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The School of Biological Sciences, The University of Manchester, England
1Correspondence: The School of Biological Sciences, 3.239 The Stopford Building, The University of Manchester, Oxford Road, Manchester, M13 9PT 275, U.K. E-mail: Gordon.Lithgow{at}man.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: aging • nematode • metallothionein • insulin signaling • stress resistance • age genes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Although ROS primarily arise from normal metabolic
activities, there is considerable evidence they have a role in the
etiology of some age-related disease states (2)
.
Caenorhabditis elegans is proving to be an excellent
experimental system for the dissection of aging processes. In this
organism, the rate of aging is determined by multiple genetic pathways,
one of which also plays a role in the formation of the dauer larvae
(3
4
5)
. Dauer larvae develop from a specialized second
larval stage (L2d) produced in response to a neuroendocrine signaling
pathway that transduces information on temperature, bacteria
concentration in the immediate environment, and the concentration of a
pheromone produced by adult and larval worms. Many genes, acting in
partially redundant pathways, control dauer formation (daf
genes).
A subset of daf genes associated with the insulin-like
signaling pathway significantly alters the life span of adult worms.
For example, mutations of age-1, daf-2, or daf-28
extend life span by up to 100% (3
, 5
, 6)
. This increased
life span is known as the Age phenotype. The daf-2 gene
encodes a protein with structural similarity to the mammalian insulin
receptor and insulin-like growth factor receptor (7)
whereas the age-1 encodes a catalytic subunit of
phosphatidylinositol-3-OH kinase (PI3K; 8
), an element
downstream from the insulin-like receptor. Mutations of either
daf-16 or daf-18 suppress both the Age phenotype
and Daf-c phenotype (dauer formation-constitutive) of age-1
and daf-2 mutations (4
, 5
, 9
10
11)
. The
daf-16 gene encodes a forkhead transcription factor that can
be suppressed by wild-type function of the insulin-like receptor
signaling pathway (12
, 13)
. The daf-18 gene has
been reported to encode a protein similar to the human PTEN protein
that has phosphatidylinositol 3,4,5-trisphosphate (PIP3) 3-phosphatase
activity (14
, 15)
, thereby possessing antagonist actions
to that of the age-1 gene.
Despite these findings, the mechanism of life span extension associated
with reduction of this pathway’s function is unknown. However,
mutations of components in this pathway also confer resistance to heat
stress (16)
, UV (17)
, and ROS
(18
19
20)
. We have proposed that life span extension
results at least in part from the coordinate overexpression of
stress-response genes (16
, 21)
. Because heavy metals
play an important role in ROS generation in biological systems and
heavy metal exposure can induce stress-related physiology associated
with aging processes (22)
, we were interested in whether
heavy metal resistance was also regulated by the insulin-like signaling
pathway. We have examined responses to both cadmium and copper ions as
they have been implicated in the generation of ROS and the subsequent
damage to proteins (23
, 24)
and DNA (25
, 26)
.
Metallothioneins are small cysteine-rich, metal binding proteins that
are implicated in protection against heavy metal toxicity
(27)
and ROS-associated damage (28)
.
Metallothionein-encoding genes are induced in response to a wide
variety of stresses involving metal ions, inflammation,
glucocorticoids, or oxidative stress (29)
. C.
elegans has two forms of metallothioneins. Metallothionein-1 (MT1)
is constitutively expressed in three cells of the posterior bulb of the
pharynx but is also induced by Cd and heat in intestinal cells. MT2
mRNA is not expressed under basal conditions and occurs only in
intestinal cells, where it is induced by Cd (30)
. The
existence of constitutive and inducible metallothioneins has been
reported in other organisms (31
32
33)
and thus may be a
conserved feature across diverse phyla.
Therefore, with a view to testing the hypothesis that Age genes regulate stress response genes, we have examined whether DAF-2 and AGE-1 determine resistance to Cd and Cu ions and influence the expression of metallothionein genes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Because
of the possible age influence on toxicity and gene expression,
age-synchronous populations were used. These synchronous populations
were prepared by sodium hypochlorite treatment, which kills adult worms
but not their eggs (35)
. The following strains were used:
wild-type C. elegans N2 (var. Bristol); TJ1052
[age-1(hx546)]; CB1370 [daf-2(e1370)]; DR26
[daf-16(m26)]; TJ3500 [daf-16(m26);
age-1(hx546)]; CB1372 [daf-7(e1372)]. All strains
were supplied by the Caenorhabditis Genetics Center
(University of Minnesota) except for TJ3500, which was a gift from
Thomas Johnson (University of Colorado at Boulder).
Cadmium and copper toxicity assays
Age-synchronous populations were prepared by transferring eggs
from a mixed population to a freshly seeded plate. Then, ten 3-day-old
adult hermaphrodites were transferred from NGM plates into Costar
24-well tissue culture plates containing 1 ml of K medium (53 mM NaCl,
32 mM KCl; 36) and Cd (6–24 mM) or Cu (1–8 mM) chloride per well.
Metal ion solutions were prepared from the appropriate chloride salts
using K medium as the diluent. Worms were incubated at 20°C for
24 ± 1 h and the number of dead worms was determined by the
absence of touch-provoked movement when probed with a platinum wire.
The tests were performed between three and nine times for each
concentration.
Cadmium and copper treatment for mRNA expression determination
After the sodium hypochlorite treatment of fertile adults,
synchronous L1 populations were put on 9 cm NGM plates and grown for
3–3.5 days until the worms developed into egg-laying adults. In one
series of experiments, the nematodes were placed into S medium (0.1 M
NaCl, 0.05 M K2PO4 of pH 6,
10 mM potassium citrate, 3 mM CaCl2, 3 mM
MgCl2, 50 µM EDTA, 25 µM
FeSO4, 10 µM MnCl2, 10
µM ZnSO4, 1 µM CuSO4,
supplemented with 5 mg/l cholesterol) containing OP50 with CdCl (0.1
mM) or CuCl (0.03 mM or 0.1 mM) or without the metal salt. In a second
series, the nematodes were transferred into K medium containing 10 mM
CdCl2 or 3 mM CuCl2 without
bacteria. For both sets of experiments, the worms were incubated at
20°C at 200 rpm in 300 ml flasks for 8 h. Then the worms were
recovered by centrifugation, washed, and live worms separated by
sucrose flotation (37)
. Approximately 1 g of
unexposed live worms was recovered and immediately frozen on dry ice
and stored at -80°C.
Competitive reverse transcriptase PCR
Precise quantification of mRNA can be achieved using
quantitative reverse transcriptase (RT) polymerase chain reaction (PCR)
where target template RNA, together with mimic RNA (which share the
same primer annealing sites but are different in length) are subjected
to cDNA synthesis and PCR. The protocols were established using general
principles of competitive RT-PCR (38)
and quantitative PCR
protocols for metallothionein (MT) determination in rat prostate tissue
(39)
. C. elegans genomic MT1 and MT2 fragments
containing introns of 120 and 57 base pairs (30)
,
respectively, were used as mimics. Mimic mRNAs were added into the
reaction at the reverse transcription stage in order to control for the
cDNA synthesis efficiency as well as for the PCR.
Genomic DNA and mimic construction
Genomic metallothionein DNA fragments, used as mimics in
the quantitative RT-PCR, were initially amplified from genomic DNA. The
genomic DNA was extracted from 20 wild-type worms in 20 µl of 10 mM
TrisHCl (pH 8.3), 50 mM KCl, and 60 µg/ml proteinase K, followed by
incubation of the mixture at -70°C for 10 min, 60°C for 1 h,
and 95°C for 15 min (40)
. The PCR mixture contained
genomic DNA template, AmpliTaq DNA polymerase (Perkin Elmer, Norwalk,
Conn.) in the presence of a 1 mM Mg2+ buffer (pH
7.9), 200 µM dNTPs and 0.4 µM each primer: MT1 (forward,
5'-GAAATCATGGCTTGCAAGTGTG-3'; reverse, 5'-TTTAATGAGCCGCAGCAGTTCC-3') or
MT2 (forward; 5'-CTCAAAAATGGTCTGCAAGTGTG-3'; reverse,
5'-AATGAGCAGCCTGAGCACATTC-3'). The PCR was carried out in the GeneAmp
9600 PCR thermal cycler, (Perkin Elmer), using 25 cycles of 94°C
30 s, 53°C 1 min, 72°C 1 min. The resulting PCR fragments were
ligated into pGEM-T Easy vector (Promega, Madison, Wis.). The plasmids
were purified from JM109 cultures using miniprep columns (Qiagen,
Chatsworth, Calif.). The identities of MT1 and MT2 fragments were
confirmed by sequencing analysis using the Big Dye Terminator
sequencing method (Perkin Elmer).
cDNA synthesis
Total RNA was extracted from 0.5 g of frozen worms using
TRIzol Reagent (Gibco BRL, Grand Island, N.Y.) after an incubation at
65°C for 30 min to lyse the cuticle. The RNA was treated with DNase I
(Promega) and extracted with phenol/chloroform. The absence of the
genomic DNA was confirmed by subjecting a sample of total RNA to a PCR
using the MT1 and 2 primers described above.
The first-strand cDNA was synthesized was using 1 µg of total RNA, M-MuLV reverse transcriptase (Fermentas, Hanover, Md.), 1 mM dNTPs, 32 µg random hexamers (Promega), and 20u of RNasin (Promega) in a 20 µl volume. The mixture was incubated at 37°C for 1 h, after which the reaction was terminated by heating the mixture at 95°C for 5 min. The PCR was carried out as described above using 1 µl of the cDNA. The identities of the amplified fragments were confirmed by sequence analyses as described previously.
cRNA preparation
One microgram of the template plasmid containing metallothionein
genomic DNA fragments was transcribed using the Riboprobe in
vitro transcription system (Promega) according to the
manufacturer’s instructions. DNA templates were removed by DNase I
treatment at 37°C for 30 min. The cRNA was subsequently purified by
phenol/chloroform extractions and stored at -80°C. The absence of
DNA contamination was established by performing PCR on the cRNA using a
40 cycle reaction.
mRNA expression reaction
cDNA synthesis was achieved using 2 µg total RNA from either
the metal ion-treated or the nontreated control worms, using the MT1
and 2 cRNAs as internal standards in the reaction. Initially, either
1.63 fM MT2 or 1.46 fM MT1 mimic cRNA and 0.2 µg of random hexamers
(Promega) were incubated at 70°C for a 5 min period. This was
followed by the addition of 40u M-MuLV reverse transcriptase and its
buffer (Fermentas) and 30u of ribonuclease inhibitor (Fermentas). The
reaction was then incubated at 25°C for 15 min, 37°C 1 h, and
95°C for 5 min and subsequently diluted to 50 µl. The PCR performed
in a 50 µl volume contained 10 µl of this diluted cDNA synthesis
reaction, AmpliTaq DNA polymerase and its buffer (1 mM
Mg2+, pH 7.9), 800 µM dNTPs, and 1 µM each of
the MT1 or MT2 primer set. The thermal cycling program was set at
94°C for 30 s, 54°C for 60 s, and 72°C for 45 s.
However, because previous experimentation established that
amplification of the products is not linear after 27–30 cycles, the
PCRs were carried out using 23 cycles. The resulting PCR amplification
products were visualized by ethidium bromide in a 1.5% agarose gel and
quantified using a ChemImager 4000i with the 3.3b program. The amount
of unknown template RNA was calculated from the ratio of template/mimic
band intensities as the amount of mRNA in attomoles (aM) per µg of
total RNA.
Statistical analysis
The lethal concentrations (LC50s) from the
toxicity assays were determined using a Probit transformation (SPSS).
The standard errors were calculated according to Finney
(41)
. Significant differences between the curves were
determined using a one-way ANOVA with a Newman-Keuls post test. As the
metallothionein mRNA levels were not expected to follow a Gaussian
distribution, the Kruskal-Wallis test was applied with Dunn’s post
test for nonparametric comparison of interstrain metallothionein level
differences. With the exception of the probit transformations, all
analyses were performed using Prism (ver 2.01) software (Graphpad
Software, San Diego, Calif.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The LC50 values for
daf-2 (23.1±0.7 mM) and age-1 (15.9±0.33 mM)
mutants were significantly (P<0.001) greater than the
values of the wild-type (10.7±0.24 mM)(Fig. 1B
). The Cd
LC50 value for daf-16 mutant (10 mM)
was significantly (P<0.05) lower than that for
daf16;age-1 double mutant (13.6±0.36 mM).
LC50 values for Cd of all strains tested (except
daf-16) were significantly (P<0.05) different
from that of the wild-type animals. This included a mutation in the
daf-7 gene that affects TGF-ß signaling. This mutant was
tested as it is Daf-c (but is non-Age) and hence is a critical test of
any correlation between heavy metal resistance and Age. The 95%
confidence intervals (CI) of the LC50 values for
the daf-7 mutants as calculated by Probit overlapped with
the CI of the wild-type, daf-16 mutants, and
daf-16;age-1 double mutants, suggesting a less robust
difference between daf-7 mutant and wild-type than that
observed for age-1 and daf-2 mutants. Only the
daf-2 and age-1 mutants possessed clearly
elevated resistance to Cd ions.
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Similarly, both daf-2 and age-1 mutants were more
resistant to Cu ions than the wild-type strain (Fig. 2A
). The LC50 values for
daf-2 mutants (6.1±0.14 mM) and age-1 mutants
(4.9±0.1 mM) were significantly (P<0.001) different from
wild-type (3.1±0.1 mM) and the rest of the mutants (Fig. 2B
). Although, the Cu LC50 for
daf-16 (3±0.36 mM) was lower than the Cu
LC50 for daf16;age-1 double mutant
(3.7±0.12 mM) (P<0.05), their Cu
LC50 95% confidence intervals overlapped. There
was also a significant difference (P<0.05) between
LC50 values of wild-type and
daf-16;age-1 mutants for Cu treatment. The daf-7
mutant was not resistant to Cu ions.
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To assess the relationship between the resistance to metal ions
(LC50 value) and longevity, regression analysis
was performed. There is a linear correlation between
LC50 values of Cd (r2=0.96)
and Cu (r2=0.94) obtained in this study of the
nematode strains and their previously published mean life spans [life
spans: daf-2, 42 days (3)
; wild-type, 20 days;
daf-16;age-1, 23 days; daf-16, 19 days
(17)
; daf-7, 20 days (4)
;
age-1, 43 days (42
].
Basal MT mRNA levels
MT mRNA levels were assessed in order to determine whether MT1 and
MT2 could be responsible for the increased resistance to Cd and Cu
ions. The toxicity bioassays indicated that daf-2 or
age-1 mutant strains were the most resistant to Cd and Cu
ions; hence, we used these mutants for MT mRNA analysis.
In wild-type worms, MT2 gene expression was not detected under basal
conditions, confirming a previously published report (30)
.
Similarly MT2 mRNA was not detected under basal conditions in
daf-2 and age-1 mutants (Fig. 3A
), and thus the basal expression of MT2 cannot account for
the differences in metal resistance. In contrast, low levels of the MT1
transcript were present in the wild-type (191.1±16.2 aM/g),
daf-2 (306.9±19.6 aM/g), and age-1 (261±12.4
aM/g) mutants under the same conditions (Fig. 4
). daf-2 mutants showed a significantly greater (1.6-fold;
P<0.005) MT1 mRNA expression than wild-type worms as
established by a Kruskal-Wallis analysis using Dunn’s post hoc test.
MT1 mRNA levels in age-1 mutants tended to be greater than
those observed in wild-type animals, though not significantly different
from either daf-2 or wild-type (Fig. 4)
. This suggests that
elevated constitutive levels of MT1 may contribute to the previously
observed metal resistance.
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Induction of MT mRNA by cadmium and copper
Initially the three strains of C. elegans were exposed
to an intermediate subtoxic Cd ion concentration of 0.1 mM, as this
concentration has been reported to be an effective inducer of MT
expression. After exposure to 0.1 mM Cd for 8 h, the level of the
MT1 mRNA increased in all strains tested (wild-type 916.6±63.6
aM/µg; daf-2 746.5±94.2 aM/µg, and 589.4±87.1 aM/µg
in age-1 mutants, Fig. 4
). The greatest increase (4.8-fold;
P<0.001) occurred in the wild-type worms. A similar
increase of fivefold under identical exposure conditions was found in
another study (37)
. Smaller but significant increases in
MT1 mRNA as compared to the basal levels of MT1 mRNA did occur in the
age-1 (2.3-fold; P<0.001) and daf-2
(2.4-fold; P<0.001) mutants.
A similar induction profile for MT2 mRNA was observed in the three strains. The mean mRNA expression of wild-type worms after exposure to 0.1 mM Cd ions increased to 1001 ± 80.6 aM/µg total RNA compared to undetectable levels under basal conditions. Mean MT2 mRNA levels of 666.1 ± 35.6 and 740 ± 81 aM/µg total RNA were observed for age-1 and daf-2 mutants, respectively. RNA levels observed in each of three strains were not significantly different from each other.
Exposure of the worms to higher concentrations of Cd similarly caused
induction of the MT genes, but the resulting mRNA levels were lower
than those observed for the 0.1 mM Cd doses. After exposure to 10 mM Cd
ions, both MT1 (Fig. 4)
and MT2 (Fig. 3)
mRNAs were expressed to a
greater extent (P<0.01 and P<0.05,
respectively) in daf-2 mutants (396.9±32.7 aM/µg of MT1
and 429±16.5 aM/µg of MT2) than in wild-type (126.9±22.6 aM/µg of
MT1 and 209.3±22.2 aM/µg of MT2). The differences between MT levels
in age-1 mutants (197.6±11.6 aM/µg of MT1 and 299.9±37.6
aM/µg of MT2) and wild-type were not significant. The MT1 transcript
levels of 396.9 ± 32.7 aM/µg total RNA (Fig. 4)
in
daf-2 mutants upon exposure of 10 mM Cd were not
significantly different from the levels of 306.9 ± 19.6 aM/µg
total RNA observed under basal conditions and were 
50% of the
levels that occurred at 0.1 mM Cd.
MT1 and MT2 mRNA levels in 10 mM exposed age-1 mutants were
intermediate between wild-type worms and daf-2 mutants.
Exposure of wild-type, daf-2, and age-1 mutants
to 10 mM Cd resulted in MT1 and MT2 mRNA levels below that observed in
worms exposed to 0.1 mM Cd (Figs. 3
, 4)
. This is most likely due to a
transcription inhibitory activity of Cd ions (43)
.
Although we only used live animals for mRNA analysis, at 10 mM Cd 50%
of the wild-type nematodes were dead, but only 20% of
daf-2 and age-1 mutants were dead. Consequently,
there may be some selection for the more resistant wild-type worms in
these experiments.
Copper failed to induce MT2 transcription in any of the three strains,
suggesting that MT2 differences cannot account for the strain
differences in Cu resistance. In addition, MT1 mRNA levels 8 h
after exposure to all three Cu concentrations 0.03 mM, 0.1 mM, and 3 mM
were not significantly different from MT1 basal expression levels
(Fig. 5
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The transforming growth factor ß (TGF-ß) -mediated pathway appears
not to be involved in heavy metal resistance. In this study, the
daf-7 gene, which encodes for a TGF-ß-like ligand and also
controls dauer formation, appears to have no influence on metal
tolerance as evidenced by the lack of response shown by the
daf-7 mutant. The failure of daf-7 mutations to
confer heavy metal resistance indicates that resistance is regulated by
the insulin signaling pathway and argues against the possibility that
resistance to metals is due to a dauer-like cuticle in Daf-c worms.
Indeed, it is known that the main route of metal uptake in the worm is
through feeding (36)
and MTs are expressed only in
intestinal and pharyngeal cells (30)
, hence it is unlikely
that resistance would depend on cuticle properties. This scenario is
mirrored for increased resistance to oxidants, where daf-7
mutants are similar to wild-type (20)
A number of models have been proposed for the life span extension
exhibited by daf-2, age-1 and daf-28 mutants. All
of these models have relied on the highly pleiotropic nature of genes,
which influence life span. Perhaps the most influential model to date
is that longevity may result from adult expression of genes usually
specific for the dauer life cycle stage in Daf-c worms
(3)
. This model is consistent with the stress
protein-associated longevity proposed previously and with the oxygen
radical theory of aging generally (16
, 21)
. Our
observation of elevated constitutive levels of metallothionein mRNA is
consistent with studies of Vanfleteren (19)
and Larsen
(18)
, who found that daf-2 and age-1
possess higher specific activities of catalase and superoxide dismutase
(SOD) in old age. However, in our study, only 3-day-old adults were
used, and the previously mentioned authors did not find significant
differences in catalase and SOD activities at this age. Honda and Honda
(20)
reported that mRNA levels of SOD3, the mitochondrial
MnSOD, were elevated in young daf-2 mutant adults. Hence,
derepressed daf-16 may induce the expression of
dauer-specific genes in adults, including those encoding stress
proteins such as the metallothioneins. The C. elegans MT1
gene promoter (30)
contains an insulin response sequence
(D. Gems, personal communication). If this is a functional sequence, it
could mean that DAF-16 directly regulates mtl-1, encoding
the basally expressed form of metallothionein and possibly contribute
to increased metal resistance. Elevated levels of metallothioneins and
antioxidant enzymes would be expected to decrease ROS-associated damage
and hence extend life span. Consequently, metallothioneins should be
considered candidate factors in life span determination.
There is considerable evidence that metallothioneins can act to
ameliorate oxidative stress. For example, embryonic cells or transgenic
mice possessing a targeted disruption of both the MT-I and -II genes
are particularly sensitive to the cytotoxic effects of Cd (44
, 45)
and to ROS-generating agents (46)
. The yeast
Saccaromyces cerevisiae strain lacking Cu-Zn SOD is
sensitive to oxidative stress and shows a number of growth defects.
However, overexpression of metallothionein using the yeast or monkey
sequences in S. cerevisiae suppressed several of the growth
defects associated with the null mutant, suggesting that the
metallothioneins could functionally substitute for Cu-Zn SOD
(47)
. Moreover, mice lacking the Cu-Zn SOD gene display a
normal phenotype (48)
but show a 10-fold induction of MT-I
and -II. Other enzymes with antioxidant activity such as Mn SOD or
cytochrome c oxidase were not induced in these animals
(49)
.
Other factors potentially regulated by the insulin signaling pathway
may account for the differences observed in resistance. Glutathione has
a major role in Cd and Cu toxicity and the regulation of ROS by direct
scavenging or through GSH peroxidase/GSH system (50
51
52)
.
It was noticed that after treatment of C. elegans with
paraquat, the expression of GSH S-transferase was elevated
(53)
. Mammalian cell line HAC600, resistant to Cu,
possessed higher levels of both GSH and GSH peroxidase relative to
nonresistant cells (50)
. The resistance to Cd in another
cell line, A549, was due to GSH but not MT, SOD, or catalase
(54)
. Another possible class of factors are the
phytochelatins [thiolate peptides
(
-Glu-Cys)n-Gly)], which have been shown to
play a major role in metal detoxification in plants and fungi.
Sequences similar to those encoding phytochelatin synthase were found
in C. elegans genome (55)
. We have recently
shown that age-1 mutants also overaccumulate a small heat
shock, HSP-16, after stress, and this to may contribute to metal
resistance.
Therefore, we propose that the insulin-like signaling pathway regulates
HSP, MT, and other stress proteins and that these stress proteins in
part determine normal life span. Support for this idea comes from the
recent demonstration that synthetic catalytic compounds that mimic
antioxidant enzyme activities extend C. elegans life span,
suggesting that oxidative stress is a factor in determining the rate of
aging (56)
. It is likely that many genes, including stress
genes, are under the control of the insulin-like signaling pathway and
that additional candidate functions for longevity will emerge. The
effects of altering the expression of stress genes should provide a
test for this proposal.
Received for publication November 15, 1999.
Revision received September 8, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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S. Nemoto and T. Finkel Redox Regulation of Forkhead Proteins Through a p66shc-Dependent Signaling Pathway Science, March 29, 2002; 295(5564): 2450 - 2452. [Abstract] [Full Text] [PDF]
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