Studies on the proteins from seeds of Croton tiglium and of Jatropha curcas

Abstract

1. Proteins extracted from the seeds of the Euphorbiaceae croton tiglium and Jatropha curcas were separated into three major peaks (I,II,and III) by Sephadex chromatography. 2. The crude protein from both seeds and peaks I and II from Croton and peak I from Jatropha were toxic to mice, to different extents. 3. The crude protein and peak I and peak II from both seeds, inhibited protein synthesis by a reticulocyte lysate; maximum inhibition was exerted by peak II from both seeds. None of these preparations affected protein synthesis in vitro by Ehrlich ascites cells.

Full text

1
Biochem.
J.
(1976)
156,
1-6
Printed
in
Great
Britain
Studies
on
the
Proteins
from
the
Seeds
of
Croton
tiglium
and
of
Jatropha
curcas
TOXIC
PROPERTIES
AND
INHIBITION
OF
PROTEIN
SYNTHESIS
IN
VITRO
By
FIORENZO
STIRPE,*
ANNALISA
PESSION-BRIZZI,*
ENZO
LORENZONI,*
PAOLA
STROCCHI,t
LUCIO
MONTANARO*
and
SIMONETTA
SPERTI*
*1stituto
diPatologia
Generale
and
tIstituto
di
Farmacologia
dell'Universita
di
Bologna,
40126
Bologna,
Italy
(Received
5
June
1975)
1.
Proteins
extracted
from
the
seeds
of
the
Euphorbiaceae
Croton
tiglium
and
Jatropha
curcas
were
separated
into
three
major
peaks
(I,
H
and
III)
by
Sephadex
chromatography.
2.
The
crude
protein
from
both
seeds
and
peaks
I
and
II
from
Croton
and
peak
I
from
Jatropha
were
toxic
to
mice,
to
different
extents.
3.
The
crude
protein
and
peak
I
and
peak
II
from
both
seeds,
inhibited
protein
synthesis
by
a
reticulocyte
lysate;
maximum
inhibition
was
exerted
by
peak
II
from
both
seeds.
None
of
these
preparations
affected
protein
synthesis
in
vitro
by
Ehrlich
ascites
cells.
The
toxicity
of
the
seeds
of
the
Euphorbiacea
Croton
tiglium
has
been
known
for
a
long
time,
and
has
been
attributed
in
part
to
their
irritating
oil
and
in
part
to
a
protein
constituent,
'crotin',
some-
times
indicated
as
consisting
of
a
'crotonglobulin'
and
a
'crotonalbumin'
(Kraemer,
1915;
Wehmer,
1931).
In
the
early
literature
crotin
was
often
associated
with
ricin
and
abrin,
the
toxic
proteins
from
Ricinus
communis
and
Abrus
precatorius
respectively
(Jacoby,
1924).
According
to
Nicolle
&
Cesari
(1913)
the
lesions
caused
in
the
rabbit
by
ricin,
abrin
and
crotin
are
identical,
and
resemble
those
brought
about
by
diphtheria
toxin.
The
less
known
'curcin',
a
toxic
protein
from
the
seeds
of
the
Euphorbiacea
Jatropha
curcas
has
also
been
included
in
the
same
group
of
toxins,
being
considered
as
similar
to
ricin
(Wehmer,
1931;
Schwarting,
1963).
More
recently
it
has
been
observed
that
ricin
and
abrin
are
powerful
inhibitors
of
protein
synthesis
in
whole
cells
(Lin
et
al.,
1971)
and
in
cell-free
systems
(Olsnes
&
Pihl,
1972).
They
act
in
an
identical
fashion
by
damaging
the
60S
ribosomal
subunit,
making
ribosomes
unable
to
form
a
stable
complex
with
elongation
factor
2
(Montanaro
et
al.,
1973,
1975;
Sperti
et
al.,
1973;
Carrasco
et
al.,
1975),
whereas
diphtheria
toxin
affects
translocation
by
in-
activating
elongation
factor
2
(see
review
by
Pappen-
heimer
&
Gill,
1973).
The
aim
of
the
present
experiments
was
to
ascertain
whether
crotin
and
curcin
inhibit
protein
synthesis,
as
do
the
other
toxins
mentioned
above.
It
was
observed
that
the
seeds
of
Croton
tiglium
and
Vol.
156
Jatropha
curcas
contain
proteins
that
are
toxic
to
animals
and
inhibit
protein
synthesis
in
a
cell-free
system,
but
not
in
whole
cells.
Experimental
Materials
Seeds
from
Croton
tiglium,
originally
from
Ceylon,
were
obtained
from
Dr.
C.
Sessa
S.p.A.,
Milan,
Italy
or
from
Mr.
F.
G.
Celo,
Zweibrucken,
West
Germany;
the
seeds
of
Jatropha
curcas,
originally
from
India,
were
also
supplied
by
Mr.
F.
G.
Celo.
Ehrlich
ascites
tumours
were
a
kind
gift
from
Dr.
F.
Spreafico,
Milan,
Italy,
and
were
transplanted
into
Swiss
mice.
L-[14C]Leucine
(specific
radioactivity
330-348mCi/mmol)
was
from
The
Radiochemical
Centre,
Amersham,
Bucks.,
U.K.;
poly-L-ornithine
hydrobromide
(mol.wt.
122000),
ATP
and
bovine
serum
albumin
were
from
Sigma
Chemical
Co.,
St.
Louis,
MO,
U.S.A.;
insolubilized
trypsin
and
chymotrypsin
(Enzyte)
and
GTP
were
from
Miles
Research
Laboratories,
Slough,
Bucks.,
U.K.;
creatine
phosphate
and
creatine
kinase
were
from
Boehringer
Mannheim
G.m.b.H.,
Mannheim,
West
Germany.
All
other
chemicals
were
of
analytical
grade.
Toxicity
experiments
Toxicity
was
evaluated
in
male
Swiss
mice
weighing
25-28
g,
kept
in
individual
cages
and
supplied
with
food
and
water
ad
libitum.
Samples
of
the
extracts,
diluted
with
0.9
%
NaCl,
were
injected
in-
traperitoneally
into
groups
of
four
animals
per
dose.
1

F.
STIRPE
AND
OTHERS
LD50
was
calculated
by
the
probit
method
(Finney,
1964).
The
general
behavioural
and
neurological
(somatic
and
vegetative)
state
of
the
animals
was
evaluated
daily
by
the
procedure
of
Irwin
(1964,
1968),
and
the
results
were
scored
on
a
semi-
quantitative
scale
or
on
a
quantal
scale
(the
frequency
of
animals
showing
a
given
symptom).
Semi-quanti-
tative
values
were
statistically
evaluated
by
the
Student's
t
test,
each
group
being
compared
with
a
control
group
on
the
same
day.
Quantal
values
were
compared
by
using
the
significance
limits
for
the
fourfold
table
test
(Diem
&
Lentner,
1972).
Protein
synthesis
in
vitro
Protein
synthesis
was
measured
with
a
lysate
of
rabbit
reticulocytes
or
with
Ehrlich
ascites
cells.
A
lysate
of
reticulocytes
was
prepared
as
described
by
Allen
&
Schweet
(1962),
and
protein
synthesis
was
assayed
as
described
by
Olsnes
&
Pihl
(1972).
After
incubation,
samples
were
withdrawn
and
added
to
1
ml
of
0.1
M-KOH.
After
30min
at
room
tem-
perature
(22-25°C),
samples
were
decolorized
with
a
drop
of
H202;
an
equal
volume
of
20%
(w/v)
tri-
chloroacetic
acid
was
added,
and
the
protein
precipi-
tate
was
collected
on
glass-fibre
filters
(Whatman
GF/C),
which
were
washed
several
times
with
5
%
trichloroacetic
acid.
Protein
synthesis
was
measured
by
the
method
of
Refsnes
et
al.
(1974)
in
Ehrlich
ascites
cells,
washed
as
described
by
the
same
authors.
Samples
were
taken
and
treated
as
described
above.
General
Radioactivity
was
determined
in
a
Packard
Tri-
Carb
liquid-scintillation
spectrometer,
with
an
external
standard:
filters
were
put
in
counting
vials
with
6ml
of
glycol
monomethyl
ether
and
lOml
of
scintillation
fluid
[0.05%
1,4-bis45-phenyloxazol-
2-yl)benzene
and
0.4%
2,5-diphenyloxazole,
in
toluene].
Counting
efficiency
was
approx.
55%.
Protein
was
determined
by
the
method
of
Lowry
et
al.
(1951),
with
bovine
serum
albumin
as
a
standard.
Results
Extraction
of
seeds
Croton
tiglium.
Portions
(up
to
lOOg)
of
decorti-
cated
seeds
were
extracted
with
8
x
250ml
of
ethyl
ether,
each
time
in
a
Waring
blender;
ether
was
removed
by
filtering,
with
suction,
through
filter
paper.
The
resulting
powder
was
allowed
to
dry
in
the
air,
and
was
then
extracted
with
1
litre
of
cold
0.005M-sodium
phosphate
buffer,
pH7.2,
containing
0.2
M-NaCI/IOOg
of
seeds.
The
extraction
was
carried
out
in
a
Waring
blender
operated
for
1
min
periods
at
5
min
intervals
for
I
h.
The
mixture
was
stirred
on
a
magnetic
stirrer
for
2-3
h,
and
was
then
left
overnight
in
the
cold-room.
After
centrifugation,
the
supernatant
was
brought
to
100%
saturation
with
solid
(NH4)2SO4,
added
slowly
with
constant
stirring,
which
was
continued
for
30-60min
after
the
addition
of
the
salt.
After
at
least
3
h
the
protein
precipitate
was
collected
by
centrifugation
at
lOOOOg
for
20min
at
2°C;
it
was
then
dissolved
in
the
minimum
amount
of
phosphate/NaCl
buffer,
and
was
dialysed
for
24-48
h
against
a
continuous
flow
of
the
same
buffer.
At
the
end
of
dialysis
a
brown
precipitate
was
present
in
the
bags,
which
was
removed
by
centrifugation.
Some
of
the
material
present
in
the
supernatant
tended
to
precipitate
on
further
cooling,
for
instance
when
transferred
from
the
cold-room
(4°C)
to
an
ice
bucket
or
to
a
cen-
trifuge
set
at
0°C;
however,
the
precipitate
redissolved
rapidly
on
increasing
the
temperature.
The
brownish
supernatant,
referred
to as
crude
crotin,
was
loaded
on
a
column
(l15cmx4.3cm)
of
Sephadex
G-100,
previously
equilibrated
with
the
phosphate/NaCl
buffer.
The
column
was
eluted
with
the
same
buffer,
and
12ml
fractions
were
collected,
the
E280
being
recorded.
Three
peaks
were
obtained,
referred
to
as
,II
and
III
in
the
order
of
elution,
with
the
maxium
emerging
at
fractions
45,
100
and
135-160
respectively.
The
fractions
from
each
peak
were
pooled,
the
protein
was
precipitated
with
(NH4)2SO4
(100%
satd.),
redissolved
in
phosphate/
NaCl
buffer
and
dialysed
against
the
same
buffer.
The
protein
from
each
peak
will
be
referred
to
as
crotin
I,
II
and
Ill,
according
to
the
peak.
The
preparations
could
be
stored
for
months
at
-25°C,
and
could
be
thawed
and
frozen
again
several
times
without
appreciable
loss
of
activity.
Attempts
to
purify
further
the
active
fractions
by
the
methods
described
for
the
purification
of
ricin
(Nicolson
&
Blaustein,
1972;
Olsnes
&
Pihli,
1973;
Hara
et
al.,
1974)
were
unsuccessful;
in
particular,
no
protein
remained
attached
to
Sepharose
4B
coluns.
Jatropha
curcas.
The
same
procedures
as
described
above
were
followed
for
the
extraction
of
the
seeds,
for
the
preparation
of
the
crude
protein
(referred
to
as
crude
curcin)
and
for
its
fractionation
on
Sephadex,
which
again
gave
three
peaks,
referred
to as
curcin
I,
II
and
Ill
in
the
order
of
elution,
similar
to
those
obtained
from
Croton.
As
com-
pared
with
Croton,
the
following
differences
were
observed:
(i)
the
precipitation
of
material
on
further
cooling
was
more
abundant;
(ii)
the
activity
(see
below)
of
curcin
II
decreased
markedly
within
a
few
days
at
-25°C;
(iii)
the
material
of
peak
III
could
not
be
precipitated
with
saturated
(NH4)2SO4,
and
was
discarded.
Toxicity
Croton
tiglium.
Crude
crotin,
crotin
I
and
II,
but
not
crotin
III
were
toxic
to
mice.
Symptoms
did
not
1976
2

TOXIC
PROPERTIES
OF
CROTIN
AND
CURCIN
begin
until
24h
after
poisoning,
and
deaths
mostly
occurred
after
48
h.
Animals
receiving
crotin
II
died
later;
at
the
lowest
doses
deaths
occurred
even
after
15
days
of
poisoning.
For
this
reason
an
acute
and
a
delayed
LD50
were
calculated
at
48
h
and
at
7
days
respectively
(Table
1).
The
LD50
of
crude
crotin
was
significantly
higher
than
expected
on
the
basis
of
the
hypothesis
of
additivity
(Finney,
1964);
thus
at
72h
crude
crotin
was
significantly
less
toxic
than
was
expected
from
the
toxicity
of
crotin
I
and
II.
Crotin
I
was
more
toxic
than
crotin
HI,
to
a
statistically
significant
extent.
The
Irwin's
tests
(Irwin,
1964,
1968)
showed
significant
alterations
of
behaviour,
such
as
(Irwin's
terminology)
transfer
arousal,
decreased
locomotor
activity
and
positional
passivity,
impairment
of
comeal
and
pinna
reflexes,
neurological
symptoms,
such
as
loss
of
grip
strength
and
impairment
of
right-
ing
reflex,
and
autonomic
symptoms,
such
as
palpe-
bral
closure
and
defecation
(presence
of
very
dark
faeces
in
and
around
the
anus).
Post-mortem
examinations
showed
alterations
of
the
liver,
which
was
enlarged,
often
yellow,
sometimes
with
red
patches
of
necrosis.
Kidneys
were
pale.
Ascites
was
present
in
some
animals
poisoned
with
crude
crotin,
more
often
in
those
treated
with
crotin
II.
Jatropha
curcas.
Crude
curcin
and
curcin
I
were
also
toxic
to
mice,
although
less
than
the
corre-
sponding
preparations
from
Croton.
As
compared
with
crotins,
their
action
was
slightly
more
rapid,
symptoms
beginning
at
12h
and
most
deaths
occurring
within
48
h
of
poisoning.
Therefore
the
acute
LD50
was
calculated
at
48h
(Table
1).
Curcin
II
was
not
toxic
up
to
6mg
of
protein
per
mouse.
The
behaviour
of
the
animals
was
similar
to
that
of
mice
treated
with
crotins,
except
for
some
neurological
symptoms
(waddling,
fine
tremors,
rocking),
which
were
present
especially
among
animals
poisoned
with
the
highest
doses
of
curcin
I;
some
of
these
animals
had
convulsions.
Post-mortem
examinations
showed
lesions
in
the
liver,
pancreas
and
spleen,
hyperaemia
of
the
intestine,
sometimes
ascites;
the
whole
picture
resembled
that
of
rats
poisoned
with
ricin
(Flexner,
1897;
Waller
et
al.,
1966;
Derenzini
et
al.,
1976.
Effect
on
protein
synthesis
Cell-free
system.
Incorporation
of
['4C]leucine
into
protein
by
a
lysate
of
rabbit
reticulocytes
was
inhibited
by
crude
crotin
(Fig.
la).
Some
inhibition
was
also
exerted
with
crotin
I
(Fig.
lb),
whereas
crotin
II
had
a
much
higher
inhibitory
activity
(Fig.
lc).
Crotin
III
inhibited
significantly
only
at
much
higher
concentrations
(about
100,ug/ml;
results
not
shown).
Similar
results
were
obtained
with
the
preparations
from
Jatropha.
Among
these,
crude
curcin
was
much
more
inhibitory
than
crude
crotin
(Fig.
2a),
curcin
I
was
less
active
(Fig.
2b),
and
the
inhibitory
activity
of
curcin
II
was
no
higher
than
that
of
the
crude
protein
(Fig.
2c).
Since
the
activity
of
curcin
II
decreases
rapidly
on
storage
at
-25°C,
it
is
possible
that
some
activity
had
already
been
lost
during
chromatography
and
dialysis.
Curcin
III
was
not
tested.
All
preparations
lost
their
inhibitory
activity
if
they
were
heated
in
a
boiling-water
bath
for
5min.
The
activity
of
crotin
I
and
II
was
unchanged
if
the
solutions
(0.5mg
of
protein/mi)
were
incubated
overnight
at
4°C
with
1%
2-mercaptoethanol,
but
was
somewhat
decreased
on
incubation
at
37°C
for
20-60min
with
insolubilized
trypsin
or
chymo-
trypsin
(10mg/mi).
Ehrlich
ascites
cells.
Crotin
and
curcin
(crude,
I
and
II)
at
concentrations
up
to
100,ug/ml
had
almost
no
effect
on
protein
synthesis
by
Ehrlich
ascites
cells
(results
not
shown).
No
inhibition
was
observed
when
crotin
H
(100,ug/ml)
was
tested
on
the
same
cells
in
the
presence
of
poly-L-ornithine
(10,ug/ml),
which
increases
uptake
by
cells
(Ryser
&
Hancok,
1965).
Table
1.
Toxicity
ofcrotin
and
curcin
Four
to
seven
scalar
doses
of
each
preparation
were
administered
to
groups
of
four
mice
for
each
dose.
The
acute
LD50
was
evaluated
at
72h
for
crotin
and
at
48h
for
curcin,
and
the
delayed
LD50
was
evaluated
at
7
days.
Other
details
are
described
in
the
Experimental
section.
Range
tested
Preparation
(mg/mouse)
Crude
crotin
Crotin
I
Crotin
Il
Crude
curcin
Curcin
I
Vol.
156
1.36-5.80
0.53-2.99
1.43-4.62
2.34-13.22
1.42-10.68
Acute
LD50
95X0
confidence
limits
Delayed
LD50
95°/
confidence
limits
(mg/mouse)
(mg/mouse)
(mg/mouse)
(mg/mouse)
3.37
1.33
4.38
9.11
6.48
2.22-5.13
0.90-1.95
2.29-8.39
6.95-11.96
5.12-8.19
1.87
0.92
1.68
5.83
2.21
1.14-3.07
0.60-1.40
0.90-3.12
3.49-9.75
1.19-2.69
3

F.
STIRPE
AND
OTHERS
20
1
(b)
0
0
10
[Crotin]
(pg/ml)
20
._
C.)
CU
CU
e
10
0
la
V
o
0
X
0
e
0
x
0I
0
[Crotin]
(ug/ml)
C>
CU
._-
'0
CU3
0
CU
4..
0
0
4-A
0
C)
0
0
(c)
5
[Crotin]
(,ug/mi)
Fig.
1.
Effect
of
crotin-on
protein
synthesis
by
a
reticulocyte
lysate
The
reaction
mixture
contained,
in
a
final
volume
of
0.5ml:
lOmM-Tris/HCI
buffer,
pH7.4,
100mM-ammonium
acetate,
2mM-magnesium
acetate,
1
mM-ATP,
0.2
mM-GTP,
15mM-creatine
phosphate,
25
jig
of
creatine
kinase,
0.05mM
amino
acids
(minus
leucine),
1.5,Ci
of
L-[14C]leucine,
the
appropriate
amount
of
inhibitor
and
0.2ml
of
reticulocyte
lysate.
Incubation
was
at
28°C
for
5
min.
Radioactivity
was
determined
on
25
lI
samples.
(a)
Crude
crotin;
(b)
crotin
I;
(c)
crotin
II.
1976
4
(a)
15
10
5
CU
'0
I-i
0
0
a
CU
0
0.
0
x
0
0

TOXIC
PROPERTIES
OF
CROTIN
AND
CURCIN
'5
.'D
10
C.o
cd
CU
'0
C-&
0
la
0
x
0
5
C-
0
x
10
(b)
1
2
0
50
[Curcin]
(pg/ml)
[Curcin]
(jig/ml)
100
(c)
0
5
[Curcin]
(,ug/ml)
Fig.
2.
Effect
of
curcin
on
protein
synthesis
by
a
reticulocyte
lysate
Experimental
conditions
were
as
described
in
the
legend
to
Fig.
1.
(a)
Crude
curcin;
(b)
curcin
I;
(c)
curcin
IT.
Discussion
The
diversity
of
the
lesions
brought
about
in
the
animals
and
the
different
properties
of
the
various
protein
peaks
obtained
from
the
Croton
tiglium
and
Jatripha
curcas
seeds
indicate
(i)
that
the
active
proteins
obtained
from
Croton
are
different
from
those
obtained
from
Jatropha,
and
(ii)
that
the
Vol.
156
active
component(s)
of
peak
I
is
different
from
that
of
peak
II
from
either
seed,
since
the
former
is
more
toxic
and
brings
about
different
symptoms
and
lesions
in
vivo,
whereas
the
latter
is
much
more
active
on
protein
synthesis
in
vitro.
The
possibility
cannot
be
excluded
that
crotin
II
and
curcin
II
are
natural
derivatives
(or
artifacts)
respectively
of
crotin
I
and
curcin
I,
which
on
Sephadex
behave
as
larger
proteins,
(a)
5
CL
-
.0
C-b
0
cq
0.
C4-
0
C)
0
CU
0
x
0
C)
-
tl
-4
0
Cd
0
._
0.
V-
0
v
3
x
0
fs
V"
0

6
F.
STIRPE
AND
OTHERS
As
outlined
in
the
introduction,
this
investigation
was
prompted
by
the
similarities
noted
in
the
past
between
the
toxic
effects
of
Croton
and
Jatropha
and
the
effects
of
Ricinus
and
Abrus.
Our
results
show
that
the
extracts
from
all
these
seeds
share
the
common
property
of
inhibiting
protein
synthesis
to
a
comparable
extent,
but
also
show
some
differences,
the
most
impressive
perhaps
being
the
degree
of
toxicity.
Crotin
II
and
curcin
II,
although
probably
not
pure,
have
an
inhibitory
effect
on
protein
synthesis
in
vitro
which
is
comparable
with
that
of
pure
ricin
and
abrin
in
the
same
system
(Olsnes
&
Pihl,
1972),
and
yet
they
are
more
than
1000
times
less
toxic
than
ricin
and
abrin
(Olsnes
et
al.,
1974a).
This
could
be
due,
at
least
in
part,
to
an
inability
of
crotin
and
curcin
to
penetrate
cell
walls,
as
indicated
by
the
fact
that
these
proteins
do
not
affect
protein
synthesis
by
Ehrlich
ascites
cells.
The
activity
of
crotin
and
curcin
in
cell-free
systems
is
not
increased
by
treatment
with
2-
mercaptoethanol,
which
greatly
enhances
the
in-
hibitory
effect
of
ricin
and
abrin
by
splitting
their
molecules
into
an
effector
(A)
and
a
carrier
(B)
moiety
(Olsnes
et
al.,
1974a);
moreover,
unlike
ricin,
crotin
and
curcin
do
not
agglutinate
Ehrlich
ascites
cells.
All
this,
and
the
fact
that
crotins,
at
least,
do
not
bind
to
Sepharose
suggests
that
crotin
and
curcin
enter
cells
with
difficulty,
because
they
lack
a
carrier
moiety
or
at
least
the
galactose-binding
groups
by
which
ricin
binds
to
cell
membranes
(Olsnes
et
al.,
1974b).
Note
Added
in
Proof
(Received
10
February
1976)
After
this
paper
had
been
accepted,
we
became
aware
that
lesions
similar
to
those
that
we
observed
had
been
described
in
mice
(Adam,
1974)
and
in
goats
(Adam
&
Magzoub,
1975)
poisoned
with
seeds
of
Jatropha
curcas.
We
thank
Professor
E.
Bonetti
for
interest
and
encouragement,
Dr.
N.
Montanaro
for
advice
and
help
with
the
statistical
calculations,
Dr.
F.
Spreafico
for
a
kind
gift
of
Ehrlich
ascites
tumours.
The
work
was
aided
by
grants
from
the
Consiglio
Nazionale
delle
Ricerche,
Rome.
References
Adam,
S.
E.
I.
(1974)
Toxicology
2,
67-76
Adam,
S.
E.
I.
&
Magzoub,
M.
(1975)
Toxicology
4,
347-
354
Allen,
E.
H.
&
Schweet,
R.
S.
(1962)
J.
Biol.
Chem.
237,
760-767
Carrasco,
L.,
Fernandez-Puentes,
C.
&
Vazquez,
D.
(1975)
Eur.
J.
Biochem.
54,
499-503
Derenzini,
M.,
Bonetti,
E.,
Marinozzi,
U.
&
Stirpe,
F.
(1976)
Virchows
Arch.
B
in
the
press
Diem,
K.
&
Lentner,
C.
(eds.)
(1972)
Scientific
Tables,
7th
edn.,
pp.
109-123,
Ciba-Geigy
S.A.,
Basle
Finney,
D.
J.
(1964)ProbitAnalysis,
2nd
edn.,
pp.
122-159,
University
Press,
Chicago
Flexner,
S.
(1897)
J.
Exp.
Med.
2,
197-216
Hara,
K.,
Ishiguro,
M.,
Funatsu,
G.
&
Funatsu,
M.
(1974)
Agric.
Biol.
Chem.
38,
65-70
Irwin,
S.
(1964)
in
Animal
and
Clinical
Pharmacologic
Techniques
(Nodine,
J.
H.
&
Siegler,
P.
E.,
eds.),
pp.
36-54,
Year
Book
Medical
Publishers,
Chicago
Irwin,
S.
(1968)
Psychopharmacologia
13,
222-257
Jacoby,
M.
(1924)
in
Pharmakologie
(Heffter,
A.,
ed.),
vol.
2,
pp.
1735-1747,
Springer,
Berlin
Kraemer,
H.
(1915)
Scientific
and
AppliedPharmacognosy,
pp.
475-476,
J.
Wiley
and
Sons,
New
York
Lin,
J.-Y.,
Liu,
K.,
Chen,
C.-C.
&
Tung,
T.-C.
(1971)
Cancer
Res.
31,
921-924
Lowry,
0.
H.,
Rosebrough,
N.
J.,
Farr,
A.
L.
&
Randall,
R.
J.
(1951)
J.
Biol.
Chem.
193,
265-275
Montanaro,
L.,
Sperti,
S.
&
Stirpe,
F.
(1973)
Biochem.
J.
136,
677-683
Montanaro,
L.,
Sperti,
S.,
Mattioli,
A.,
Testoni,
G.
&
Stirpe,
F.
(1975)
Biochem.
J.
146,
127-131
Nicolle,
M.
&
Cesari,
E.
(1913)
Ann.
Inst.
Pasteur
Paris
27,
358-365
Nicolson,
G.
L.
&
Blaustein,
J.
(1972)
Biochim.
Biophys.
Acta
266,
543-547
Olsnes,
S.
&
Pihl,
A.
(1972)
FEBSLett.
20,327-329
Olsnes,
S.
&
Pihl,
A.
(1973)
Biochemistry
12,
3121-3126
Olsnes,
S.,
Refsnes,
K.
&
Pihl,
A.
(1974a)
Nature
(London)
249,
627-631
Olsnes,
S.,
Saltvedt,
E.
&
Pihl,
A.
(1974b)
J.
Biol.
Chem.
249,
803-810
Pappenheimer,
A.
M.,
Jr.
&
Gill,
D.
M.
(1973)
Science
182,
353-358
Refsnes,
K.,
Olsnes,
S.
&
Pihl,
A.
(1974)
J.
Biol.
Chem.
249,
3557-3562
Ryser,
H.
J.-P.
&
Hancok,
R.
(1965)
Science
150,
501-503
Schwarting,
A.
E.
(1963)
Prog.
Chem.
Toxicol.
1,
385-401
Sperti,
S.,
Montanaro,
L.,
Mattioli,
A.
&
Stirpe,
F.
(1973)
Biochem.
J.
136,
813-815
Wailer,
G.
R.,
Ebner,
K.
E.,
Scroggs,
R.
A.,
Das
Gupta,
B.
R.
&
Corcoran,
J.
B.
(1966)
Proc.
Soc.
Exp.
Biol.
Med.
121,
685-691
Wehmer,
C.
(1931)
Die
Pflanzenstoffe,
vol.
2,
pp.
668-700,
Fischer,
Jena
1976