Báo cáo khoa học: Calix[4]arene methylenebisphosphonic acids as inhibitors of fibrin polymerization doc

Calix[4]arene methylenebisphosphonic acids as inhibitors
of fibrin polymerization
Eduard V. Lugovskoy
1
, Pavel G. Gritsenko
1
, Tatyana A. Koshel
1
, Ievgen O. Koliesnik
1
,
Serhey O. Cherenok
2
, Olga I. Kalchenko
2
, Vitaliy I. Kalchenko
2
and Serhey V. Komisarenko
1
1 Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine, Kyiv, Ukraine
2 Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine
Introduction
Fibrinogen is a glycoprotein (MW $344 kDa) com-
posed of two monomeric units connected by disulfide
bonds. Each monomer consists of three non-identical
polypeptide chains Aa,Bb and c, also connected by
disulfide bridges [1]. The NH
2
-terminal ends of all six
polypeptide chains are situated in the central region of
fibrinogen known as the E-domain. Two peripheral

regions of the molecule historically are called the D-
domains. When blood coagulation system is activated,
thrombin is formed from protrombin and attacks
fibrinogen, splitting off two fibrinopeptides A (Aa1–
16), and thereby exposing fibrin polymerization sites
‘A’ (Aa17–19) [2]. Removal of fibinopeptides A leads
to a form of fibrinogen deemed ‘desAA’, which poly-
merizes spontaneously to form two-molecule thick
protofibrils. Removal of fibrinopeptides B (‘desA-
Keywords
calix[4]arene methylenebisphosphonic acid;
fibrin; fibrinogen; inhibition; polymerization
Correspondence
E. Lugovskoy, Palladin Institute of
Biochemistry, National Academy of
Sciences of Ukraine, 9 Leontovicha Street,
01601, Kyiv, Ukraine
Fax: +38 044 2796365
Tel.: +38 044 2343354
E-mail:
(Received 30 September 2010, revised 23
December 2010, accepted 2 February 2011)
doi:10.1111/j.1742-4658.2011.08045.x
Calix[4]arenes bearing two or four methylenebisphosphonic acid groups at
the macrocyclic upper rim have been studied with respect to their effects
on fibrin polymerization. The most potent inhibitor proved to be calix[4]ar-
ene tetrakis-methylene-bis-phosphonic acid (C-192), in which case the maxi-
mum rate of fibrin polymerization in the fibrinogen + thrombin reaction
decreased by 50% at concentrations of 0.52 · 10
)6

M (IC
50
). At this con-
centration, the molar ratio of the compound to fibrinogen was 1.7 : 1. For
the case of desAABB fibrin polymerization, the IC
50
was 1.26 · 10
)6
M at
a molar ratio of C-192 to fibrin monomer of 4 : 1. Dipropoxycalix[4]arene
bis-methylene-bis-phosphonic acid (C-98) inhibited fibrin desAABB poly-
merization with an IC
50
= 1.31 · 10
)4
M. We hypothesized that C-192
blocks fibrin formation by combining with polymerization site ‘A’ (Aa17–
19), which ordinarily initiates protofibril formation in a ‘knob-hole’ man-
ner. This suggestion was confirmed by an HPLC assay, which showed a
host–guest inclusion complex of C-192 with the synthetic peptide Gly-Pro-
Arg-Pro, an analogue of site ‘A’. Further confirmation that the inhibitor
was acting at the initial step of the reaction was obtained by electron
microscopy, with no evidence of protofibril formation being evident. Calix-
arene C-192 also doubled both the prothrombin time and the activated
partial thromboplastin time in normal human blood plasma at concentra-
tions of 7.13 · 10
)5
M and 1.10 · 10
)5
M, respectively. These experiments

demonstrate that C-192 is a specific inhibitor of fibrin polymerization and
blood coagulation and can be used for the design of a new class of anti-
thrombotic agents.
Abbreviations
1, para-hydroxyphenyl-methylene-bis-phosphonic acid; 2, methylenel-bis-phosphonic acid; APTT, activated partial thromboplastin time; C-98,
dipropoxycalix[4]arene bis-methylene-bis-phosphonic acid; C-192, calix[4]arene tetrakis-methylene-bis-phosphonic acid; PT, prothrombin time.
1244 FEBS Journal 278 (2011) 1244–1251 ª 2011 The Authors Journal compilation ª 2011 FEBS
ABB’) encourages lateral associations that lead to
fibrils [3,4]. The fibrils continue to associate, branching
and forming a 3D network: the framework of the
blood thrombus [5]. It is widely accepted that the ini-
tial step of fibrin polymerization (protofibril forma-
tion) is carried out by the intermolecular pairing of
‘A’ and ‘a’ polymerization sites of fibrin monomers.
Site ‘a’ is a cavity (‘hole;) that includes amino acid res-
idues cGln329, cAsp330, cHis340 and cAsp364, and is
situated in the cC-domain of the fibrinogen ⁄ fibrin
molecule [6].
Recently, calixarenes, comprising nanosize cup-
shaped compounds, have aroused keen interest as a
result of their various effects on biochemical processes
[7,8]. They are widely used as the template for artificial
receptors designed for the recognition and binding of
bioactive compounds: biometals, amino acids, dipep-
tides, proteins, etc. [9,10]. Because they are capable of
forming host–guest supramolecular complexes with
biologically important molecules, calixarenes have the
potential to influence a variety of biochemical pro-
cesses and can serve as molecular platforms for drug
design [11–13].

Antithrombotic properties of calixarenes were ini-
tially demonstrated in 1995 [14]. Subsequently,
Da Silva et al. [15] showed that two para-sulfonato-
calix[8]arenes essentially increase an activated partial
thromboplastin time (APTT) and thrombin time.
Recently, Coleman et al. [16] demonstrated anticoagu-
lant activity for derivatives of two para-octanoylca-
lix[8]arenes. An attempt was made to elucidate the
mechanism of the antithrombotic activity of these
calixarene derivatives [15,16]. It was found that 49-
mono-(2-carboxymethoxy)-5,11,17,23,29,35,41,47-octa-
sulfonato-calix[8]arene (C8SMA) indirectly inhibited
thrombin via interaction with heparin cofactor II (as
dermatan sulphate) but not via interaction with
antithrombin in an heparinoid-like manner. However,
the effect of the calixarenes on the final step of
blood coagulation (i.e. fibrin polymerization) was not
investigated.
The present study aimed to investigate the anticoag-
ulant properties of phosphorus contained calyx[n]ar-
enes in last two steps of blood coagulation:
thrombin + fibrinogen reaction and fibrin monomer
polymerization. In particular, we have focused on
compounds in which the molecular scaffold is deco-
rated with methylene-bis-phosphonic acid groups. One
of these, calix[4]arene tetrakis-methylene-bis-phosphon-
ic acid (C-192), has four such substituent groups.
Another compound, dipropoxycalix[4]arene bis-methy-
lene-bis-phosphonic acid (C-98), has two such substitu-
tents, as well as internal propyl groups (Fig. 1).

Results and Discussion
C-192 was studied with respect to its effects on fibrin
polymerization in two kinds of assay. In the first assay,
the formation of fibrin was followed directly after the
addition of thrombin. In the second assay, previously
prepared fibrin was dispersed and then allowed to
repolymerize under appropriate conditions. In both
cases, fibrin formation was gauged by turbidity mea-
surements. Turbidity analysis showed that the com-
pound decreased the maximum rate of fibrin
polymerization in the thrombin–fibrinogen reaction by
50% at a molar ratio of compound to starting fibrino-
gen of 1.7 : 1 (Fig. 2). The final turbidity of clots was
decreased by 50% at a molar ratio of 4.3 : 1 (com-
pound : starting fibrinogen) (Fig. 2C). The lag-time
was also increased strongly in the presence of C-192,
indicating either an decrease of the rate of protofibril
formation or, conceivably, an increase of protofibril
critical length (Fig. 2B). Similar results were obtained
when calixarene C-192 inhibited the re-association of
dispersed desAABB fibrin (Fig. 3A–C); in this case,
IC
50
= 1.26 · 10
)6
m.
Such a strong and specific inhibition by calixarene
C-192 of both the thrombin–fibrinogen reaction and
the re-association of fibrin desAABB indicates that cal-
ixarene retards clotting not as a result of the inhibition

of thrombin, but entirely because of the blocking of
fibrin polymerization sites.
We also performed electron microscopy studies to
determine the stage of fibrin polymerization that was
inhibited by C-192. Transmission electron microscopy
of the thrombin + fibrinogen media showed that fibrin
remained in monomer state in the presence of calixa-
rene C-192 at its final concentration of 10
)5
m,
whereas, at the same time, mature fibrils were formed
in the absence of C-192 (Fig. 4).
Fig. 1. Structural formulas of C-192 and C-98.
E. V. Lugovskoy et al. Calix[4]arene methylenebisphosphonic acids
FEBS Journal 278 (2011) 1244–1251 ª 2011 The Authors Journal compilation ª 2011 FEBS 1245
Fig. 2. Turbidity analysis of the influence of C-192 on fibrin polymerization in the fibrinogen + thrombin reaction. The dependence on calixar-
ene C-192 concentration is shown for (A) the maximum rate of fibrin polymerization Vmax, (B) the lag-time t and (C) the final turbidity of
fibrin clots Dh.
Fig. 3. Turbidity analysis of the influence of C-192 on fibrin desAABB polymerization. The dependence on calixarene C-192 concentration is
shown for (A) the maximum rate of fibrin polymerization Vmax, (B) the lag-time t and (C) the final turbidity of fibrin clots Dh.
A
30 s
B
85 s
D
200 s
C
85 s
Fig. 4. Electron micrographs of
fibrinogen + thrombin reaction media in the

absence of C-192 (A, B), as well as in its
presence (C, D). Scale bar = 100 nm.
Calix[4]arene methylenebisphosphonic acids E. V. Lugovskoy et al.
1246 FEBS Journal 278 (2011) 1244–1251 ª 2011 The Authors Journal compilation ª 2011 FEBS
The results of turbidity analysis and electron micros-
copy indicate that the inhibition by C-192 occurs at
the first stage of fibrin polymerization, presumably by
blocking one of the sites of protofibril formation.
We also investigated the inhibitory effects of two
structural fragments of C-192: para-hydroxyphenylm-
ethylene-bis-phosphonic acid (1) and methylene-bis-
phosphonic acid (2) (Fig. 5). The results showed that
these constituents inhibit fibrin polymerization with
considerably less activity: the IC
50
value was
> 1.0 · 10
)4
m for 1 and 0.72 · 10
)4
m for 2.
It is of interest that the inhibitory activity of C-98,
which has the two methylene-bis-phosphonic acid
substituents, is much less (Table 1) (IC
50
=
1.31 · 10
)4
m), indicating that the calixarene scaffold
and the number of phosphoryl groups in the molecule

play a crucial role in the inhibitory effect.
Furthermore, calixarene C-192 doubles both the
prothrombin time (PT) and APTT in normal human
blood plasma at concentrations of 7.13 · 10
)5
m and
1.10 · 10
)5
m, respectively (Fig. 6). The molar ratios
of C-192 to plasma fibrinogen were approximately
21 : 1 and 3.3 : 1 for the PT and APTT assays, respec-
tively. The delays in clotting times in the blood plasma
coagulation experiments are what would be expected
by the inhibition of the fibrin polymerization from
fibrinogen after the activation of the blood coagulation
system.
Electron microscopy confirmed that C-192 inhibits
the first stage of fibrin polymerization (i.e. the forma-
tion of protofibrils). Because this stage is fulfilled
through the intermolecular binding of the complemen-
tary sites ‘A’–’a’, it appeared to be certain that
this inhibition is a result of the blocking of site ‘A’
(Aa17-19, GlyProArg) by the calixarene in a ‘knob-
hole’ manner. To confirm that this was the case, we
employed HPLC to study complex formation between
C-192 and the synthetic peptide Gly-Pro-Arg-Pro, a
synthetic analogue of the A knob; the free amino acids
Gly, Pro and Arg were used as controls. Association
constants of calixarene C-192 complexes with amino
acids Gly, Pro, Arg and tetrapeptide Gly-Pro-Arg-Pro

in methanol–water mobile phase (50 : 50, v ⁄ v) were
determined as previously described [17,18]. The addi-
tion of calixarene C-192 to the mobile phase decreased
the capacity factor, k ¢, of the guest molecules
(Table 2). This confirms the formation of inclusion
host–guest complexes. There is linear dependence of
1 ⁄ k¢ versus the concentration of C-192 in the mobile
phase (Fig. 7); the correlation coefficient is 0.98–0.99,
indicating a 1 : 1 ratio of calixarene to Gly-Pro-Arg-
Pro in the complex.
Fig. 5. Two structural fragments of C-192: para-hydroxyphenyl-
methylene-bis-phosphonic acid (1) and methylene-bis-phosphonic
acid (2).
Table 1. Concentration values of compounds that cause 50% inhi-
bition of the maximum rate of the polymerization of fibrin produced
in the fibrinogen + thrombin reaction.
Compound IC
50
C-192 1.26 · 10
)6
M
C-98 1.31 · 10
)4
M
1,para-hydroxyphenyl-methylene-bis-
phosphonic acid
> 1.0 · 10
)4
M
2,methylenel-bis-phosphonic acid 0.72 · 10

)4
M
Fig. 6. The dependence of the PT and APTT ratios on the calixa-
rene C-192 concentration.
Table 2. Values 1 ⁄ k¢ of the guests and association constants K
A
(M
)1
) for their complexes with calixarene C-192. RSD, relative stan-
dard deviation.
Guest
Calixarene concentration
K
A
,M
)1
(RSD, %)
0.0 1.48 2.52 3.54 5.00
1 ⁄ k ¢
Gly 0.302 0.313 0.324 0.331 0.349 280 (10)
Pro 0.294 0.318 0.367 0.396 0.403 814 (26)
Arg 0.311 0.395 0.532 0.592 0.794 2576 (21)
Gly-Pro-Arg-Pro 1.287 1.754 2.453 3.015 3.693 3395 (19)
E. V. Lugovskoy et al. Calix[4]arene methylenebisphosphonic acids
FEBS Journal 278 (2011) 1244–1251 ª 2011 The Authors Journal compilation ª 2011 FEBS 1247
In accordance with the molecular modelling data
(Fig. 8A,B), there are two modes of C-192–Gly-
Pro-Arg-Pro complexation. In the first mode (A),
cooperative electrostatic interactions of two proximal
negatively-charged phosphonyl groups with the Gly

a-amino terminal group and the Arg guanidinium
residue play a principal role in complex formation.
Most of the tetrapeptide molecule is exposed outside
the calixarene cavity. In the second mode (B), the
hydrophobic part of Gly-Pro-Arg-Pro molecule is dee-
ply embedded into the calixarene cavity. The complex
is stabilized by P-O
)
H
3
N
+
electrostatic interactions
of the phosphonyl group with the Gly amino acid resi-
due, as well as by CH-p interactions of CH
2
-group in
the Pro residue with the calixarene aromatic ring.
Hydrophobic forces can additionally stabilize the com-
plex in a water solution.
Thus, we have shown for the first time that C-192 is
a powerful and specific inhibitor of the final step of
blood coagulation, fibrin polymerization, and can be
used as the basis for the design of new class of anti-
thrombotic agents. We found that the mechanism of
C-192 inhibition involves its effect on the first step of
fibrin polymerization, protofibril formation, which is
carried out via intermolecular interactions of comple-
mentary polymerization sites ‘A’ and ‘a’ of fibrin mole-
cules.

We have also shown that C-192 forms complex with
synthetic peptide Gly-Pro-Arg-Pro, which imitates
polymerization site ‘A’ (Aa17 Gly-Pro-Arg), suggesting
that the inhibitory effect of C-192 may be a result of
the blocking of site ‘A’ by this calixarene.
The results obtained in the present study suggest
that the other types of calixarenes noted above [15,16]
have the same mechanism of inhibitory action on
blood clotting.
Materials and methods
1
H and
31
P NMR spectra were recorded on a VXP 300
spectrometer (Varian Inc., Palo Alto, CA, USA) operating
at 300 MHz and 121.5 MHz, respectively. Chemical shifts
are reported using internal tetramethylsilane and external
85% H
3
PO
4
as references. Melting points were determined
on a Boetius apparatus and are uncorrected. Bromotrimeth-
ylsilane was freshly distilled. All reactions were carried out
under dry argon. Tetraformylcalix[4]arene 3 was synthe-
sized as described previously [19].
Methylene-bis-phosphonic acid (2) was purchased from
Aldrich (St Louis, MO, USA). Para-hydroxyphenylmethyl-
ene-bis-phosphonic acid (1) and tetrapropoxycalixarene bis-
methylene-bis-phosphonic acid C-98 were prepared by a

method described previously [20]. Calixarene tetrakis-meth-
ylene-bis-phosphonic acid C-192 was synthesized by the
same method with a sequence of transformations as shown
in the scheme outlined in Fig. 9. The reaction of tetra-
formylcalix[4]arene 3 with a large excess of (iPrO)
2
PONa in
diisopropylphosphite ⁄ dioxane solution affords quantita-
tively calixarene tetrakis-methylene-bis-phosphonate 4. The
standard dealkylation of calixarene phosphonate 4 with
Fig. 8. Two modes of energy minimized
structures of calixarene C-192 complexed
with GlyProArgPro. (A) Cooperative
electrostatic interactions of two proximal
negatively charged phosphonyl groups of
C-192 molecule with Gly a-amino terminal
group and Arg guanidinium residue. (B) The
hydrophobic part of GlyProArgPro molecule
is embedded into the calixarene cavity.
Fig. 7. Dependence of 1 ⁄ k ¢ for Gly, Pro, Arg and Gly-Pro-Arg-Pro
on the C-192 concentration in the mobile phase.
Calix[4]arene methylenebisphosphonic acids E. V. Lugovskoy et al.
1248 FEBS Journal 278 (2011) 1244–1251 ª 2011 The Authors Journal compilation ª 2011 FEBS
Me
3
SiBr and subsequent methanolysis gives quantitatively
C-192.
5,11,17,23-Tetrakis[bis(diisopropylphosphoryl)-
methyl]calix[4]arene
Sodium metal (0.69 g, 29 mmol) was carefully added in

small portions to diisopropyl phosphite (15 mL) at room
temperature. The solution formed was diluted with dioxane
(15 mL). Tetraformylcalixarene 3 (1 g, 1.86 mmol) was
added to the resulting solution, as appropriate. The reac-
tion mixture was stirred at room temperature for 48 h and
then quenched with water (100 mL) and extracted with
chloroform (3 · 50 mL). The chloroform layer was concen-
trated under vacuum and the residue was washed with hex-
ane and dried in vacuum. White powder, 2.9 g, yield 99%,
melting point 65–67 °C (from hexane). Found: C, 53.69; H,
7.73; P, 13.65. C
81
H
138
O
2
8P
8
requires C, 53.82; H, 7.69; P,
13.71%.
1
H (300 MHz; CDCl
3
;Me
4
Si) d 7.18 (s, 8H,
PhOH); 4.85 (m, 8H, OCH); 4.38 (m, 8H, OCH); 3.95
(wide s, 8H, ArCH
2
); 3.45 (t, 4H, J

PH
= 25 Hz, PCH);
1.35, 1.25, 1.18, 0.95 (four d, 30H+30H+18H+18H);
31
P
NMR d 19.5; m ⁄ z (FAB MS) 1794 ([M + H]
+
, 100%).
5,11,17,23-Tetrakis [bis(dihydroxyphospho-
ryl)methyl]calix[4]arene
An eight-fold molar excess of bromotrimethylsilane per
phosphonate group (5.46 g, 35 mmol) was added to a solu-
tion of tetrakis-bisphosphonate 4 (1 g, 0.55 mmol) in dry
chloroform (5 mL). The reaction mixture was stirred at
room temperature for 30 h and then was concentrated
under reduced pressure. The residue was dissolved in abso-
lute methanol (15 mL), the resulting mixture stirred at
50 °C for 2 h, and then concentrated and dried in vacuum
(0.05 mmHg) for 10 h. Light powder, 0.59 g, yield 98%,
melting point > 100 °C (decomposition). Found: C, 50.61;
H, 5.12; P, 14.52. C
32
H
40
O
28
P
8
requires C, 34.30; H, 3.60;
P, 22.12%.

1
H (300 MHz; DMSO-d
6
;Me
4
Si) d 7.45 (s, 8H,
PhOH); 4.25 (d, 4H, J
HH
= 13 Hz, ArCH
2
); 3.65 (d, 4H,
J
HH
= 13 Hz, ArCH
2
); 3.55 (t, 4H, J
PH
= 25 Hz, PCH);
31
P NMR d 16.5.
Preparation of fibrinogen, fibrin desAABB
Human fibrinogen was prepared by sodium sulphate precip-
itation from human plasma [21] DesAABB fibrin monomer
was prepared as described previously [22].
Turbidity analysis of fibrin polymerization
The effects of compounds on fibrin polymerization were
studied spectrophotometrically at 350 nm as described pre-
viously [23]. The curve of increasing turbidity during fibrin
clotting shows the parameters: s, lag time, which corre-
sponds to the time of protofibril formation; V

max
, maxi-
mum rate of fibrin polymerization, which was defined by
graphic calculation of the angle of the tangent to the tur-
bidity increase curve at the point of maximum steepness;
and Dh, final turbidity of fibrin clots. The polymerization
of desAABB fibrin was studied at its final concentration
0.1 mgÆmL
)1
in the polymerization medium containing
0.05 m ammonium acetate (pH 7.4) with 0.1 m NaCl and
1 · 10
)4
m CaCl
2
. The polymerization of fibrin formed in
the fibrinogen + thrombin reaction was investigated at a
final concentration of fibrinogen of 0.1 mgÆmL
)1
and
thrombin of 0.4 NIH unitsÆmL
)1
in the same polymeriza-
tion medium.
Electron microscopy
The samples of polymerizing fibrin produced in the throm-
bin–fibrinogen reaction in the absence or the presence of
calixarene C-192 (10
)5
m) were taken out of the reaction

medium at various times, placed on a carbon-coated grid
for 2 min and then stained with 1% (w ⁄ v) uranyl acetate
for 1 min. Transmission electron microscopy was performed
with an H-600 electron microscope (Hitachi, Tokyo, Japan)
operated at 75 kV. Electron micrographs were obtained at
·50 000 magnification.
Fig. 9. Scheme presenting the sequence of transformations during the synthesis of C-192.
E. V. Lugovskoy et al. Calix[4]arene methylenebisphosphonic acids
FEBS Journal 278 (2011) 1244–1251 ª 2011 The Authors Journal compilation ª 2011 FEBS 1249
The determination of association constants by
the RP-HPLC method
The HPLC consisted of a high-pressure pump (type HPP
4001) (Laboratorni Pristroje, Prague, Czech Republic) con-
nected to a Rheodyne sample 7120 injector (Rheodyne,
Berkeley, CA, USA) and an ultraviolet-visible detector
(type LCD 2563) (Laboratorni Pristroje). The column
(150 · 3.3 mm inner diameter) was packed with Separon
SGX CN (Lachema, Prague, Czech Republic). The mobile
phase was a mixture of methanol–water in the ratio 50 : 50
(v ⁄ v), with the calixarene C-192 additive at a concentration
in the range 1.48 · 10
)4
to 5 · 10
)4
m. The flow rate was
0.6 mLÆmin
)1
. The concentration of the guests ⁄ analytes in
solution for analysis was 10
)5

m. The solvent was identical
to the mobile phase composition. The amount of the sam-
ple injected was 0.5 lL. Each of the samples was analyzed
five times. All chromatograms were obtained at 20 °C.
Association constants of the calixarene complexes with
amino acids Gly, Pro, Arg and tetrapeptyde Gly-Pro-Arg-
Pro (280–3395 m
)1
) were calculated from the dependence of
the 1 ⁄ k¢ value versus the calixarene concentration [CA] in
the mobile phase by Eqn (1) (Table 1):
K
A
¼
k
0
0
ð1=k
0
À 1=k
0
0
Þ
½CA
ð1Þ
where k
0
¢ and k¢ are the capacity factors determined in the
absence and presence of the calixarene in the mobile phase
and [CA] is the calixarene concentration in the mobile

phase.
PT and APTT assays
The effects of calixarene C-192 on the coagulation of
human blood plasma were studied using a coagulometer
(CT 2410 ‘Solar’, Minsk, Belarus). Reagents from Renam
(Moscow, Russia) were used to calculate PT and APTT.
PT and APTT ratios were calculated using the formula:
t
c
⁄ t
o
, where t
o
is the time of clot formation in blood plasma
without calixarene C-192 and t
c
is the time of clot forma-
tion in blood plasma with calixarene C-192.
Acknowledgements
We are grateful to Professor Russell Doolittle (Center
for Molecular Genetics, University of California, San
Diego, CA, USA) for useful discussion of the results
obtained.
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