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Genet. Mol. Biol. vol.27 no.1 S?o Paulo
http://dx.doi.org/10.-00007
ANIMAL GENETICS
RESEARCH ARTICLE
Hepatic mRNA expression
and plasma levels of insulin-like growth factor-I (IGF-I) in broiler chickens
selected for different growth rates
Poliana Fernanda GiachettoI; Eduardo
Correa RiedelI; Jane Eyre GabrielI; Maria In&s Tiraboschi
FerroI; S&nia Marli Zingaretti Di MauroI; Marcos
MacariII; Jesus Aparecido FerroI
IUniversidade Estadual Paulista, Faculdade
de Ci&ncias Agr&rias e Veterin&rias, Departamento de Tecnologia,
Laborat&rio de Bioqu&mica e Biologia Molecular, Jaboticabal, SP,
IIUniversidade Estadual Paulista, Faculdade de Ci&ncias Agr&rias
e Veterin&rias, Departamento de Morfologia e Fisiologia Animal, Jaboticabal,
SP, Brazil
The hepatic expression and plasma concentrations
of IGF-I were investigated in three broiler chicken strains selected for different
growth rates (HP-Hubbard-Pettersen, a NN-Naked-neck, a
strain with an intermediate growth rate and a heterozygous genotype, and C-Caipira,
a slow growing crossbred strain). The chickens were studied at 1, 21 and 42
days of age and had free access to food throughout the study. Hepatic IGF-I
mRNA expression was assessed by dot blot analysis using a randomly labeled chicken
IGF-I cDNA as the probe and plasma IGF-I concentrations were assayed by radioimmunoassay.
The hepatic levels of IGF-I mRNA increased from 1 to 21 days of age in all strains,
with NN chickens showing a higher (p&&&0.05) IGF-I expression than
the other strains. Plasma IGF-I concentrations increased (p&&&0.05)
with broiler chicken age, but there were no significant differences among the
strains. These results indicate that despite differences in the growth rates
among the strains, the changes in the expression of IGF-I mRNA in liver and
in the plasma levels of IGF-I were independent of broiler chicken strain, but
varied with chicken age.
Key words: IGF-I mRNA, broiler chicken,
growth performance, plasma IGF-I.
Introduction
Insulin-like growth factor-I (IGF-I) is a highly
conserved, 70 amino acid, single-chain polypeptide that plays an important role
in the control of growth and metabolism in chickens and mammals (Dawe et
al., 1988; Florini et al., 1996). Growth and differentiation are
stimulated when exogenous IGF-I is injected into chicken embryos (Girbau et
al., 1987), but after hatching, the gene expression and plasma concentration
of IGF-I increase with age and then decline between 6 and 7 weeks of age (Huybrechts
et al., 1985; Johnson et al., 1990; McGuinness and Cogburn, 1990).
The plasma levels of IGF-I vary with the nutritional state since chickens fed
a low protein diet have a low plasma content of this polypeptide (Rosebrough
et al., 1992; Rosebrough and McMurtry, 1993).
Although much is known about the mechanisms involved
in the synthesis and secretion of GH and its regulatory effect on IGF-I production,
various aspects about the role of GH and its interactions with its receptors
require clarification, especially in recently improved broiler chicken strains
selected for fast growth or food conversion. Contradictory data have been reported
about the positive correlation between IGF-I plasma levels and growth rate.
Scanes et al. (1989) reported higher IGF-I plasma levels in strains selected
for heavy body weight and Beccavin et al. (2001) found an association
between high growth rates and higher levels of hepatic IGF-I mRNA and circulating
IGF-I. However, Goddard et al. (1988) and Leenstra et al. (1991)
found no difference in plasma IGF-I levels in strains selected for food conversion
or weight gain.
In this work, we examined the hypothesis that
broiler chickens selected for fast growth have higher liver expression of IGF-I
mRNA and a higher plasma concentration of this polypeptide during growth.
Material and Methods
Selection, housing and management of chickens
Six hundred one-day-old male and female broiler
chicks from three strains (200 birds per strain) were used. The strains were:
Hubbard-Pettersen (HP), a commercial strain selected for fast growth, Naked-neck
(NN), a strain with the heterozygous genotype Nana selected for medium
weight gain and Caipira (C), a native crossbred strain with a very low growth
The birds were housed on the floor in separate
boxes at a temperature close to thermoneutrality for each age interval. Thus,
the initial temperature was 33 °C and decreased at a rate of 2 °C per week to
23 °C at 35 days of age. The birds were fed ad libitum with diets containing
3,100&kcal of metabolizable energy (ME)/kg that consisted of corn and soybean
meal with a vitamin-mineral supplement (). From
1-21 and 22-42 days of age, the birds were fed diets containing 21.8% and 19.8%
of crude protein (CP), respectively.
The growth curve for each strain was determined
by weighing the birds at 1, 7, 21, 35 and 42 days of age. Food intake was also
measured, and food conversion was calculated for each strain for the initial
(1-21 days), final (22-42 days) and total (1-42 days) periods.
Liver sampling
To assess IGF-I mRNA expression, three males
of each strain were sacrificed by cervical dislocation at 1, 21 and 42 days
of age and 1 gram of liver was quickly collected and frozen in liquid nitrogen.
The samples were stored at -80 °C until total RNA isolation.
Blood sampling and IGF-I assay
Blood samples (5 mL) from male chickens (n =
12) were obtained by venipuncture (brachial vein) using a heparinized syringe.
The blood was centrifuged at 3,000&rpm and 4 °C for 10 min. The plasma
was collected and stored at -20 °C until assayed for IGF-I. Following the precipitation
of plasma binding proteins (IGFBPs) with acidified ethanol, plasma IGF-I was
quantified using a heterologous radioimmunoassay (RIA) previously validated
for chicken plasma (Huybrechts et al. 1985).
For IGFBP precipitation, 400 mL
of an ethanol-HCl solution (2 N HCl, 95% ethanol) were added to a tube containing
100 mL of plasma. After vortex-mixing, the tubes
were incubated at room temperature for 30 min and then centrifuged at 3,000
x g for 30 min, at 4 °C. After centrifugation, an aliquot (200 mL)
of each supernatant was collected without disturbing the precipitates and was
thoroughly mixed with 80 mL of neutralizing solution
(0.855 M Tris) followed by incubation for 60 min at room temperature. After
a further centrifugation (3,000 x g, 60&min, 4&°C), a 100 mL
aliquot was removed for IGF-I quantification.
For RIA, 1 mg of recombinant
human IGF-I (hIGF-I) in 0,1% acetic acid was iodinated with 1 mCi [125I]
NaI (Amersham Biosciences) by adding chloramine T (0.1&mg/mL in 0.3 M sodium
phosphate buffer, pH 7.6) in a stepwise manner over a period of 4.5 min. The
iodination reaction was terminated by adding to the reaction mixture
150 mL of a solution containing 1.0 M NaI, 100 mM
KH2PO4, 0,1% BSA and 0.02% NaN3. Iodinated
peptide was separated from free 125I by chromatography on a Sephadex
G-50 column pre-equilibrated and eluted with 30&mM NaH2PO4
containing 0.25% BSA. The specific activity of the purified [125I]-hIGF-I
was ~ 320 mCi/mg. Sodium
phosphate buffer (30 mM, pH 7.5), containing 10 mM EDTA, 0.02% protamine sulphate
and 0.02% sodium azide was used to dilute the primary and secondary antibodies
and the hormone standard for the assay. Standard hormone (hIGF-I) and tracer
(125I-labelled hIGF-I) were initially dissolved, diluted and stored
in phosphate buffer containing 0,1% BSA. The RIA diluent was the phosphate buffer
described above containing 0.05% (v/v) Tween 20.
The assay was done under nonequilibrium conditions.
On day 1, RIA diluent (250 mL) plus a 50 mL
sample of the acid-ethanol extract and 100 mL of
tracer containing 125I-labelled hIGF-I (6,000 cpm) were added to
RIA tubes containing 100 mL of primary antibody (1:16,000
working dilution), followed by mixing and incubation for 24 h at room temperature.
On day 2, secondary antibody (100 mL of goat anti-rabbit
IgG diluted 1:25 in phosphate buffer) plus 100 mL
of carrier solution (49 mL of RIA buffer, 1 mL of normal rabbit serum, 1.5 g
of polyethylene glycol) were added to each tube, followed by vortex-mixing and
centrifugation at 2,000 x g for 30 min at 4 °C. Subsequently, 1 mL of 1 M NaCl
and 6% polyethylene glycol were added and incubated at 4 °C for 10 min followed
by centrifugation at 2,000 x g for 30 min. The resulting supernatant was aspirated
and the pellet counted in a g-counter. The intra-assay
coefficient of variation was 6.9%.
Dot blot analysis of IGF-I mRNA expression
Frozen hepatic tissue (1 g/bird) was homogenized,
and total RNA was isolated by the guanidinium thiocyanate method of Chomczynski
and Sacchi (1987). Total RNA was quantified by the absorbance at 260 nm and
the relative IGF-I mRNA content was determined by dot blot hybridization. Briefly,
20 mg of total RNA were dried under vacuum and dissolved
in 25 mL of 50% DMSO (dimethyl sulphoxide), 10 mM
sodium phosphate, pH 7.0 and 1.0 M glyoxal. After incubation at 50 °C for 1
h, the samples were placed on dry ice. Two dilutions of 5 mg
and 10 mg of RNA were prepared in a final volume
of 400 mL of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
and dotted (Bio-Dot Microfiltration Apparatus, Bio-Rad) onto nylon membranes
(Hybond N, Amersham Pharmacia Biotech). The membranes were pre-hybridized for
2 h at 65 °C in a pre-hybridization solution (1% BSA fraction V, 7% sodium dodecyl
sulphate, 1 mM EDTA, 0.5 M sodium phosphate, pH 7.2), with 20 mL per 100 cm2,
and hybridized for 16 h at 65 °C with the same solution containing a randomly
labeled (Feinberg and Vogelstein, 1983) cDNA probe for chicken IGF-I (cloned
into the pGEM-3Z plasmid and kindly provided by Dr. Peter S. Rotwein (School
of Medicine, Washington University)). After hybridization, the membranes were
washed twice in wash A solution (0.5% BSA, 5% SDS, 1 mM EDTA, 40 mM sodium phosphate,
pH 7.2) at 65 °C for 30 min. Autoradiography was done using Kodak X-OMAT X-ray
film and Dupont Cronex Lightening Plus intensifying screens. For re-probing,
the membranes were washed in 10 mM Tris-HCl at 90 °C for 20 min and then re-hybridized
with a cDNA probe for rat 28S RNA under the same conditions as used for IGF-I
cDNA. The signals obtained in the dots were quantified by densitometry (GS 300
densitometer, Hoefer Scientific Instruments) and the values were normalized
according to the 28S RNA hybridization signal, used as an RNA quantity and quality
control for each sample. The densitometric values obtained were within the linear
range of the method.
Statistical analysis
The experiments were done using a split-plot
design with strains as parcels and age as sub-parcels. The data were analyzed
using the general linear model procedure (GLM) and means were compared by the
Tukey test using SAS software (SAS Institute, 2000).
shows the mean body
weight from 1 to 42 days of age for the three strains studied. As expected,
broiler chickens of the fast growing strain (HP) grew faster than the NN and
C strains (NN also grew faster than C). The hatching body weight was higher
(p&&&0.05) in HP broilers than in the other strains (HP&&&NN
= C). This difference could be attributed not only to genotype, but also to
the nutritional status and age of the chickens. From seven days of age, the
three strains differed in body weight (p&&&0.05), with HP birds
always being the heaviest, followed by NN and C birds.
The weight gain of the HP strain paralleled the
better food conversion values (), but only during
the initial phase (1-21 days), when HP had a significantly lower food conversion
than NN birds (1.50 vs. 1.65, p&&&0.05). During the final
phase (22-42 days), food conversion was not different (2.18 vs. 2.24,
p&&&0.05) between the HP and NN strains, but both were different
(p&&&0.05) from C birds.
shows the autoradiographic
signals of the dot blots for hepatic IGF-I mRNA and 28S cDNA, the latter used
as an internal control. The dots correspond to ages of 1, 21 and 42 days. The
relative densitometric values for the dots at the three different ages (expressed
as arbitrary units) are shown in . Regardless of
the broiler strain, hepatic IGF-I mRNA expression increased with age, from hatching
to 21 days old. From 21 to 42 days, a decrease (p&&&0.05) in IGF-I
mRNA expression was seen in the NN strain. A comparative analysis among strains
showed that NN had a much higher hepatic IGF-I mRNA expression than the other
two strains at the age of 21 days (NN&&&HP&&&C).
The plasma IGF-I concentrations for the three
strains at different ages are shown in . The broiler
strain did not affect the levels of circulating IGF-I but, as observed for IGF-I
mRNA expression, plasma IGF-I levels increased from 1 to 21 days of age with
no significant change thereafter (days 21 to 42).
Discussion
As expected, the HP and NN birds had better growth
performances than the C strain. At 42 days of age, body weight was significantly
different among the three strains, thus confirming the growth potential of the
genetically improved strains. Despite the increase in body weight, food conversion
was not different for HP and NN strains after 21 days. Thus, the regulatory
mechanisms involved in growth seem to be dependent on the genetic characteristics
of the strains and also on the age of the birds.
Burnside and Cogburn (1992) reported that the
hepatic expression of IGF-I mRNA in broiler chickens peaked at 28 days of age,
and Beccavin et al. (2001) found that high growth rate birds had a five-fold
increase in IGF-I expression from the first to sixth week of life. The plasma
concentration of IGF-I was reported to increase progressively until the third
week of age, and reached a plateau by the seventh week (McGuiness and Cogburn,
1990; Beccavin et al., 2001). As already observed by others (Kikuchi
et al., 1991; Burnside and Cogburn, 1992; Radecki et al., 1997;
Beccavin et al., 2001), the hepatic IGF-I mRNA expression and IGF-I plasma
levels in the present study also increased from hatching to day 21, with hepatic
expression increasing at least three-fold and plasma levels showing a six- to
seven-fold increase. Nevertheless, at 42 days of age, both the expression and
plasma IGF-I levels showed no significant increase (p&&&0.05) when
compared to 21 days, although other reports have indicated that this increase
persisted until six weeks of age (McGuiness and Cogburn, 1990; Burnside and
Cogburn, 1992; Beccavin et al. 2001).
The most interesting finding of this study was
that the patterns of increase in IGF-I expression and production were the same,
irrespective of the broiler chicken strain, except for the NN strain, which
had higher values at all ages, but also showed reduced IGF-I mRNA expression
from 21 to 42 days of age. Huybrechts et al. (1987) and Goddard et
al. (1988) observed no positive correlation between growth rate and absolute
plasma IGF-I levels in chickens. Our results also showed that there was no clear
correlation between the growth of the birds and hepatic IGF-I mRNA expression
and plasma IGF-I levels.
In mice, complete disruption of the liver IGF-I
gene in the post-natal period decreased the circulating IGF-I levels by 75%,
while the growth rate was normal (Yakar et al., 1999). According to Oudin
et al. (1998), there were no differences in the number, affinity or tyrosine
kinase activity of IGF-I receptors purified from the muscles of fast and slow
growth chickens at one to seven weeks of age. Thus, the difference in body weight
among strains may be related to the hypothalamic-pituitary feedback mechanism
that interferes with GH synthesis and release. This aspect requires further
investigation, as does the genetic variation in muscle IGF-I gene expression,
especially since a paracrine action of IGF-I on chicken muscle has been reported
(Duclos et al., 1999).
The temporal regulation of IGF-I expression in
broiler chickens of unrelated genotypes and different growth rates has been
studied (Goddard et al., 1988; Ballard et al., 1990; Johnson et
al., 1990), but no influence of strain was reported. In contrast, in work
with chickens of different genotypes and high or low growth rates, Scanes et
al. (1989) found that plasma IGF-I levels were lower in the low compared
to high weight strains. Becavin et al. (2001) also found that high growth
rate birds had higher levels of hepatic IGF-I mRNA and plasma IGF-I. Thus, the
increased hepatic IGF-I mRNA expression observed here, which was associated
with age but not strain, could be related to the complex regulatory mechanisms
of growth in birds. Liver GHR (growth hormone receptor) expression is low after
hatching, but increases with age (Burnside and Cogburn, 1992; Scanes et al.,
1996; Mao et al., 1998). In fast growing broilers, increased hepatic
GHR expression was observed between the first and the fourth week of age and
coincided with an increase in IGF-I mRNA expression and plasma IGF-I levels
(Leung et al., 1987). The subsequent decrease in plasma GH, also described
by McCann-Levorse et al. (1993), does not appear to interfere with the
hepatic GHR since the number and affinity of these receptors compensates for
the low level of circulating GH, thereby maintaining the steady-state expression
of IGF-I mRNA.
These results show that the ontogenetic pattern
of hypothalamic-pituitary axis activity after hatching is apparently independent
of broiler chicken strain, despite the magnitude of the difference in their
growth response. Together with previous reports (Leili et al. 1997; Beccavin
et al. ), our findings indicate that additional temporal regulatory
mechanisms related to nutrition and age are involved in modulating the expression
of hepatic IGF-I mRNA and the consequent increase in plasma IGF-I levels.
Acknowledgments
This work was supported by grants from FAPESP,
PADCT and CNPq. E. C. Riedel was supported by a master's fellowship from CNPq.
References
Ballard FJ, Johnson RJ, Owens PC, Francis GL,
Upton FM, McMurtry JP and Wallace JC (1990) Chicken insulin-like growth factor-I:
amino acid sequence, radioimmunoassay, and plasma levels between strains and
during growth. Gen Comp Endocrinol 79:459-468.&&&&&&&&[  ]Beccavin C, Chevalier B, Simon J and Duclos MJ
(1999) Circulating insulin-like growth factors (IGF-I and II) and IGF binding
proteins in divergently selected fat or lean chickens: effect of prolonged fasting.
Growth Horm IGF Res 9:187-194.&&&&&&&&[  ]Beccavin C, Chevalier B, Cogburn LA, Simon J
and Duclos MJ (2001) Insulin-like growth factors and body growth in chickens
divergently selected for high and low growth rate. J Endocrinol 168:297-306.&&&&&&&&[  ]Burnside J and Cogburn LA (1992) Developmental
expression of hepatic growth hormone receptor and insulin-like growth factor-I
mRNA in the chicken. Mol Cell Endocrinol 89:91-96.&&&&&&&&[  ]Chomczynski P and Sacchi N (1987) Single-step
method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162:156-159.&&&&&&&&[  ]Dawe SR, Francis GL, McNamara PJ, Wallace JC
and Ballard FJ (1988) Purification, partial sequences and properties of chicken
insulin-like growth factors. J Endocrinol 117:173-181.&&&&&&&&[  ]Duclos MJ, Beccavin C and Simon J (1999) Genetic
models for the study of insulin-like growth factors (IGF) and muscle development
in birds compared to mammals. Domest Anim Endocrinol 17:231-243.&&&&&&&&[  ]Feinberg AP and Vogelstein B (1983) A technique
for radiolabeling DNA restriction endonuclease fragments to high specific activity.
Anal Biochem 132:6-13.&&&&&&&&[  ]Florini JR, Ewton DZ and Coolican SA (1996) Growth
hormone and the insulin-like growth factor system in myogenesis. Endocr Rev
17:481-517.&&&&&&&&[  ]Girbau M, Gomez JA, Lesniak MA and de Pablo F
(1987) Insulin and insulin-like growth factor I both stimulate metabolism, growth,
and differentiation in the postneurula chick embryo. Endocrinology 121:.&&&&&&&&[  ]Goddard C, Wilkie RS and Dunn IC (1988) The relationship
between insulin-like growth factor-1, growth hormone, thyroid hormones and insulin
in chickens selected for growth. Domest Anim Endocrinol 5:165-176.&&&&&&&&[  ]Huybrechts LM, King DB, Lauterio TJ, Marsh J
and Scanes CG (1985) Plasma concentrations of somatomedin-C in hypophysectomized,
dwarf and intact growing domestic fowl as determined by heterologous radioimmunoassay.
J Endocrinol 104:233-239.&&&&&&&&[  ]Huybrechts LM, Kuhn ER, Decuypere E, Merat P
and Scanes CG (1987) Plasma concentrations of growth hormone and somatomedin
C in dwarf and normal chickens. Reprod Nutr Dev 27:547-553.&&&&&&&&[  ]Johnson RJ, McMurtry JP and Ballard FJ (1990)
Ontogeny and secretory patterns of plasma insulin-like growth factor-I concentrations
in meat-type chickens. J Endocrinol 124:81-87.&&&&&&&&[  ]Kikuchi K, Buonomo FC, Kajimoto Y and Rotwein
P (1991) Expression of insulin-like growth factor-I during chicken development.
Endocrinology 128:.&&&&&&&&[  ]Leenstra FR, Decuypere E, Beuving G, Buyse J,
Berghman L and Herremans M (1991) Concentrations of hormones, glucose, triglycerides
and free fatty acids in the plasma of broiler chickens selected for weight gain
or food conversion. Br Poult Sci 32:619-632.&&&&&&&&[  ]Leili S, Buonomo FC and Scanes CG (1997) The
effects of dietary restriction on insulin-like growth factor (IGF)-I and II,
and IGF-binding proteins in chicken. Proc Soc Exp Biol Med 216:104-111.&&&&&&&&[  ]Leung FC, Styles WJ, Rosenblum CI, Lilburn MS
and Marsh JA (1987) Diminished hepatic growth hormone receptor binding in sex-linked
dwarf broiler and leghorn chickens. Proc Soc Exp Biol Med 184:234-238.&&&&&&&&[  ]Mao JNC, Burnside J, Postel-Vinay MC, Pesek JD,
Chambers JR and Cogburn, LA (1998) Ontogeny of growth hormone receptor gene
expression in tissue of growth-selected strains of broiler chickens. J Endocrinol
156:67-75.&&&&&&&&[  ]McCann-Levorse LM, Radecki SV, Donoghue DJ, Malamed
S, Foster DN and Scanes CG (1993) Ontogeny of pituitary growth hormone and growth
hormone mRNA in the chicken. Proc Soc Exp Biol Med 202:109-113.&&&&&&&&[  ]McGuinness MC and Cogburn LA (1990) Measurement
of developmental changes in plasma insulin-like growth factor-I levels of broiler
chickens by radioreceptor assay and radioimmunoassay. Gen Comp Endocrinol 79:446-458.&&&&&&&&[  ]Oudin A, Chevalier B and Simon J (1998) Muscle
insulin-like growth factor-I (IGF-I) receptors in chickens with high and low
body weight: effects of age and muscle fiber type. Growth Horm IGF Res 8:243-250.&&&&&&&&[  ]Radecki SV, Capdevielle MC, Buonomo FC and Scanes
CG (1997) Ontogeny of insulin-like growth factors (IGF-I and IGF-II) and IGF-binding
proteins in the chicken following hatching. Gen Comp Endocrinol 107:109-117.&&&&&&&&[  ]Rosebrough RW and McMurtry JP (1993) Protein
and energy relationships in the broiler chicken. Effects of protein quantity
and quality on metabolism. Br J Nutr 70:667-678.&&&&&&&&[  ]Rosebrough RW, McMurtry JP and Vasilatos-Younken
R (1992) In vitro lipid metabolism, growth and metabolic hormone concentrations
in hyperthyroid chickens. Br J Nutr 68:667-676.&&&&&&&&[  ]SAS Institute (2000). SAS (Statistical Analysis
System): users' Guide. SAS Institute, Cary, 496 pp.&&&&&&&&[  ]Scanes CG, Dunnington EA, Buonomo FC, Donoghue
DJ and Siegel PB (1989) Plasma concentrations of insulin like growth factors
(IGF-I) and IGF-II in dwarf and normal chickens of high and low weight selected
strains. Growth Dev Aging 53:151-157.&&&&&&&&[  ]Scanes CG, Radecki, SV and Aramburo C (1996)
Growth hormone and growth factors in avian development. Poult Avian Biol Rev
7:89-98.&&&&&&&&[  ]Yakar S, Liu JL, Stannard B, Butler A, Accili
D, Sauer B and LeRoith D (1999) Normal growth and development in the absence
of hepatic insulin-like growth factor I. Proc Natl Acad Sci USA 96:.&&&&&&&&[  ]&
Editor: Klaus Hartfelder
Correspondence to
Jesus Aparecido Ferro
Universidade Estadual Paulista
Faculdade de Ci&ncias Agr&rias e Veterin&rias
Departamento de Tecnologia
Via de Acesso Prof. Paulo Donato Castellane, s/n
Jaboticabal, SP, Brazil
Received: April 3, 2003; Accepted: September
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