Vol. 276, Issue 3, R659-R664, March 1999
Effect of prostanoids and their precursors on the aggregation
of rainbow trout thrombocytes
D. J.
Hill1,
M. B.
Hallett2, and
A. F.
Rowley1
1 School of Biological
Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP;
and 2 Department of Surgery,
University of Wales College of Medicine, Heath Park, Cardiff CF4
4XN, United Kingdom
 |
ABSTRACT |
The role of
prostanoids and their precursor fatty acids in the aggregatory response
of thrombocytes (platelet equivalents of fish) from the rainbow trout,
Oncorhynchus mykiss, was studied. Aggregation of these cells was induced by the thromboxane mimetic U-46619 or arachidonic acid (AA) in the presence of human or trout fibrinogen. The production of
TXB2/3 by thrombocytes in response to stimulation with AA was inhibited by aspirin, ibuprofen, and indomethacin. However, thrombocyte aggregation in response to AA
stimulation was not significantly altered by these agents at the
concentrations tested (10-100 µM), with the exception of
indomethacin at 20 and 40 µM. Effects on cytosolic calcium
concentration have been suggested as an alternative mechanism for the
inhibitory action of indomethacin on human platelet aggregation. The
present study, however, failed to identify this as a mechanism for the inhibition of U-46619-induced trout thrombocyte aggregation by indomethacin. The polyunsaturated fatty acids docosahexaenoic acid and
eicosapentaenoic acid both exhibited an inhibitory effect on
U-46619-induced thrombocyte aggregation similar to that observed with
mammalian platelets. Unlike the case in mammalian hemostasis, prostacyclin inhibited thrombocyte aggregation only at high
concentrations (>5 µM). Prostaglandin
E2, however, inhibited thrombocyte
aggregation at much lower concentrations (>0.01 µM), suggesting
that it may be the major inhibitory eicosanoid in trout.
eicosanoids; platelet equivalent; hemostasis; fish
| |
INTRODUCTION |
SINCE THE DISCOVERY OF PROSTANOIDS, such as thromboxane
(TX) A2, prostaglandin (PG)
E2, and prostacyclin
(PGI2), the role of eicosanoids
in mammalian platelet function has been well characterized (8, 18).
TXA2 is the most potent platelet
activator (28), whereas PGE2 has
been shown to exert a biphasic effect on these cells (34). At low
concentrations, it potentiates platelet aggregation in response to
various stimuli, whereas at high concentrations, it has an inhibitory
effect (34). The strongest naturally occurring inhibitor of platelet
aggregation is PGI2. Synthesis of
this compound occurs not in platelets but mainly in the endothelial
cell layer of the vasculature. The inhibitory effect of
PGI2 is brought about by an
increase in cAMP levels in platelets that decreases the cytosolic
calcium concentration and thus prevents activation of calcium-dependent
processes (31). PGD2 is also a
potent inhibitor of platelet function but is produced in relatively
small amounts and therefore not considered a major contributor to
hemostatic regulation (5).
Precursor fatty acids also play a role in platelet regulation in the
hemostatic response. All the aforementioned prostanoids are derived
from the polyunsaturated fatty acid (PUFA) arachidonic acid (AA). This
PUFA has no direct effect on aggregation; rather its action is exerted
via conversion to proaggregatory prostanoids such as
TXA2 (32). It has been noted that
Eskimos have a much lower incidence of thrombosis than is found in many
Western cultures. This observation has been attributed to the high
dietary intake of fish oils, which contain high levels of
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (4, 33).
Dietary trials have been performed in which volunteers were given
higher levels of EPA and/or DHA for a period of weeks. In all
cases, a higher proportion of these PUFAs were incorporated into the
membrane phospholipids of the subject's platelets at the expense of AA (15, 19, 24). A decrease in platelet aggregation in response to
collagen and thrombin stimulation occurred, and
TXB2 (nonenzymatic hydrolysis
product of TXA2) production was
reduced in platelets of subjects on the high-EPA diet (12, 15, 30). EPA
competes with AA as a substrate for the enzyme cyclooxygenase and is
metabolized to generate the three series eicosanoids,
TXA3, and
PGH3, which have little if any
biological activity (29). PGI3,
however, is equipotent to PGI2 in
inhibiting platelet function (27). It is possible that DHA can act
synergistically with EPA to inhibit platelet function (3). Recently,
however, it has been indicated that diets rich in DHA or AA alone had
no significant effect on the function of platelets from these subjects
(20, 21).
Fish blood lacks platelets but possesses nucleated cells termed
thrombocytes, which are thought to serve a similar hemostatic function
(10). In mammalian systems, the predominant PUFA used as substrate for
the biosynthesis of eicosanoids is AA. In fish, however, higher levels
of DHA and EPA, rather than AA, are present in the major phospholipids
(23), and hence a wider range of eicosanoids are generated in such
animals (23). The prostanoid-generating capacity of thrombocytes from
different species of fish has been shown to be variable. For example,
thrombocytes from flounder, red sea bream, black sea bream, and black
rockfish have been reported to convert AA to
PGF2
,
PGE2, and
PGD2, whereas EPA was only converted to PGE3 and
PGD3 by black rockfish
thrombocytes (16). Furthermore, DHA was not converted to eicosanoids in
any of the four species studied (16). Leucocytes from the dogfish,
Scyliorhinus canicula, have been shown
to generate PGF2,
PGE2, and
PGD2 on stimulation with the
calcium ionophore A-23187, and smaller amounts of
TXB2 were also produced (25).
Purified thrombocytes from carp have also been reported to synthesize
PGF2,
PGE2, and
PGD2; however, no
TXB2 generation was observed (13).
Conversely, whole blood and purified thrombocytes from the rainbow
trout, Oncorhynchus mykiss, generated
more TXB2 than other eicosanoids on stimulation with calcium ionophore (14, 22). Given that TXA2 is the most potent inducer of
human platelet aggregation, the lack of generation in the blood cells
of certain fish may indicate that it is not as important in inducing
thrombocyte aggregation in those species.
Although our understanding of the hemostatic mechanisms in fish is
improving (see Ref. 26 for review), little work has been performed
regarding the role of eicosanoids and their precursors in this process.
The present study was therefore undertaken to study the effect a range
of prostanoids and their precursor fatty acids have on the hemostatic
response of thrombocytes from the rainbow trout, O. mykiss.
| |
MATERIALS AND METHODS |
Fish and chemicals.
Adult rainbow trout, O. mykiss, were
obtained from Lliw Mill Trout Farm, Pont Lliw, South Wales. They were
maintained in large external tanks and fed ad libitum on Mainstream
expanded trout diet (BP Nutrition, Cheshire, UK). The fatty acids AA,
DHA, and EPA, the TX mimetic U-46619
(9,11-dideoxy-9
,11-methanoepoxy PGF2), and
PGE2 were obtained from Cascade
Biochem (Reading, UK). PGI2 was
supplied by Alexis Biochemicals (Nottingham, UK). Fura 2 acetoxymethyl
ester (AM) was purchased from Molecular Probes (Eugene, OR). All other
chemicals were of the highest grade available commercially.
Thrombocyte isolation.
Before blood collection, fish were given terminal anesthesia by
immersion in MS-222 (final concentration 0.1 g/l) for 10-15 min.
Blood (~7 ml) was collected from the caudal vessel into a syringe
containing heparin (final concentration ~10 IU/ml). A two-step
Percoll density gradient centrifugation procedure was used to isolate
thrombocytes from the peripheral blood of rainbow trout, as described
previously (9, 14). Each fish yielded 5-7 × 107 thrombocytes of ~86%
purity. The contaminating cells were mainly lymphocytes, neutrophilic
granulocytes, and erythrocytes.
Thrombocyte aggregation studies.
The aggregatory response of trout thrombocyte suspensions purified on
Percoll gradients was measured turbidimetrically by a method similar to
Born (1), using a Payton Minigator II aggregometer (Payton Associates,
Scarborough, ON, Canada). Data were collected by a Macintosh LCII
computer via a MacLab/2e interface using Chart software (ADInstruments,
London, UK). Aggregometers measure cell aggregation by changes in light
transmittance. As cells aggregate, the light transmittance through a
cell suspension increases, which is measured by a detector in the
aggregometer. Thrombocyte suspensions [500 µl aliquots; 1 × 107 cells/ml in
Ca2+-Mg2+
containing Hanks' balanced salt solution (HBSS) with 5 mM MOPS] were aggregated by the addition of the
TXA2 mimetic U-46619 (0.5 µM) in
the presence of trout or human fibrinogen (400 µg/ml). The percentage
aggregation was determined against a cell-free blank. To assess the
involvement of various nonsteroidal anti-inflammatory drugs (NSAIDs),
aspirin, ibuprofen (both 0-100 µM), and indomethacin (0-40
µM) were preincubated with thrombocyte suspension for 15 min before
the induction of aggregation with U-46619 and fibrinogen. The effect of
the AA, DHA, and EPA (0-10 µM) was studied by their addition
immediately before the induction of aggregation. To assess the effect
of PGE2 and
PGI2 on aggregation, these
prostanoids (0.001-1.0 and 0.1-20 µM, respectively) were
added to thrombocyte suspensions alone or immediately before the
induction of aggregation with U-46619 and fibrinogen. Because
PGI2 is unstable in aqueous media,
particularly in the presence of cells, it was prepared immediately
before its addition and all experiments performed within a few hours of
its preparation.
TXB2 EIA.
TXB2/3 production by thrombocyte
preparations was determined using a Biotrak enzyme immunoassay (EIA)
system (Amersham Life Sciences, Little Chalfont, UK). Assay reagents
were prepared and used according to the manufacturer's instructions.
The sensitivity of this assay was 3.6 pg/ml, with cross-reactivity of
100% with TXB3, 60.5% with
2,3-dinor TXB2, 0.18% with
PGD2, and <0.01% with PGE2.
Measurement of cytosolic
Ca2+
concentration.
Thrombocytes (~1 × 107
cells/ml) were loaded with fura 2 by incubation with fura 2-AM (1 µM)
for 40 min at 12°C. The cells were then centrifuged and resuspended
in
Ca2+-Mg2+-containing
HBSS (1.8 mM CaCl2) and kept at
4°C before use. Dual-excitation fluorescence measurements at 340 and 380 nm were achieved using a Spex Fluorolog dual-wavelength
fluorimeter (Glen Spectra, Stanmore, UK) on cell suspensions maintained
at 18°C challenged with U-46619 (0.3-20 µM) in the presence
of indomethacin (10-40 µM) or ethanol (1 µl) as a control.
Maximum and minimum 340/380 ratio values were determined in every
experiment using digitonin (50 µM) and EGTA (20 mM), respectively,
and the cytosolic free Ca2+
concentration was calculated assuming a dissociation
constant of 183 nM, as described previously (7).
Statistical analysis.
Statistical analyses were performed with Instat2 (Graphpad Software)
using Student's t-tests or the
Student-Newman-Keuls multiple comparison test as appropriate. All
experiments reported were performed using at least three different
batches of thrombocytes each from individual animals.
| |
RESULTS |
Previous studies have shown that trout thrombocytes aggregate in the
presence of human fibrinogen (400 µg/ml) and the
TXA2 mimetic U-46619 (0.03-10
µM) in a dose-dependent manner (9). They also aggregate on
stimulation with AA, but not EPA or DHA (10).
The proaggregatory response of thrombocytes in the presence of AA could
have been due to its conversion to eicosanoids such as
PGG2,
PGH2, or
TXA2 rather than a direct effect.
To ascertain if this was the case, the effect of three NSAIDs (which
inhibit cyclooxygenase, the enzyme central to prostanoid synthesis),
namely aspirin, ibuprofen, and indomethacin, on thrombocyte aggregation was studied. In these experiments, aspirin, ibuprofen (10-100 µM), or indomethacin (20-40 µM) were preincubated with
thrombocyte suspensions before the addition of U-46619 (0.5 µM) or AA
(10 µM) in the presence of human fibrinogen (400 µg/ml). Aspirin
inhibited U-46619-induced aggregation, although this was only
statistically significant at a concentration of 100 µM (Fig.
1A).
Similarly, ibuprofen caused a significant decrease in U-46619-induced
aggregation at a concentration of 100 µM (Fig.
1B). The most inhibitory NSAID was
indomethacin (Fig. 1C), with
significant inhibition of U-46619- and AA-induced aggregation at 20 and
40 µM.
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Fig. 1.
Effect of aspirin (A), ibuprofen
(B), and indomethacin
(C) on thrombocyte aggregation
induced by U-46619 (0.5 µM) or arachidonic acid (AA; 10 µM) in
presence of human fibrinogen (400 µg/ml).
* P < 0.05, ** P < 0.01, *** P < 0.001 compared with
appropriate control. Mean values ± SE,
n = 3-6.
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The effects of the three NSAIDs tested in inhibiting thrombocyte
aggregation induced by both AA and U-46619 suggested the involvement of
prostanoid formation in the mechanism of action of both agonists. To
test this, the levels of immunoreactive TXB (stable breakdown
product of TXA) produced by thrombocytes in response to U-46619 (0.5 µM) or AA (10 µM) were determined (Fig. 2). The addition of exogenous AA alone
resulted in the generation of a significant amount of immunoreactive
TXB, whereas the presence of each inhibitor significantly decreased the
amount of this product released by thrombocytes (Fig. 2).
Indomethacin exhibited a significantly greater inhibition of TXB
produced by the addition of AA than seen with aspirin or ibuprofen
(P < 0.05). The addition of U-46619 (0.5 µM) to thrombocyte suspensions resulted in minimal (not above background) generation of immunoreactive TXB that was not significantly affected by the NSAIDs tested.
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Fig. 2.
Effect of aspirin (aspn), ibuprofen (ibup), and indomethacin (indo) on
thromboxane (TX) B immunoreactive material production by thrombocytes
stimulated by AA (10 µM) or U-46619 (0.5 µM).
* P < 0.05, ** P < 0.01 compared with
control. Mean values ± SE, n = 3.
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Inhibition of calcium uptake has been implicated in the action of
indomethacin on purified human platelets, and this has been suggested
as a mechanism of its antiaggregatory activity (6). To ascertain if
this inhibitor affected calcium ion movements in trout thrombocytes,
cytosolic free Ca2+ ion
concentration measurements were performed. After incorporation of fura
2, thrombocytes were challenged with U-46619 and an increase in
cytosolic free Ca2+ ion
concentration was observed (Fig. 3) that
was largely dose dependent. However, there was no significant effect of
indomethacin (40 µM) on the U-46619-induced
Ca2+ signal (data not shown).
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Fig. 3.
Representative trace to show effect of U-46619 (10 µM) on cytosolic
free Ca2+ ion concentration
([Ca2+]i)
in fura 2-loaded thrombocytes in presence of 1.8 mM extracellular
CaCl2. Agonist was added at
arrow.
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AA has been shown to induce the aggregation of thrombocytes in the
presence of human fibrinogen (9). However, because the prominent PUFAs
in trout are DHA and EPA (20), the effect of these fatty acids
(0.03-10 µM) on trout thrombocyte preparations in the presence
of fibrinogen was studied. Neither induced aggregation (data not
shown). However, the addition of DHA to thrombocyte preparations
immediately before the addition of U-46619 resulted in a significant
dose-dependent decrease in the amount of aggregation observed that was
saturable at concentrations above 0.3 µM (Fig. 4). Experiments with EPA also indicated a
similar inhibitory action on U-46619-induced thrombocyte aggregation
(data not shown).
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Fig. 4.
Effect of docosahexanenoic acid (DHA) on U-46619-induced thrombocyte
aggregation in presence of trout fibrinogen (200 µg/ml). Mean values ± SE, n = 3.
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As well as TXB2 production by
trout thrombocytes, another cyclooxygenase product,
PGE2, is produced in significant
amounts by both whole trout blood and purified thrombocytes (14, 22). Exogenous PGE2 (0.001-1 µM)
exhibited no proaggregatory affect on trout thrombocytes in the
presence of fibrinogen. The addition of
PGE2 immediately before the
addition of U-46619 resulted in a dose-dependent decrease in the
resulting thrombocyte aggregation that was significant above
concentrations of 0.01 µM (Fig. 5). As in
mammals, PGI2 is the most potent
inhibitor of platelet function (18); therefore, the effect of
PGI2 on trout thrombocyte
aggregation was studied. The addition of
PGI2 (5 and 10 µM)
immediately before stimulation with U-46619 (0.5 µM) in the presence
of human fibrinogen resulted in a significant decrease of thrombocyte
aggregation (Fig. 6). No significant effect
was observed at the other concentrations tested.
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Fig. 5.
Effect of prostaglandin (PG) E2
thrombocyte aggregation induced by U-46619 (0.3 µM) in presence of
trout fibrinogen (200 µg/ml).
** P < 0.01, *** P < 0.001 compared with
control. Mean values ± SE, n = 4.
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Fig. 6.
Effect of PGI2 on thrombocyte
aggregation induced by U-46619 (0.5 µM) in presence of human
fibrinogen (400 µg/ml). * P < 0.05 compared with appropriate control. Mean values ± SE,
n = 4.
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| |
DISCUSSION |
Both TXA2 and its precursor fatty
acid, AA, have been shown to induce human platelet aggregation (11).
Similarly, trout thrombocytes aggregate in response to AA in the
presence of fibrinogen. In human platelets, inhibition of
cyclooxygenase, the central enzyme responsible for the initial
conversion of AA to PGs, abolishes platelet responses to AA. This
suggests that AA has no direct effect on human platelet hemostatic
responses. In the present study, the effect of three NSAIDs, namely
aspirin, ibuprofen, and indomethacin, on trout thrombocyte aggregation
varied. Both aspirin and ibuprofen significantly inhibited
U-46619-induced aggregation at the highest concentration tested (100 µM). Conversely, these inhibitors were without significant effect on
AA-induced aggregation. Finally, indomethacin significantly inhibited
both AA and U-46619-induced aggregation at all concentrations used. The
inhibition of U-46619-induced aggregation by the NSAIDs tested is of
interest because as a stable mimetic of
TXA2, inhibition of cyclooxygenase
activity would not be expected to affect its action. It is possible,
therefore, that U-46619 elicits its proaggregatory effect on trout
thrombocytes by stimulating further eicosanoid production rather than
by a direct action, although in the present study this agent at 0.5 µM failed to induce significant generation of TXB immunoreactive
material. Previous studies using higher concentrations of U-46619 (10 µM) have, however, been shown to cause the generation of TXB and
other eicosanoids by trout thrombocytes (14). A further
alternative is that the NSAIDs used in the present study may inhibit
U-46619-induced aggregation by a route other than the inhibition of
cyclooxygenase. Both indomethacin and ibuprofen have been shown to
inhibit the uptake of calcium ions in ionophore- or
epinephrine-stimulated human platelets by a mechanism other than the
inhibition of cyclooxgenase (6). In the present study, however,
indomethacin had no effect on calcium ion flux, hence implying that
this is not an explanation for the effect of this inhibitor on thrombocytes.
Although AA produced a proaggregatory response in trout thrombocytes,
other prominent PUFAs in fish, EPA and DHA, failed to induce
aggregation. These findings correlate with the case in human platelets,
in which EPA is converted to TXA3,
which is biologically inactive (12). DHA has been shown to inhibit the
activity of cyclooxygenase on AA in human platelets (3). In the present study, U-46619-induced thrombocyte aggregation was inhibited by exogenous DHA; however, the mechanism of inhibition was not further investigated. These results are unexpected due to the high levels of
DHA and EPA present in fish. Release of these fatty acids by enzymes
such as phospholipase A2 would
lead to the generation of products with antiaggregatory activity.
Therefore, a mechanism may exist to control the release of pro- and
antiaggregatory PUFAs.
PGE2 showed a marked inhibition of
thrombocyte aggregation induced by U-46619 in the present study. In
human platelets, however, PGE2 has
been shown to exert a dual effect on aggregation, being inhibitory at
high concentrations (>50 µM) and stimulatory at low concentrations
(5-500 nM) (34). The stimulatory effect of PGE2 on human platelets is due to
an inhibition of adenylate cyclase and the priming of protein kinase C
to activation by other agonists such as
TXA2 (17, 34), whereas the
inhibitory effect of PGE2 has been
attributed to its nonspecific binding to
PGI2 receptors on the surface of
platelets (34). The inhibition of trout thrombocyte aggregation
observed in the present study by
PGE2 occurs within a concentration
range that would be produced by these cells under normal physiological
conditions (14), suggesting that under such conditions it would be
antiaggregatory in vivo as well as in vitro.
PGI2 is the most potent inhibitor
of platelet aggregation, with an
IC50 of ~1 nM for human
platelet-rich plasma and 10 nM for sheep platelet-rich plasma (35). In
the present study, trout thrombocyte aggregation was significantly
inhibited by PGI2, but only at
concentrations of 5 and 10 µM. Although we cannot totally discount
the possibility of some breakdown of
PGI2 to 6-keto
PGF1 (biologically inactive
breakdown product), this is unlikely to have happened to any great
extent due to the precautions used in our studies. Hence we conclude
that, unlike the observed case with mammalian platelets,
PGI2 is not the most potent
inhibitor of thrombocyte aggregation in trout and that
PGE2 has a more prominent role in
the regulation of thrombocyte aggregation. This is also the situation
in chickens, in which PGE2 is a
more potent inhibitor of aggregation than
PGI2 (2).
In summary, the present study identified both similarities and
differences between the responses of trout thrombocytes and mammalian
platelets to various prostanoids and their precursors. These and other
studies (2, 10, 24) do, however, show that at a relatively early stage
in the evolution of vertebrates, a mechanism of platelet-thrombocyte
aggregation evolved that was dependent on eicosanoids for its induction
and regulation. From the present study, the mechanism of action for the
inhibition of thrombocyte aggregation by DHA, EPA,
PGE2,
PGI2, and the three NSAIDs tested
remains to be elucidated. Ultimately, such studies should lead to a
greater knowledge of hemostasis in stress-related disorders of
commercially important fish and provide a better understanding of the
origins of the mammalian hemostatic mechanism.
Perspectives
This paper is part of a wider study on the evolution of hemostatic
mechanisms. Lower animals, such as invertebrates, have a range of
hemostatic mechanisms, including plasma gelation and blood cell
aggregation. Initial studies in this laboratory have investigated if
eicosanoids are involved in the aggregatory response of coelomocytes
(blood cells) in echinoderms (C. E. Ray and A. F. Rowley, unpublished
observations). These studies showed that the TX mimetic
U-46619 had no effect on the aggregatory response of these cells,
suggesting that in echinoderms, at least, the mechanism of aggregation
is independent of TX and potentially of eicosanoids in general. Perhaps
this may be the case in all invertebrates, but clearly studies with one
species are unlikely to be representative of the diverse range of
invertebrate phyla. Furthermore, to our knowledge, no one has shown
that invertebrates generate TXA2/3
in significant amounts, and this may explain the lack of effect of
U-46619 on blood cell aggregation. The evolution of the first
vertebrates was accompanied by the appearance of a hemostatic mechanism
involving thrombocytes in which aggregation is initiated by TX
generation. The present study showed striking similarities in the role
of eicosanoids in initiating and controlling the aggregation of both
fish thrombocytes and mammalian platelets. This implies that this is a
relatively ancient mechanism that was established with the evolution of
teleost fish. Clearly, it would be of interest to see if similar
mechanisms also operate in cartilaginous fish such as sharks and rays
as well as the evolutionarily more primitive agnathan fish (lampreys
and hagfishes). Finally, although we know the basis by which
eicosanoids are involved in the aggregatory response of trout
thrombocytes, we still have little idea of the underlying mechanisms at
the molecular level.
| |
ACKNOWLEDGEMENTS |
These studies were supported by the Biotechnology and Biological
Sciences Research Council and the Leverhulme Trust (F/391/K).
| |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: A. F. Rowley, School of Biological
Sciences, Univ. of Wales Swansea, Singleton Park, Swansea SA2 8PP,
UK.
Received 26 May 1998; accepted in final form 2 November 1998.
| |
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Abstract
Introduction
