NBD-PC incorporated
was measured
0.2'
in probe
experiements.
The fluorescent
allowed
suspended
of NBD-PC emission
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
LOG
3.30
3.40
3.50
3.60
3 (OK )-'
4’0
T
9!F:
Plot of the fluorescence intensity of the NBD-PC phospholipid pro e incorporated into the plasma membranes of control (closed circles) and cystic fibrosis (open circles) fibroblasts as a function of the reciprocal of the absolute temperature. The exciting wavelength was 465 nm and emission was observed at 525 nm. Excitation and emission slits were 10 and 5 nm respectively. Experimental conditions are the same as those descri ed in the text. The cell concentration in each case was approximately 5 x 10 1 cells/ml in a 3 ml cuvette. The phase transition diagrams of the cystic fibrosis fibroblasts indicate both a different slope and widely different phase transition points from the normal cells. Phase transitions occur in these cells at 13.5' and 8.7' C respectively. Normal fibroblasts under identical conditions show phase transitions at 21.2 and 17.7 C respectively. All values are the average of triplicate determinations.
intensity types.
when the This
figure
shows
and cystic
fibroblasts
The normal
cells
abnormal previously
cells
with
in incorporated
a distinct respect
show discontinuities apparently
stated,
in the overall
fluorochrome
undergo
such differences
composition
of their
difference to their at 21.2' transitions could
between thermal
normal
of both
C and 17.7'
be a reflection cytoplasmic
cell
fibroblasts
transition
at 13.5'
respective
969
in the membrane
characteristics. C while
C and 8.7' of the
the C.
As
changes
membranes,
or
VoL79,No.
BIOCHEMICAL
3,1977
250
300
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
400
350
450
5w
X(nm) of fluorescence of the NBD-Cl phospholipid probe in Figure 2: Polarization the membrane of both normal and abnormal cell types measured as a function of the excitation wavelength. Emission was observed at 525 nm and excitation and emission slits were 10 and 5 nm respectively. The normal fibroblasts (closed circles) show a consistently higher degree of polarization than do Each data point represents the the cystic fibrosis cells (triangles). average of eight separate determinations in the case of normal fibroblasts and three separate determinations in the case of the cystic fibrosis cell.
alterations at this
in the time,
CF fibroblast Figure between
these
organization
of the membrane's
we do not
know what
membranes
might
two cell
types.
spectra
polarization
of the
be explained
by more restricted
in
the normal
a much lower
account
2 shows the differences
polarization
probe
specific
degree
different normal
cells
fibroblasts. of polarization.
for in the
These
data
lipid
difference(s) our
show that
is
than
970
the
mobility
The abnormal These
between
polarization
the two cell
rotational
(9).
However, normal
observation.
between
higher
layer
of fluorescence
types
but that
CF cells. of the
cells, data
are the
not only
appear
This
the may
fluorescent
however,
indicate
to be in agreement
and
BIOCHEMICAL
Vol. 79, No. 3, 1977
with
the
usually
transition indicate
a higher
temperatures.
transition
The data
presented
patients
higher
between
with
cystic
proteins
expect in these
chromosomal
markers
is
cystic
basis
sufficiently
sensitive
by utilizing
the
the
with
fibrosis
higher
indicate
and fibroblasts
the
muscular
obtained using
mobility
dystrophy,
would
contain
physical-
(lo-14),
surface
Our cwn data
a recessively
are present
it
inherited
which
themselves
is in the early that
to clearly
fibroblasts
(14).
is,
in the
of polarization.
appear
from patients
to be made, or the carriers
suggest
That
of
and more
suggest
regions
of
that
cellular
of greater
in normal cells.
membranes fibrosis
to change
lower
with
Cystic
membranes
finding
from than
report
transition polarization
temperature. the degree
temperatures
the
have reported
in cells
persons
to find
investigators
membranes
on a prenatal
do lower
Other
disease
findings
than
fibrosis.
Huntingdon's
area
viscosity
normal
recently,
in this
transition
probe
in this
techniques,
fluidity
higher
one might
the
differences
spin-label
since
as does the transition temperature,
membrane
membrane
of the phospholipid
same direction
chemical
data,
Consequently,
fluorescence
from
temperature
AND BIOPHYSICAL RESEARCH COMMUNlCATlONS
stage
cells
instrumentation,
phospholipid
allow
and no known
either
an in utero
to be identified.
of development,
may eventually
by obtaining
would
disease
Although
we believe
be possible from amniotic identify
fluorescent
fluid,
the carriers
probe--fibroblast
our work
that
to diagnose
diagnosis
our
the
disease
and using of the disease membrane
technique. ACKNOWLEDGEMENTS We thank Ms. Maureen Lynch Caste and Mr. Jerome technical assistance, and to W. 0. Romine for typing work was supported by NIMH grant 5ROl DA 01235-02.
McCombs for expert the manuscript. This
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