from the cells into the extracellular space in a merocrine manner. We suggest that it is involved in regulat- ing the bacteria with which the sponge is symbiotically.
0022-1554/86/$3.30 The Journal of Histochemistry Copyright © 1986 by The
and Cytochemistry Histochemical Society.
Vol.
CORNELIUS Institutfur mora
MULLER,2
SOBEL,
Physio/ogische
i oceanografiju,
Received
for
BARBEL
Kotor
publication
BECHTOLD,
Universitdt,
Yugloslavia
February
and
and
6500
Institut
in revised
form
May
avarol, a potent cytostatic and hydroquinone, is present in large amounts only in the sponge Dysidea avra (2.7 g avarol/1 kg of fresh material). The present study was designed to determine the storage site of this compound within the organism. Light and transmission electron microscopic studies revealed that avarol is probably stored only in spherular cells. The compound is compartmented in intracellular cytoplasmic vesides in a paracrystalline form, and therefore can have no inhibitory effect on the sponge
are the simplest
a state
of
of an
of fusion
live
bacteria
(Muller
hypothesis
that cells
and
symbiosis
et al. , 1986).
the
symbiotic
sponge al.,
Dysidea
1985b),
agent dure,
which
Stniegler,
limited with
has its foundation
contains
avara was
fungi,
low
level
the
fact
that
bryozoae,
evidence between
and
supports sponge
a growth-promoting
principle we discovered
(cytostatic that the
amounts to be
in
This
in both
large
determined
by
algae,
sponge
coexisting
1979).
Experimental relationship
(e.g., lectin) and a growth-inhibiting (Muller et al. , 1981a). Recently,
A single
organisms
is additionally
in obligate
non-sponge
organisms.
of individual
(Weissenfels
individualization
sponges
multicellular
aggregate
of avarol
a highly
the and agent) marine
(Muller
active
KLJAJK,
West
Universitdt,
14,
accepted
1986;
cytostatic
May
metabolites;
Spherular
influence
Correspondence
iologische Germany.
Chemie,
to: Prof.
Dr.
Universit#{228}t,
W.E.G. Duesbergweg,
Muller,
Institut 6500
f#{252}r Phys-
Mainz,
West
biologiju
(AD).
(6A0663).
cells;
Sponges.
natural
product.
age of avarol in Dysidea avara. dence that avarol is synthesized been
shown
pounds
that
these
(MUller
et
in the
probable
present
study
temporary
ston-
can
contain
biologically
Thompson
et
al.,
cvialready
active
com-
1984).
Methods
collected
of the
marine
by dredging
sponge
in the
Dysidea
Bay of Kotor
avara
(Dictyocer-
(Yugoslavia)
in Sep-
1985.
Avarol.
This
compound
(MUller et al., of avarol, see
They
thoroughly
from
Rr electron ofsponge
2% specimens were
tetroxide,
hr. After
dehydration,
in 0.05
then
cut
into
in the small
in cacodylate 1%
in 0.05
cubes
M cacodylate
the specimens
were
as previously and chemical et al. (1985b).
preparations
1 cm thick
buffer,
fixative
buffer.
avara
phyiscal MUller
microscopic about
M cacodylate
remained
overnight
osmium
pieces
Dysidea
For details of the et al., (1974) and
fresh
in glutaraldehyde, The
was isolated 1985b). Minale
of Sections. sections,
sucrose. ature.
and
We provide some experimental in spherular cells. It has
1981b;
Specimens
were
tember,
cells
al.,
and
Animals. atida)
Therefore,
the site of synthesis
Preparation
2
1986
Germany
has a potent cytostatic effect on eukaryotic cells, the sponge protects itself against the inhibitory
of this
we determined
semi-thick Supported in part by a grant from the Deutsche Krebshilfe e.V. and by a grant from the Bundesministerium f#{252}r Forschung und Technologie (German-Yugoslavic cooperation program) under the coordination of the Internationales B#{252}no GKSS, Geesthacht.
24,
West
u
described properties
I
Mainz,
DORN
CS, AB); Zavodza
BD-S,
cells. Quantitative analysis utilizing high-pressure liquid chromatography revealed that avarol is present at a concentration of 3.2 tg/1O6 spherular cells. It appears that avarol is released from the cells into the extracellular space in a merocrine manner. We suggest that it is involved in regulating the bacteria with which the sponge is symbiotically associated. Hisrochem Cyrochem 34:1687, 1986) KEY WORDS: Avarol; Antimitotic agent; Dys:dea avara; Secondary
(Muller et al., 1985a). Using the appropriate isolation proce2.7 g of avarol can be isolated from 1 kg of fresh material.
(WEGM)
AUGUST
(iVEGM, 6500
Materials et
and
Germany
Because avarol we must ask how
Introduction is composed
Mainz,
f#{252}r Zoologie,
The secondary metabolite antibacterial sesquiterpenoid
Sponges
1986
USA.
Biochemical of the Antimitotic
ZORAN
Duesbergweg,
(ZK);
3, 1986
1687-1690,
DIEHL-SEIFERT,
ANDREAS
Chemie,
12, pp.
Article
Sponge Secondary Metabolites: and Ultrastructural Localization Agent Avarol in Dysidea avara1 E. G.
No.
Pnntedrn
Original
WERNER
34.
Inc.
pH
for 3 days of about
embedded
7.4,
and
pre-fixed with
at room
1 mm
Post-fixation buffer,
were
and
0.2 washed
was carried pH
7.4,
in araldite.
M
temperout
at 4’C Thin
in
for
2
sec-
1687
1688
MULLER,
lions
DIEHL-SELFERT,
A)
(700
studied tim)
with were
stained
Isolation and
were
fixed
.. .#-
.
: .
S
centrifuged The
model
ODS cm)
Hypensil filled
the
the
H20
under
injected;
(5 tm;
with
methanol: the
and
(85:15,
peaks
molar
same
5000
was
the
the
organic
in a Gynkotec wavelength
steel
detec-
column
filled (0.3
Elution
was
performed
done
at 300
nm.
was
proportional
coefficient
g) were mm,
a pre-column
used.
Detection were
30
and
analyzed
a variable
In addition,
material
for
g; 20CC)
cm stainless
25
Shandon). v/v).
gradient
cells (0.5
stirring
was
with x
1 cm cubes
dissociation, density
Spherular x
obtained
a 0.3
of avarol
extinction
mechanical
After
equipped
65)
and (0.5
observations.
Ficoll
of Avarol.
material
citrate
1979).
acetate. mm;
DORN
sections
was cut into
by discontinuous
(10
lead
microscopic
After
Konigsmann,
of ethyl
ton (Beckman 0.5
#{149}c
was
ml
chromatograph
planes
S
0.5
was collected.
with
qq:;t.
and
300 CS liquid with
‘
obtained
and Identification with
suspension
.
were
for light
water.
and Semi-thick
The fresh sponge
formaldehyde-sea
cells
Extraction
phase
blue
Cells.
KLJAJK,
acetate
microscope.
methylene
(Bretting
extracted
-‘a
with
in 3.5%
the spherulan
in uranyl
EM 9A electron
ofSpherular
centnifugation
.
double-stained
a Zeiss
BECHTOLD,
SOBEL,
to the
at 300 nm
x
The
concentrations
was determined
to be
3820.
Results , ..
----
-
----:--
0
‘a.
...
-
i.
.
#{182}, .
..,
Earlier
studies
contain
either
(Muller .
had
already
et al. , 1981b).
.Th
Figures 1 and 2. Spherular cells of Dysidea avara. Figure 1 . Semi-thick section through the mesohyl. Arrows markthe spherular cells(light micrograph)Original magnification x 650. Figure 2. Isolated, whole spherular cells (light micrograph) Original magnification x 750. Bars = 20 tm.
cells
of
Dysidea
revealed
antibiotics
avara
that
spherular
(Thompson Therefore,
also
et
we wished
contain
the
al. ,
cells 1983)
to determine
secondary
in sponges or
lectins whether
metabolite
avarol.
Figures 3-6. Transmission electron micrographs of spherular cells. Figure 3. cell with vacuoles, containing lamellar (f) or granular (g) inclusions. Original magnification x 5400. Figure 4. Inclusions are packed as paracrystalline material (p); n, nucleus. Original magnification x 8500. Figure 5. Higher magnification of the paracrystalline inclusion content (p). Original magnification x 17,000. Figure 6. Release of the paracrystalline material (arrow); n, nucleus. Original magnification x 5000. Bars 2 tm.
=
OF AVAROL
LOCALIZATION
Light
Microscopic
Semi-thick
cells
ranges and
very
9-15
appearance
1.5-4.0
in the
After
almost
the
Their
step, They
size
of
1) that
diameter
dissociation
population.
2);
(Figure
mesohyl.
a mechanical centnifugation
pure
(Figure
revealed
the
proce-
these
have
cells
can
a mulberry-
vesicles
ranges
from
p.tm.
Transmission
Electron
After
sectioning,
cells
became
fibnillan .tm
of
cases,
the
documented
avarol
for the
tract
from
only
one
This
component
ofthe
that
vary
cells
(Figures
assume
contain
internal
4 and
5).
inclusions these
or
between
In
rare
could
internal
be
“grains” Figure
existance
spherulan major
of avarol
cells
was
analyzed
with
3.2
it cannot
contain
7). and
at a detection
co-eluted that
in spherulan
(Figure
component
However, also
We
vesicles
of single
chromatography
revealed
cells.
the
spherulan
granular
paracnystals.
by liquid
analysis
30%
contents
6).
in the either
of Avarol
proof
tamed
of
structure
of the
Identification One
About
vesicles
containing
diameters
3-6).
(Figure
represent
3),
a panacrystalline
release
Studies
cytoplasmic
(Figure The
(Figures
“grains”
Microscopic
intracellular invisible
inclusions.
0.3-4
cells
sponge
abundant
gradient
as an
H
the
tm.
a subsequent
be obtained like
through
are
between
dune
Observation
sections
spherular
1689
.tg
The
ethyl
determined
authentic
at present
from
Tethya
ob-
tions
ofthe
cx-
is present
to contain of
avarol.
is present
was
acetate
wavelength
of avarol
be excluded
cells
in
that
300
8. Chemical
nm.
(MUller
is usually
sponge
In
the
In
1955,
cytostatic
Bergman
and
agents;
Bunk
discovered
that
1--D-arabinofuranosylthymine
sponges was
that
contain
cells
that
live
in
(Bergquist,
1978).
quantitative
analysis
DNA
100
5 x
lated
to be localized
C
is apparently this
C’)
na
presented
This
in the
means,
that
in the
sponge,
because
As found paracrystals
0
2
4
6
8 10
‘12 14 16 18
20 22 24 26
t (mm) Figure 7. Identification of avarol pressure liquid chromatography.
in spherular Standards:
bly
represent yet
lan
space.
species cells of Dysidea avara by high1, authentic avarol; 2, avarone.
know
cells
avarol, the
are
However,
(Alcaligenes
sponge (in preparation), regulating the bacterial
is present
fact
sponge that
by bacteal-
unable
strain
to
that
we
results). (Donadey,
that
very
manner. in the only
extracellulan
it is possible that flora. This suggestion
1982),
which
ofavarol
in the
the
calcu-
1 kg Dysidea
were
in a menocnine
the
From was
be produced
we
avara,
ifany,
of
unlikely
a bacterial
sponges
of Dysidea
considering sp.)
to
(unpublished
are 10i2
Conse-
in the
It is very
known
from
other
role,
in
present
Moreover,
broth
released
functional
10%.
8) is synthesized
are
cells.
avara
present
avanol cells.
terpenes
in some
in spherular
not
cells
all the
x
percentage
be
1.6 g of avarol
by eukaryotic
already
The to
cells
0.16
in 100 g oftissue.
(Figure
detect any avarol in a culture have isolated from Dysidea
cell;
in Results,
compound
exclusively
mg).
semi-
lO
cells exist spherular
as cells
content,
pen
80
The
microgranular
5 x
estimated
in spherulan
sesquiterpenoid
most
tissue;
almost
sequestered
classified
DNA
content
was
spherular
avarol.
be
approximately
(DNA g of
as
with
identified
to be
can
of
evidence bacteria
were
interpreted
basis
tissue
of Streptomyces
cells
appear
that
of fresh compound
by the
“grains”
the
100
the
data
E 0 0
per in
not
particular,
they
revealed
lOb
experimental
cases
deter-
0.3% active
experimental
and
were
agent
We
al. , 1969).
some
internal
g of sponge
cells
quently,
these
rare
amounts.
broths
cells
investiga-
a cytostatic
comprises
et
provide
In
On
content
avara.
we
these
isolated
spherular
avarol
fermentation
symbiosis.
in
high
(Bernard
paracrystals;
cells;
in our
that
of a biologically
by sponge
contain
in
that
in
study,
spherulan
g;
1985b)
only
contain
present
in extremely
is produced
they
surprised
to discover
antibiotics
present
avarol
we were
a concentration
obtained
which
Discussion
et al.,
producing
that
However,
organism
Such
strains
of avarol.
Dysideaavara
in this
mined
Quantitative
other
crypta. sponge
materials.
106 spherular
avarol.
structure
the
probaWe
do
extracellu-
one space
bacterial of this
avarol is involved in is supported by our
1690
MULLER,
recent
finding
that
avarol
antifungal activity In preliminary cytostatic preparation). tected basis
arrest
in some
way against
Moreover,
in
by
these a form
penimental
data cells
the most
Literature
a
of avarol. avarol special
avarol
functional the
is stored
mechanism
is binding
as
No
by which
avarol. ofthe
the
vacuoles.
inertness.
extracellular
or soluble
On
is func-
cxthe
At present,
compound
to cx-
macromolecules.
Cited
PR (1978):
Sponges.
H, Konigsmann
cells in the sponge
Axinella
Contribution of sponges. London,
C, Manal
M,Jacquillat
K (1979):
to the study Chem
Investigations
polypoides
ofmanine 20:1501
J Org Hutchinson
University
R (1969): on the
(Schmidt).
Donadey C (1982): Les cellules pigmentaires l’#{233}ponge Cacospongia scalaris (Demosponge 4:67
and causes
effect within
about
explanation
BernardJ, Paul R, Boiron Berlin, Springer-Verlag Bretting
avarol
intracellular
organelles
against
Bergman W, Bunk DC (1955): ucts; XXXIX. The nucleosides Bergquist
that
guarantees
are available
structures
that
cytostatic
cytoplasmic
are protected
plausible
tracellular
the
sequestration
that
antibacterial
cells of Dysidea avara in vitro (in the sponge cells in vivo must be pro-
it is likely
inactivated
paracrystals, sponge
data,
considerable
et al., 1985). we determined
of dissociated Consequently,
of available
tionally
displays
(Seibert studies,
prod-
Rubidomycin.
lectin-producing
Cell Tissue Res 201:487
et les cellules Dictyocenatide).
a inclusions
de Vie Marine
KLJAJI, DORN
BECHTOLD,
Minale L, Ricco R, Sodano G (1974): Avarol, a novel sesquiterpenoid hydroquinone with a rearranged drimane skeleton from the sponge Dysidea avara. Tetrahedron Lett 38:3401 Muller WEG, Maidhof A, Eich E, Schn#{246}der HC (1986): Potent kemic and antimicrobial activity of the novel antimitotic agents and avarol. In Proceedings of the First International Congress Adjuvant Chemotherapy. Paris, Nov 7-9, 1985, in press.
antileuavanone on Neo-
MUller WEG, Maidhof A, Zahn RK, Schn#{246}der HC, Gasif MJ, Heidemann D, Bernd A, Kurelec B, Eich E, Seibert G (1985a): Potent antileukemic activity of the novel cytostatic agent avarone and its analogues in vitro and in vivo. Cancer Res 45:4822 Muller WEG, Zahn RK, Gasif Diehl-Seifert B, Eich E (1985b): from the marine sponge Dysidea
MJ, Dogovif N, Maidhof A, Becker C, Avarol, a cytostatically active compound avana. Comp Biochem Physiol 80C:47
Muller (1981a): sponges.
WEG, Zahn RK, Kurelec Lectin, a possible basis J Bacteniol 145:548
MUllen (1981b): tification
WEG, Zahn RK, Muller I, Kunelec B, Uhlenbruck G, Cell aggregation of the marine sponge Geodia cydonium. of lectin-producing cells. EurJ Cell Biol 24:28
Seibent Library
DIEHI,-SEffERT, SOBEL,
(1985):
Bakt
G, Raether Antibacterial Hyg 260A:379
B, Lucu for
W, Dogovi N, and antifungal
C, MUller
symbiosis
between
I, Uhlenbruck bacteria
G and
Vaith
P
Iden-
Gasif MJ, Zahn RK, Muller WEG activity of avarone and avanol. Zbl
ThompsonJE, Barrow KD, Faulkner DJ (1984): Localization oftwo brominated metabolites, aerothionin and homoaerothionin, in sphenulous cells of a marine demosponge, Aplysina fistularis (Verongia thiona). Acta Zool 27:410
Weissenfels schwammes problem.
N,
Stniegler
Ephydatia Zoomorphol
B
(1979):
fluviatilis 92:49
Bau
und
L. (Ponifera).
Funktion VI.
Das
des SUsswasserIndividualit#{228}ts-
