From the Mason Eye Institute, Department of
Ophthalmology and the § Department of Biochemistry,
University of Missouri, Columbia, Missouri 65212
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ABSTRACT |
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The hydrophobic sites in -crystallin
were evaluated using a fluorescent probe
1,1'-bi(4-anilino)naphthalenesulfonic acid (bis-ANS). Approximately one
binding site/subunit of
-crystallin at 25 °C was estimated by
equilibrium binding and Scatchard analysis (Kd = 1.1 µM). Based on fluorescence titration, the
dissociation constant was 0.95 µM. The number of bis-ANS
binding sites nearly doubled upon heat treatment of the protein at
60 °C. Likewise, the exposure of
-crystallin to 2-3
M urea resulted in increased binding of bis-ANS. Above 3 M urea there was a rapid loss in the fluorescence
indicating the loss of interaction between bis-ANS and protein. The
-crystallin refolded from 6 M urea showed tryptophan fluorescence emission similar to the native
-crystallin. However, the refolded
-crystallin showed a 60% increase in bis-ANS binding, suggesting distinct changes on the protein surface resulting from exposure to urea similar to the changes occurring due to heat treatment. The fluorescence of tryptophan in native
-crystallin was
quenched by the addition of bis-ANS. The quenching was inversely related to the amount of bis-ANS bound to
-crystallin. Additionally, the binding of bis-ANS reduced the chaperone-like activity of the
protein. Photolysis of bis-ANS-
-crystallin complex resulted in
incorporation of the probe to both A- and B-subunits, indicating that
both subunits in native
-crystallin contribute to the surface hydrophobicity of the protein.
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INTRODUCTION |
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,
-, and
-crystallins constitute the major portion of the
eye lens fiber cells (1). Among the crystallins,
-crystallin is the
most abundant protein, existing as a polydisperse aggregate with the
average molecular mass of 800 kDa (2).
-Crystallin is made up of two
types of subunits, designated
A and
B with molecular masses
19,832 and 20,079 kDa, respectively (2). The sequences of the subunits
of
-crystallin have high homology to small heat shock proteins (3,
4).
-Crystallin subunits, once thought to be lens-specific, are now
widely known to be present in other tissues as well (5-8), and
increased expression of
B-crystallin has been documented in some
neurological disorders (6, 9, 10).
Recently, the ability of native -crystallin to suppress the
aggregation of heat-denatured (11-26), UV-irradiated (26, 27), and
chemically denatured (28) proteins and enzymes has been demonstrated.
Complex formation between
-crystallin and denatured proteins and
enzymes or
- and
-crystallins has been demonstrated (14, 18). On
the basis of these in vitro data, it has been proposed that
-crystallin acts as a chaperone in vivo to maintain the
lens clarity and that
-crystallin loses this ability during aging.
Consistent with this hypothesis, a decreased chaperone-like activity
has been observed for the
-crystallin present in high molecular mass
aggregates from aged bovine and human lens (29, 30).
It has been proposed that surface hydrophobic sites in the native
-crystallin aggregate are involved in binding of target proteins to
-crystallin during chaperone-like activity display (17). A direct
correlation between the extent of
-crystallin hydrophobicity and
chaperone-like activity has been demonstrated (31-34). Liang and
co-workers (35) in their recent study used recombinant
A- and
B-homopolymers and reported that the relative fluorescence
enhancement of ANS1 is
greater with
B compared with
A and concluded that
B has higher
hydrophobicity. However, so far the amino acid sequences that
contribute to the hydrophobic site(s) have not been identified. In a
recent report, Smulders and de Jong (36) described that the N-terminal
domain of recombinant murine
B-crystallin binds hydrophobic probe
bis-ANS. We have recently reported that amino acid residues 57-69 and
93-107 of
B-crystallins interact with heat-denaturing alcohol
dehydrogenase (37). Liang and Li (38) reported that there are about 40 ANS binding sites/native
-crystallin. Stevens and Augusteyn (39)
have disputed the study of Liang and Li and reported that there is one
ANS binding site/24 subunits of
-crystallins. It is rather difficult
to explain the stoichiometry of ANS binding to
-crystallin in view
of the proposed complex but ordered structure for
-crystallin
(2).
In the present study we have determined the binding of bis-ANS to
-crystallin by equilibrium dialysis. The data presented here show
the binding of bis-ANS to both A- and B-subunits of
-crystallin and
transfer of the energy from protein tryptophan to the bound
fluorophore. Furthermore, we show that prior binding of bis-ANS to
-crystallin can affect the chaperone-like activity.
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EXPERIMENTAL PROCEDURES |
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Materials--
bis-ANS was obtained from Molecular Probes, Inc.
(Junction City, OR). The stock solutions of bis-ANS were prepared in
95% alcohol, and the concentration was determined by absorbance at 385 nm using an extinction coefficient, 385 = 16,790 cm
1 M
1 (40). Ultra pure urea
was purchased from U. S. Biochemical Corp. Yeast alcohol dehydrogenase
(ADH) was obtained from Sigma. All other chemicals were of the
highest grade commercially available.
Preparation of -Crystallin--
-Crystallin was isolated
from young bovine lens cortex by gel filtration on Sephadex G-200 and
ion exchange chromatography on trimethylaminoethyl-fractogel column
(EM-Separations) as described earlier (21). The
-crystallin thus
obtained was >99% pure as judged by SDS-PAGE and used in this
study.
Equilibrium Binding of bis-ANS to -Crystallin--
Bovine
-crystallin (0.3 µM) was incubated with 0-40
µM bis-ANS in 50 mM sodium phosphate buffer,
pH 7.0, containing 100 mM NaCl (buffer A) for 1 h at
25 °C. The entire sample was then subjected to equilibrium dialysis
in microdialysis cells (Technilab Instruments Inc.) using 1.2-ml
volumes of
-crystallin and bis-ANS. Following equilibration for
24 h with stirring, the concentration of bis-ANS in each chamber
was measured by its absorbance at 385 nm. The number of bis-ANS binding
sites/
-crystallin was determined by Scatchard analysis (41).
Fluorescence Titration of bis-ANS Binding to
-Crystallin--
Fluorescence measurements were made on a
Perkin-Elmer Spectrofluorimeter model 650-40 with a 3600 data station.
The concentration of protein used was approximately 0.125 µM. The excitation and emission slit width were set to 4 nm. Samples with bis-ANS were excited at 390 nm, and the emission was
measured at 490 nm in a cuvette with a 1-cm path length or recorded
between 400 and 600 nm. Briefly, bis-ANS at several fixed
concentrations was mixed with
-crystallin, and the fluorescence was
measured. All the measurements were made at 25 °C. The fluorescence
intensities of the samples were corrected for the absorption of the dye
by the relation (42), Fcorr = Fobs antilog (ODex + ODem/2), where ODex and
ODem are the optical densities at excitation and
emission wavelengths, respectively. To see the effect of urea on
bis-ANS binding to
-crystallin, a known amount of protein was mixed
with 0-6 M urea in buffer A. After 14 h at 5 °C,
bis-ANS was added, and the fluorescence spectra was recorded as above.
Blanks without protein were prepared in same buffer.
Photoincorporation of bis-ANS into
-Crystallin--
-Crystallin (1 mg) was mixed with 100 µg of
bis-ANS in 1 ml of buffer A, and the excess bis-ANS was removed by
dialysis using buffer A. After dialysis the sample was taken in a glass
Petri dish. The Petri dish was kept on ice, and a hand-held UV lamp (UVL-56 from Ultra-violet Products, Inc., San Gabriel, CA) was placed
approximately 2 cm above the sample (45). The sample was illuminated
for 10 min with constant stirring. The sample was analyzed by HPLC and
SDS-PAGE after photolysis.
Separation of bis-ANS-labeled A- and
B-Crystallin--
The
-crystallin-bis-ANS complex was treated with 5 mM
dithiothreitol for 2 h and filtered. The
A- and
B-subunits
were separated from one another by HPLC using a C18 column (Vydac C18,
218TP1010 from The Separation Group, Hesparia, CA) and linear gradient
formed between 0.065% trifluoroacetic acid in water and 0.065%
trifluoroacetic acid in acetonitrile. The elution was monitored by
absorbance at 280 nm. All the fractions were tested for fluorescence
(excitation, 350 nm; emission, 450 nm) in a Perkin-Elmer
Spectrofluorimeter. The identities of
A- and
B-subunit peaks were
confirmed by SDS-PAGE and pooled separately. The fluorescence spectrum
of both the
A- and
B-crystallin fractions was obtained
(excitation, 390 nm).
Thermal Denaturation and Light Scattering Assay--
The
capacity of the bis-ANS treated -crystallin to protect against
heat-induced aggregation of ADH was determined according to the
procedure described earlier (19). Briefly, 400 µg of ADH was heated
in 50 mM PO4, pH 7.0, and 0.1 M
NaCl (buffer A) in the presence or the absence of different amounts of
the bis-ANS-
-crystallin in a final volume of 1.0 ml.
bis-ANS-
-crystallin was prepared by saturating bis-ANS binding sites
in
-crystallin at 48 °C and removing excess bis-ANS by dialysis.
The aggregation of proteins at the specified temperature was followed
by recording the increase in light scattering as a function of time in
a Perkin-Elmer Lambda 3 spectrophotometer equipped with multicell
transporter attached to a circulating water bath.
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RESULTS |
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Interaction of bis-ANS with -Crystallin--
bis-ANS is an
environment-sensitive probe that shows an emission maximum at 533 nm in
aqueous medium (46). When bis-ANS binds to a hydrophobic protein such
as
-crystallin, its fluorescence intensity increases severalfold,
and the emission maxima is blue shifted (32). Titration of
-crystallin (0.125 µM) with bis-ANS (0-10
µM) gave a hyperbolic plot as shown in Fig.
1, suggesting the saturation of bis-ANS
binding sites in
-crystallin. The titration data, when analyzed by a
double reciprocal plot (47), gave a Kd value of 0.95 µM for the binding of bis-ANS to
-crystallin.
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Effect of Heat Treatment on bis-ANS Binding to
-Crystallin--
Earlier reports have shown that interaction of ANS
or bis-ANS with
-crystallin is temperature-dependent
(31-34). When
-crystallin was incubated with excess of bis-ANS at
48 or 70 °C for 1 h and dialyzed to remove the unbound dye,
considerably higher amounts of bis-ANS remained bound to
-crystallin
compared with bis-ANS-
-crystallin incubated at 25 °C.
-Crystallin incubated at 48 °C was able to bind 80 ± 4 mol
of bis-ANS, whereas prior heat treatment of
-crystallin at 70 °C
resulted in binding of 110 ± 6 mol of bis-ANS. This translated to
2 and 2.7 binding sites/subunit of
-crystallin at 48 and 70 °C, respectively.
Effect of Urea on bis-ANS Binding to -Crystallin--
The
effect of varying concentration of urea on bis-ANS binding to
-crystallin is shown in Fig. 3.
Maximum dye binding to
-crystallin was observed in the presence of
3.0 M urea. The increase in bis-ANS fluorescence at low
urea concentrations and the sharp decrease in the fluorescence above 3 M urea suggest an initial exposure of the buried binding
sites at low urea concentration and the destruction of protein
quaternary, tertiary, and secondary structures at higher urea
concentration. The bis-ANS emission maximum also red shifted in samples
with urea (data not shown). Fig. 3 also shows the change in tryptophan
emission maximum at various concentrations of urea used. The graph
depicts a partial exposure of buried tryptophan fluorescence (maximum
emission, 346 nm) at 2.5 M urea concentration, which also
showed maximal bis-ANS fluorescence. In another experiment,
-crystallin that had been denatured and unfolded in 6 M
urea was allowed to refold and renature by dialysis and tested for
bis-ANS binding by equilibrium dialysis method at 25 °C. The
urea-denatured and renatured
-crystallin and the native
-crystallin, however, showed similar tryptophan emission maxima at
336 nm (data not shown). However, the
-crystallin exposed to urea
was found to bind 60% more bis-ANS after refolding compared with the
native
-crystallin (Fig. 4).
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Energy Transfer from Tryptophan to Bound bis-ANS--
Energy
transfer was detected from the overlap of the tryptophan fluorescence
emission spectrum of -crystallin and excitation spectrum of bis-ANS.
The emission spectra of
-crystallin and the
-crystallin-bis-ANS
complex when excited at 295 nm are shown in Figs.
5 and 6. In
the absence of bis-ANS, the fluorescence was emitted maximally at 336 nm due to the excitation of tryptophan residues in
-crystallin. Upon
addition of bis-ANS to
-crystallin there was a decrease in the
tryptophan fluorescence at 336 nm concurrent with an increase in the
fluorescence at 488 nm. Because free bis-ANS does not fluoresce when
excited at 295 nm, the 488 nm band represents emission due to
tryptophan-excited bis-ANS bound to
-crystallin. The isoemmissive
point was 420 nm. A Kd value of 1.4 µM
was obtained for the binding of bis-ANS by the analysis of tryptophan
quenching data (44). This value is not significantly different from the
Kd value obtained by equilibrium dialysis method. A
similar value was obtained when the tryptophan quenching data were
analyzed by a modified Stern-Volmer method (48, 49). The maximum
transfer efficiency of 0.9 was obtained when the quenching data were
analyzed by the method of Wallach et al. (50). The transfer
efficiency was also calculated by the method described by Stryer (43)
and found to be 0.83.
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Effect of bis-ANS Binding on Chaperone-like Activity of
-Crystallin--
The effect of prior binding of bis-ANS to
-crystallin on its chaperone-like activity is shown in Fig.
7. Only a marginal decrease in the
-crystallin chaperone-like activity was observed when tested with
ADH.
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bis-ANS Incorporation to -Crystallin--
It has been shown
that bis-ANS can be incorporated to specific hydrophobic sites in
proteins by UV activation and that the dye incorporated into the
proteins remains sensitive to the polarity of its general environment
(45). Furthermore, it has been shown that bis-ANS binding sequences in
molecular chaperones can be identified by photocross-linking of the dye
to the protein, peptide mapping, and sequencing (45). During this study
bis-ANS was initially allowed to bind to
-crystallin by the addition
of saturating amounts of the probe. The excess probe was removed by
dialysis. The
-crystallin-bis-ANS complex was photolyzed by UV-A
light (366 nm). Fig. 8 shows the SDS-PAGE
of the bis-ANS-
-crystallin complex subjected to a 10-min photolysis.
The fluorescence seen in the 20-kDa region (Fig. 8, lane 3,
left panel) was due to the covalently bound bis-ANS.
Lane 2 of Fig. 8 contains the unphotolyzed
-crystallin-bis-ANS complex. The
A- and
B-subunits of
photolyzed
-crystallin-bis-ANS complex were separated by HPLC.
SDS-PAGE of
A and
B is shown in Fig. 8 (lanes 4 and
5). Both lanes show fluorescence in
A- and
B-crystallin protein band region. These data suggest that both
A-
and
B-subunits may be contributing to the bis-ANS binding sites in
-crystallin.
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DISCUSSION |
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Although the presence of surface hydrophobic sites on
-crystallin has been known for a number of years (51), only recently has considerable interest been shown in the hydrophobic sites within
-crystallins because these sites have been implicated in
chaperone-like function of the protein (17, 31-36, 38, 39). There is,
however, disagreement with respect to the number of hydrophobic sites
available within
-crystallin as determined by the
environment-sensitive probe ANS (38, 39). We used bis-ANS instead of
ANS to determine the nature of hydrophobic sites in
-crystallin.
Consistent with the report published earlier (32), our data show that
-crystallin has hydrophobic sites capable of binding
environment-sensitive probe bis-ANS. bis-ANS binds to
-crystallin
very tenaciously. Unlike ANS-
-crystallin, dialysis of
bis-ANS-
-crystallin for 2 days in buffer A did not dissociate the
protein bound bis-ANS. We determined the affinity of bis-ANS to
-crystallin by three methods. Although the fluorescence titration method gave a Kd of 0.95 µM (Fig. 1),
the equilibrium dialysis method and the tryptophan quenching studies
gave Kd and Kd app
values of 1.15 µM (Fig. 2) and 1.4 µM (Fig. 6), respectively. The difference in dissociation constants determined by the three methods was not significant. These values are five to
eight times lower than the Kd value for ANS binding to
-crystallin reported recently (39).
We determined the number of bis-ANS binding sites/subunit of
-crystallin by equilibrium dialysis and measuring the absorbance of
bound bis-ANS at 385 nm rather than the fluorescence titration method
for the following reasons. The titration method is likely to give
incorrect values if all the binding sites are not homogenous. The
quantum yield of the dye bound at different sites in subunits may vary
significantly. It has also been shown recently that
A- and
B-crystallins have different ANS binding characteristics (35). From
earlier studies we know that there is no constant stoichiometry between
the two types of subunits (2). Therefore if we do not know the
contribution of each type of binding site to the total sites, a
reliable estimate of the total binding sites cannot be made by
fluorescence titration studies. In view of this an estimate of binding
sites determined earlier by fluorescence titration is likely to include
significant error. The equilibrium dialysis method we employed involves
the direct estimation of the bound dye on the basis of molar
absorbance. The absorbance of bis-ANS is not altered upon binding to
proteins. Our estimate of one bis-ANS binding site/
-crystallin
subunit at 25 °C is much higher than one ANS binding site/24
subunits estimated by titration method (39). The estimation of one
binding site/24 subunits of
-crystallin cannot be explained by any
of the proposed structural models (2) for the protein. The increase in
the bis-ANS binding sites at elevated temperature, the earlier
demonstration that
-crystallin displays higher chaperone-like
activity if exposed to elevated temperatures (32-34), and the
estimated stoichiometry of interactions between the target protein and
-crystallin during chaperoning (18) suggest that one to three
hydrophobic sites may be involved in binding a target protein.
Although we could not quantify the extent of A- and B-subunit
contribution to the total bis-ANS binding sites on -crystallin, the
photocross-linking experiments (Fig. 8) show that both
A- and
B-subunits in
-crystallin bind bis-ANS. By cross-linking studies
we have recently shown that both A- and B-subunits of
-crystallin
participate in chaperone-like activity display (37). As has been
reported for rat
B-crystallin (36), bovine
-crystallin also
showed a decrease in chaperone-like activity after complexing with
bis-ANS. Although only a partial loss in chaperone-like activity of
-crystallin was observed when it was complexed with bis-ANS (Fig.
7), the data support the hypothesis that hydrophobic sites in
A and
B are involved in chaperone activity. The high residual chaperone-like activity observed for bis-ANS-
-crystallin may be due
to the retention of the hydrophobic nature of the site subsequent to
the binding of the probe. We have also observed that glycated
-crystallin binds less ANS and displays lower chaperone-like activity. The residual chaperone-like activity of the glycated
-crystallin was directly proportional to the residual ANS
binding.2 These data suggest
the involvement of same site/amino acid residues in
-crystallin
during ANS binding, glycation, and chaperone-like activity.
The binding of ANS or bis-ANS to -crystallin also increases after
heat treatment (31-34), partial unfolding by urea (Fig. 3), or urea
denaturation and refolding (Fig. 4). Although earlier studies have
demonstrated that increased bis-ANS or ANS fluorescence is seen with
-crystallin at 25 °C that has been exposed once to higher
temperature (32-34), there are no reports on the number of newly
formed hydrophobic sites. Our data show that approximately 40 new
bis-ANS binding sites are formed per
-crystallin molecule when it is
heated to 48 °C. Retention of the additional bis-ANS sites by
-crystallin exposed to at higher temperatures (48 and 72 °C)
points to the inability of
-crystallin to regain its native structure after cooling. It is unlikely that in our experiments bis-ANS
was trapped in
-crystallin at higher temperature because experiments
where bis-ANS was added after cooling
-crystallin also showed
increased bis-ANS binding. The increased binding of bis-ANS to
urea-denatured and renatured
-crystallin suggests that although the
refolded
-crystallin displays intrinsic fluorescence similar to the
native protein as reported earlier (52), there is a measurable
difference with respect to hydrophobic sites between the native protein
and renatured protein (Fig. 4). Therefore caution should be exercised
in interpreting the recombinant
-crystallin chaperone-like activity
data if the protein isolation step involves urea.
The increased binding of bis-ANS to structurally perturbed
-crystallin reported here is similar to that observed with molecular chaperone GroEL (49, 53, 54), but the number of bis-ANS that can bind
to native or partially unfolded
-crystallin is greater. Although the
oligomeric GroEL (800 kDa), binds one or two bis-ANS molecules in its
native form and about 14 bis-ANS molecules in the presence of ~2.5
M urea (54), we estimated that 40 and 65 bis-ANS molecules
bind to
-crystallin in its native form and in the presence of 2.5 M urea, respectively. The high number of available surface
hydrophobic sites are probably responsible for the increased capacity
of
-crystallin to bind denaturing proteins compared with GroEL,
which is believed to bind one protein at a time (55).
The -crystallin A-subunit has one Trp (Trp-9), whereas the B-subunit
has two tryptophans (Trp-9 and 60) (2). The fluorescence emission
studies have shown that two tryptophans near the N terminus are
relatively buried and the other Trp is near the surface (56). The
fluorescence quenching studies reported here suggest that the bis-ANS
binding sites are relatively closer to the Trp. During our studies to
identify the
-crystallin amino acid sequences involved in
chaperone-like function, we observed that one of the binding sequences,
APSWIDTGLSEMR (37), contained the Trp-60 in
B-crystallin. The
sequence is also a hydrophobic sequence predicted by hydropathy plot
and deuterium exchange studies (57). Therefore we hypothesize that
Trp-60 of
B-crystallin forms the part of chaperone binding site as
well as bis-ANS binding site. While this work was in progress, Smulders
and de Jong (36) reported that the bis-ANS binding site is on the
N-terminal domain of the recombinant rat
B-crystallin. However, it
is not known whether the bis-ANS binding to rat
B-crystallin was
influenced by the urea used during the isolation of the recombinant
protein.
It remains to be determined which amino acid residues form the bis-ANS
binding site(s) in native, heat-treated, and refolded -crystallin.
Once the chaperone binding sites are identified in both A- and
B-subunits of
-crystallin, the extent of involvement of bis-ANS
binding sites in chaperoning can be determined.
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ACKNOWLEDGEMENT |
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We thank Dr. B. J. Ortwerth for helpful discussions on this project and critically reading the manuscript.
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FOOTNOTES |
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* This work was supported in part by a grant-in-aid from the Fight For Sight Research Division of Prevent Blindness America and Research to Prevent Blindness, Inc.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. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Mason Eye Inst., University of Missouri, Columbia, One Hospital Drive, Columbia, MO 65212. Fax: 573-884-4100; E-mail: opthks{at}showme.missouri.edu.
1 The abbreviations used are: ANS, 1-anilinonaphthalene-8-sulfonic acid; bis-ANS, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid; ADH, alcohol dehydrogenase; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.
2 K. K. Sharma, unpublished data.
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REFERENCES |
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